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Journal: Biotechnology and bioengineering


Thermostability is an important feature in industrial enzymes: it increases biocatalyst lifetime and enables reactions at higher temperatures, where faster rates and other advantages ultimately reduce the cost of biocatalysis. Here we report the thermostabilization of a chimeric fungal family 6 cellobiohydrolase (HJPlus) by directed evolution using random mutagenesis and recombination of beneficial mutations. Thermostable variant 3C6P has a half-life of 280 minutes at 75°C and a T(50) of 80.1°C, a ∼15°C increase over the thermostable Cel6A from H. insolens (HiCel6A) and a ∼20°C increase over that from H. jecorina (HjCel6A). Most of the mutations also stabilize the less-stable HjCel6A, the wild-type Cel6A closest in sequence to 3C6P. During a 60-hour Avicel hydrolysis, 3C6P released 2.4 times more cellobiose equivalents at its optimum temperature (T(opt) ) of 75°C than HiCel6A at its T(opt) of 60°C. The total cellobiose equivalents released by HiCel6A at 60°C after 60 hours is equivalent to the total released by 3C6P at 75°C after ∼6 hours, a 10-fold reduction in hydrolysis time. A binary mixture of thermostable Cel6A and Cel7A hydrolyzes Avicel synergistically and released 1.8 times more cellobiose equivalents than the wild-type mixture, both mixtures assessed at their respective T(opt) . Crystal structures of HJPlus and 3C6P, determined at 1.5Å and 1.2Å resolution, indicate that the stabilization comes from improved hydrophobic interactions and restricted loop conformations by introduced proline residues. Biotechnol. Bioeng. © 2013 Wiley Periodicals, Inc.

Concepts: Mutation, Adenosine triphosphate, Enzyme, Starch, Cellulose, Hydrolysis, Enzymes, Cellulase


Plant expansin proteins induce plant cell wall extension and have the ability to extend and disrupt cellulose. In addition, these proteins show synergistic activity with cellulases during cellulose hydrolysis. BsEXLX1 originating from Bacillus subtilis is a structural homolog of a β-expansin produced by Zea mays (ZmEXPB1). The Langmuir isotherm for binding of BsEXLX1 to microcrystalline cellulose (i.e., Avicel) revealed that the equilibrium binding constant of BsEXLX1 to Avicel was similar to those of other Type A surface-binding carbohydrate-binding modules (CBMs) to microcrystalline cellulose, and the maximum number of binding sites on Avicel for BsEXLX1 was also comparable to those on microcrystalline cellulose for other Type A CBMs. BsEXLX1 did not bind to cellooligosaccharides, which is consistent with the typical binding behavior of Type A CBMs. The preferential binding pattern of a plant expansin, ZmEXPB1, to xylan, compared to cellulose was not exhibited by BsEXLX1. In addition, the binding capacities of cellulose and xylan for BsEXLX1 were much lower than those for CtCBD3. Biotechnol. Bioeng. 2013; 110: 401-407. © 2012 Wiley Periodicals, Inc.

Concepts: Cell, Bacteria, Cell wall, Polysaccharide, Bacillus, Cellulose, Hydrolysis, Firmicutes


Anion exchange membrane adsorbers are used for contaminant removal in flow-through polishing steps in the manufacture of biopharmaceuticals. This contribution describes the clearance of minute virus of mice, DNA, and host cell proteins by three commercially available anion-exchange membranes: Sartobind Q, Mustang Q, and ChromaSorb. The Sartobind Q and Mustang Q products contain quaternary amine ligands; whereas, ChromaSorb contains primary amine based ligands. Performance was evaluated over a range of solution conditions: 0-200 mM NaCl, pH 6.0-9.0, and flow rates of 4-20 membrane volumes/min in the presence and absence of up to 50 mM phosphate and acetate. In addition contaminant clearance was determined in the presence and absence of 5 g/L monoclonal antibody. The quaternary amine based ligands depend mainly on Coulombic interactions for removal of negatively charged contaminants. Consequently, performance of Sartobind Q and Mustang Q was compromised at high ionic strength. Primary amine based ligands in ChromaSorb enable high capacities at high ionic strength due to the presence of secondary, hydrogen bonding interactions. However, the presence of hydrogen phosphate ions leads to reduced capacity. Monoclonal antibody recovery using primary amine based anion-exchange ligands may be lower if significant binding occurs due to secondary interactions. The removal of a specific contaminant is affected by the level of removal of the other contaminants. The results of this study may be used to help guide selection of commercially available membrane absorbers for flow-through polishing steps. Biotechnol. Bioeng. 2013; 110: 500-510. © 2012 Wiley Periodicals, Inc.

Concepts: DNA, Protein, Acid, Ammonia, Hydrogen, Uranium, Ion, Salt


Mammalian cell cultures typically exhibit an energy inefficient phenotype characterized by the consumption of large quantities of glucose and the concomitant production of large quantities of lactate. Under certain conditions, mammalian cells can switch to a more energy efficient state during which lactate is consumed. Using a metabolic model derived from a mouse genome scale model we performed flux balance analysis of Chinese hamster ovary cells before and after a metabolic switch from lactate production (in the presence of glucose) to lactate consumption (after glucose depletion). Despite a residual degree of freedom after accounting for measurements, the calculated flux ranges and associated errors were narrow enough to enable investigation of metabolic changes across the metabolic switch. Surprisingly, the fluxes through the lower part of the TCA cycle from oxoglutarate to malate were very similar (around 60 µmol/gDW/h) for both phases. A detailed analysis of the energy metabolism showed that cells consuming lactate have an energy efficiency (total ATP produced per total C-mol substrate consumed) six times greater than lactate producing cells. Biotechnol. Bioeng. 2013; 110: 660-666. © 2012 Wiley Periodicals, Inc.

Concepts: Metabolism, Adenosine triphosphate, Organism, Mitochondrion, Cellular respiration, Cell culture, Chinese hamster ovary cell, Citric acid cycle


Since their introduction in 2001 miniaturized bioreactor systems have made great advances in function and performance. In this paper the dissolved oxygen (DO) transfer performance of sub millilitre microbioreactors, and 1 to 10 mL minibioreactors was examined. Microbioreactors have reached k(L) a values of 460 hr(-1) , and are offering instrumentation and some functionality comparable to production systems, but at high throughput screening volumes. Minibioreactors, aside from one 1440 hr(-1) k(L) a system, have not offered as high rates of DO transfer, but have demonstrated superior integration with automated fluid handling systems. Microbioreactors have been typically limited to studies with E. coli, while minibioreactors have offered greater versatility in this regard. Further, mathematical relationships confirming the applicability of k(L) a measurements across all scales have been derived, and alternatives to fluorescence lifetime DO sensors have been evaluated. Finally, the influence on reactor performance of oxygen uptake rate (OUR), and the possibility of its real time measurement have been explored. Biotechnol. Bioeng. © 2012 Wiley Periodicals, Inc.

Concepts: Time, Fluorescence, Oxygen, Function, Measurement, Biotechnology, 2001


This work critically reviews modeling concepts for standard activated sludge wastewater treatment processes (e.g., hydrolysis, growth and decay of organisms, etc.) for some of the most commonly used models. Based on a short overview on the theoretical biochemistry knowledge this review should help model users to better understand (i) the model concepts used; (ii) the differences between models, and (iii) the limits of the models. The seven analyzed models are: (1) ASM1; (2) ASM2d; (3) ASM3; (4) ASM3 + BioP; (5) ASM2d + TUD; (6) Barker & Dold model; and (7) UCTPHO+. Nine standard processes are distinguished and discussed in the present work: hydrolysis; fermentation; ordinary heterotrophic organisms (OHO) growth; autotrophic nitrifying organisms (ANO) growth; OHO & ANO decay; poly-hydroxyalkanoates (PHA) storage; polyphosphate (polyP) storage; phosphorus accumulating organisms PAO) growth; and PAO decay. For a structured comparison, a new schematic representation of these processes is proposed. Each process is represented as a reaction with consumed components on the left of the figure and produced components on the right. Standardized icons, based on shapes and color codes, enable the representation of the stoichiometric modeling concepts and kinetics. This representation allows highlighting the conceptual differences of the models, and the level of simplification between the concepts and the theoretical knowledge. The model selection depending on their theoretical limitations and the main research needs to increase the model quality are finally discussed. Biotechnol. Bioeng. 2013; 110: 24-46. © 2012 Wiley Periodicals, Inc.

Concepts: Bacteria, Organism, Truth, Sewage treatment, Model, Heterotroph, Activated sludge, Autotroph


The capability to print three-dimensional (3D) cellular tubes is not only a logical first step towards successful organ printing but also a critical indicator of the feasibility of the envisioned organ printing technology. A platform-assisted 3D inkjet bioprinting system has been proposed to fabricate 3D complex constructs such as zigzag tubes. Fibroblast (3T3 cell)-based tubes with an overhang structure have been successfully fabricated using the proposed bioprinting system. The post-printing 3T3 cell viability of printed cellular tubes has been found above 82% (or 93% with the control effect considered) even after a 72-h incubation period using the identified printing conditions for good droplet formation, indicating the promising application of the proposed bioprinting system. Particularly, it is proved that the tubular overhang structure can be scaffold-free fabricated using inkjetting, and the maximum achievable height depends on the inclination angle of the overhang structure. As a proof-of-concept study, the resulting fabrication knowledge helps print tissue-engineered blood vessels with complex geometry. Biotechnol. Bioeng. 2012; 109: 3152-3160. © 2012 Wiley Periodicals, Inc.

Concepts: Printing, Inkjet printer, Ink, Printing press, Lithography, Printmaking, Giclée, Old master print


3-Hydroxypropionaldehyde (3HPA) is an important specialty chemical which can be produced from glycerol using resting cells of Lactobacillus reuteri. This biocatalytic route, however, suffers from substrate- and product-mediated loss of enzyme activity within 2 h of biotransformation. In order to overcome the inhibitory effects of 3HPA, complex formation with sodium bisulfite was investigated, optimized and applied for in situ capture of the aldehyde during biotransformation of glycerol in a fed-batch process. As a result, the activity of the cells was maintained for at least 18 h. The 3HPA produced per gram cell dry weight was increased 5.7 times compared to the batch production process, and 2.2 times compared to fed-batch process without in situ complex formation. This approach may have potential for production and in situ removal of 3HPA after further process development. Biotechnol. Bioeng. © 2012 Wiley Periodicals, Inc.

Concepts: Bacteria, Enzyme, Microbiology, Biotechnology, Probiotic, Lactobacillus, Chemical engineering, Lactobacillus reuteri


Most mAb platform purification processes consist of an affinity capture step followed by 1 or 2 polishing steps. An understanding of the performance linkages between the unit operations can lead to robust manufacturing processes. In this study, a weak-partitioning anion-exchange chromatography polishing step used in a mAb purification process was characterized through high throughput screening (HTS) experiments, small-scale experiments including a cycling study performed on qualified scale-down models, and large-scale manufacturing runs. When material from a Protein A column that had been cycled less than 10 times was loaded on the AEX resin, early breakthrough of impurities and premature loss of capacity was observed. As the cycle number on the Protein A resin increased, the capacity of the subsequent AEX step increased. Different control strategies were considered for preventing impurity breakthrough and improving AEX resin lifetimes. Depth filtration of the Protein A peak pool significantly improved the AEX resin capacity, robustness and lifetime. Further, the turbidity of the Protein A pool has the potential for use as an in-process control parameter for monitoring the performance of the AEX step. Biotechnol. Bioeng. © 2012 Wiley Periodicals, Inc.

Concepts: Improve, Water purification, Analytical chemistry, Control theory, Performance, Ion exchange, Failure assessment, Impurity


The goal of this study is the selective oxyfunctionalization of aliphatic compounds under mild and environmentally-friendly conditions using a low-cost enzymatic biocatalyst. This could be possible taking advantage from a new peroxidase type that catalyzes monooxygenase reactions with H2 O2 as the only cosubstrate (peroxygenase). With this purpose, recombinant peroxygenase, from gene mining in the sequenced genome of Coprinopsis cinerea and heterologous expression using an industrial fungal host, is tested for the first time on aliphatic substrates. The reaction on free and esterified fatty acids and alcohols, and long-chain alkanes was followed by gas chromatography, and the different reaction products were identified by mass spectrometry. Regioselective hydroxylation of saturated/unsaturated fatty acids was observed at the ω-1 and ω-2 positions (only at the ω-2 position in myristoleic acid). Alkyl esters of fatty acids and monoglycerides were also ω-1 or ω-2 hydroxylated, but di- and triglycerides were not modified. Fatty alcohols yielded hydroxy derivatives at the ω-1 or ω-2 positions (diols) but also fatty acids and their hydroxy derivatives. Interestingly, the peroxygenase was able to oxyfunctionalize alkanes giving, in addition to alcohols at positions 2 or 3, dihydroxylated derivatives at both sides of the molecule. The predominance of mono- or dihydroxylated derivatives seems related to the higher or lower proportion of acetone, respectively, in the reaction medium. The recombinant C. cinerea peroxygenase appears as a promising biocatalyst for alkane activation and production of aliphatic oxygenated derivatives, with better properties than the previously reported peroxygenase from Agrocybe aegerita, and advantages related to its recombinant nature for enzyme engineering and industrial production. Biotechnol. Bioeng. © 2013 Wiley Periodicals, Inc.

Concepts: Alcohol, Metabolism, Fatty acid, Enzyme, Fatty acid metabolism, Ester, Alkane, Fatty alcohol