The stabilizing effects of immobilization in D-amino acid oxidase from Trigonopsis variabilis

<p>Abstract</p> <p>Background</p> <p>Immobilization of <it>Trigonopsis variabilis </it>D-amino acid oxidase (<it>Tv</it>DAO) on solid support is the key to a reasonably stable performance of this enzyme in the industrial process for the conversion of cephalosporin C as well as in other biocatalytic applications.</p> <p>Results</p> <p>To provide a mechanistic basis for the stabilization of the carrier-bound oxidase we analyzed the stabilizing effects of immobilization in <it>Tv</it>DAO exposed to the stress of elevated temperature and operational conditions. Two different strategies of immobilization were used: multi-point covalent binding to epoxy-activated Sepabeads EC-EP; and non-covalent oriented immobilization of the enzyme through affinity of its N-terminal <it>Strep</it>-tag to <it>Strep</it>-Tactin coated on insoluble particles. At 50°C, the oriented immobilizate was not stabilized as compared to the free enzyme. The structure of <it>Tv</it>DAO was stabilized via covalent attachment to Sepabeads EC-EP but concomitantly, binding of the FAD cofactor was weakened. FAD release from the enzyme into solution markedly reduced the positive effect of immobilization on the overall stability of <it>Tv</it>DAO. Under conditions of substrate conversion in a bubble-aerated stirred tank reactor, both immobilization techniques as well as the addition of the surfactant Pluronic F-68 stabilized <it>Tv</it>DAO by protecting the enzyme from the deleterious effect of gas-liquid interfaces. Immobilization of <it>Tv</it>DAO on Sepabeads EC-EP however stabilized the enzyme beyond this effect and led to a biocatalyst that could be re-used in multiple cycles of substrate conversion.</p> <p>Conclusion</p> <p>Multi-point covalent attachment of <it>Tv</it>DAO on an isoluble porous carrier provides stabilization against the denaturing effects of high temperature and exposure to a gas-liquid interface. Improvement of binding of the FAD cofactor, probably by using methods of protein engineering, would further enhance the stability of the immobilized enzyme.</p

Altering the coenzyme preference of xylose reductase to favor utilization of NADH enhances ethanol yield from xylose in a metabolically engineered strain of Saccharomyces cerevisiae

<p>Abstract</p> <p>Background</p> <p>Metabolic engineering of <it>Saccharomyces cerevisiae </it>for xylose fermentation into fuel ethanol has oftentimes relied on insertion of a heterologous pathway that consists of xylose reductase (XR) and xylitol dehydrogenase (XDH) and brings about isomerization of xylose into xylulose via xylitol. Incomplete recycling of redox cosubstrates in the catalytic steps of the NADPH-preferring XR and the NAD⁺-dependent XDH results in formation of xylitol by-product and hence in lowering of the overall yield of ethanol on xylose. Structure-guided site-directed mutagenesis was previously employed to change the coenzyme preference of <it>Candida tenuis </it>XR about 170-fold from NADPH in the wild-type to NADH in a Lys²⁷⁴→Arg Asn²⁷⁶→Asp double mutant which in spite of the structural modifications introduced had retained the original catalytic efficiency for reduction of xylose by NADH. This work was carried out to assess physiological consequences in xylose-fermenting <it>S. cerevisiae </it>resulting from a well defined alteration of XR cosubstrate specificity.</p> <p>Results</p> <p>An isogenic pair of yeast strains was derived from <it>S. cerevisiae </it>Cen.PK 113-7D through chromosomal integration of a three-gene cassette that carried a single copy for <it>C. tenuis </it>XR in wild-type or double mutant form, XDH from <it>Galactocandida mastotermitis</it>, and the endogenous xylulose kinase (XK). Overexpression of each gene was under control of the constitutive TDH3 promoter. Measurement of intracellular levels of XR, XDH, and XK activities confirmed the expected phenotypes. The strain harboring the XR double mutant showed 42% enhanced ethanol yield (0.34 g/g) compared to the reference strain harboring wild-type XR during anaerobic bioreactor conversions of xylose (20 g/L). Likewise, the yields of xylitol (0.19 g/g) and glycerol (0.02 g/g) were decreased 52% and 57% respectively in the XR mutant strain. The xylose uptake rate per gram of cell dry weight was identical (0.07 ± 0.02 h^-1) in both strains.</p> <p>Conclusion</p> <p>Integration of enzyme and strain engineering to enhance utilization of NADH in the XR-catalyzed conversion of xylose results in notably improved fermentation capabilities of recombinant <it>S. cerevisiae</it>.</p

Orthophosphate binding at the dimer interface of Corynebacterium callunae starch phosphorylase: mutational analysis of its role for activity and stability of the enzyme

<p>Abstract</p> <p>Background</p> <p>Orthophosphate recognition at allosteric binding sites is a key feature for the regulation of enzyme activity in mammalian glycogen phosphorylases. Protein residues co-ordinating orthophosphate in three binding sites distributed across the dimer interface of a non-regulated bacterial starch phosphorylase (from <it>Corynebacterium callunae</it>) were individually replaced by Ala to interrogate their unknown function for activity and stability of this enzyme.</p> <p>Results</p> <p>While the mutations affected neither content of pyridoxal 5'-phosphate cofactor nor specific activity in phosphorylase preparations as isolated, they disrupted (Thr²⁸→Ala, Arg¹⁴¹→Ala) or decreased (Lys³¹→Ala, Ser¹⁷⁴→Ala) the unusually strong protective effect of orthophosphate (10 or 100 mM) against inactivation at 45°C and subunit dissociation enforced by imidazole, as compared to wild-type enzyme. Loss of stability in the mutated phosphorylases appeared to be largely due to weakened affinity for orthophosphate binding. Binding of sulphate mimicking the crystallographically observed "non-covalent phosphorylation" of the phosphorylase at the dimer interface did not have an allosteric effect on the enzyme activity.</p> <p>Conclusions</p> <p>The phosphate sites at the subunit-subunit interface of <it>C. callunae </it>starch phosphorylase appear to be cooperatively functional in conferring extra kinetic stability to the native dimer structure of the active enzyme. The molecular strategy exploited for quaternary structure stabilization is to our knowledge novel among dimeric proteins. It can be distinguished clearly from the co-solute effect of orthophosphate on protein thermostability resulting from (relatively weak) interactions of the ligand with protein surface residues.</p

Rules for biocatalyst and reaction engineering to implement effective, NAD(P)H-dependent, whole cell bioreductions

AbstractAccess to chiral alcohols of high optical purity is today frequently provided by the enzymatic reduction of precursor ketones. However, bioreductions are complicated by the need for reducing equivalents in the form of NAD(P)H. The high price and molecular weight of NAD(P)H necessitate in situ recycling of catalytic quantities, which is mostly accomplished by enzymatic oxidation of a cheap co-substrate. The coupled oxidoreduction can be either performed by free enzymes in solution or by whole cells. Reductase selection, the decision between cell-free and whole cell reduction system, coenzyme recycling mode and reaction conditions represent design options that strongly affect bioreduction efficiency. In this paper, each option was critically scrutinized and decision rules formulated based on well-described literature examples. The development chain was visualized as a decision-tree that can be used to identify the most promising route towards the production of a specific chiral alcohol. General methods, applications and bottlenecks in the set-up are presented and key experiments required to “test” for decision-making attributes are defined. The reduction of o-chloroacetophenone to (S)-1-(2-chlorophenyl)ethanol was used as one example to demonstrate all the development steps. Detailed analysis of reported large scale bioreductions identified product isolation as a major bottleneck in process design

From wheat straw to bioethanol: integrative analysis of a separate hydrolysis and co-fermentation process with implemented enzyme production

BACKGROUND: Lignocellulosic ethanol has a high potential as renewable energy source. In recent years, much research effort has been spent to optimize parameters involved in the production process. Despite that, there is still a lack of comprehensive studies on process integration. Single parameters and process configurations are, however, heavily interrelated and can affect the overall process efficiency in a multitude of ways. Here, we present an integrative approach for bioethanol production from wheat straw at a representative laboratory scale using a separate hydrolysis and co-fermentation (SHCF) process. The process does not rely on commercial (hemi-) cellulases but includes enzyme production through Hypocrea jecorina (formerly Trichoderma reesei) on the pre-treated feedstock as key unit operation. Hydrolysis reactions are run with high solid loadings of 15% dry mass pre-treated wheat straw (DM WS), and hydrolyzates are utilized without detoxification for mixed glucose-xylose fermentation with the genetically and evolutionary engineered Saccharomyces cerevisiae strain IBB10B05. RESULTS: Process configurations of unit operations in the benchtop SHCF were varied and evaluated with respect to the overall process ethanol yield (Y(Ethanol-Process)). The highest Y(Ethanol-Process) of 71.2 g ethanol per kg raw material was reached when fungal fermentations were run as batch, and the hydrolysis reaction was done with an enzyme loading of 30 filter paper units (FPU)/g(DM WS). 1.7 ± 0.1 FPU/mL were produced, glucose and xylose were released with a conversion efficiency of 67% and 95%, respectively, and strain IBB10B05 showed an ethanol yield of 0.4 g/g(Glc + Xyl) in 15% hydrolyzate fermentations. Based on the detailed process analysis, it was further possible to identify the enzyme yield, the glucose conversion efficiency, and the mass losses between the unit operations as key process parameters, exhibiting a major influence on Y(Ethanol-Process). CONCLUSIONS: Y(Ethanol-Process) is a measure for the efficiency of the lignocellulose-to-bioethanol process. Based on mass balance analysis, the correlations between single process parameters and Y(Ethanol-Process) were elucidated. The optimized laboratory scale SHCF process showed efficiencies similar to pilot scale plants. The herein presented process analysis can serve as effective and simple tool to identify key process parameters, bottlenecks, and future optimization targets. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13068-015-0232-0) contains supplementary material, which is available to authorized users

Monitoring and control of the release of soluble O2 from H2O2 inside porous enzyme carrier for O2 supply to an immobilized d-amino acid oxidase

While O(2) substrate for bio‐transformations in bulk liquid is routinely provided from entrained air or O(2) gas, tailored solutions of O(2) supply are required when the bio‐catalysis happens spatially confined to the microstructure of a solid support. Release of soluble O(2) from H(2)O(2) by catalase is promising, but spatiotemporal control of the process is challenging to achieve. Here, we show monitoring and control by optical sensing within a porous carrier of the soluble O(2) formed by an immobilized catalase upon feeding of H(2)O(2). The internally released O(2) is used to drive the reaction of d‐amino acid oxidase (oxidation of d‐methionine) that is co‐immobilized with the catalase in the same carrier. The H(2)O(2) is supplied in portions at properly timed intervals, or continuously at controlled flow rate, to balance the O(2) production and consumption inside the carrier so as to maintain the internal O(2) concentration in the range of 100–500 µM. Thus, enzyme inactivation by excess H(2)O(2) is prevented and gas formation from the released O(2) is avoided at the same time. The reaction rate of the co‐immobilized enzyme preparation is shown to depend linearly on the internal O(2) concentration up to the air‐saturated level. Conversions at a 200 ml scale using varied H(2)O(2) feed rate (0.04–0.18 mmol/min) give the equivalent production rate from d‐methionine (200 mM) and achieve rate enhancement by ∼1.55‐fold compared to the same oxidase reaction under bubble aeration. Collectively, these results show an integrated strategy of biomolecular engineering for tightly controlled supply of O(2) substrate from H(2)O(2) into carrier‐immobilized enzymes. By addressing limitations of O(2) supply via gas‐liquid transfer, especially at the microscale, this can be generally useful to develop specialized process strategies for O(2)‐dependent biocatalytic reactions

Host cell and expression engineering for development of an E. coli ketoreductase catalyst: Enhancement of formate dehydrogenase activity for regeneration of NADH

<p>Abstract</p> <p>Background</p> <p>Enzymatic NADH or NADPH-dependent reduction is a widely applied approach for the synthesis of optically active organic compounds. The overall biocatalytic conversion usually involves <it>in situ </it>regeneration of the expensive NAD(P)H. Oxidation of formate to carbon dioxide, catalyzed by formate dehydrogenase (EC 1.2.1.2; FDH), presents an almost ideal process solution for coenzyme regeneration that has been well established for NADH. Because isolated FDH is relatively unstable under a range of process conditions, whole cells often constitute the preferred form of the biocatalyst, combining the advantage of enzyme protection in the cellular environment with ease of enzyme production. However, the most prominent FDH used in biotransformations, the enzyme from the yeast <it>Candida boidinii</it>, is usually expressed in limiting amounts of activity in the prime host for whole cell biocatalysis, <it>Escherichia coli</it>. We therefore performed expression engineering with the aim of enhancing FDH activity in an <it>E. coli </it>ketoreductase catalyst. The benefit resulting from improved NADH regeneration capacity is demonstrated in two transformations of technological relevance: xylose conversion into xylitol, and synthesis of (<it>S</it>)-1-(2-chlorophenyl)ethanol from <it>o</it>-chloroacetophenone.</p> <p>Results</p> <p>As compared to individual expression of <it>C. boidinii </it>FDH in <it>E. coli </it>BL21 (DE3) that gave an intracellular enzyme activity of 400 units/g_CDW, co-expression of the FDH with the ketoreductase (<it>Candida tenuis </it>xylose reductase; XR) resulted in a substantial decline in FDH activity. The remaining FDH activity of only 85 U/g_CDWwas strongly limiting the overall catalytic activity of the whole cell system. Combined effects from increase in FDH gene copy number, supply of rare tRNAs in a Rosetta strain of <it>E. coli</it>, dampened expression of the ketoreductase, and induction at low temperature (18°C) brought up the FDH activity threefold to a level of 250 U/g_CDWwhile reducing the XR activity by just 19% (1140 U/g_CDW). The <it>E. coli </it>whole-cell catalyst optimized for intracellular FDH activity showed improved performance in the synthesis of (<it>S</it>)-1-(2-chlorophenyl)ethanol, reflected in a substantial, up to 5-fold enhancement of productivity (0.37 g/g_CDW) and yield (95% based on 100 mM ketone used) as compared to the reference catalyst. For xylitol production, the benefit of enhanced FDH expression was observed on productivity only after elimination of the mass transfer resistance caused by the cell membrane.</p> <p>Conclusions</p> <p>Expression engineering of <it>C. boidinii </it>FDH is an important strategy to optimize <it>E. coli </it>whole-cell reductase catalysts that employ intracellular formate oxidation for regeneration of NADH. Increased FDH-activity was reflected by higher reduction yields of D-xylose and <it>o</it>-chloroacetophenone conversions provided that mass transfer limitations were overcome.</p