Engineered whole-cell biocatalyst-based detoxification and detection of neurotoxic organophosphate compounds

The development of efficient tools is required for the eco-friendly detoxification and effective detection of neurotoxic organophosphates (OPs). Although enzymes have received significant attention as biocatalysts because of their high specific activity, the uneconomic and labor-intensive processes of enzyme production and purification make their broad use in practical applications difficult. Because whole-cell systems offer several advantages compared with free enzymes, including high stability, a reduced purification requirement, and low preparation cost, they have been suggested as promising biocatalysts for the detoxification and detection of OPs. To develop efficient whole-cell biocatalysts with enhanced activity and a broad spectrum of substrate specificity, several factors have been considered, namely the selected strains, the chosen OP-hydrolyzing enzymes, where enzymes are localized in a cell, and which enhancer will assist the expression, function, and folding of the enzyme. In this article, we review the current investigative progress in the development of engineered whole-cell biocatalysts with excellent OP-hydrolyzing activity, a broad spectrum of substrate specificity, and outstanding stability for the detoxification and detection of OPs.     

Chemical modification of proteolytic enzymes by low molecular weight agents

Low-molecular modification of proteolytic enzymes with aldehydes and anhydrides of carboxylic acids as well as with 2,4,6-trinitrobenzene sulphonic acid was studied. Specific activities of the enzymes were found to be dependent on the modification degree of their amino groups. The retaining of high activities in the region of low extents of enzyme modification enabled biocatalysts with activities similar to those of the native enzymes to be prepared.     

Commercial scale biocatalysis: myths and realities

The unique ways in which enzymes are differentiated from other catalysts translate into special advantages. Understanding these advantages is the key toward better matching of biocatalysts needs in industrial chemistry. Specific cases where enzymes and biotransformations have been used successfully at the production scale are examined, permitting the realities of using biocatalysts to be separated from the misconceptions and myths. Five such misconceptions will be examined in the context of examples of some commercially-successful biocatalytic processes.     

生物催化剂立体选择性的基因工程改造

野生型生物催化剂对于其天然底物通常具有较好的反应活性和选择性,但生物催化剂在有机合成中应用时多数情况下是非天然底物,这就要求对野生型生物催化剂进行改造,以提高其对非天然底物的反应活性、稳定性和选择性(包括区域选择性和立体选择性)。以下根据酶催化剂的类型总结了近几年来通过基因工程改变生物催化剂的立体选择性的最新进展,盼望起到抛砖引玉的作用,以此促进我国在这一领域的快速发展。     

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.     

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.     

Engineering natural products using combinatorial biosynthesis and biocatalysis

Many biologically active natural products are produced by the host organisms using dedicated biosynthetic pathways. The programming rules of these pathways may be rationally manipulated through combinatorial biosynthesis to produce natural products that contain structural variations or enhanced pharmacological properties. Furthermore, these pathways contain enzymes that can be harvested as powerful biocatalysts for the synthesis of both new drugs and existing blockbuster therapeutics. This review will highlight recent advances in exploring natural product biosynthetic pathways for new compounds, novel enzymes and useful biocatalysts.     

Surface-modified polymeric nanogranules containing entrapped enzymes: A novel biocatalyst for use in organic media

Summary A novel biocatalytic system for use in organic solvents, based on enzymes entrapped into surface-modified polymeric nanogranules, is suggested. Nanogranulated biocatalysts are soluble in organic solvents of different polarity, possess high stability and catalytic activity, and can be used continuously in membrane reactors.     

The search for the ideal biocatalyst.

While the use of enzymes as biocatalysts to assist in the industrial manufacture of fine chemicals and pharmaceuticals has enormous potential, application is frequently limited by evolution-led catalyst traits. The advent of designer biocatalysts, produced by informed selection and mutation through recombinant DNA technology, enables production of process-compatible enzymes. However, to fully realize the potential of designer enzymes in industrial applications, it will be necessary to tailor catalyst properties so that they are optimal not only for a given reaction but also in the context of the industrial process in which the enzyme is applied.     

Enzymatic and whole cell catalysis: Finding new strategies for old processes

The use of enzymes and whole bacterial cells has allowed the production of a plethora of compounds that have been used for centuries in foods and beverages. However, only recently we have been able to master techniques that allow the design and development of new biocatalysts with high stability and productivity. Rational redesign and directed evolution have lead to engineered enzymes with new characteristics whilst the understanding of adaptation mechanisms in bacterial cells has allowed their use under new operational conditions. Bacteria able to thrive under the most extreme conditions have also provided new and extraordinary catalytic processes. In this review, the new tools available for the improvement of biocatalysts are presented and discussed.     

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.     

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.     

生物催化剂发现与改造的研究进展

生物催化是指将酶或生物有机体用于有用的化学转化的过程,在人们对传统化学催化的环境影响抱有忧虑的情况下,生物催化提供了一种有吸引力的选择。在过去的几十年里,对生物催化剂的研究每出现一次大的进步,生物催化的发展就会出现一次高潮。因此,生物催化剂的发现与改造已成为当今研究的热点。宏基因组文库技术的出现克服了许多微生物不可培养的障碍,人们能够从自然资源中获得丰富的潜在的生物催化剂。而基于理性设计的分子改造技术的发展,可以使得人们对潜在的生物催化剂进行快速而有效的改造以满足工业化生产的需求。随着生物催化剂发现与改造的手段不断进步,更多的优良生物催化剂得到了广泛的应用,生物催化在工业生产中也得到了更深入的应用。结合作者的研究工作,总结了生物催化剂发现与改良的一些研究进展,以为获得更多优良的、能够实现工业应用的生物催化剂奠定理论基础。     

Integrating enzyme immobilization and protein engineering: An alternative path for the development of novel and improved industrial biocatalysts

Enzyme immobilization often achieves reusable biocatalysts with improved operational stability and solvent resistance. However, these modifications are generally associated with a decrease in activity or detrimental modifications in catalytic properties. On the other hand, protein engineering aims to generate enzymes with increased performance at specific conditions by means of genetic manipulation, directed evolution and rational design. However, the achieved biocatalysts are generally generated as soluble enzymes, ?thus not reusable- and their performance under real operational conditions is uncertain.Combined protein engineering and enzyme immobilization approaches have been employed as parallel or consecutive strategies for improving an enzyme of interest. Recent reports show efforts on simultaneously improving both enzymatic and immobilization components through genetic modification of enzymes and optimizing binding chemistry for site-specific and oriented immobilization. Nonetheless, enzyme engineering and immobilization are usually performed as separate workflows to achieve improved biocatalysts.In this review, we summarize and discuss recent research aiming to integrate enzyme immobilization and protein engineering and propose strategies to further converge protein engineering and enzyme immobilization efforts into a novel “immobilized biocatalyst engineering” research field. We believe that through the integration of both enzyme engineering and enzyme immobilization strategies, novel biocatalysts can be obtained, not only as the sum of independently improved intrinsic and operational properties of enzymes, but ultimately tailored specifically for increased performance as immobilized biocatalysts, potentially paving the way for a qualitative jump in the development of efficient, stable biocatalysts with greater real-world potential in challenging bioprocess applications.     

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.     

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.     

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.     

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.     

Immobilized Biocatalysts for the Production of Nucleosides and Nucleoside Analogues by Enzymatic Transglycosylation Reactions

The recombinant enzymes uridine phosphorylase (UP) and purine nucleoside phosphorylase (PNP) were over-expressed in high-biomass bacterial fermentations and co-immobilized, without previous purification, on epoxy-activated solid supports by covalent linkages. These preparations are efficient biocatalysts of transglycosylation reactions and have been developed for producting natural and modified nucleosides of pharmaceutical interest in the field of antiviral and antitumoral agents. The new biocatalysts described in this work are suitable for both laboratory and industrial scale applications due to the maintainance of high catalytic efficiency, thermal and solvent stability, reusability and ease of operation in batch as well as in continuous reactions.     

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.