Pictet–Spenglerases in alkaloid biosynthesis: Future applications in biocatalysis

Pictet–Spenglerases provide a key role in the biosynthesis of many biologically active alkaloids. There is increasing use of these biocatalysts as an alternative to traditional organic synthetic methods as they provide stereoselective and regioselective control under mild conditions. Products from these enzymes also contain privileged drug scaffolds (such as tetrahydroisoquinoline or β-carboline moieties), so there is interest in the characterization and use of these enzymes as versatile biocatalysts to synthesize analogs of the corresponding natural products for drug discovery. This review discusses all known Pictet–Spenglerase enzymes and their applications as biocatalysts.     

The use of molecular modelling in the understanding of configurational specificity (R or S) in asymmetric reactions catalyzed by Saccharomyces cerevisiae or isolated dehydrogenases

This method gives a general ideal how to use crystallographic information of enzymes to understand reactions catalyzed by these biocatalysts, commonly used by biochemists to produce chiral products. The interactions of three acetoacetic esters with the enzymes L-lactate dehydrogenase and alcohol dehydrogenase were studied through molecular modelling computer program. These artificial substrates have been widely used to produce chiral synthons. Through this methodology it was possible to understand the conformational specificity of these enzymes with respect to the products and how these enzymes can be inhibited by modifying the structures of the artificial substrates. Also, it was possible to predict whether some type of artificial substrate will suffer reduction by cells that contain these dehydrogenases and what kind of configuration (R or S) the final product will have.     

Screening for novel biocatalysts

This review attempts to demonstrate the importance of goal-orientated screening for new biocatalysts. Examples of enzymes and microorganisms that have been developed and that have acquired commercial applications are described so as to illustrate the technological potential of biocatalysts. A survey of screening techniques and recently reported examples of screening from food, chemical, pharmaceutical and waste disposal applications etc. are also presented to demonstrate the feasibility of this approach for generating new biocatalysts. An appreciation of some of the difficulties involved, the achievements of Japanese researchers and some examples of the cornucopia of largely unrecognized and potentially valuable microbial activities are also given. An increased effort in screening would have the following benefits: an increased range of biocatalysts with different enzyme activities would be available and more biocatalysts with improved characteristics, suitable for use under industrial conditions, such as resistance to elevated temperatures, extremes of pH and organic solvents would be discovered. Secondly the manpower and other resources required to carry out screening programmes would be reduced, for instance by developing automated techniques. Thirdly, screening procedures would be made much more accessible to non-specialists. Fourthly, improved efforts and expertise in screening would supplement other emerging techniques such as protein engineering. The development of selective, non-random, goal-orientated screening techniques, methods of evaluating biocatalyst performance under operational conditions, and an approach that is more orientated towards commercially desirable goals are essential if these objectives are to be achieved. Screening of naturally occurring microorganisms still appears to be the best way to obtain new strains and/or enzymes for commercial applications. However, two major problems appear to exist. Firstly in identifying applications that are technically feasible and that have sufficient commercial potential to justify the research and development required to generate a new commercially viable biocatalyst and secondly the relatively small number of scientists outside Japan with skill and experience in screening for biocatalysts.     

Recent advances in heme biocatalysis engineering

Heme enzymes have the potential to be widely used as biocatalysts due to their capability to perform a vast variety of oxidation reactions. In spite of their versatility, the application of heme enzymes was long time-limited for the industry due to their low activity and stability in large scale processes. The identification of novel natural biocatalysts and recent advances in protein engineering have led to new reactions with a high application potential. The latest creation of a serine-ligated mutant of BM3 showed an efficient transfer of reactive carbenes into C═C bonds of olefins reaching total turnover numbers of more than 60,000 and product titers of up to 27 g/L⁻¹. This prominent example shows that heme enzymes are becoming competitive to chemical syntheses while being already advantageous in terms of high yield, regioselectivity, stereoselectivity and environmentally friendly reaction conditions. Advances in reactor concepts and the influencing parameters on reaction performance are also under investigation resulting in improved productivities and increased stability of the heme biocatalytic systems. In this mini review, we briefly present the latest advancements in the field of heme enzymes towards increased reaction scope and applicability.     

宏基因组学在微生物活性物质筛选中的应用

采用传统分离培养筛选微生物新活性物质的方法受到很大制约,自然界99%以上的微生物不能培养,其资源开发受到很大限制。环境微生物宏基因组技术应用避开了微生物分离纯培养问题,极大拓展了微生物资源的利用空间,增加获得新活性物质的机会和途径。本文着重介绍宏基因组的概念、研究策略包括DNA提取、文库构建与筛选等及在微生物活性物质筛选中的应用,并对宏基因组研究中存在的问题进行探讨。     

Enzymatic Modifications of Cephalosporins by Cephalosporin Acylase and Other Enzymes

ABSTRACT

Semisynthetic cephalosporins are important antibacterials in clinical practice. Semisynthetic cephalosporins are manufactured by derivatizing 7-aminocephalosporanic acid (7-ACA) and its desacetylated form. Microbial enzymes such as D-amino acid oxidase, glutaryl-7-ACA acylase and cephalosporin esterase are being used as biocatalysts for the conversion of cephalosporin C (CEPH-C) to 7-ACA and its desacetylated derivatives. Recent developments in the field of enzymatic modifications of cephalosporin with special emphasis on group of enzymes called as cephalosporin acylase is discussed in this review. Aspects related to screening methods, isolation and purification, immobilization, molecular cloning, gene structure and expression and protein engineering of cephalosporin acylases have been covered. Topics pertaining to enzymatic modifications of cephalosporin by D-amino acid oxidase, cephalosporin methoxylase and β -lactamase are also covered.     

Metagenomics: an inexhaustible access to nature's diversity

The chemical industry has an enormous need for innovation. To save resources, energy and time, currently more and more established chemical processes are being switched to biotechnological routes. This requires white biotechnology to discover and develop novel enzymes, biocatalysts and applications. Due to a limitation in the cultivability of microbes living in certain habitats, technologies have to be established which give access to the enormous resource of uncultivated microbial diversity. Metagenomics promises to provide new and diverse enzymes and biocatalysts as well as bioactive molecules and has the potential to make industrial biotechnology an economic, sustainable success.     

Glycosynthases: new enzymes for oligosaccharide synthesis

The mutation of putative acid/base and nucleophile of the active sites of retaining glycosyl hydrolases, together with kinetic analysis of the mutants, and stereochemical identification of products lead to useful information for the understanding of the reaction mechanism of these enzymes. This was the preliminary and fundamental step toward the preparation of new enzymatic activities called glycosynthases. Direct exploitation of this information has been possible, leading to the design of four new enzymes for oligosaccharides synthesis. The interest for these biocatalysts rises from the fact that the yield of the reaction can be increased and selectivity can be interpreted as key characteristic of the transfer reaction instead of a balance of hydrolytic and transferring pathways followed either by substrates and products. These new biocatalysts possess different specificities and are promising and useful tools in the construction of oligosaccharide molecules of great biological interest. This short review focused the attention on different glycosynthases obtained from four glycosyl hydrolases highlighting on the preparation and development of these new enzymes.     

Bioconversions in aqueous two-phase systems.

Bioconversions involving enzymes and/or microbial cells in aqueous two-phase systems are reviewed. The partitioning of biocatalysts, substrates, and products is discussed in relation to their size. The efficiency of retaining biocatalysts in aqueous two-phase systems is summarized in relation to other methods of recirculating. The influence of phase components on the activity and the stability of enzymatic biocatalysts is exemplified with penicillin acylase and the cellulolytic enzyme system, and the effect of phase components on biocatalytic living cells is exemplified with the production of alpha-amylase with Bacillus sp. Process design costs in bioconversions in aqueous two-phase systems are briefly summarized.     

Biocatalytic synthesis of atorvastatin intermediates

Atorvastatin is a 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor, and this drug leads to decreased levels of low density lipoprotein (LDL) cholesterol. Lower LDL cholesterol has direct relationship in reducing mortality from coronary heart diseases. Lipitor® (atorvastatin calcium) was the first drug to reach the annual sales of 10 billion dollars in USA and currently is the top selling pharmaceutical product globally. Atorvastatin has a side chain containing two chiral centers as its pharmacophore and it can be synthesized either from chiral pool precursors, by using metal catalysts; or more preferably by the application of free or immobilized enzymes and whole cell biocatalysts for carrying out either asymmetric synthesis or racemic resolution. Biocatalytic synthesis methods for chiral atorvastatin intermediates employ a wide variety of biocatalysts such as alcohol dehydrogenase, 2-deoxy-d-ribose 5-phosphate aldolase, nitrilase, lipase, etc. and each of these biocatalytic processes is discussed in detail in this paper.     

Microbial lipolytic fusion enzymes: current state and future perspectives

Genetic fusion of coding ORFs or connection of proteins in a post translational process are rather novel techniques to build products called fusion proteins that possess combined characteristics of their parental biomolecules. This attractive strategy used to create new enzymes not only diversifies their functionality by improving thermostability, thermo- and catalytic activity, substrate specificity, regio- or enantio-selectivity but also facilitates their purification and increases their yield. Many examples of microbial synthetic fusion biocatalysts are associated with fused enzymes that are involved in biomass degradation. However, one of the leading production segments is occupied by microbial lipolytic enzymes (lipases and esterases). As powerful biocatalysts these enzymes found their application in detergent, food, oil and fat, pulp and paper, leather, textile, cosmetics, biodiesel production industries. Moreover, lipolytic enzymes market is predicted to maintain leadership up to the year of 2024 and exceed millions of dollars. Recently, creation of lipolytic fusion biocatalysts for industrial applications gained more attention since it is not only a way of achievement of enzymes with improved properties but also a way to reduce industrial energy costs and ensure other economic benefits. This paper provides a comprehensive review on current state of microbial lipolytic fusion enzymes and their future potential.     

Recent trends and novel concepts in cofactor-dependent biotransformations

Cofactor-dependent enzymes catalyze a broad range of synthetically useful transformations. However, the cofactor requirement also poses economic and practical challenges for the application of these biocatalysts. For three decades, considerable research effort has been devoted to the development of reliable in situ regeneration methods for the most commonly employed cofactors, particularly NADH and NADPH. Today, researchers can choose from a plethora of options, and oxidoreductases are routinely employed even on industrial scale. Nevertheless, more efficient cofactor regeneration methods are still being developed, with the aim of achieving better atom economy, simpler reaction setups, and higher productivities. Besides, cofactor dependence has been recognized as an opportunity to confer novel reactivity upon enzymes by engineering their cofactors, and to couple (redox) biotransformations in multi-enzyme cascade systems. These novel concepts will help to further establish cofactor-dependent biotransformations as an attractive option for the synthesis of biologically active compounds, chiral building blocks, and bio-based platform molecules.     

Novel biocatalysts for white biotechnology

White Biotechnology uses microorganisms and enzymes to manufacture a large variety of chemical products. Therefore, the demand for new and useful biocatalysts is steadily and rapidly increasing. We have developed methods for the isolation of new enzyme genes, constructed novel expression systems, and optimized existing enzymes for biotechnological applications by methods of directed evolution. Furthermore, we have isolated and characterized biocatalysts relevant for the preparation of enantiopure compounds.     

High throughput screening methods for ω-transaminases

Recently, ω-transaminases have been increasingly used to synthesize amine compounds by reductive amination of prochiral ketones which are of high pharmacological significance. However, the conventional methods for evaluating these enzymes are time consuming and have often been regarded as a bottle neck in developing these enzymes as industrial biocatalysts. In the past few years, several high throughput screening methods have been developed for fast evaluation and identification of ω-transaminase. This review summarizes the various methodologies developed for rapidly screening ω-transaminases.     

Potential biocatalysts originating from sea environments

This review is intended to give an account of the knowledge about known enzymes of marine origin described in literature thus stimulating future applications in biocatalysis that these biocatalysts can offer to a large spectra of end-users. The uniqueness of marine biocatalysts is not only based on habitat-related properties such as salt tolerance, hyperthermostability, barophilicity, cold adaptivity, etc. A marine enzyme in fact may carry more, e.g. novel chemical and stereochemical properties. This “chemical biodiversity” increases interest in this field; substrate specificity and affinity are evolved properties linked to the metabolic functions of the enzymes and to ecological asset related to the natural source and this is an important aspect in the bioprospecting for new biocatalysts. The importance of all examples reported should be sufficient to trigger the attention of the biocatalytically oriented scientific community towards marine environment as source of biocatalysts, and this could in turn enhance both new discovery and improvement of marine enzymes.     

Laccase immobilization and insolubilization: from fundamentals to applications for the elimination of emerging contaminants in wastewater treatment

Over the last few decades many attempts have been made to use biocatalysts for the biotransformation of emerging contaminants in environmental matrices. Laccase, a multicopper oxidoreductase enzyme, has shown great potential in oxidizing a large number of phenolic and non-phenolic emerging contaminants. However, laccases and more broadly enzymes in their free form are biocatalysts whose applications in solution have many drawbacks rendering them currently unsuitable for large scale use. To circumvent these limitations, the enzyme can be immobilized onto carriers or entrapped within capsules; these two immobilization techniques have the disadvantage of generating a large mass of non-catalytic product. Insolubilization of the free enzymes as cross-linked enzymes (CLEAs) is found to yield a greater volume ratio of biocatalyst while improving the characteristics of the biocatalyst. Ultimately, novel techniques of enzymes insolubilization and stabilization are feasible with the combination of cross-linked enzyme aggregates (combi-CLEAs) and enzyme polymer engineered structures (EPESs) for the elimination of emerging micropollutants in wastewater. In this review, fundamental features of laccases are provided in order to elucidate their catalytic mechanism, followed by different chemical aspects of the immobilization and insolubilization techniques applicable to laccases. Finally, kinetic and reactor design effects for enzymes in relation with the potential applications of laccases as combi-CLEAs and EPESs for the biotransformation of micropollutants in wastewater treatment are discussed.     

Enzyme cascade converting cyclohexanol into ε-caprolactone coupled with NADPH recycling using surface displayed alcohol dehydrogenase and cyclohexanone monooxygenase on E. coli

The application of enzymes as biocatalysts in industrial processes has great potential due to their outstanding stereo-, regio- and chemoselectivity. Using autodisplay, enzymes can be immobilized on the cell surface of Gram-negative bacteria such as Escherichia coli. In the present study, the surface display of an alcohol dehydrogenase (ADH) and a cyclohexanone monooxygenase (CHMO) on E. coli was investigated. Displaying these enzymes on the surface of E. coli resulted in whole-cell biocatalysts accessible for substrates without further purification. An apparent maximal reaction velocity V_MAX(app) for the oxidation of cyclohexanol with the ADH whole-cell biocatalysts was determined as 59.9 mU ml⁻¹. For the oxidation of cyclohexanone with the CHMO whole-cell biocatalysts a V_MAX(app) of 491 mU ml⁻¹ was obtained. A direct conversion of cyclohexanol to ε-caprolactone, which is a known building block for the valuable biodegradable polymer polycaprolactone, was possible by combining the two whole-cell biocatalysts. Gas chromatography was applied to quantify the yield of ε-caprolactone. 1.12 mM ε-caprolactone was produced using ADH and CHMO displaying whole-cell biocatalysts in a ratio of 1:5 after 4 h in a cell suspension of OD_578nm 10. Furthermore, the reaction cascade as applied provided a self-sufficient regeneration of NADPH for CHMO by the ADH whole-cell biocatalyst.     

催化元件和途径的人工设计与组装

催化元件以及由多个催化元件组成的合成途径的设计与组装为人工合成体系的建立奠定了基础,是合成生物学的重要研究内容。除从自然生物中挖掘大量的天然酶和途径可供人工合成体系使用外,将计算生物学、蛋白质工程以及组合生物合成等技术相结合,理性地、有目的地进行催化元件和途径的人工设计与组装,将提供新功能酶以及新物质合成途径。介绍了催化元件和合成途径人工设计与组装的研究策略和最新进展。     

Advances in recovery of novel biocatalysts from metagenomes

Metagenomics has accelerated the process of discovery of novel biocatalysts by enabling scientists to tap directly into the entire diversity of enzymes held within natural microbial populations. Their characterization has revealed a great deal of valuable information about enzymatic activity in terms of factors which influence their stability and activity under a wide range of conditions. Many of the biocatalysts have particular properties making them suitable for biotechnological applications. A diverse array of strategies has been developed to optimize each step of the process of generating and screening metagenomic libraries for novel biocatalysts. This review covers the diversity of metagenome-derived enzymes characterized to date, and the strategies currently being developed to optimize discovery of novel metagenomic biocatalysts.     

Insect Transgenesis: Mechanisms,Applications, and Ecological Safety

Abstract

Glycosylation is considered to be an important reaction for the chemical modification of compounds with useful biological activities. Glycoside hydrolases are biotechnologically attractive enzymes which can be used in synthetic reactions for assembling glycosidic linkages with absolute stereoselectivity at an anomeric centre. Most of these enzymes are commercially available but there is great interest in the search for new biocatalysts with original catalytic characteristics. The marine environment has shown to be a very interesting source for new glycosyl hydrolases for both hydrolytic and synthetic aspects. In particular, Aplysia fasciata a marine herbivorous mollusc has been shown to be a potent producer of a library of glycoside hydrolases applied to the synthesis of glycosidic bonds. The impressive assortment of glycosidases in marine organisms clearly indicates that the potential biodiversity of these enzymes is still largely unexplored and that potential applications of biocatalysts from the sea will increase in the near future.