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。     

Application of whole cell rhodococcal biocatalysts in acrylic polymer manufacture

Rhodococci are ubiquitous in nature and their ability to metabolise a wide range of chemicals, many of which are toxic, has given rise to an increasing number of studies into their diverse use as biocatalysts. Indeed rhodococci have been shown to be especially good at degrading aromatic and aliphatic nitriles and amides and thus they are very useful for waste clean up where these toxic chemicals are present.The use of biocatalysts in the chemical industry has in the main been for the manufacture of high-value fine chemicals, such as pharmaceutical intermediates, though investigations into the use of nitrile hydratase, amidase and nitrilase to convert acrylonitrile into the higher value products acrylamide and acrylic acid have been carried out for a number of years. Acrylamide and acrylic acid are manufactured by chemical processes in vast tonnages annually and they are used to produce polymers for applications such as superabsorbents, dispersants and flocculants. Rhodococci are chosen for use as biocatalysts on an industrial scale for the production of acrylamide and acrylic acid due to their ease of growth to high biomass yields, high specific enzyme activities obtainable, their EFB class 1 status and robustness of the whole cells within chemical reaction systems.Several isolates belonging to the genus Rhodococcus have been shown in our studies to be among the best candidates for acrylic acid preparation from acrylonitrile due to their stability and tolerance to high concentrations of this reactive and disruptive substrate. A critical part of the selection procedure for the best candidates during the screening programme was high purity product with very low residual substrate concentrations, even in the presence of high product concentrations. Additionally the nitrile and amide substrate scavenging ability which enables rhodococci to survive very successfully in the environment leads to the formation of biocatalysts which are suitable for the removal of low concentrations of acrylonitrile and acrylamide in waste streams and for the removal of impurities in manufacturing processes.     

Immobilization of biocatalysts with poly(vinyl alcohol) supports.

Two polymer materials, poly(vinyl alcohol) (PVA) superfine fibers and photocrosslinkable PVA bearing styrylpyridinium groups, have been developed to immobilize biocatalysts. The former has a large surface consisting of relatively large-size pores and the fibers can immobilize a large amount of biocatalyst on their surface by ionic interaction. The latter entraps many kinds of biocatalysts by cyclodimerization caused by visible light irradiation. The biocatalysts on/in these supports maintain high activity and thermal stability. These materials can easily be formed into various shapes suitable for various applications. A new bioreactor system was constructed for evaluating a variety of biocatalysts and supports.     

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.     

Developing microbial biofilm as a robust biocatalyst and its challenges

Biocatalysts, such as bacteria, yeast, fungi and the enzymes they produce, have been used for many industrial applications since they function as effective and environmentally friendly tools. Whole cells have also been used in many sophisticated bioprocesses since a number of sequential reactions can be catalyzed within the cells. However, the use of whole cells in suspension in batch, fed-batch and continuous processes has some limitations. For instance, the cultures are non-reusable, they are sometimes sensitive to the toxicity of substrates or products, there can be issues with short-term stability, and each of these issues can impede biocatalyst regeneration, perturbing the downstream process and causing complexity in running large scale continuous culture. Recently, biofilms have emerged as a new generation of biocatalysts to solve these limitations in the production of many bio-based materials, including chemicals, antibiotics, enzymes, bioethanol, biohydrogen, and electricity production via microbial fuel cells. The establishment of industrial processes using biofilms has the potential for high benefit in terms of low-cost cell immobilization without the necessity of added polymers or chemicals. Many small-scale biofilm reactors have been developed for the production of value-added products, and it may be challenging to establish it on an industrial scale.     

Prospecting for biocatalysts and drugs in the genomes of non-cultured microorganisms

Modern biotechnology has a steadily increasing demand for vitamins, antibiotics and, in particular, novel biocatalysts for use in the production of flavors, agrochemicals, pharmaceuticals and high-value fine chemicals. Novel experimental approaches are being developed in attempts to identify such molecules. However, it is known that up to 99.8% of the microbes present in many environments are not readily culturable; hence, they cannot be exploited for biotechnology. The 'metagenome technology' offers a solution to this problem by developing culture-independent methods to isolate, clone and express environmental DNA. So far, metagenome-based approaches have led to the isolation of many novel biocatalysts and a variety of other molecules with a high potential for downstream applications.     

Candida spp. redox machineries: An ample biocatalytic platform for practical applications and academic insights

The use of oxidoreductases as biocatalysts for the production of a wide number of chiral building blocks is presently a mature (bio-)technology. In this context some industrial applications are currently performed by means of those enzymatic approaches, and new examples are expected to be realized. Moreover, oxidoreductases provide an interesting academic platform to undertake fundamental research in enzymology, to acquire a better understanding on catalytic mechanisms, and to facilitate the development of new biocatalytic applications. Within this area, a wide number of oxidoreductases from genus Candida spp. have been characterized and used as biocatalysts. These enzymes are rather diverse, and are able to carry out many useful reactions, like highly (enantio)selective keto-reductions, (de)racemizations and stereoinversions, and promiscuous catalytic imine reductions. In addition, some Candida spp. dehydrogenases are very useful for regenerating the cofactors, with the aid of sacrificial substrates. Addressing those features, the present paper aims to give an overview of these enzymes, by focusing on practical applications that these biocatalysts can provide. Furthermore, when possible, academic insights on the enzymatic performances will be discussed as well.     

Incorporation of non-natural modules into proteins: structural features beyond the genetic code

The biotechnological application of enzymes necessitates a permanent quest for new biocatalysts. Among others, improvement of catalytic activity, modification of substrate specificity, or increase in stability of the enzymes are desirable goals. The exploration of homologous enzymes from various sources or DNA-based methods, like site-directed mutagenesis or directed evolution, yield an incredible variety of biocatalysts but they all rely on the restricted number of canonical amino acids. Chemistry offers an almost unlimited palette of additional modifications which can endow the proteins with improved or even completely new properties. Numerous techniques to furnish proteins with non-natural amino acids or non-proteinogenic modules have been introduced and are reviewed with special focus on expressed protein ligation, a method that combines the potential of protein biosynthesis and chemical synthesis. An erratum to this article can be found at     

Preparative use of isolated CYP102 monooxygenases -- a critical appraisal

Isolated P450 monooxygenases have for long been neglected catalysts in enzyme technology. This is surprising as they display a remarkable substrate specificity catalyzing reactions, which represent a challenge for classic organic chemistry. On the other hand, many P450 monooxygenases are membrane bound, depend on rather complicated electron transfer systems and require expensive cofactors such as NAD(P)H. Their activities are low, and stability leaves much to be desired. The use of bacterial P450 monooxygenases from CYP102 family allows overcoming some of these handicaps. They are soluble and their turnovers are high, presumably because their N-terminal heme monooxygenase and their C-terminal diflavin reductase domain are covalently linked. In recent years, protein engineering approaches have been successfully used to turn CYP102 monooxgenases into powerful biocatalysts.     

Screening for novel enzymes from metagenome and SIGEX,as a way to improve it

Metagenomics has been successfully applied to isolate novel biocatalysts from the uncultured microbiota in the environment. Two types of screening have been used to identify clones carrying desired traits from metagenomic libraries: function-based screening, and sequence-based screening. Both function- and sequence- based screening have individual advantages and disadvantages, and they have been applied successfully to discover biocatalysts from metagenome. However, both strategies are laborious and tedious because of the low frequency of screening hits. A recent paper introduced a high throughput screening strategy, termed substrate-induced gene-expression screening (SIGEX). SIGEX is designed to select the clones harboring catabolic genes induced by various substrates in concert with fluorescence activated cell sorting (FACS). This method was applied successfully to isolate aromatic hydrocarbon-induced genes from a metagenomic library. Although SIGEX has many limitations, it is expected to provide economic advantages, especially to industry.     

Improving the ‘tool box’ for robust industrial enzymes

The speed of sequencing of microbial genomes and metagenomes is providing an ever increasing resource for the identification of new robust biocatalysts with industrial applications for many different aspects of industrial biotechnology. Using ‘natures catalysts’ provides a sustainable approach to chemical synthesis of fine chemicals, general chemicals such as surfactants and new consumer-based materials such as biodegradable plastics. This provides a sustainable and ‘green chemistry’ route to chemical synthesis which generates no toxic waste and is environmentally friendly. In addition, enzymes can play important roles in other applications such as carbon dioxide capture, breakdown of food and other waste streams to provide a route to the concept of a ‘circular economy’ where nothing is wasted. The use of improved bioinformatic approaches and the development of new rapid enzyme activity screening methodology can provide an endless resource for new robust industrial biocatalysts.This mini-review will discuss several recent case studies where industrial enzymes of ‘high priority’ have been identified and characterised. It will highlight specific hydrolase enzymes and recent case studies which have been carried out within our group in Exeter.

     

极端微生物：一种新型的酶资源

极端微生物具有自身独特的特点和代谢产物 ,在食品工业、化工、药用工业和环境生物技术领域都有潜在的应用。一些酶已经得到纯化 ,其基因在宿主中已成功克隆。主要介绍和讨论极端微生物的类型、基因组及极端酶类的生产、分离与应用。     

Progress in biocatalysis with immobilized viable whole cells: systems development,reaction engineering and applications

Viable microbial cells are important biocatalysts in the production of fine chemicals and biofuels, in environmental applications and also in emerging applications such as biosensors or medicine. Their increasing significance is driven mainly by the intensive development of high performance recombinant strains supplying multienzyme cascade reaction pathways, and by advances in preservation of the native state and stability of whole-cell biocatalysts throughout their application. In many cases, the stability and performance of whole-cell biocatalysts can be highly improved by controlled immobilization techniques. This review summarizes the current progress in the development of immobilized whole-cell biocatalysts, the immobilization methods as well as in the bioreaction engineering aspects and economical aspects of their biocatalytic applications.     

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.     

Cofactor regeneration for sustainable enzymatic biosynthesis

Oxidoreductases are attractive catalysts for biosynthesis of chiral compounds and polymers, construction of biosensors, and degradation of environmental pollutants. Their practical applications, however, can be quite challenging since they often require cofactors such as nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP). These cofactors are generally expensive. Efficient regeneration of cofactors is therefore critical to the economic viability of industrial-scale biotransformations using oxidoreductases. The chemistry of cofactor regeneration is well known nowadays. The challenge is mostly regarding how to achieve the regeneration with immobilized enzyme systems which are preferred for industrial processes to facilitate the recovery and continuous use of the catalysts. This has become a great hurdle for the industrialization of many promising enzymatic processes, and as a result, most of the biotransformations involving cofactors have been traditionally performed with living cells in industry. Accompanying the rapidly growing interest in industrial biotechnology, immobilized enzyme biocatalyst systems with cofactor regeneration have been the focus for many studies reported since the late 1990s. The current paper reviews the methods of cofactor retention for development of sustainable and regenerative biocatalysts as revealed in these recent studies, with the intent to complement other reviewing articles that are mostly regeneration chemistry-oriented. We classify in this paper the methods of sustainable cofactor regeneration into two categories, namely membrane entrapment and solid-attachment of cofactors.     

Metagenomics for the development of new biocatalysts to advance lignocellulose saccharification for bioeconomic development

The world economy is moving toward the use of renewable and nonedible lignocellulosic biomasses as substitutes for fossil sources in order to decrease the environmental impact of manufacturing processes and overcome the conflict with food production. Enzymatic hydrolysis of the feedstock is a key technology for bio-based chemical production, and the identification of novel, less expensive and more efficient biocatalysts is one of the main challenges. As the genomic era has shown that only a few microorganisms can be cultured under standard laboratory conditions, the extraction and analysis of genetic material directly from environmental samples, termed metagenomics, is a promising way to overcome this bottleneck. Two screening methodologies can be used on metagenomic material: the function-driven approach of expression libraries and sequence-driven analysis based on gene homology. Both techniques have been shown to be useful for the discovery of novel biocatalysts for lignocellulose conversion, and they enabled identification of several (hemi)cellulases and accessory enzymes involved in (hemi)cellulose hydrolysis. This review summarizes the latest progress in metagenomics aimed at discovering new enzymes for lignocellulose saccharification.     

<Emphasis Type="Italic">Acinetobacter</Emphasis> lipases: molecular biology,biochemical properties and biotechnological potential

Lipases (EC 3.1.1.3) have received increased attention recently, evidenced by the increasing amount of information about lipases in the current literature. The renewed interest in this enzyme class is due primarily to investigations of their role in pathogenesis and their increasing use in biotechnological applications [38]. Also, many microbial lipases are available as commercial products, the majority of which are used in detergents, cosmetic production, food flavoring, and organic synthesis. Lipases are valued biocatalysts because they act under mild conditions, are highly stable in organic solvents, show broad substrate specificity, and usually show high regio- and/or stereo-selectivity in catalysis. A number of lipolytic strains of Acinetobacter have been isolated from a variety of sources and their lipases possess many biochemical properties similar to those that have been developed for biotechnological applications. This review discusses the biology of lipase expression in Acinetobacter, with emphasis on those aspects relevant to potential biotechnology applications.     

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.     

Cell-free protein synthesis enables one-pot cascade biotransformation in an aqueous-organic biphasic system

Biocatalytic cascade reactions have become increasingly important and useful for chemical synthesis. However, biocatalysts are often incompatible with organic solvents, which prohibits many cascade reactions involving nonpolar substrates. In this study, we used cell-free protein synthesis (CFPS) to express enzymes in an aqueous-organic biphasic system for the construction of an artificial enzymatic pathway. CFPS-expressed enzymes without purification performed efficiently to convert styrene (below 20 mM) to (S)-1-phenyl-1,2-ethanediol (two steps in one pot) with 100% conversion. In addition, our CFPS system showed great tolerance to different organic solvents, and, importantly, the entire biocatalytic system can be consistently scaled up without a reduction of the substrate conversion rate. We, therefore, anticipate that our cell-free approach will make a possible cost-effective, high-yielding synthesis of valuable chemicals.     

Biocatalysis for the synthesis of pharmaceuticals and pharmaceutical intermediates

Biocatalysis has been increasingly used for pharmaceutical synthesis in an effort to make manufacturing processes greener and more sustainable. Biocatalysts that possess excellent activity, specificity, thermostability and solvent-tolerance are highly sought after to meet the requirements of practical applications. Generating biocatalysts with these specific properties can be achieved by either discovery of novel biocatalysts or protein engineering. Meanwhile, chemoenzymatic routes have also been designed and developed for pharmaceutical synthesis on an industrial scale. This review discusses the recent discoveries, engineering, and applications of biocatalysts for the synthesis of pharmaceuticals and pharmaceutical intermediates. Key classes of biocatalysts include reductases, oxidases, hydrolases, lyases, isomerases, and transaminases.