Biotechnology and Bioengineering via MedWorm.com

Wall effects in continuous microfluidic magneto-affinity cell separation

Biotechnology and Bioengineering — Tue, 09 Mar 2010 00:00:00 +0100

We present a theoretical and experimental study that provides a quantitative description of hydrodynamic wall interactions and wall rolling velocity of cells. A transient convection model describes the transport of cells in two-phase microfluidic flow under the influence of an external magnetic field. Transport of cells along the microchannel walls is also considered via an additional equation. Results show the variation of cell flux in the fluid phases and the wall as a function of a dimensionless parameter arising in the equations. Our results suggest that conditions may be optimized to maximize cell separation while minimizing contact with the wall surfaces. Experimentally measured cell rolling velocities on the wall indicate the presence of other near-wall forces in addition to fluid s...

Analysis of viral clearance unit operations for monoclonal antibodies

Biotechnology and Bioengineering — Fri, 05 Mar 2010 00:00:00 +0100

We report that the diversity of model viruses tested expands as products transition to late-phase. We also present averages and ranges of viral clearance results by Protein A and ion exchange chromatography steps, low pH chemical inactivation, and virus filtration, focusing on retro- and parvoviruses. For most unit operations, an average log reduction value (LRV, a measure of clearance power) for retrovirus of >4 log10 were measured. Cases where clearance data fell outside of the anticipated range (i.e., outliers) were rationally explained. Lastly, a historical analysis did not find evidence of any improvement trend in viral clearance over time. The data collectively suggest that many unit operations in general can reliably clear viruses. Biotechnol. Bioeng. Published 2010 Wiley Periodical...

A process for microbial hydrocarbon synthesis: Overproduction of fatty acids in Escherichia coli and catalytic conversion to alkanes

Biotechnology and Bioengineering — Fri, 26 Feb 2010 00:00:00 +0100

The development of renewable alternatives to diesel and jet fuels is highly desirable for the heavy transportation sector, and would offer benefits over the production and use of short-chain alcohols for personal transportation. Here, we report the development of a metabolically engineered strain of Escherichia coli that overproduces medium-chain length fatty acids via three basic modifications: elimination of [beta]-oxidation, overexpression of the four subunits of acetyl-CoA carboxylase, and expression of a plant acyl-acyl carrier protein (ACP) thioesterase from Umbellularia californica (BTE). The expression level of BTE was optimized by comparing fatty acid production from strains harboring BTE on plasmids with four different copy numbers. Expression of BTE from low copy number plasmids...

3D bio-printing of multi-layered cell-laden fluidic hydrogel scaffold

Biotechnology and Bioengineering — Wed, 24 Feb 2010 00:00:00 +0100

No Abstract. (Source: Biotechnology and Bioengineering)

A site-specific gene amplification system on cellular genome

Biotechnology and Bioengineering — Wed, 24 Feb 2010 00:00:00 +0100

No Abstract. (Source: Biotechnology and Bioengineering)

Biosensor detects few hundred mycoplasma cells in cell culture

Biotechnology and Bioengineering — Wed, 24 Feb 2010 00:00:00 +0100

No Abstract. (Source: Biotechnology and Bioengineering)

Taming of the shrew - engineering the morphology of filamentous fungi for improved production performance

Biotechnology and Bioengineering — Wed, 24 Feb 2010 00:00:00 +0100

No Abstract. (Source: Biotechnology and Bioengineering)

Increasing the speed and throughput of recombinant molecular probe development

Biotechnology and Bioengineering — Fri, 19 Feb 2010 00:00:00 +0100

No Abstract. (Source: Biotechnology and Bioengineering)

A new look at bacterial adhesion and its effects on metabolic activity

Biotechnology and Bioengineering — Fri, 19 Feb 2010 00:00:00 +0100

No Abstract. (Source: Biotechnology and Bioengineering)

How strong are bacterial biofilms?

Biotechnology and Bioengineering — Fri, 19 Feb 2010 00:00:00 +0100

No Abstract. (Source: Biotechnology and Bioengineering)

Biotechnological process for achieving very low phosphate concentrations

Biotechnology and Bioengineering — Fri, 19 Feb 2010 00:00:00 +0100

No Abstract. (Source: Biotechnology and Bioengineering)

Reaching the depth of the Chinese hamster ovary cell transcriptome

Biotechnology and Bioengineering — Sat, 13 Feb 2010 00:00:00 +0100

The high-throughput DNA sequencing Illumina Solexa GAII platform was employed to characterize the transcriptome of an antibody-producing Chinese hamster ovary (CHO) cell line. More than 55 million sequencing reads were generated and mapped to an existing set of CHO unigenes derived from expressed sequence tags (ESTs), as well as several public sequence databases. A very significant fraction of sequencing reads has not been previously seen. The frequency with which fragments of a unigene were sequenced was taken as an estimate of the abundance level of the corresponding transcripts. A wide dynamic range of transcript abundance levels was observed, spanning six orders of magnitude. However, the distribution of coverage across transcript lengths was found to vary, from relatively uniform to h...

The effect of isolated lignins, obtained from a range of pretreated lignocellulosic substrates, on enzymatic hydrolysis

Biotechnology and Bioengineering — Tue, 09 Feb 2010 00:00:00 +0100

The influence of the residual lignin remaining in the cellulosic rich component of pretreated lignocellulosic substrates on subsequent enzymatic hydrolysis was assessed. Twelve lignin preparations were isolated by two isolation methods (protease treated lignin (PTL) and cellulolytic enzymatic lignin (CEL)) from three types of biomass (corn stover, poplar, and lodgepole pine) that had been pretreated by two processes (steam and organosolv pretreatments). Comparative analysis of the isolated lignin showed that the CEL contained lower amounts of carbohydrates and protein than did the PTL and that the isolated lignin from corn stover contained more carbohydrates than did the lignin derived from the poplar and lodgepole pine. The lower yields of acid insoluble lignin (AIL) obtained from the cor...

Disulfide trapping of protein complexes on the yeast surface

Biotechnology and Bioengineering — Tue, 02 Feb 2010 00:00:00 +0100

Protein complexes are common in nature and play important roles in biology, but studying the quaternary structure formation in vitro is challenging since it involves lengthy and expensive biochemical steps. There are frequent technical difficulties as well with the sensitivity and resolution of the assays. In this regard, a technique that can analyze protein-protein interactions in high throughput would be a useful experimental tool. Here, we introduce a combination of yeast display and disulfide trapping that we refer to as stabilization of transient and unstable complexes by engineered disulfide (STUCKED) that can be used to detect the formation of a broad spectrum of protein complexes on the yeast surface using fluorescence labeling. The technique uses an engineered intersubunit disulfi...

Development and testing of a novel microcantilever technique for measuring the cohesive strength of intact biofilms

Biotechnology and Bioengineering — Mon, 01 Feb 2010 00:00:00 +0100

Cohesive strength is an important parameter for understanding and modeling the mechanics of biomass detachment from bacterial biofilms. It is challenging to measure the mechanical properties of biofilms, however, because biofilms may desiccate when removed from liquid medium and they are inherently fragile. Poppele and Hozalski (Poppele and Hozalski, 2003, J Microb Methods 55:607-615) presented a microcantilever method for measuring the tensile strength of detached biofilm fragments while submersed in liquid medium. Here we present a modification of the microcantilever method to quantify the strength of intact bacterial biofilms. Initial testing was performed on Pseudomonas aeruginosa biofilms and on Staphylococcus epidermidis biofilms grown in rotating disk reactors. The cohesive strength...

Impact of recycling stillage on conversion of dilute sulfuric acid pretreated corn stover to ethanol

Biotechnology and Bioengineering — Fri, 29 Jan 2010 00:00:00 +0100

The objective of this study was to understand the impact of recycling stillage on conversion of corn stover to ethanol. Sugars in a dilute-acid pretreated corn stover hydrolysate were fermented to ethanol by the glucose-xylose fermenting bacteria Zymomonas mobilis 8b. Three serial fermentations were performed at two different initial sugar concentrations using either 10% or 25% of the stillage as makeup water for the next fermentation in the series. Serial fermentations were performed to achieve near steady state concentration of inhibitors and other compounds in the corn stover hydrolysate. Little impact on ethanol yields was seen at sugar concentrations equivalent to pretreated corn stover slurry at 15% (w/w) with 10% recycle of the stillage. However, ethanol yields became progressively ...

Anaerobic mixed-culture fermentation of aqueous ammonia-treated sugarcane bagasse in consolidated bioprocessing

Biotechnology and Bioengineering — Wed, 20 Jan 2010 00:00:00 +0100

The MixAlco process is an example of consolidated bioprocessing (CBP) in which anaerobic mixed-culture fermentation biochemically converts any biodegradable feedstock into carboxylate salts. Downstream processing thermochemically transforms the resulting salts into mixed alcohol fuels or gasoline. To enhance digestibility, sugarcane bagasse was treated under mild conditions (55°C, 24 h, and 30% aqueous ammonia solution with a loading of 10 mL/g dry biomass). Using NH4HCO3 buffer, the feedstock (80% ammonia-treated sugarcane bagasse/20% chicken manure) was anaerobically fermented by a mixed culture of marine microorganisms at 55°C. Four-stage countercurrent fermentations were performed at various volatile solids loading rates (VSLRs) and liquid residence times (LRTs). The highest acid pro...

Inhibitors of the Pseudomonas aeruginosa quorum-sensing regulator, QscR

Biotechnology and Bioengineering — Wed, 20 Jan 2010 00:00:00 +0100

QscR is a quorum-sensing (QS) signal receptor that controls expression of virulence genes in the prevalent opportunistic pathogen, Pseudomonas aeruginosa. Unlike the previously reported LuxR-type QS receptor proteins, that is, LasR and TraR, QscR can be obtained as an apo-protein that can reversibly form an active complex in vitro with its cognate signal molecule, 3-oxododecanoyl-homoserine lactone (3OC12-HSL), and subsequently bind to target promoter DNA sequences. To search for potential QS inhibitors, an in vitro gel retardation assay was developed using the purified QscR. Both the in vitro assay and the in vivo cell-based assay using QscR-overproducing recombinant strains were applied in the screening process. Furanones were chosen for testing the activity as QS inhibitors because they...

Photoregulated assembly/disassembly of DNA-templated protein arrays using modified oligonucleotide carrying azobenzene side chains

Biotechnology and Bioengineering — Wed, 20 Jan 2010 00:00:00 +0100

DNA-templated self-assembly of nanomaterials provides great potential for applications including biosensors, nanoelectronics, and programmable and autonomous molecular machines. To switch or regulate the activities of those nanobiotechnological devices, non-invasive methods to assemble and disassemble specific nanoscale components are needed. Here, we describe photocontrol of assembly of DNA-templated protein arrays in solution, by using photo-controlled duplex formation of oligonucleotides carrying azobenzene. As a proof of concept prototype, we designed a one-dimensional protein array system that consists of a scaffold of DNA and two kinds of anchor DNA that were conjugated with fluorescent proteins (CFP and YFP, respectively). The scaffold DNA was modified to carry multiple azobenzene s...

Enhancement of farnesyl diphosphate pool as direct precursor of sesquiterpenes through metabolic engineering of the mevalonate pathway in Saccharomyces cerevisiae

Biotechnology and Bioengineering — Wed, 20 Jan 2010 00:00:00 +0100

The mevalonate pathway in the yeast Saccharomyces cerevisiae was deregulated in order to enhance the intracellular pool of farnesyl diphosphate (FPP), the direct precursor for the biosynthesis of sesquiterpenes. Over-expression of the catalytic domain of HMG1, both from the genome and plasmid, resulted in higher production of cubebol, a plant originating sesquiterpene, and increased squalene accumulation. Down-regulation of ERG9 by replacing its native promoter with the regulatable MET3 promoter, enhanced cubebol titers but simultaneous over-expression of tHMG1 and repression of ERG9 did not further improve cubebol production. Furtheremore, the concentrations of squalene and ergosterol were measured in the engineered strains. Unexpectedly, significant accumulation of squalene and restoring...

A low shear stress modular bioreactor for connected cell culture under high flow rates

Biotechnology and Bioengineering — Wed, 20 Jan 2010 00:00:00 +0100

A generic "system on a plate" modular multicompartmental bioreactor array which enables microwell protocols to be transferred directly to the bioreactor modules, without redesign of cell culture experiments or protocols is described. The modular bioreactors are simple to assemble and use and can be easily compared with standard controls since cell numbers and medium volumes are quite similar. Starting from fluid dynamic and mass transport considerations, a modular bioreactor chamber was first modeled and then fabricated using "milli-molding," a technique adapted from soft lithography. After confirming that the shear stress was extremely low in the system in the range of useful flow rates, the bioreactor chambers were tested using hepatocytes. The results show that the bioreactor chambers c...

THIOMABs: How to decrease triple light chain (3LC) antibody formation?

Biotechnology and Bioengineering — Wed, 20 Jan 2010 00:00:00 +0100

No Abstract. (Source: Biotechnology and Bioengineering)

Novel access to an emerging biopolymer

Biotechnology and Bioengineering — Wed, 20 Jan 2010 00:00:00 +0100

No Abstract. (Source: Biotechnology and Bioengineering)

Systematic engineering for efficient arsenic removal

Biotechnology and Bioengineering — Wed, 20 Jan 2010 00:00:00 +0100

No Abstract. (Source: Biotechnology and Bioengineering)

Continuous long-term biotransformations using microbial biofilms

Biotechnology and Bioengineering — Wed, 20 Jan 2010 00:00:00 +0100

No Abstract. (Source: Biotechnology and Bioengineering)

Dissolved oxygen and pH profile evolution after cryovial thaw and repeated cell passaging in a T-75 flask

Biotechnology and Bioengineering — Wed, 20 Jan 2010 00:00:00 +0100

In this study, we show that on-line monitoring of dissolved oxygen (DO) and pH in a T-75 flask-based PSD can be achieved during cell passaging and that this information can be linked to different cellular metabolic states. In this case, on-line monitoring of DO and pH show three distinctive metabolic regions in passages 1-18, 19-28, 29-54 and in particular, the shift in the pH curve, the specific oxygen uptake rate (qO2), and the lactate production rate to the oxygen consumption rate yield (YLac/ox) confirm the existence of these distinctive metabolic regions. These findings are particularly useful because they show that sensor equipped PSDs can help to monitor cell culture behavior after thaw, in pre- and seed culture prior to scale-up and in development/optimization studies. Such routine...

Hyperosmotic stress induces autophagy and apoptosis in recombinant Chinese hamster ovary cell culture

Biotechnology and Bioengineering — Tue, 19 Jan 2010 00:00:00 +0100

During recombinant Chinese hamster ovary (rCHO) cell culture, various events, such as feeding with concentrated nutrient solutions or the addition of base to maintain an optimal pH, increase the osmolality of the medium. To determine the effect of hyperosmotic stress on two types of programmed cell death (PCD), apoptosis and autophagy, of rCHO cells, two rCHO cell lines, producing antibody and erythropoietin, were subjected to hyperosmotic stress resulting from NaCl addition (310-610 mOsm/kg). For both rCHO cell lines, hyperosmolality up to 610 mOsm/kg increased cleaved forms of PARP, caspase-3, caspase-7, and fragmentation of chromosomal DNA, confirming the previous observation that apoptosis was induced by hyperosmotic stress. Concurrently, hyperosmolality increased the level of accumula...

A sustainable pathway of cellulosic ethanol production integrating anaerobic digestion with biorefining

Biotechnology and Bioengineering — Mon, 18 Jan 2010 00:00:00 +0100

This report shows that AD also changes the composition of manure fiber and makes it suitable as a cellulosic feedstock for ethanol production. Anaerobically digested manure fiber (AD fiber) contains less hemicellulose (11%) and more cellulose (32%) than raw manure, and has better enzymatic digestibility than switchgrass. Using the optimal dilute alkaline pretreatment (2% sodium hydroxide, 130°C, and 2 h), enzymatic hydrolysis of 10% (dry basis) pretreated AD fiber produces 51 g/L glucose at a conversion rate of 90%. The ethanol fermentation on the hydrolysate has a 72% ethanol yield. The results indicate that 120 million dry tons of cattle manure available annually in the U.S. can generate 63 million dry tons of AD fiber that can produce more than 1.67 billion gallons of ethanol. Integrat...

Novel micro-bioreactor high throughput technology for cell culture process development: Reproducibility and scalability assessment of fed-batch CHO cultures

Biotechnology and Bioengineering — Wed, 13 Jan 2010 00:00:00 +0100

This study describes the use of the SimCellTM micro-bioreactor technology for fed-batch cultivation of a GS-CHO transfectant expressing a model IgG4 monoclonal antibody. Cultivations were conducted in gas-permeable chambers based on a micro-fluidic design, with six micro-bioreactors (MBs) per micro-bioreactor array (MBA). Online, non-invasive measurement of total cell density, pH and dissolved oxygen (DO) was performed. One hundred fourteen parallel MBs (19 MBAs) were employed to examine process reproducibility and scalability at shake flask, 3- and 100-L bioreactor scales. The results of the study demonstrate that the SimCellTM platform operated under fed-batch conditions could support viable cell concentrations up to least 12 × 106 cells/mL. In addition, both intra-MB (MB to MB) as well...

Functional comparison of hemoglobin purified by different methods and their biophysical implications

Biotechnology and Bioengineering — Wed, 13 Jan 2010 00:00:00 +0100

In this study, bovine and human Hb purified by tangential flow filtration (TFF) was compared to commercial preparations of human Hb (Hemosol, Inc., Toronto, Canada) and bovine Hb (Biopure, Inc., Cambridge, MA). Purified Hbs were characterized by measuring their overall purity (SDS-PAGE, SEC, and ESI-MS), susceptibility to oxidation (kox), responses to physiological conditions (pH, [Cl-], [IHP], and T), and ligand binding kinetics (O2, NO, and CO). All Hbs evaluated possessed comparable biophysical properties, however, a small amount of catalase was detected in the TFF-purified Hbs that reduced the rate of autoxidation. Mass changes observed by mass spectrometry suggest that structural alterations may be introduced into Hbs that are purified by extensive chromatographic separations. These r...

TCR-[beta] repertoire analysis of antigen-specific single T cells using a high-density microcavity array

Biotechnology and Bioengineering — Wed, 13 Jan 2010 00:00:00 +0100

In this study, we applied a high-density microcavity array enabling target cell screening of several thousands of single cells for identification of functional TCR-[beta] gene repertoires specific to melanoma (gp100) and cytomegalovirus (CMV) antigens. T cells expressing TCRs with the ability to recognize fluorescent-labeled antigen peptide tetramers were isolated by using a micromanipulator under microscopy. Regularly arranged cells on the microcavity array eased detection and isolation of target single cells from a polyclonal T-cell population. The isolated single cells were then directly utilized for RT-PCR. By sequencing the amplified PCR products, antigen-specific TCR-[beta] repertoires for gp100 and human cytomegalovirus antigens were successfully identified at the single-cell level....

Three-dimensional cell culture microarray for high-throughput studies of stem cell fate

Biotechnology and Bioengineering — Tue, 12 Jan 2010 00:00:00 +0100

We have developed a novel three-dimensional (3D) cellular microarray platform to enable the rapid and efficient tracking of stem cell fate and quantification of specific stem cell markers. This platform consists of a miniaturized 3D cell culture array on a functionalized glass slide for spatially addressable high-throughput screening. A microarray spotter was used to deposit cells onto a modified glass surface to yield an array consisting of cells encapsulated in alginate gel spots with volumes as low as 60 nL. A method based on an immunofluorescence technique scaled down to function on a cellular microarray was also used to quantify specific cell marker protein levels in situ. Our results revealed that this platform is suitable for studying the expansion of mouse embryonic stem (ES) cells...

Predictive modeling of non-viral gene transfer

Biotechnology and Bioengineering — Tue, 12 Jan 2010 00:00:00 +0100

We present a mathematical model of transfection, where a stochastic, two-step process is assumed, with the first being the low-probability entry step of complexes into the nucleus, followed by the subsequent release and activation of a small number of plasmids from a delivered complex. This conceptually simple model consistently predicts the observed fraction of transfected cells, the cotransfection ratio and the expression level distribution. It yields the number of efficient plasmids per complex and elucidates the origin of the associated noise, consequently providing a platform for evaluating and improving non-viral vectors. Biotechnol. Bioeng. © 2009 Wiley Periodicals, Inc. (Source: Biotechnology and Bioengineering)

Alteration of bacterial surface electrostatic potential and pH upon adhesion to a solid surface and impacts to cellular bioenergetics

Biotechnology and Bioengineering — Sat, 09 Jan 2010 00:00:00 +0100

In our previous study [Hong Y, Brown DG (2009) Appl Environ Microbiol 75(8):2346-2353], the adenosine triphosphate (ATP) level of adhered bacteria was observed to be 2-5 times higher than that of planktonic bacteria. Consequently, the proton motive force ([Delta]p) of adhered bacteria was approximately 15% greater than that of planktonic bacteria. It was hypothesized that the cell surface pH changes upon adhesion due to the charge-regulated nature of the bacterial cell surface and that this change in surface pH can propagate to the cytoplasmic membrane and alter [Delta]p. In the current study, we developed and applied a charge regulation model to bacterial adhesion and demonstrated that the charge nature of the adhering surface can have a significant effect on the cell surface pH and ultim...

Complex responses to culture conditions in Pseudomonas syringae pv. tomato DC3000 continuous cultures: The role of iron in cell growth and virulence factor induction

Biotechnology and Bioengineering — Thu, 07 Jan 2010 00:00:00 +0100

The growth of a model plant pathogen, Pseudomonas syringae pv. tomato DC3000, was investigated using a chemostat culture system to examine environmentally regulated responses. Using minimal medium with iron as the limiting nutrient, four different types of responses were obtained in a customized continuous culture system: (1) stable steady state, (2) damped oscillation, (3) normal washout due to high dilution rates exceeding the maximum growth rate, and (4) washout at low dilution rates due to negative growth rates. The type of response was determined by a combination of initial cell mass and dilution rate. Stable steady states were obtained with dilution rates ranging from 0.059 to 0.086 h-1 with an initial cell mass of less than 0.6 OD600. Damped oscillations and negative growth rates ar...

Generation of a triple-gene knockout mammalian cell line using engineered zinc-finger nucleases

Biotechnology and Bioengineering — Thu, 31 Dec 2009 00:00:00 +0100

Mammalian cells with multi-gene knockouts could be of considerable utility in research, drug discovery, and cell-based therapeutics. However, existing methods for targeted gene deletion require sequential rounds of homologous recombination and drug selection to isolate rare desired events - a process sufficiently laborious to limit application to individual loci. Here we present a solution to this problem. Firstly, we report the development of zinc-finger nucleases (ZFNs) targeted to cleave three independent genes with known null phenotypes. Mammalian cells exposed to each ZFN pair in turn resulted in the generation of cell lines harboring single, double, and triple gene knockouts, that is, the successful disruption of two, four, and six alleles. All three biallelic knockout events were ob...

High-level rapid production of full-size monoclonal antibodies in plants by a single-vector DNA replicon system

Biotechnology and Bioengineering — Thu, 31 Dec 2009 00:00:00 +0100

Plant viral vectors have great potential in rapid production of important pharmaceutical proteins. However, high-yield production of hetero-oligomeric proteins that require the expression and assembly of two or more protein subunits often suffers problems due to the "competing" nature of viral vectors derived from the same virus. Previously we reported that a bean yellow dwarf virus (BeYDV)-derived, three-component DNA replicon system allows rapid production of single recombinant proteins in plants (Huang et al., 2009. Biotechnol Bioeng 103: 706-714). In this article, we report further development of this expression system for its application in high-yield production of oligomeric protein complexes including monoclonal antibodies (mAbs) in plants. We showed that the BeYDV replicon system p...

Covalent dimerization of camelidae anti-human TNF-alpha single domain antibodies by the constant kappa light chain domain improves neutralizing activity

Biotechnology and Bioengineering — Thu, 31 Dec 2009 00:00:00 +0100

The tumor necrosis factor-alpha (TNF[alpha]) plays an important role in a number of chronic inflammatory disorders. Monoclonal camelidae variable heavy chain domain-only antibodies (VHH) have been developed to antagonize the action of human TNF[alpha] (anti-TNF-VHH). Here we describe a strategy to obtain functional dimeric anti-TNF-VHH molecules, based on the C-terminal fusion of a [kappa] light chain domain to the anti-TNF-VHH. The resulting fusion protein was transiently expressed by use of viral vectors in Nicotiana benthamiana(Nb) leaves and purified. Competitive ELISA and cell cytotoxicity assays revealed that the dimerized anti-NbTNF-VHHC[kappa] proteins blocked TNF[alpha]-activity more effectively than either the monomeric Escherichia coli(Ec) produced anti-EcTNF-VHH or the monomeri...

Influence of the hydrostatic pressure and pH on the asymmetric 2-hydroxyketone formation catalyzed by Pseudomonas putida benzoylformate decarboxylase and variants thereof

Biotechnology and Bioengineering — Thu, 31 Dec 2009 00:00:00 +0100

Benzoylformate decarboxylase (BFD) from Pseudomonas putida is a thiamine diphosphate-dependent (ThDP) enzyme that catalyzes the asymmetric C[bond]C bond formation to (S)-2-hydroxypropiophenone [(S)-HPP] starting from benzaldehyde and acetaldehyde. The enantioselectivity of BFD was shown to be a function of temperature and substrate concentration. It can additionally be changed by site-directed mutagenesis on hot spot positions in the active site. In this article, we present the effect of hydrostatic pressure up to 250 MPa on the enantioselectivity for the recombinant wtBFD as well as for the variants BFD F464I, BFD A460I, and BFD A460I-F464I. A general tendency toward lower amounts of (S)-HPP could be observed at increasing pressures. For two of these variants an increase in pressure even ...

Effects of culture conditions on N-glycolylneuraminic acid (Neu5Gc) content of a recombinant fusion protein produced in CHO cells

Biotechnology and Bioengineering — Mon, 28 Dec 2009 00:00:00 +0100

CHO cells express glycoproteins containing both the N-acetylneuraminic acid (Neu5Ac) and minor amounts of the N-glycolylneuraminic acid (Neu5Gc) forms of sialic acid. As Neu5Gc is not expressed in humans and can be recognized as a foreign epitope, there is the potential for immunogenicity issues for glycoprotein therapeutics. During process development of a glycosylated fusion protein expressed by CHO cells, a number of culture conditions were identified that affected the Neu5Gc content of the recombinant glycoprotein. Sodium butyrate (SB), a well-known additive reported to enhance recombinant protein productivity in specific cases, minimally affected product titers here, but did decrease Neu5Gc levels by 50-62%. A shift in culture temperature to a lower value after the exponential growth ...

Simultaneous saccharification and ethanol fermentation at high corn stover solids loading in a helical stirring bioreactor

Biotechnology and Bioengineering — Thu, 24 Dec 2009 00:00:00 +0100

In this study, a bioreactor with a novel helical impeller was designed and applied to the SSF operation of the steam explosion pretreated corn stover under different solids loadings and different enzyme dosages. The performances using the helical impeller and the common Rushton impeller were compared and analyzed by measuring rheological properties and the mixing energy consumption. The results showed that the new designed stirring system had better performances in the saccharification yield, ethanol titer, and energy cost than those of the Rushton impeller stirring. The mixing energy consumption under different solids loadings and enzyme dosages during SSF operation were analyzed and compared to the thermal energy in the ethanol produced. A balance for achieving the optimal energy cost be...

Constructing tissue constructs by assembling tissue building blocks on the surface

Biotechnology and Bioengineering — Wed, 23 Dec 2009 00:00:00 +0100

No Abstract. (Source: Biotechnology and Bioengineering)

Biofilm streamers and drag of flexible structures in flow

Biotechnology and Bioengineering — Wed, 23 Dec 2009 00:00:00 +0100

No Abstract. (Source: Biotechnology and Bioengineering)

HBOCs and PFCs synergistically oxygenate hollow fiber bioreactors

Biotechnology and Bioengineering — Wed, 23 Dec 2009 00:00:00 +0100

No Abstract. (Source: Biotechnology and Bioengineering)

A rapid way of tracking changes in cell wall polymers during biofuel production

Biotechnology and Bioengineering — Wed, 23 Dec 2009 00:00:00 +0100

No Abstract. (Source: Biotechnology and Bioengineering)

Mathematical modeling to aid in drug formulation

Biotechnology and Bioengineering — Mon, 21 Dec 2009 00:00:00 +0100

No Abstract. (Source: Biotechnology and Bioengineering)

DIGE approach to investigate the effects of Bcl-xL overexpression

Biotechnology and Bioengineering — Mon, 21 Dec 2009 00:00:00 +0100

No Abstract. (Source: Biotechnology and Bioengineering)

2D-DIGE: A new paradigm in profiling host cell proteins during bioprocess development

Biotechnology and Bioengineering — Mon, 21 Dec 2009 00:00:00 +0100

No Abstract. (Source: Biotechnology and Bioengineering)

Delivering high capacity, yield, and resolution

Biotechnology and Bioengineering — Mon, 21 Dec 2009 00:00:00 +0100

No Abstract. (Source: Biotechnology and Bioengineering)

Degassing-assisted patterning of cell culture surfaces

Biotechnology and Bioengineering — Sat, 19 Dec 2009 00:00:00 +0100

We developed an alternative patterning technique which is capable of producing both topographic and biochemical features for cell culture studies. This technique is based on microaspiration induced with a degassed poly (dimethylsiloxane) (PDMS) mold. After degassing in a rough vacuum chamber and placed on a sample surface, liquid solution can be aspired through channels and cavities created in the PDMS mold. Depending on the composition of the solution and the associated drying or incubation processes, a variety of surface patterns can be produced without applying external pressure. For demonstration, we fabricated agarose gel microwells and biomolecule patterns either on a glass plate or in a cell culture Petri dish, both applicable for cell culture studies. Biotechnol. Bioeng. © 2009 Wi...

Semi-permeable membrane retention of synovial fluid lubricants hyaluronan and proteoglycan 4 for a biomimetic bioreactor

Biotechnology and Bioengineering — Tue, 15 Dec 2009 00:00:00 +0100

The objectives were to analyze expanded polytetrafluoroethylene membranes with pore sizes of 50 nm, 90 nm, 170 nm, and 3 µm in terms of (1) HA and PRG4 secretion rates by adherent synoviocytes, and (2) the extent of HA and PRG4 retention with or without synoviocytes adherent on the membrane. Experiment 1: Synoviocytes were cultured on tissue culture (TC) plastic or membranes ± IL-1[beta] + TGF-[beta]1 + TNF-[alpha], a cytokine combination that stimulates lubricant synthesis. HA and PRG4 secretion rates were assessed by analysis of medium. Experiment 2: Bioreactors were fabricated to provide a BF compartment enclosed by membranes ± adherent synoviocytes, and an external compartment of nutrient fluid (NF). A solution with HA (1 mg/mL, MW ranging from 30 to 4,000 kDa) or PRG4 (50 µg/mL) w...

Hypoxia enhances proliferation of mouse embryonic stem cell-derived neural stem cells

Biotechnology and Bioengineering — Tue, 15 Dec 2009 00:00:00 +0100

Neural stem (NS) cells can provide a source of material with potential applications for neural drug testing, developmental studies, or novel treatments for neurodegenerative diseases. Herein, the ex vivo expansion of a model system of mouse embryonic stem (mES) cell-derived NS cells was characterized and optimized, cells being cultivated under adherent conditions. Culture was first optimized in terms of initial cell plating density and oxygen concentration, known to strongly influence brain-derived NS cells. To this end, the growth of cells cultured under hypoxic (2%, 5%, and 10% O2) and normoxic (20% O2) conditions was compared. The results showed that 2-5% oxygen, without affecting multipotency, led to fold increase values in total cell number about twice higher than observed under 20% o...

Influence of substrate stiffness on the phenotype of heart cells

Biotechnology and Bioengineering — Tue, 15 Dec 2009 00:00:00 +0100

Adult cardiomyocytes (CM) retain little capacity to regenerate, which motivates efforts to engineer heart tissues that can emulate the functional and mechanical properties of native myocardium. Although the effects of matrix stiffness on individual CM have been explored, less attention was devoted to studies at the monolayer and the tissue level. The purpose of this study was to characterize the influence of substrate mechanical stiffness on the heart cell phenotype and functional properties. Neonatal rat heart cells were seeded onto collagen-coated polyacrylamide (PA) substrates with Young's moduli of 3, 22, 50, and 144 kPa. Collagen-coated glass coverslips without PA represented surfaces with effectively "infinite" stiffness. The local elastic modulus of native neonatal rat heart tissue ...

Sensitive multiplex RNA quantification using capillary electrophoresis-based single-strand conformation polymorphism

Biotechnology and Bioengineering — Tue, 15 Dec 2009 00:00:00 +0100

Quantification of RNA provides information crucial for various biological studies, including analysis of mRNA expression and that of microRNAs. Reverse transcription (RT) coupled with real-time polymerase chain reaction (PCR) is known to be the most accurate method for quantifying nucleic acids, and thus represents the state-of-the-art for RNA quantification. However, the use of real-time PCR for RNA quantification is limited to a single target per analytical run because of reductions in quantification power and limitations of fluorescence dyes associated with multiplex applications. Here, we report a novel multiplex RNA quantification method that uses capillary electrophoresis single-strand conformation polymorphism (CE-SSCP) coupled with modified RT and asymmetric PCR. The reverse transc...

Industrial bioprocesses: Beyond routine applications of established methodologies

Biotechnology and Bioengineering — Fri, 11 Dec 2009 00:00:00 +0100

The subject matter of manuscripts by industrial authors has primarily focused on elements with perceived commercial or regulatory significance. Once published, this information interacted and ultimately influenced manuscripts from authors of other affiliations, creating the rapid advancements that culminated in the current multi-billion dollar worldwide biotechnology industry. This paper discusses trends in "solely industrial" articles published in the specific journal of Biotechnology and Bioengineering over the past five decades of this journal's lifetime. "Solely industrial" articles were defined as papers in which all the authors were affiliated with industry. Data were gathered concerning "solely industrial" article distribution and frequency, authoring companies, subject classificati...

Effect of cell lysates on retroviral transduction efficiency of cells in suspension culture

Biotechnology and Bioengineering — Fri, 11 Dec 2009 00:00:00 +0100

This study investigates the effect of cell lysis products, a common cell culture by-product, on the transduction of suspension cells by gammaretroviral vectors. Cell lysates derived from human and murine suspension cell lines significantly increased the transduction of human TF-1 and K-562 cell lines by gibbon ape leukemia virus-pseudotyped retroviral vectors without altering tropism. The transduction efficiency of TF-1 cells increased as a function of lysate concentration and decreased with increasing target cell concentrations. This was adequately predicted using a saturation equation based on the lysed-to-target cell concentration ratio, R, where:Lysate completely masked the effects of fibronectin when the two were added in combination. With protamine sulfate, the transduction efficienc...

Quantification of non-specific binding of magnetic micro- and nanoparticles using cell tracking velocimetry: Implication for magnetic cell separation and detection

Biotechnology and Bioengineering — Fri, 11 Dec 2009 00:00:00 +0100

The maturation of magnetic cell separation technology places increasing demands on magnetic cell separation performance. While a number of factors can cause sub-optimal performance, one of the major challenges can be non-specific binding of magnetic nano- or microparticles to non-targeted cells. Depending on the type of separation, this non-specific binding can have a negative effect on the final purity, the recovery of the targeted cells, or both. In this work, we quantitatively demonstrate that non-specific binding of magnetic nanoparticles can impart a magnetization to cells such that these cells can be retained in a separation column and thus negatively impact the purity of the final product and the recovery of the desired cells. Through experimental data and theoretical arguments, we ...

Sensitive and selective detection of mycoplasma in cell culture samples using cantilever sensors

Biotechnology and Bioengineering — Fri, 11 Dec 2009 00:00:00 +0100

This study shows that PEMC sensor can be used for sensitive and rapid mycoplasma detection in cell culture samples. Biotechnol. Bioeng. © 2009 Wiley Periodicals, Inc. (Source: Biotechnology and Bioengineering)

Enhancing glycoprotein sialylation by targeted gene silencing in mammalian cells

Biotechnology and Bioengineering — Fri, 11 Dec 2009 00:00:00 +0100

In this study, CHO cells overexpressing recombinant human interferon gamma (hIFN[gamma]) were treated using short interfering RNA (siRNA) and short-hairpin RNA (shRNA) to reduce expression of two newly identified sialidase genes, Neu1 and Neu3. By knocking down expression of Neu3 we achieved a 98% reduction in sialidase function in CHO cells. The recombinant hIFN[gamma] was examined for sialic acid content that was found to be increased 33% and 26% respectively with samples from cell stationary phase and death phase as compared to control. Here, we demonstrate an effective targeted gene silencing strategy to enhance protein sialylation using RNA interference (RNAi) technology. Biotechnol. Bioeng. © 2009 Wiley Periodicals, Inc. (Source: Biotechnology and Bioengineering)

Micropatterning contractile C2C12 myotubes embedded in a fibrin gel

Biotechnology and Bioengineering — Fri, 11 Dec 2009 00:00:00 +0100

Contractile C2C12 myotube line patterns embedded in a fibrin gel have been developed to afford a physiologically relevant and stable bioassay system. The C2C12 myotube/fibrin gel system was prepared by transferring a myotube monolayer from a glass substrate to a fibrin gel while retaining the original line patterns of myotubes. To endow the myotubes with contractile activity, a series of electrical pulses was applied through a pair of carbon electrodes placed at either side of a fibrin gel separately. The frequency and magnitude of myotube contraction were functions of the pulse frequency and duration, respectively. We found that the myotubes supported by an elastic fibrin gel maintained their line patterns and contractile activities for a longer period of time (1 week) than myotubes adher...

Micro-Raman spectroscopy of algae: Composition analysis and fluorescence background behavior

Biotechnology and Bioengineering — Mon, 07 Dec 2009 00:00:00 +0100

Preliminary feasibility studies were performed using Stokes Raman scattering for compositional analysis of algae. Two algal species, Chlorella sorokiniana (UTEX #1230) and Neochloris oleoabundans (UTEX #1185), were chosen for this study. Both species were considered to be candidates for biofuel production. Raman signals due to storage lipids (specifically triglycerides) were clearly identified in the nitrogen-starved C. sorokiniana and N. oleoabundans, but not in their healthy counterparts. On the other hand, signals resulting from the carotenoids were found to be present in all of the samples. Composition mapping was conducted in which Raman spectra were acquired from a dense sequence of locations over a small region of interest. The spectra obtained for the mapping images were filtered f...

Modeling predation processes in activated sludge

Biotechnology and Bioengineering — Mon, 07 Dec 2009 00:00:00 +0100

Predation by protozoa plays an important role in activated sludge. In this work, the kinetics for protozoan predation of active bacteria (XH), extracellular polymeric substances (EPS), and intracellular storage products (XSTO) are added into a previously expanded unified model that describes the dynamics of EPS, XSTO, and soluble microbial products (SMP). The new biomass growth-decay-predation model describes the biomass fractions, soluble organic components, and oxygen-uptake rates considering EPS, XSTO, and predators during dynamic operating conditions in activated sludge. Model calibration using batch experimental data provides the new parameter values for predation processes and insights into mechanisms involving predators. The calibrated value of the maximum specific growth rate for t...

Aerated Shewanella oneidensis in continuously fed bioelectrochemical systems for power and hydrogen production

Biotechnology and Bioengineering — Mon, 07 Dec 2009 00:00:00 +0100

We studied the effects of aeration of Shewanella oneidensis on potentiostatic current production, hydrogen production in a microbial electrolysis cell, and electric power generation in a microbial fuel cell (MFC). The potentiostatic performance of aerated S. oneidensis was considerably enhanced to a maximum current density of 0.45 A/m2 or 80.3 A/m3 (mean: 0.34 A/m2, 57.2 A/m3) compared to anaerobically grown cultures. Biocatalyzed hydrogen production rates with aerated S. oneidensis were studied within the applied potential range of 0.3-0.9 V and were highest at 0.9 V with 0.3 m3 H2/m3 day, which has been reported for mixed cultures, but is [sim]10 times higher than reported for an anaerobic culture of S. oneidensis. Aerated MFC experiments produced a maximum power density of 3.56 W/m3 at ...

Production of biocommodities and bioelectricity by cell-free synthetic enzymatic pathway biotransformations: Challenges and opportunities

Biotechnology and Bioengineering — Mon, 07 Dec 2009 00:00:00 +0100

Cell-free synthetic (enzymatic) pathway biotransformation (SyPaB) is the assembly of a number of purified enzymes (usually more than 10) and coenzymes for the production of desired products through complicated biochemical reaction networks that a single enzyme cannot do. Cell-free SyPaB, as compared to microbial fermentation, has several distinctive advantages, such as high product yield, great engineering flexibility, high product titer, and fast reaction rate. Biocommodities (e.g., ethanol, hydrogen, and butanol) are low-value products where costs of feedstock carbohydrates often account for [sim]30-70% of the prices of the products. Therefore, yield of biocommodities is the most important cost factor, and the lowest yields of profitable biofuels are estimated to be ca. 70% of the theore...

Multi-species nitrifying biofilm model (MSNBM) including free ammonia and free nitrous acid inhibition and oxygen limitation

Biotechnology and Bioengineering — Mon, 07 Dec 2009 00:00:00 +0100

A multi-species nitrifying biofilm model (MSNBM) is developed to describe nitrite accumulation by simultaneous free ammonia (FA) and free nitrous acid (FNA) inhibition, direct pH inhibition, and oxygen limitation in a biofilm. The MSNBM addresses the spatial gradient of pH with biofilm depth and how it induces changes of FA and FNA speciation and inhibition. Simulations using the MSNBM in a completely mixed biofilm reactor show that influent total ammonia nitrogen (TAN) concentration, bulk dissolved oxygen (DO) concentration, and buffer concentration exert significant control on the suppression of nitrite-oxidizing bacteria (NOB) and shortcut biological nitrogen removal (SBNR), but the pH in the bulk liquid has a weaker influence. Ammonium oxidation increases the nitrite concentration and ...

Potential hydrophobic interaction between two cysteines in interior hydrophobic region improves thermostability of a family 11 xylanase from Neocallimastix Patriciarum

Biotechnology and Bioengineering — Mon, 07 Dec 2009 00:00:00 +0100

In this study, we employed directed evolution and site-directed mutagenesis to screen thermostable mutants of a family 11 xylanase from Neocallimastix patriciarum, and found that the thermostability and specific activity are both enhanced when mutations (G201C and C60A) take place in the interior hydrophobic region of the enzyme. Far-ultraviolet circular dichroism analysis showed that the melting temperatures (Tm) of the G201C and C60A-G201C mutants are higher than that of the wild type by about 10 and 12°C, respectively. At 72°C, their specific activities are about 4 and 6 times as that of the wild type, respectively. Homology modeling and site-directed mutagenesis demonstrated that the enhanced thermostability of the G201C and C60A-G201C mutants may be mainly attributed to a potential ...

An accumulative site-specific gene integration system using cre recombinase-mediated cassette exchange

Biotechnology and Bioengineering — Mon, 07 Dec 2009 00:00:00 +0100

The Cre-loxP system is frequently used for site-specific recombination in animal cells. The equilibrium and specificity of the recombination reaction can be controlled using mutated loxPs. In the present study, we designed an accumulative site-specific gene integration system using Cre recombinase and mutated loxPs in which the Cre-mediated cassette exchange reaction is infinitely repeatable for target gene integration into loxP target sites. To evaluate the feasibility and usefulness of this system, a series of integration reactions were repeated and confirmed in vitro using Cre recombinase protein and plasmids. Accumulative gene integration was also performed on the genome of Chinese hamster ovary (CHO) cells. The results indicated that the system was applicable for repeated gene integra...

Metabolite stress and tolerance in the production of biofuels and chemicals: Gene-expression-based systems analysis of butanol, butyrate, and acetate stresses in the anaerobe Clostridium acetobutylicum

Biotechnology and Bioengineering — Mon, 07 Dec 2009 00:00:00 +0100

Metabolite accumulation has pleiotropic, toxic, or beneficial effects on cell physiology, but such effects are not well understood at the molecular level. Cells respond and adapt to metabolite stress by mechanisms largely unexplored, especially in the context of multiple and simultaneous stresses. Solventogenic and related clostridia have an inherent advantage for production of biofuels and chemicals directly from cellulosic material and other complex carbohydrates, but issues of product/metabolite tolerance and related culture productivities remain. Using DNA microarray-based gene expression analysis, the transcriptional-stress responses of Clostridium acetobutylicum to fermentation acids acetate and butyrate and the solvent product butanol were analyzed and compared in the context of cel...

Enhanced degradation of phenanthrene in a solid-liquid two-phase partitioning bioreactor via sonication

Biotechnology and Bioengineering — Mon, 07 Dec 2009 00:00:00 +0100

The current article examined the feasibility of inducing improved delivery and degradation of phenanthrene in a solid-liquid partitioning bioreactor system at bench scale by means of ultrasonic energy input. Initial degradation rates of phenanthrene by a microbial consortium, delivered from Desmopan, were improved 2.7-fold in the presence of sonication relative to unsonicated controls. Results demonstrated that an operating window involving on/off sonication cycling improved substrate delivery and rational selection of ultrasound cycling profiles could lead to even further enhancements. Additionally, all results were obtained in a conventional bioreactor with commercial ultrasonic equipment and a commercially available polymer. Subsequent DGGE analysis demonstrated that the sonication cycl...

Biohydrogen production from wheat straw hydrolysate by dark fermentation using extreme thermophilic mixed culture

Biotechnology and Bioengineering — Mon, 07 Dec 2009 00:00:00 +0100

Hydrolysate was tested as substrate for hydrogen production by extreme thermophilic mixed culture (70°C) in both batch and continuously fed reactors. Hydrogen was produced at hydrolysate concentrations up to 25% (v/v), while no hydrogen was produced at hydrolysate concentration of 30% (v/v), indicating that hydrolysate at high concentrations was inhibiting the hydrogen fermentation process. In addition, the lag phase for hydrogen production was strongly influenced by the hydrolysate concentration, and was prolonged from approximately 11 h at the hydrolysate concentrations below 20% (v/v) to 38 h at the hydrolysate concentration of 25% (v/v). The maximum hydrogen yield as determined in batch assays was 318.4 ± 5.2 mL-H2/g-sugars (14.2 ± 0.2 mmol-H2/g-sugars) at the hydrolysate concentrat...

A validated model of GAG deposition, cell distribution, and growth of tissue engineered cartilage cultured in a rotating bioreactor

Biotechnology and Bioengineering — Fri, 04 Dec 2009 00:00:00 +0100

In this work a new phenomenological model of growth of cartilage tissue cultured in a rotating bioreactor is developed. It represents an advancement of a previously derived model of deposition of glycosaminoglycan (GAG) in engineered cartilage by (i) introduction of physiological mechanisms of proteoglycan accumulation in the extracellular matrix (ECM) as well as by correlating (ii) local cell densities and (iii) tissue growth to the ECM composition. In particular, previously established predictions and correlations of local oxygen concentrations and GAG synthesis rates are extended to distinguish cell secreted proteoglycan monomers free to diffuse in cell surroundings and outside from the engineered construct, from large aggrecan molecules, which are constrained within the ECM and practic...

Creation of arrays of cell aggregates in defined patterns for developmental biology studies using dielectrophoresis

Biotechnology and Bioengineering — Tue, 01 Dec 2009 00:00:00 +0100

It is shown that dielectrophoresis - the movement of particles in non-uniform electric fields - can be used to create engineered skin with artificial placodes of different sizes and shapes, in different spatial patterns. Modeling of the electric field distribution and image analysis of the cell aggregates produced showed that the aggregation is highly predictable. The cells in the aggregates remain viable, and reorganization and compaction of the cells in the aggregates occurs when the artificial skin is subsequently cultured. The system developed could be of considerable use for the in vitro study of developmental processes where local variations in cell density and direct cell-cell contacts are important. Biotechnol. Bioeng. © 2009 Wiley Periodicals, Inc. (Source: Biotechnology and Bioe...

Morphology engineering of Aspergillus niger for improved enzyme production

Biotechnology and Bioengineering — Tue, 01 Dec 2009 00:00:00 +0100

Supplementation with silicate microparticles was used as novel approach to control the morphological development of Aspergillus niger, important as the major world source of citric acid and higher-value enzymes, in submerged culture. With careful variation of size and concentration of the micromaterial added, a number of distinct morphological forms including pellets of different size, free dispersed mycelium, and short hyphae fragments could be reproducibly created. Aluminum oxide particles similarly affected morphology, showing that this effect is largely independent of the chemical particle composition. Image analysis of morphological development of A. niger during the cultivation process showed that the microparticles influence the morphology by collision-induced disruption of conidia ...

A multichamber fluidic device for 3D cultures under interstitial flow with live imaging: Development, characterization, and applications

Biotechnology and Bioengineering — Tue, 01 Dec 2009 00:00:00 +0100

We present a nine-chamber radial flow device that allows simultaneous 3D fluidic experiments for relatively long-term culture with live imaging capabilities. Flow velocity profiles were characterized by fluorescence recovery after photobleaching (FRAP) for flow uniformity and estimating the hydraulic conductivity. We demonstrate lymphatic and blood capillary morphogenesis in fibrin gels over 10 days, comparing flow with static conditions as well as the effects of an engineered variant of VEGF that binds fibrin via Factor XIII. We also demonstrate the culture of contractile fibroblasts and co-cultures with tumor cells for modeling the tumor microenvironment. Therefore, this device is useful for studies of capillary morphogenesis, cell migration, contractile cells like fibroblasts, and multi...

On-demand three-dimensional freeform fabrication of multi-layered hydrogel scaffold with fluidic channels

Biotechnology and Bioengineering — Tue, 01 Dec 2009 00:00:00 +0100

We report a three-dimensional (3D) direct printing technique to construct hydrogel scaffolds containing fluidic channels. Cells can also be printed on to and embedded in the scaffold with this technique. Collagen hydrogel precursor was printed and subsequently crosslinked via nebulized sodium bicarbonate solution. A heated gelatin solution, which served as a sacrificial element for the fluidic channels, was printed between the collagen layers. The process was repeated layer-by-layer to form a 3D hydrogel block. The printed hydrogel block was heated to 37°C, which allowed the gelatin to be selectively liquefied and drained, generating a hollow channel within the collagen scaffold. The dermal fibroblasts grown in a scaffold containing fluidic channels showed significantly elevated cell viab...

Development of a bionanotechnological phosphate removal system with thermostable ferritin

Biotechnology and Bioengineering — Tue, 01 Dec 2009 00:00:00 +0100

Phosphate removal to ecologically desired levels of (Source: Biotechnology and Bioengineering)

Anoxic oxidation of arsenite linked to chemolithotrophic denitrification in continuous bioreactors

Biotechnology and Bioengineering — Tue, 01 Dec 2009 00:00:00 +0100

In this study, the anoxic oxidation of arsenite (As(III)) linked to chemolithotrophic denitrification was shown to be feasible in continuous bioreactors. Biological oxidation of As(III) was stable over prolonged periods of operation ranging up to 3 years in continuous denitrifying bioreactors with granular biofilms. As(III) was removed with a high conversion efficiency (>92%) to arsenate (As(V)) in periods with high volumetric loadings (e.g., 3.5-5.1 mmol As Lreactor-1 day-1). The maximum specific activity of sampled granular sludge from the bioreactors was 0.98 ± 0.04 mmol As(V) formed g-1 VSS day-1 when determined at an initial concentration of 0.5 mM As(III). The microbial population adapted to high influent concentrations of As(III) up to 5.2 mM. However, the As(III) oxidation process...

Effect of the size of yeast flocs and zinc supplementation on continuous ethanol fermentation performance and metabolic flux distribution under very high concentration conditions

Biotechnology and Bioengineering — Tue, 01 Dec 2009 00:00:00 +0100

Taking continuous ethanol fermentation with the self-flocculating yeast SPSC01 under very high concentration conditions as an example, the fermentation performance of the yeast flocs and their metabolic flux distribution were investigated by controlling their average sizes at 100, 200, and 300 µm using the focused beam reflectance online measurement system. In addition, the impact of zinc supplementation was evaluated for the yeast flocs at the size of 300 µm grown in presence or absence of 0.05 g L-1 zinc sulfate. Among the yeast flocs with different sizes, the group with the average size of 300 µm exhibited highest ethanol production (110.0 g L-1) and glucose uptake rate (286.69 C mmol L-1 h-1), which are in accordance with the increased flux from pyruvate to ethanol and decreased flu...

Flow cytometry-based methods for assessing soluble scFv activities and detecting antigens in solution

Biotechnology and Bioengineering — Tue, 01 Dec 2009 00:00:00 +0100

Novel methods are reported for evaluating and utilizing single chain fragment variable (scFv) antibodies derived from yeast-display libraries. Yeast-display was used to select scFv specific to invariant surface glycoproteins (ISG) of Trypanosoma brucei. A limiting step in the isolation of scFv from non-immune libraries is the conversion of highly active yeast-displayed scFv into soluble antibodies that can be used in standard immunoassays. Challenges include limited solubility or activity following secretion and purification of scFv. For this reason, few scFv derived from yeast-display platforms have moved into development and implementation as diagnostic reagents. To address this problem, assays were developed that employ both yeast-displayed and -secreted scFv as analytical reagents. The...

Effects from shear stress on morphology and growth of early stages of Norway spruce somatic embryos

Biotechnology and Bioengineering — Mon, 30 Nov 2009 00:00:00 +0100

The shear stress effect on directional expansion of pro embryogenic masses (PEMs) and suspensor cell development of somatic embryos of Norway spruce (Picea abies) at the proliferation stage was studied by a direct and quantitative image analysis system. The experimental system allowed for detailed observations of the effect of hydrodynamic shear stress in rotating and deforming liquid cultures of proliferating Norway spruce somatic embryos. Briefly, somatic embryos at an early development stage comprised only of clusters of meristematic cells without suspensor cells were fixed on an alginate film. The alginate film was affixed on the bottom of a flow cell and the somatic embryos were subjected to laminar flow through the chamber of the flow cell. Magnified images of the cell clusters were ...

Metabolic engineering of Escherichia coli for the production of polylactic acid and its copolymers

Biotechnology and Bioengineering — Mon, 23 Nov 2009 00:00:00 +0100

In this study, the metabolic pathways of E. coli were further engineered by knocking out the ackA, ppc, and adhE genes and by replacing the promoters of the ldhA and acs genes with the trc promoter based on in silico genome-scale metabolic flux analysis in addition to rational approach. Using this engineered strain, PLA homopolymer could be produced up to 11 wt% from glucose. Also, P(3HB-co-LA) copolymers containing 55-86 mol% lactate could be produced up to 56 wt% from glucose and 3HB. P(3HB-co-LA) copolymers containing up to 70 mol% lactate could be produced to 46 wt% from glucose alone by introducing the Cupriavidus necator [beta]-ketothiolase and acetoacetyl-CoA reductase genes. Thus, the strategy of combined metabolic engineering and enzyme engineering allowed efficient bio-based one-...

Biosynthesis of polylactic acid and its copolymers using evolved propionate CoA transferase and PHA synthase

Biotechnology and Bioengineering — Mon, 23 Nov 2009 00:00:00 +0100

For the synthesis of polylactic acid (PLA) and its copolymers by one-step fermentation process, heterologous pathways involving Clostridium propionicum propionate CoA transferase (PctCp) and Pseudomonas sp. MBEL 6-19 polyhydroxyalkanoate (PHA) synthase 1 (PhaC1Ps6-19) were introduced into Escherichia coli for the generation of lactyl-CoA endogenously and incorporation of lactyl-CoA into the polymer, respectively. Since the wild-type PhaC1Ps6-19 did not efficiently accept lactyl-CoA as a substrate, site directed mutagenesis as well as saturation mutagenesis were performed to improve the enzyme. The wild-type PctCp was not able to efficiently convert lactate to lactyl-CoA and was found to exert inhibitory effect on cell growth, random mutagenesis by error-prone PCR was carried out. By employ...

Enzymatic hydrolysis of waste cellulose

Biotechnology and Bioengineering — Mon, 23 Nov 2009 00:00:00 +0100

No Abstract. (Source: Biotechnology and Bioengineering)

Deceptive shine: Protein aggregation induced by stainless steel

Biotechnology and Bioengineering — Mon, 23 Nov 2009 00:00:00 +0100

No Abstract. (Source: Biotechnology and Bioengineering)

Process model for glycopeptide antibiotic production

Biotechnology and Bioengineering — Mon, 23 Nov 2009 00:00:00 +0100

No Abstract. (Source: Biotechnology and Bioengineering)

Hypothermic culturing results in more accurate protein folding?

Biotechnology and Bioengineering — Mon, 23 Nov 2009 00:00:00 +0100

No Abstract. (Source: Biotechnology and Bioengineering)

Plastics from E. coli: Metabolic engineering for PLA and its copolymers

Biotechnology and Bioengineering — Mon, 23 Nov 2009 00:00:00 +0100

No Abstract. (Source: Biotechnology and Bioengineering)

Hydrodynamic modulation of embryonic stem cell differentiation by rotary orbital suspension culture

Biotechnology and Bioengineering — Thu, 19 Nov 2009 00:00:00 +0100

Embryonic stem cells (ESCs) can differentiate into all somatic cell types, but the development of effective strategies to direct ESC fate is dependent upon defining environmental parameters capable of influencing cell phenotype. ESCs are commonly differentiated via cell aggregates referred to as embryoid bodies (EBs), but current culture methods, such as hanging drop and static suspension, yield relatively few or heterogeneous populations of EBs. Alternatively, rotary orbital suspension culture enhances EB formation efficiency, cell yield, and homogeneity without adversely affecting differentiation. Thus, the objective of this study was to systematically examine the effects of hydrodynamic conditions created by rotary orbital shaking on EB formation, structure, and differentiation. Mouse E...

Enhancement of ethanol and biogas production from high-crystalline cellulose by different modes of NMO pretreatment

Biotechnology and Bioengineering — Fri, 13 Nov 2009 00:00:00 +0100

Pretreatment of high-crystalline cellulose with N-methyl-morpholine-N-oxide (NMO or NMMO) to improve bioethanol and biogas production was investigated. The pretreatments were performed at 90 and 120°C for 0.5-15 h in three different modes, including dissolution (85% NMO), ballooning (79% NMO), and swelling (73% NMO). The pretreated materials were then enzymatically hydrolyzed and fermented to ethanol or anaerobically digested to biogas (methane). The pretreatment at 85% NMO, 120°C and 2.5 h resulted in 100% yield in the subsequent enzymatic hydrolysis and around 150% improvement in the yield of ethanol compared to the untreated and water-treated material. However, the best results of biogas production were obtained when the cellulose was treated with swelling and ballooning mode, which g...

CHO gene expression profiling in biopharmaceutical process analysis and design

Biotechnology and Bioengineering — Fri, 06 Nov 2009 00:00:00 +0100

Increase in both productivity and product yields in biopharmaceutical process development with recombinant protein producing mammalian cells can be mainly attributed to the advancements in cell line development, media, and process optimization. Only recently, genome-scale technologies enable a system-level analysis to elucidate the complex biomolecular basis of protein production in mammalian cells promising an increased process understanding and the deduction of knowledge-based approaches for further process optimization. Here, the use of gene expression profiling for the analysis of a low titer (LT) and high titer (HT) fed batch process using the same IgG producing CHO cell line was investigated. We found that gene expression (i) significantly differed in HT versus LT process conditions ...

Plasmid-free T7-based Escherichia coli expression systems

Biotechnology and Bioengineering — Wed, 04 Nov 2009 00:00:00 +0100

In order to release host cells from plasmid-mediated increases in metabolic load and high gene dosages, we developed a plasmid-free, T7-based E. coli expression system in which the target gene is site-specifically integrated into the genome of the host. With this system, plasmid-loss, a source of instability for conventional expression systems, was eliminated. At the same time, system leakiness, a challenging problem with recombinant systems, was minimized. The efficiency of the T7 RNA polymerase compensates for low gene dosage and provides high rates of recombinant gene expression without fatal consequences to host metabolism. Relative to conventional pET systems, this system permits improved process stability and increases the host cell's capacity for recombinant gene expression, resulti...

Industrial systems biology

Biotechnology and Bioengineering — Wed, 04 Nov 2009 00:00:00 +0100

The chemical industry is currently undergoing a dramatic change driven by demand for developing more sustainable processes for the production of fuels, chemicals, and materials. In biotechnological processes different microorganisms can be exploited, and the large diversity of metabolic reactions represents a rich repository for the design of chemical conversion processes that lead to efficient production of desirable products. However, often microorganisms that produce a desirable product, either naturally or because they have been engineered through insertion of heterologous pathways, have low yields and productivities, and in order to establish an economically viable process it is necessary to improve the performance of the microorganism. Here metabolic engineering is the enabling techn...

High affinity peptides for the recognition of the heart disease biomarker troponin I identified using phage display

Biotechnology and Bioengineering — Wed, 04 Nov 2009 00:00:00 +0100

In this study we have used polyvalent phage display to isolate unique linear peptide motifs which recognize both the human and rat homologs of troponin I. The peptide specific for human troponin I has a sequence of FYSHSFHENWPS and the peptide specific for the rat troponin I has a sequence of FHSSWPVNGSTI. Enzyme-linked immunosorbent assays (ELISAs) were used to evaluate the binding interactions, and the two phage-displayed peptides exhibited some cross-reactivity, but they were both more specific for the troponin I homolog they were selected against. The binding affinities of the phage-displayed peptides were decreased by the presence of complex tissue culture media (MEM), and the addition of 10% calf serum further interfered with the binding of the target proteins. Kinetic indirect phage...

Prediction of the enantioselectivity of lipases and esterases by molecular docking method with modified force field parameters

Biotechnology and Bioengineering — Wed, 04 Nov 2009 00:00:00 +0100

In the work, molecular docking method was applied to extensively predict the enantioselectivity of lipases and esterases. A ligand library consisted of 69 chiral substrates was docked to four lipases and two esterases to set up the prediction model. During the docking process, necessary modification was carried out on van de Waals and hydrogen bond parameters of enzyme/substrate pair so that the ligands were able to adopt productive geometry in the enzymes. The docking results correctly indicated the enantiopreference for 91% (63/69) of docking pairs and the docking energy difference between substrate enantiomers ([Delta][Delta]Gdocking) was significantly (correlation coefficient = 0.72, P < 0.05) correlated with the activation free energy difference ([Delta][Delta]G[ne]) that was quantita...

Capturing of cell culture-derived modified Vaccinia Ankara virus by ion exchange and pseudo-affinity membrane adsorbers

Biotechnology and Bioengineering — Wed, 04 Nov 2009 00:00:00 +0100

This study compares different ion exchange and pseudo-affinity membrane adsorbers (MA) to capture chicken embryo fibroblast cell-derived MVA-BN® after cell homogenization and clarification. In parallel, the overall performance of classical bead-based resin chromatography (Cellufine® sulfate and Toyopearl® AF-Heparin) was investigated. The two tested pseudo-affinity MA (i.e., sulfated cellulose and heparin) were superior over the applied ion exchange MA in terms of virus yield and contaminant depletion. Furthermore, studies confirmed an expected increase in productivity resulting from the increased volume throughput of MA compared to classical bead-based column chromatography methods. Overall virus recovery was [sim]60% for both pseudo-affinity MA and the Cellufine® sulfate resin. Deple...

Modeling the influence of cyclodextrins on oral absorption of low solubility drugs: II. Experimental validation

Biotechnology and Bioengineering — Sun, 01 Nov 2009 00:00:00 +0100

A model was developed for predicting the influence of cyclodextrins (CDs) delivered as a physical mixture with drug on oral absorption. CDs are cyclic oligosaccharides which form inclusion complexes with many drugs and are often used as solubilizing agents. The purpose of this work is to compare the simulation predictions with in vitro as well as in vivo experimental results to test the model's ability to capture the influence of CD on key processes in the gastrointestinal (GI) tract environment. Dissolution and absorption kinetics of low solubility drugs (Naproxen and Nifedipine) were tested in the presence and absence of CD in a simulated gastrointestinal environment. Model predictions were also compared with in vivo experimental results (Glibenclamide and Carbamazepine) from the literat...

Model-based high cell density cultivation of Rhodospirillum rubrum under respiratory dark conditions

Biotechnology and Bioengineering — Fri, 30 Oct 2009 00:00:00 +0100

The potential of facultative photosynthetic bacteria as producers of photosynthetic pigments, vitamins, coenzymes and other valuable products has been recognized for decades. However, mass cultivation under photosynthetic conditions is generally inefficient due to the inevitable limitation of light supply when cell densities become very high. The previous development of a new cultivation process for maximal expression of photosynthetic genes under semi-aerobic dark conditions in common bioreactors offers a new perspective for utilizing the facultative photosynthetic bacterium Rhodospirillum rubrum for large-scale applications. Based on this cultivation system, the present study aimed in determining the maximal achievable cell density of R. rubrum in a bioreactor, thereby providing a major ...

Drug release and its relationship with kinetic and thermodynamic parameters of drug sorption onto starch acetate fibers

Biotechnology and Bioengineering — Fri, 30 Oct 2009 00:00:00 +0100

Drug release and its relationship with kinetic and thermodynamic parameters of drug sorption onto starch acetate (SA) fibers have been studied using Diclofenac, 5-Fluorouracil (5-Fu), and Metformin as model drugs. The sorption method is more flexible and can avoid limitations or problems which occur with molten or dissolution methods. To understand drug release of sorption loading, kinetic and apparent thermodynamic parameters, such as diffusion coefficient, activation energy for diffusion, affinity, and sorption enthalpy and entropy, have been investigated. The quantitative relationship between drug release and drug-loading concentration, affinity, and activation energy for diffusion has been established to predict the initial burst and subsequent release of the drugs. Up to 12% of Diclof...

Dynamic flux responses in riboflavin overproducing Bacillus subtilis to increasing glucose limitation in fed-batch culture

Biotechnology and Bioengineering — Fri, 30 Oct 2009 00:00:00 +0100

How do intracellular fluxes respond to dynamically increasing glucose limitation when the physiology changes from strong overflow metabolism near to exclusively maintenance metabolism? Here we investigate this question in a typical industrial, glucose-limited fed-batch cultivation with a riboflavin overproducing Bacillus subtilis strain. To resolve dynamic flux changes, a novel approach to 13C flux analysis was developed that is based on recording 13C labeling patterns in free intracellular amino acids. Fluxes are then estimated with stationary flux ratio and iterative isotopomer balancing methods, for which a decomposition of the process into quasi-steady states and estimation of isotopic steady state 13C labeling patterns was necessary. By this approach, we achieve a temporal resolution ...

Bioreactor expansion of human neural precursor cells in serum-free media retains neurogenic potential

Biotechnology and Bioengineering — Fri, 30 Oct 2009 00:00:00 +0100

Human neural precursor cells (hNPCs), harvested from somatic tissue and grown in vitro, may serve as a source of cells for cell replacement strategies aimed at treating neurodegenerative disorders such as Parkinson's disease (PD), Huntington's disease (HD), and intractable spinal cord pain. A crucial element in a robust clinical production method for hNPCs is a serum-free growth medium that can support the rapid expansion of cells while retaining their multipotency. Here, we report the development of a cell growth medium (PPRF-h2) for the expansion of hNPCs, achieving an overall cell-fold expansion of 1013 over a period of 140 days in stationary culture which is significantly greater than other literature results. More importantly, hNPC expansion could be scaled-up from stationary culture ...

Protein disulfide isomerase does not control recombinant IgG4 productivity in mammalian cell lines

Biotechnology and Bioengineering — Fri, 30 Oct 2009 00:00:00 +0100

Post-translational limitations in the endoplasmic reticulum during recombinant monoclonal antibody production are an important factor in lowering the capacity for synthesis and secretion of correctly folded proteins. Mammalian protein disulfide isomerase (PDI) has previously been shown to have a role in the formation of disulfide bonds in immunoglobulins. Several attempts have been made to improve the rate of recombinant protein production by overexpressing PDI but the results from these studies have been inconclusive. Here we examine the effect of (a) transiently silencing PDI mRNA and (b) increasing the intracellular levels of members of the PDI family (PDI, ERp72, and PDIp) on the mRNA levels, assembly and secretion of an IgG4 isotype. Although transiently silencing PDI in NS0/2N2 cells...

An unbiased sensitivity analysis reveals important parameters controlling periodicity of circadian clock

Biotechnology and Bioengineering — Wed, 28 Oct 2009 00:00:00 +0100

In this study, we propose a computational approach to determine the relative importance of parameters controlling the performance of the circadian clock in Drosophila. While previous attempts to sensitivity analysis largely depend on the knowledge of model parameters which are generally unknown, our study depicts a consistent picture of sensitivity assessment for a large number of parameters, even when the values of these parameters are not available in vivo. The resulting parametric sensitivity analysis suggests that PER/TIM negative loop is critical to maintain the stable periodicity of the circadian clock, which is consistent to the previously experimental and computational findings. Furthermore, our analysis generates a rich hypothesis of important parameters in the circadian clock tha...

Micro-scale bioreactors that predict production scale

Biotechnology and Bioengineering — Mon, 26 Oct 2009 00:00:00 +0100

No Abstract. (Source: Biotechnology and Bioengineering)

Overcoming diffusional limitations in vascular tissue engineering with transmural flow

Biotechnology and Bioengineering — Mon, 26 Oct 2009 00:00:00 +0100

No Abstract. (Source: Biotechnology and Bioengineering)

Old wine in new skins

Biotechnology and Bioengineering — Mon, 26 Oct 2009 00:00:00 +0100

No Abstract. (Source: Biotechnology and Bioengineering)

Predictive biodegradation made feasible

Biotechnology and Bioengineering — Mon, 26 Oct 2009 00:00:00 +0100

No Abstract. (Source: Biotechnology and Bioengineering)

Development of an Ibuprofen-releasing biodegradable PLA/PGA electrospun scaffold for tissue regeneration

Biotechnology and Bioengineering — Wed, 21 Oct 2009 23:00:00 +0100

Our aim was to develop a biodegradable fibrous dressing to act as a tissue guide for in situ wound repair while releasing Ibuprofen to reduce inflammation in wounds and reduce pain for patients on dressing changes. Dissolving the acid form of Ibuprofen (from 1% to 10% by weight) in the same solvent as 75% polylactide, 25% polyglycolide (PLGA) polymers gave uniformly loaded electrospun fibers which gave rapid release of drug within the first 8 h and then slower release over several days. Scaffolds with 10% Ibuprofen degraded within 6 days. The Ibuprofen released from these scaffolds significantly reduced the response of fibroblasts to major pro-inflammatory stimulators. Fibroblast attachment and proliferation on scaffolds was unaffected by the addition of 1-5% Ibuprofen. Scaffolds loaded wi...

Regioselective hydroxylation of daidzein using P450 (CYP105D7) from Streptomyces avermitilis MA4680

Biotechnology and Bioengineering — Tue, 20 Oct 2009 00:00:00 +0100

Regiospecific 3[prime]-hydroxylation reaction of daidzein was performed with CYP105D7 from Streptomyces avermitilis MA4680 expressed in Escherichia coli. The apparent Km and kcat values of CYP105D7 for daidzein were 21.83 ± 6.3 µM and 15.01 ± 0.6 min-1 in the presence of 1 µM of CYP105D7, putidaredoxin (CamB) and putidaredoxin reductase (CamA), respectively. When CYP105D7 was expressed in S. avermitilis MA4680, its cytochrome P450 activity was confirmed by the CO-difference spectra at 450 nm using the whole cell extract. When the whole-cell reaction for the 3[prime]-hydroxylation reaction of daidzein was carried out with 100 µM of daidzein in 100 mM of phosphate buffer (pH 7.5), the recombinant S. avermitilis grown in R2YE media overexpressing CYP105D7 and ferredoxin FdxH (SAV7470) sh...

Characterization of a biofilm membrane reactor and its prospects for fine chemical synthesis

Biotechnology and Bioengineering — Tue, 20 Oct 2009 00:00:00 +0100

Biofilms are known to be robust biocatalysts. Conventionally, they have been mainly applied for wastewater treatment, however recent reports about their employment for chemical synthesis are increasingly attracting attention. Engineered Pseudomonas sp. strain VLB120[Delta]C biofilm growing in a tubular membrane reactor was utilized for the continuous production of (S)-styrene oxide. A biofilm specific morphotype appeared in the effluent during cultivation, accounting for 60-80% of the total biofilm irrespective of inoculation conditions but with similar specific activities as the original morphotype. Mass transfer of the substrate styrene and the product styrene oxide was found to be dependent on the flow rate but was not limiting the epoxidation rate. Oxygen was identified as one of the m...

Coaxial electrohydrodynamic spraying of plasmid DNA/polyethylenimine (PEI) polyplexes for enhanced nonviral gene delivery

Biotechnology and Bioengineering — Tue, 20 Oct 2009 00:00:00 +0100

DNA/polyethylenimine (PEI) polyplexes are an important class of nonviral vectors. Although the conventional preparation method, bulk mixing, is straightforward, the formation of the DNA/PEI polyplexes is not well controlled. This work explores coaxial electrohydrodynamic spraying (EHDS) as a novel, alternative method to produce DNA/PEI polyplexes in a more controlled manner. Both pGFP/PEI and pSEAP/PEI polyplexes were produced by EHDS with a coaxial needle setup. The size of the polyplexes was determined using dynamic light scattering, and their ability to transfect NIH 3T3 cells was observed by using an inverted fluorescence microscope (pGFP) or quantified by measuring the activity level of alkaline phosphatase (pSEAP). At nitrogen to phosphate ratio (N/P) of 6.7, the polyplexes produced ...

Triple light chain antibodies: Factors that influence its formation in cell culture

Biotechnology and Bioengineering — Tue, 20 Oct 2009 00:00:00 +0100

THIOMABs are recombinant antibodies engineered with reactive cysteines, which can be covalently conjugated to drugs of interest to generate targeted therapeutics. During the analysis of THIOMABs secreted by stably transfected Chinese Hamster Ovary (CHO) cells, we discovered the existence of a new species - Triple Light Chain Antibody (3LC). This 3LC species is the product of a disulfide bond formed between an extra light chain and one of the engineered cysteines on the THIOMAB. We characterized the 3LC by size exclusion chromatography, mass spectrometry, and microchip electrophoresis. We also investigated the potential causes of 3LC formation during cell culture, focusing on the effects of free light chain (LC) polypeptide concentration, THIOMAB amino acid sequence, and glutathione (GSH) p...

Transient expression of human TorsinA enhances secretion of two functionally distinct proteins in cultured Chinese hamster ovary (CHO) cells

Biotechnology and Bioengineering — Tue, 20 Oct 2009 00:00:00 +0100

Cultured mammalian cells, particularly Chinese hamster ovary (CHO) cells, are widely exploited as hosts for the production of recombinant proteins, but often yields are limiting. Such limitations may be due in part to the misfolding and subsequent degradation of the heterologous proteins. Consequently we have determined whether transiently co-expressing yeast and/or mammalian chaperones that act to disaggregate proteins, in CHO cell lines, improve the levels of either a cytoplasmic (Fluc) or secreted (Gluc) form of luciferase or an immunoglobulin IgG4 molecule. Over-expression of the yeast 'protein disaggregase' Hsp104 in a CHO cell line increased the levels of Fluc more significantly than for Gluc although levels were not further elevated by over-expression of the yeast or mammalian Hsp70...

Systematic engineering of phytochelatin synthesis and arsenic transport for enhanced arsenic accumulation in E. coli

Biotechnology and Bioengineering — Tue, 20 Oct 2009 00:00:00 +0100

Phytochelatin (PC) is a naturally occurring peptide with high affinity towards arsenic (As). In this article, we demonstrated the systematic engineering of PC-producing E. coli for As accumulation by addressing different bottlenecks in PC synthesis as well as As transport. Phytochelatin synthase from Schizosaccharomyces pombe (SpPCS) was expressed in E. coli resulting in 18 times higher As accumulation. PC production was further increased by co-expressing a feedback desensitized [gamma]-glutamylcysteine synthetase (GshI*), resulting in 30-fold higher PC levels and additional 2-fold higher As accumulation. The significantly increased PC levels were exploited further by co-expressing an arsenic transporter GlpF, leading to an additional 1.5-fold higher As accumulation. These engineering step...

Profiling of host cell proteins by two-dimensional difference gel electrophoresis (2D-DIGE): Implications for downstream process development

Biotechnology and Bioengineering — Mon, 19 Oct 2009 23:00:00 +0100

This article describes the use of a comparative proteomic profiling method viz. two-dimensional difference gel electrophoresis (2D-DIGE) to examine HCP composition in the harvest stream of CHO cell culture. The effect of upstream process parameters such as cell culture media, bioreactor control strategy, feeding strategy, and cell culture duration/cell viability on HCP profile was examined using this technique. Among all the parameters studied, cell viability generated the most significant changes on the HCP profile. 2D-DIGE was also used to compare the HCP differences between monoclonal antibody producing and null cell cultures. The HCP species in production cell culture was found to be well represented in null cell culture, which confirms the suitability of using the null cell culture fo...

Dynamic measurement of the volumetric oxygen transfer coefficient in fermentation systems

Biotechnology and Bioengineering — Sun, 18 Oct 2009 23:00:00 +0100

No Abstract. (Source: Biotechnology and Bioengineering)

Enhanced tubular scaffold design with gradient layers

Biotechnology and Bioengineering — Sun, 18 Oct 2009 23:00:00 +0100

No Abstract. (Source: Biotechnology and Bioengineering)

Observation without provocation: Using electrochemical impedance spectroscopy to understand bacterial respiration in microbial fuel cells

Biotechnology and Bioengineering — Sun, 18 Oct 2009 23:00:00 +0100

No Abstract. (Source: Biotechnology and Bioengineering)

Improved subtilase variant better suited for industrial applications

Biotechnology and Bioengineering — Sun, 18 Oct 2009 23:00:00 +0100

No Abstract. (Source: Biotechnology and Bioengineering)

Enzymatic synthesis of specialty surfactants

Biotechnology and Bioengineering — Sun, 18 Oct 2009 23:00:00 +0100

No Abstract. (Source: Biotechnology and Bioengineering)

A DIGE approach for the assessment of differential expression of the CHO proteome under sodium butyrate addition: Effect of Bcl-xL overexpression

Biotechnology and Bioengineering — Tue, 13 Oct 2009 23:00:00 +0100

Bcl-xL, a member of the Bcl-2 family, is known to inhibit apoptosis of recombinant Chinese hamster ovary (rCHO) cells induced by the addition of sodium butyrate (NaBu), which is used for the elevated expression of recombinant protein. In order to understand the intracellular effects of Bcl-xL overexpression on CHO cells treated with NaBu, changes to the proteome caused by controlled Bcl-xL expression in rCHO cells producing erythropoietin (EPO) in the presence of 3 mM NaBu were evaluated using two-dimensional differential in-gel electrophoresis (2D-DIGE) and MS analysis. The consequences of Bcl-xL overexpression were not limited to the apoptotic signaling pathway. Out of eight proteins regulated significantly by Bcl-xL overexpression in 3 mM NaBu addition culture, four proteins were relate...

Microfluidic biochip for the perifusion of precision-cut rat liver slices for metabolism and toxicology studies

Biotechnology and Bioengineering — Thu, 08 Oct 2009 23:00:00 +0100

This article describes a novel in vitro system for the incubation of precision-cut liver slices (PCLS) under flow conditions, based on a poly(dimethylsiloxane) (PDMS) device containing 25-µL microchambers for integration of the slices. The microdevice is coupled to a perifusion system, which enables a constant delivery of nutrients and oxygen and a continuous removal of waste products. Both a highly controlled incubation environment and high metabolite detection sensitivity could be achieved using microfluidics. Liver slices were viable for at least 24 h in the microdevice. The compound, 7-ethoxycoumarin (7-EC), was chosen to test metabolism, since its metabolism includes both phase I and phase II metabolism and when tested in the conventional well plate system, correlates well with the i...

White biotechnology for cellulose manufacturing - The HoLiR concept

Biotechnology and Bioengineering — Thu, 08 Oct 2009 00:00:00 +0100

A variety of approaches are available for generation of bacteria-produced nanocellulose (BNC) in different forms. BNC production under static cultivation conditions usually results in fleeces or foils, characterized by a homogeneous, three-dimensional network of nanofibers and a uniform surface. However, under static cultivation conditions in batch vessels, the widths and the lengths of the BNC sheets cultured are determined by the dimensions of the culture vessel. In this contribution, a novel, efficient process for a (semi-)continuous cultivation of planar BNC fleeces and foils with a freely selectable length and an adjustable height is presented. By means of comprehensive investigations, the comparability of the BNC harvested to that gained from static cultivation under batch conditions...

Behavior of Marinobacter hydrocarbonoclasticus SP17 cells during initiation of biofilm formation at the alkane-water interface

Biotechnology and Bioengineering — Thu, 08 Oct 2009 00:00:00 +0100

In this study we focused on the interactions of cells with the alkane-water interface occurring during initiation of biofilm development. The behavior of cells at the interface was apprehended by investigating alterations of the mechanical properties of the interface during cell adsorption, using dynamic drop tensiometry measurements. It was found that after having reached the hexadecane-water interface, by a purely thermal diffusion process, cells released surface-active compounds (SACs) resulting in the formation of an interfacial visco-elastic film. Release of SACs was an active process requiring protein synthesis. This initial interaction occurred on metabolizable as well as non-metabolizable alkanes, indicating that at this stage cells are not affected by the nature of the alkane form...

IgG-single chain Fv fusion protein therapeutic for alzheimer's disease: Expression in CHO cells and pharmacokinetics and brain delivery in the rhesus monkey

Biotechnology and Bioengineering — Thu, 08 Oct 2009 00:00:00 +0100

In conclusion, the HIRMAb-ScFv fusion protein is a new class of antibody-based therapeutic for AD that has been specifically engineered to cross the human BBB. Biotechnol. Bioeng. © 2009 Wiley Periodicals, Inc. (Source: Biotechnology and Bioengineering)

PEGDA hydrogels with patterned elasticity: Novel tools for the study of cell response to substrate rigidity

Biotechnology and Bioengineering — Thu, 08 Oct 2009 00:00:00 +0100

The ability of cells to migrate in response to mechanical gradients (durotaxis) and differential cell behavior in adhesion, spreading, and proliferation in response to substrate rigidity are key factors both in tissue engineering, in which materials must be selected to provide the appropriate mechanical signals, and in studies of mechanisms of diseases such as cancer and atherosclerosis, in which changes in tissue stiffness may inform cell behavior. Using poly(ethylene glycol) diacrylate hydrogels with varying polymer chain length and photolithographic patterning techniques, we are able to provide substrates with spatially patterned, tunable mechanical properties in both gradients and distinct patterns. The hydrogels can be patterned to produce anisotropic structures and exhibit patterned ...

Considerations for the design and construction of a synthetic platform cell for biotechnological applications

Biotechnology and Bioengineering — Thu, 08 Oct 2009 00:00:00 +0100

The design and construction of an artificial bacterial cell could revolutionize biotechnological processes and technologies. A functional platform cell that can be easily customized for a pre-defined task would be useful for applications from producing therapeutics to decontaminating waste streams. The platform cell must be robust and highly efficient. A biotechnological platform cell is related to the concept of a minimal cell, but several factors beyond those necessary for a minimal cell must be considered for a synthetic organism designed for biotechnological applications. Namely, a platform cell must exhibit robust cell reproduction, decreased genetic drift, a physically robust cell envelope, efficient and simplified transcription and translation controls, and predictable metabolic int...

Genome-scale analysis of library sorting (GALibSo): Isolation of secretion enhancing factors for recombinant protein production in Pichia pastoris

Biotechnology and Bioengineering — Thu, 08 Oct 2009 00:00:00 +0100

A method combining fluorescence activated cell sorting (FACS) and DNA microarray assisted clone identification was developed and termed Genome-Scale Analysis of Library Sorting (GALibSo). Genes enhancing the production of secreted heterologous proteins in Pichia pastoris were identified out of a cDNA library by cell surface display and FACS. The trends of gene enrichment during consecutive FACS rounds were monitored by DNA microarrays. In a case study a P. pastoris cDNA library was co-expressed in a strain secreting the Fab fragment of a monoclonal antibody against human immunodeficiency virus type 1 as a model protein. Three genes were identified, increasing the relative expression level of the surface-displayed model protein up to 45%. While one of these genes had a positive effect on th...

Mixtures of hemoglobin-based oxygen carriers and perfluorocarbons exhibit a synergistic effect in oxygenating hepatic hollow fiber bioreactors

Biotechnology and Bioengineering — Thu, 08 Oct 2009 00:00:00 +0100

Hepatic hollow fiber (HF) bioreactors are being developed for use as bioartificial liver assist devices (BLADs). In general, BLADs suffer from O2 limited transport, which reduces their performance. This modeling study seeks to investigate if O2 carrying solutions consisting of mixtures of hemoglobin-based oxygen carriers (HBOCs) and perfluorocarbons (PFCs) can enhance O2 transport to hepatocytes cultured in the extra capillary space (ECS) of HF bioreactors. We simulated supplementing the circulating cell culture media stream of the HF bioreactor with a mixture containing these two types of oxygen carriers (HBOCs and PFCs). A mathematical model was developed based on the dimensions and physical characteristics of a commercial HF bioreactor. The resulting set of partial differential equation...

Global metabolic profiling of plant cell wall polysaccharide degradation by Saccharophagus degradans

Biotechnology and Bioengineering — Mon, 05 Oct 2009 00:00:00 +0100

This study demonstrates that the gas chromatography-time of flight mass spectrometry-based global metabolomics are useful for understanding microbial metabolism and evaluating its fermentation characteristics. Biotechnol. Bioeng. © 2009 Wiley Periodicals, Inc. (Source: Biotechnology and Bioengineering)

Isomerization in the CDR2 of a monoclonal antibody: Binding analysis and factors that influence the isomerization rate

Biotechnology and Bioengineering — Mon, 05 Oct 2009 00:00:00 +0100

Isomerization of a monoclonal antibody is one of the common routes of protein degradation. An isomerization in the complementarity-determining region (CDR) was found previously and is investigated in depth in this work. Affinity analysis proves that the antibody with one isomerized heavy chain has lower binding. Binding constants were determined, and exhibited a slower on-rate in conjunction with a faster off-rate for this isomerization. To determine the role of the buffer on the rate of isomerization, this antibody was incubated in various matrices and the amount of isomerized antibody was determined by hydrophobic interaction chromatography (HIC). The rate was found to be dependent on the pH as well as the net negative charge of the buffer components that can act as proton acceptors. An ...

Investigating the role of native propionyl-CoA and methylmalonyl-CoA metabolism on heterologous polyketide production in Escherichia coli

Biotechnology and Bioengineering — Mon, 05 Oct 2009 00:00:00 +0100

In this study, the sbm, ygfG, and ygfH genes were individually deleted and over-expressed to investigate their effect on heterologous 6dEB production. Our results indicate that the deletion and over-expression of sbm did not influence 6dEB production; ygfG over-expression reduced 6dEB production by fourfold while ygfH deletion increased 6dEB titers from 65 to 129 mg/L in shake flask experiments. It was also found that native E. coli metabolism could support 6dEB biosynthesis in the absence of exogenous propionate and the substrate provision pcc genes. Lastly, the effect of the ygfH deletion was tested in batch bioreactor cultures in which 6dEB titers improved from 206 to 527 mg/L. Biotechnol. Bioeng. © 2009 Wiley Periodicals, Inc. (Source: Biotechnology and Bioengineering)

Theoretical investigation of the sequential reductive dechlorination pathways of chlorobenzenes and chloroanilines

Biotechnology and Bioengineering — Mon, 05 Oct 2009 00:00:00 +0100

The distribution of product isomers during the sequential reductive dechlorination of pentachloroaniline (PCA) and pentachlorobenzene (PeCB) was examined based on calculated thermodynamic, chromatographic, and electronic properties and then compared to the product distribution achieved by enrichment cultures. The dechlorination pathway analysis based on free energy considerations matched 78% and 67% of the experimental results for the sequential reductive dechlorination of chlorobenzenes (CBs) and chloroanilines (CAs), respectively. Chromatographic properties of CBs and CAs were able to explain some but not all of the reactions in the observed dechlorination pathways. Correlations between the observed dechlorination pattern and electronic properties of the parent compounds were able to exp...

Maintenance of pluripotency in human embryonic stem cells cultured on a synthetic substrate in conditioned medium

Biotechnology and Bioengineering — Thu, 01 Oct 2009 23:00:00 +0100

Realizing the potential clinical and industrial applications of human embryonic stem cells (hESCs) is limited by the need for costly, labile, or undefined growth substrates. Here we demonstrate that trypsin passaging of the hESC lines, HUES7 and NOTT1, on oxygen plasma etched tissue culture polystyrene (PE-TCPS) in conditioned medium is compatible with pluripotency. This synthetic culture surface is stable at room temperature for at least a year and is readily prepared by placing polystyrene substrates in a radio frequency oxygen plasma generator for 5 min. Modification of the polystyrene surface chemistry by plasma etching was confirmed by X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS), which identified elemental and molecular changes ...

Power production in MFCs inoculated with Shewanella oneidensis MR-1 or mixed cultures

Biotechnology and Bioengineering — Mon, 28 Sep 2009 00:00:00 +0100

Power densities and oxidation-reduction potentials (ORPs) of MFCs containing a pure culture of Shewanella oneidensis MR-1 were compared to mixed cultures (wastewater inoculum) in cube shaped, 1-, 2-, and 3-bottle batch-fed MFC reactor configurations. The reactor architecture influenced the relative power produced by the different inocula, with the mixed culture generating 68-480% more power than MR-1 in each MFC configuration. The mixed culture produced the maximum power density of 858 ± 9 mW m-2 in the cubic MFC, while MR-1 produced 148 ± 20 mW m-2. The higher power by the mixed culture was primarily a result of lower internal resistances than those produced by the pure culture. Power was a direct function of ohmic resistance for the mixed culture, but not for strain MR-1. ORP of the an...

Modeling of flow-induced shear stress applied on 3D cellular scaffolds: Implications for vascular tissue engineering

Biotechnology and Bioengineering — Mon, 28 Sep 2009 00:00:00 +0100

Novel tissue-culture bioreactors employ flow-induced shear stress as a means of mechanical stimulation of cells. We developed a computational fluid dynamics model of the complex three-dimensional (3D) microstructure of a porous scaffold incubated in a direct perfusion bioreactor. Our model was designed to predict high shear-stress values within the physiological range of those naturally sensed by vascular cells (1-10 dyne/cm2), and will thereby provide suitable conditions for vascular tissue-engineering experiments. The model also accounts for cellular growth, which was designed as an added cell layer grown on all scaffold walls. Five model variants were designed, with geometric differences corresponding to cell-layer thicknesses of 0, 50, 75, 100, and 125 µm. Four inlet velocities (0.5, ...

High-pressure systems for gas-phase free continuous incubation of enriched marine microbial communities performing anaerobic oxidation of methane

Biotechnology and Bioengineering — Mon, 28 Sep 2009 00:00:00 +0100

Novel high-pressure biotechnical systems that were developed and applied for the study of anaerobic oxidation of methane (AOM) are described. The systems, referred to as high-pressure continuous incubation system (HP-CI system) and high-pressure manifold-incubation system (HP-MI system), allow for batch, fed-batch, and continuous gas-phase free incubation at high concentrations of dissolved methane and were designed to meet specific demands for studying environmental regulation and kinetics as well as for enriching microbial biomass in long-term incubation. Anoxic medium is saturated with methane in the first technical stage, and the saturated medium is supplied for biomass incubation in the second stage. Methane can be provided in continuous operation up to 20 MPa and the incubation syste...

Degradation of an Fc-fusion recombinant protein by host cell proteases: Identification of a CHO cathepsin D protease

Biotechnology and Bioengineering — Sat, 26 Sep 2009 23:00:00 +0100

This study is an example of how quality and stability of therapeutic recombinant molecules are strongly influenced by cell culture parameters. Biotechnol. Bioeng. © 2009 Wiley Periodicals, Inc. (Source: Biotechnology and Bioengineering)

Computational framework for predictive biodegradation

Biotechnology and Bioengineering — Fri, 25 Sep 2009 23:00:00 +0100

As increasing amounts of anthropogenic chemicals are released into the environment, it is vital to human health and the preservation of ecosystems to evaluate the fate of these chemicals in the environment. It is useful to predict whether a particular compound is biodegradable and if alternate routes can be engineered for compounds already known to be biodegradable. In this work, we describe a computational framework (called BNICE) that can be used for the prediction of novel biodegradation pathways of xenobiotics. The framework was applied to 4-chlorobiphenyl, phenanthrene, [gamma]-hexachlorocyclohexane, and 1,2,4-trichlorobenzene, compounds representing various classes of xenobiotics with known biodegradation routes. BNICE reproduced the proposed biodegradation routes found experimentall...

Surface-directed assembly of cell-laden microgels

Biotechnology and Bioengineering — Wed, 23 Sep 2009 00:00:00 +0100

Cell-laden microscale hydrogels (microgels) can be used as tissue building blocks and assembled to create 3D tissue constructs with well-defined microarchitecture. In this article, we present a bottom-up approach to achieve microgel assembly on a patterned surface. Driven by surface tension, the hydrophilic microgels can be assembled into well-defined shapes on a glass surface patterned with hydrophobic and hydrophilic regions. We found that the cuboidic microgels ([sim]100-200 µm in width) could self-assemble into defined shapes with high fidelity to the surface patterns. The microgel assembly process was improved by increasing the hydrophilicity of the microgels and reducing the surface tension of the surrounding solution. The assembled microgels were stabilized by a secondary crosslink...

Computational study of the drag and oscillatory movement of biofilm streamers in fast flows

Biotechnology and Bioengineering — Wed, 23 Sep 2009 00:00:00 +0100

In this study, a state of the art two-dimensional fluid-structure interaction model of biofilm streamers is developed, which implements a transient coupling between the fluid and biofilm mechanics. Hereby, it is clearly shown that formation of a Kármán vortex street behind the streamer body is the main source of the periodic oscillation of the streamers. Additionally it is shown that the formation of streamers reduces the fluid forces which biofilm surface experiences. Biotechnol. Bioeng. © 2009 Wiley Periodicals, Inc. (Source: Biotechnology and Bioengineering)

Analysis of exposed cellulose surfaces in pretreated wood biomass using carbohydrate-binding module (CBM)-cyan fluorescent protein (CFP)

Biotechnology and Bioengineering — Wed, 23 Sep 2009 00:00:00 +0100

In enzymatic saccharification of lignocellulosics, the access of the enzymes to exposed cellulose surfaces is a key initial step in triggering hydrolysis. However, knowledge of the structure-hydrolyzability relationship of the pretreated biomass is still limited. Here we used fluorescent-labeled recombinant carbohydrate-binding modules (CBMs) from Clostridium josui as specific markers for crystalline cellulose (CjCBM3) and non-crystalline cellulose (CjCBM28) to analyze the complex surfaces of wood tissues pretreated with NaOH, NaOH-Na2S (kraft pulping), hydrothermolysis, ball-milling, and organosolvolysis. Japanese cedar wood, one of the most recalcitrant softwood species was selected for the analysis. The binding analysis clarified the linear dependency of the exposure of crystalline and ...

High-throughput microarray profiling of cell wall polymers during hydrothermal pre-treatment of wheat straw

Biotechnology and Bioengineering — Wed, 23 Sep 2009 00:00:00 +0100

Lignocellulosic plant material is potentially a sustainable source of fermentable sugars for bioethanol production. However, a barrier to this is the high resistance or recalcitrance of plant cell walls to be hydrolyzed. Therefore, a detailed knowledge of the structural features of plant cell walls that contribute to recalcitrance is important for improving the efficiency of bioethanol production. In this work we have used a technique known as Comprehensive Microarray Polymer Profiling (CoMPP) to analyze wheat straw before and after being subjected to hydrothermal pre-treatments at four different temperatures. The CoMPP technique combines the specificity of monoclonal antibodies with the high-throughput capacity of microarrays. Changes in the relative abundance of cell wall polysaccharides...

Serum-free, chemically defined medium with TGF-[beta]3 enhances functional properties of nucleus pulposus cell-laden carboxymethylcellulose hydrogel constructs

Biotechnology and Bioengineering — Wed, 23 Sep 2009 00:00:00 +0100

In this study, a standard serum-containing medium formulation was compared to a chemically defined, serum-free medium to determine the effect on matrix elaboration and functional properties of NP cell-laden carboxymethylcellulose (CMC) hydrogels. Additionally, both media were further supplemented with transforming growth factor-beta 3 (TGF-[beta]3). Glycosaminoglycan (GAG) content increased in both TGF-[beta]3-treated groups and was highest for treated, serum-free constructs (9.46 ± 1.51 µg GAG/mg wet weight), while there were no quantifiable GAGs in untreated serum-containing samples. Histology revealed uniform, interterritorial staining for chondroitin sulfate proteoglycan throughout the treated, serum-free constructs. Type II collagen content was greater in both serum-free groups and ...

High stability of self-assembled peptide nanowires against thermal, chemical, and proteolytic attacks

Biotechnology and Bioengineering — Wed, 23 Sep 2009 00:00:00 +0100

In this study, we investigated the stability of self-assembled peptide nanowires (PNWs) and nanotubes (PNTs) against thermal, chemical, proteolytic attacks, and their conformational changes upon heat treatment. PNWs and PNTs were grown by the self-assembly of diphenylalanine (Phe-Phe), a peptide building block, on solid substrates at different chemical atmospheres and temperatures. The incubation of diphenylalanine under aniline vapor at 150°C led to the formation of PNWs, while its incubation with water vapor at 25°C produced PNTs. We analyzed the stability of peptide nanostructures using multiple tools, such as electron microscopy, thermal analysis tools, circular dichroism, and Fourier-transform infrared spectroscopy. Our results show that PNWs are highly stable up to 200°C and remai...

Metabolic flux analysis for recombinant protein production by Pichia pastoris using dual carbon sources: Effects of methanol feeding rate

Biotechnology and Bioengineering — Wed, 23 Sep 2009 00:00:00 +0100

The intracellular metabolic fluxes through the central carbon pathways in the bioprocess for recombinant human erythropoietin (rHuEPO) production by Pichia pastoris (Mut+) were calculated to investigate the metabolic effects of dual carbon sources (methanol/sorbitol) and the methanol feed rate, and to obtain a deeper understanding of the regulatory circuitry of P. pastoris, using the established stoichiometry-based model containing 102 metabolites and 141 reaction fluxes. Four fed-batch operations with (MS-) and without (M-) sorbitol were performed at three different constant specific growth rates (h-1), and denoted as M-0.03, MS-0.02, MS-0.03, and MS-0.04. Considering the methanol consumption pathway, the M-0.03 and MS-0.02 conditions produced similar effects and had >85% of formaldehyde ...

Integrated solution to purification challenges in the manufacture of a soluble recombinant protein in E. coli

Biotechnology and Bioengineering — Wed, 23 Sep 2009 00:00:00 +0100

Apolipoprotein A 1 Milano (ApoA-1M), the protein component of a high-density lipoprotein (HDL) mimic with promising potential for reduction of atherosclerotic plaque, is produced at large scale by expression in E. coli. Significant difficulty with clearance of host cell proteins (HCPs) was experienced in the original manufacturing process despite a lengthy downstream purification train. Analysis of purified protein solutions and intermediate process samples led to identification of several major HCPs co-purifying with the product and a bacterial protease potentially causing a specific truncation of ApoA-1M found in the final product. Deletion of these genes from the original host strain succeeded in substantially reducing the levels of HCPs and the truncated species without adversely affec...

BAK and BAX deletion using zinc-finger nucleases yields apoptosis-resistant CHO cells

Biotechnology and Bioengineering — Wed, 23 Sep 2009 00:00:00 +0100

Anoxic and metabolic stresses in large-scale cell culture during biopharmaceutical production can induce apoptosis. Strategies designed to ameliorate the problem of apoptosis in cell culture have focused on mRNA knockdown of pro-apoptotic proteins and over-expression of anti-apoptotic ones. Apoptosis in cell culture involves mitochondrial permeabilization by the pro-apoptotic Bak and Bax proteins; activity of either protein is sufficient to permit apoptosis. We demonstrate here the complete and permanent elimination of both the Bak and Bax proteins in combination in Chinese hamster ovary (CHO) cells using zinc-finger nuclease-mediated gene disruption. Zinc-finger nuclease cleavage of BAX and BAK followed by inaccurate DNA repair resulted in knockout of both genes. Cells lacking Bax and Bak...

Perturbation-independent community development in low-temperature anaerobic biological wastewater treatment bioreactors

Biotechnology and Bioengineering — Tue, 22 Sep 2009 23:00:00 +0100

The reproducibility and stability of low- temperature anaerobic wastewater treatment systems undergoing transient perturbations was investigated. Three identical anaerobic expanded granular sludge bed-based bioreactors were used to degrade a volatile fatty acid and glucose-based wastewater under sub-ambient (15°C) conditions. The effect of a variety of environmental perturbations on bioreactor performance was assessed by chemical oxygen demand removal. Temporal microbial community development was monitored by denaturation gradient gel electrophoresis (DGGE) of 16S rRNA genes extracted from sludge granules. Methanogenic activity was monitored using specific methanogenic activity assays. Bioreactor performance and microbial population dynamics were each well replicated between both experime...

Aeration in fermentations

Biotechnology and Bioengineering — Sun, 20 Sep 2009 23:00:00 +0100

No Abstract. (Source: Biotechnology and Bioengineering)

3-hydroxypropionic acid, a value-added bulk chemical from glycerol

Biotechnology and Bioengineering — Sun, 20 Sep 2009 23:00:00 +0100

No Abstract. (Source: Biotechnology and Bioengineering)