Remarkable activation of enzymes in nonaqueous media by denaturing organic cosolvents

The rates of transesterification reactions catalyzed by the protease subtilisin Carlsberg suspended in various anhydrous solvents at 30 degrees C can be increased more than 100-fold by the addition of denaturing organic cosolvents (dimethyl sulfoxide or formamide); in water, the same cosolvents exert no enzyme activation. At 4 degrees C, the activation effect on the lyophilized protease is even higher, reaching 1000-fold. Marked enhancement of enzymatic activity in anhydrous solvents by formamide is also observed for two other enzymes, alpha-chymotrypsin and Rhizomucor miehei lipase, and is manifested in two transesterification reactions. In addition to lyophilized subtilisin, crosslinked crystals of subtilisin are also amenable to the dramatic activation by the denaturing cosolvents. In contrast, subtilisin solubilized in anhydrous media by covalent modification with poly(ethylene glycol) exhibits only modest activation. These observations are rationalized in terms of a mechanistic hypothesis based on an enhanced protein flexibility in anhydrous millieu brought about by the denaturing organic cosolvents. The latter exert their lubricating effect largely at the interfaces between enzyme molecules in a solid preparation, thus easing the flexibility constraints imposed by protein-protein contacts. (c) 1996 John Wiley & Sons, Inc.     

非水酶学和非水相生物催化研究进展

近年来工业生物技术飞速发展,酶学和生物催化领域也取得突破性进展,特别在酶在非水相中活性及稳定性研究,耐溶剂生物催化剂的筛选、构建、修饰和改造,生物相容性和环境相容性好的绿色介质等方面取得了较大的进展。最近的研究热点和未来几年的研究方向主要为:基于基因组信息的耐溶剂酶的虚拟筛选和构建;基于自然界筛选新酶基因的耐溶剂酶重构和改造;离子液体等环境友好的绿色介质系统等几个方面。     

Carrier-free immobilized enzymes for biocatalysis

Methods for the preparation of carrier-free insoluble enzymes are reviewed. The technology of cross-linked enzyme aggregates has now been applied to a range of synthetically useful activities. Fusion proteins are also gaining momentum because they allow a relatively selective aggregation or even a specific self-assembly of the desired enzyme activity into insoluble particles in the absence of potentially denaturing chemicals required for precipitation and cross-linking. Recycling of insoluble protein particles for multiple rounds of batchwise reaction has been demonstrated in selected biotransformations. However, for application in a fully continuous biocatalytic process, low resistance to mechanical stress and high compressibility are issues for consideration on carrier-free enzyme particles.     

Enzyme stabilization by covalent binding in nanoporous sol-gel glass for nonaqueous biocatalysis

A unique nanoporous sol-gel glass possessing a highly ordered porous structure (with a pore size of 153 A in diameter) was examined for use as a support material for enzyme immobilization. A model enzyme, alpha-chymotrypsin, was efficiently bound onto the glass via a bifunctional ligand, trimethoxysilylpropanal, with an active enzyme loading of 0.54 wt%. The glass-bound chymotrypsin exhibited greatly enhanced stability both in aqueous solution and organic solvents. The half-life of the glass-bound alpha-chymotrypsin was >1000-fold higher than that of the native enzyme, as measured either in aqueous buffer or anhydrous methanol. The enhanced stability in methanol, which excludes the possibility of enzyme autolysis, particularly reflected that the covalent binding provides effective protection against enzyme inactivation caused by structural denaturation. In addition, the activity of the immobilized alpha-chymotrypsin was also much higher than that of the native enzyme in various organic solvents. From these results, it appears that the glass-enzyme complex developed in the present work can be used as a high-performance biocatalyst for various chemical processing applications, particularly in organic media. Published by John Wiley & Sons     

Directed evolution of enzymes for applied biocatalysis

Directed evolution has rapidly emerged as a powerful new strategy for improving the characteristics of enzymes in a targeted manner. By coupling various protocols for generating large variant libraries of genes, together with high-throughput screens that select for specific properties of an enzyme, such as thermostability, catalytic activity and substrate specificity, it is now possible to optimize biocatalysts for specific applications. However, further work is required to broaden the range of screens that can be used, particularly in terms of reaction type, such as hydroxylation and carbon-carbon bond formation, and functional characteristics, such as enantioselectivity and regioselectivity, so that directed evolution can be used in a routine manner for biocatalyst development.     

Activation and fragmentation of Bacillus thuringiensis delta-endotoxin by high concentrations of proteolytic enzymes

Commercial enzymes and insect gut juice at various concentrations were used to digest Bacillus thuringiensis subsp. sotto Cry1Aa protoxin and examine the fragmentation pattern and effect on insecticidal activity. Trypsin at both high (5 mg/mL) and low (0.05 mg/mL) concentrations converted protoxin to toxin with no difference in insecticidal activity against Bombyx mori larvae. In both cases, the toxin protein had an apparent M(r) of 58.4 kDa (SDS-PAGE). Active toxin of identical M(r) was also produced with low concentrations of Pronase and subtilisin, but at high concentration, it was degraded into two protease-resistant fragments of apparent M(r) 31.8 and 29.6 kDa, and exhibited no insecticidal activity. Sequencing data established the primary cleavage site to be in domain II, the receptor-binding region of the toxin, in an exposed loop between two beta-sheet strands. Fragmentation was not observed, however, when the digests were analyzed by native protein techniques, but rather the toxin molecule appeared to be intact. The amount of activated toxin produced by Choristoneura fumiferana gut juice was markedly reduced when the gut-juice concentration was increased from 1 to 50% and correlated with a loss in insecticidal activity. However, no lower M(r) protease-resistant fragments were evident in the SDS-PAGE of these digests.     

Directed evolution of enzymes and pathways for industrial biocatalysis

Directed evolution has become a powerful tool for developing enzyme and whole cell based biocatalysts. Significant recent advances include the creation of novel enzyme functions and the development of several new efficient directed evolution methods. The combination of directed evolution and rational design promises to accelerate the development of biocatalysts for applications in the pharmaceutical, chemical and food industries.     

Exploitation of carbohydrate processing enzymes in biocatalysis

Exploitation of enzymes in biocatalytic processes provides scope both in the synthesis and degradation of molecules. Enzymes have power not only in their catalytic efficiency, but their chemoselectivity, regioselectivity, and stereoselectivity means the reactions they catalyze are precise and reproducible. Focusing on carbohydrate processing enzymes, this review covers advances in biocatalysis involving carbohydrates over the last 2–3 years. Given the notorious difficulties in the chemical synthesis of carbohydrates, the use of enzymes for synthesis has potential for significant impact in the future. The use of catabolic enzymes in the degradation of biomass, which can be exploited in the production of biofuels to provide a sustainable and greener source of energy, and the synthesis of molecules that have a range of applications including in the pharmaceutical and food industries will be explored.     

Emerging strategies for expanding the toolbox of enzymes in biocatalysis

Expanding the repertoire of reactions available to enzymes is an enduring challenge in biocatalysis. Owing to the synthetic versatility of transition metals, metalloenzymes have been favored targets for achieving new catalytic functions. Although less well explored, enzymes lacking metal centers can also be effective catalysts for non-natural reactions, providing access to reaction modalities that compliment those available to metals. By understanding how these activation modes can reveal new functions, strategies can be developed to access novel biocatalytic reactions. This review will cover discoveries in the last two years which access catalytic reactions that go beyond the native repertoire of metal-free biocatalysts.     

High-throughput strategies for the discovery and engineering of enzymes for biocatalysis

Bioprocess and Biosystems Engineering - Innovations in novel enzyme discoveries impact upon a wide range of industries for which biocatalysis and biotransformations represent a great challenge,...     

Engineering Pichia pastoris for biocatalysis: co-production of two active enzymes

High levels of active glycolate oxidase from spinach (GO) and active catalase T from Saccharomyces cerevisiae (catT) have been co-produced in the methylotrophic yeast Pichia pastoris (Pp). In sequential rounds of transformation using two selectable markers, multiple copies of the genes encoding GO and catT were integrated into the Pp chromosome under control of the methanol inducible alcohol oxidase I promoter, resulting in a strain designated MSP8.6. MSP8.6 is a second-generation biocatalyst used for the conversion of glycolate to glyoxylate in the presence of a reaction component which inhibits endogenous Pp catalase. This work demonstrates a significant advance in the utility of recombinant Pp for commercial bioprocess development.     

Inhibition of urea cycle enzymes by lysine and saccharopine

Crude and purified preparations of argininosuccinate synthetase, argininosuccinate lyase and arginase were subjected to inhibition studies with L-lysine and saccharopine. Saccharopine proved to be the more potent inhibitor of argininosuccinate synthetase and lyase, whereas lysine had more effect on arginase. Similar results were found with pure enzyme and crude preparations. Computer analysis of the results suggested that inhibition of urea cycle enzymes by saccharopine and lysine might have contributed to the high levels of citrulline found in a human patient with saccharopinuria, a defect of saccharopine metabolism, but that this was unlikely to be the sole explanation.     

Activation of the latent human neutrophil gelatinase by urea.

The mechanism of activation of the latent human neutrophil gelatinase by urea has been studied in greater detail. After dialysis of the latent gelatinase against increasing concentrations of urea a considerable increase of its activity was observed. Moreover, the results indicate a progressive conversion of the latent 94,000 Da gelatinase into a proteolytically active fragment of 80,000 Da, which was subsequently processed to a few species of lower molecular mass inactive against gelatin. This conversion was completely inhibited by EDTA, suggesting an autocatalytic reaction. The inhibition was reversed by Zn2+ or Co2+. Thus, urea alters both the enzymatic and physical characteristics of the latent gelatinase which suggests that conformational changes may induce autoactivation of the latent enzyme.     

Activation of vitronectin (serum spreading factor) binding of heparin by denaturing agents

Vitronectin (serum spreading factor), a cell-adhesive glycoprotein present in mammalian serum, has previously been the subject of conflicting reports concerning its binding to heparin. Vitronectin purified from human plasma does not bind to heparin under physiological conditions, but it does so after treatment with denaturing agents including 8 M urea or 6 M guanidine-HC1, or heating at 100 degrees C for 5 min. These treatments seem to expose a heparin-binding site in vitronectin; this finding thus resolves the conflicts concerning this function.