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.     

Cytochrome P450 monooxygenases: an update on perspectives for synthetic application

Cytochrome P450 monooxygenases (P450s) are versatile biocatalysts that catalyze the regio- and stereospeci?c oxidation of non-activated hydrocarbons under mild conditions, which is a challenging task for chemical catalysts. Over the past decade impressive advances have been achieved via protein engineering with regard to activity, stability and specificity of P450s. In addition, a large pool of newly annotated P450s has attracted much attention as a source for novel biocatalysts for oxidation. In this review we give a short up-to-date overview of recent results on P450 engineering for technical applications including aspects of whole-cell biocatalysis with engineered recombinant enzymes. Furthermore, we focus on recently identified P450s with novel biotechnologically relevant properties.     

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.     

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。     

Aspects Of Biocatalyst Stability In Organic Solvents

The stability of biocatalysis in systems containing organic solvents is reviewed. Among the examples presented are homogeneous mixtures of water and water-miscible organic solvents, aqueous/organic two-phase systems, solid biocatalysts suspended in organic solvents, enzymes in reverse micelles and modified enzymes soluble in water immiscible solvents. The stability of biocatalysts in organic solvents depends very much on the conditions. The hydrophobicity or the polarity of the solvent is clearly of great importance. More hydrophobic solvents (higher log P values) are less harmful to enzymes than less hydrophobic solvents. The water content of the system is a very important parameter. Some water is essential for enzymatic activity; however, the stability of enzymes decreases with increasing water content. Mechanisms of enzyme inactivation are discussed.     

Effect of immiscible organic solvents on activity/stability of native chymotrypsin and immobilized-stabilized derivatives

We have developed different activity/stability tests to evaluate the possibilities of fully dispersed chymotrypsin derivatives as industrial catalysts in biphasic systems. We have tested different immiscible organic solvents (log P ranged from 0.65 to 2.8) and used different enzyme derivatives (soluble chymotrypsin and one-point and multipoint covalent attached derivatives). Special emphasis has been given to the role of the "exact composition of the aqueous phase."High phosphate concentrations largely protect every hymotrypsin derivative from the distorting effects of dissolved solvent molecules. The effects on the activity and stability of soluble chymotrypsin due to saturating solvent concentrations in an aqueous solution, and the much more severe effects of contact with the phase interface in a stirred biphasic system, all show the opposite trend for the influence of solvent polarity to that generally observed for biocatalysts. For example, deleterious effects decline in the order chloroform, dichloromethane, ethyl acetate. On the contrary, with or without stirring, our stabilized chymotrypsin-agarose derivatives are much more stable against these water-immiscible solvents, and their relative effects follow the normal trend. From these integrated activity and stability tests we can conclude that fully dispersed immobilized-stabilized derivatives seem to be an interesting alternative to develop industrial biphasic processes catalyzed by chymotrypsin.     

Immobilized yeast membranes as biocatalysts for sucrose inversion

Yeast membranes were obtained by autolysis of various strains with relatively high invertase activity. Heterogeneous biocatalysts for sucrose inversion were made of the yeast membranes and granulated carbon-containing supports made of common natural materials: expanded clay aggregate (ECA), sapropel, and lignin. The properties of these biocatalysts were studied. It was shown that the biocatalyst activity and stability of the immobilized yeast membranes increased with reference to the initial ECA, independent of the structure of the carbon layer synthesized on the support surface. Heterogeneous biocatalysts prepared by adsorption of yeast membranes on sapropel had the greatest activity and stability, whereas lignin-based biocatalysts were relatively unstable.     

Maximizing the stability of metabolic engineering‐derived whole‐cell biocatalysts

A systematic and powerful knowledge‐based framework exists for improving the activity and stability of chemical catalysts and for empowering the commercialization of respective processes. In contrast, corresponding biotechnological processes are still scarce and characterized by case‐by‐case development strategies. A systematic understanding of parameters affecting biocatalyst efficiency, that is, biocatalyst activity and stability, is essential for a rational generation of improved biocatalysts. Today, systematic approaches only exist for increasing the activity of whole‐cell biocatalysts. They are still largely missing for whole‐cell biocatalyst stability. In this review, we structure factors affecting biocatalyst stability and summarize existing, yet not completely exploited strategies to overcome respective limitations. The factors and mechanisms related to biocatalyst destabilization are discussed and demonstrated inter alia based on two case studies. The factors are similar for processes with different objectives regarding target molecule or metabolic pathway complexity and process scale, but are in turn highly interdependent. This review provides a systematic for the stabilization of whole‐cell biocatalysts. In combination with our knowledge on strategies to improve biocatalyst activity, this paves the way for the rational design of superior recombinant whole‐cell biocatalysts, which can then be employed in economically and ecologically competitive and sustainable bioprocesses.     

微生物脂肪酶蛋白质工程*

微生物脂肪酶催化的化学反应具有严格的立体选择性、位点选择性等专一性,催化活性高而副反应少,催化反应不需要辅助因子等特点,因此广泛应用于工农业生产中的诸多领域。利用蛋白质工程技术,提高微生物脂肪酶的特异性、活性和稳定性,对提高微生物脂肪酶制剂产品的市场竞争能力,扩大微生物脂肪酶的应用领域,具有重要的意义。综述了蛋白质工程技术在微生物脂肪酶改性方面的应用现状、存在问题及前景。     

Immobilization of a recombinant strain producing glucose isomerase on SiO2-xerogel and properties of prepared biocatalysts

An original method of immobilization of nongrowing microorganism cells on xerogel of silicon dioxide containing insoluble hydroxyl compounds of cobalt(III) has been developed. A recombinant strain producing glucose isomerase has been constructed on the basis of Escherichia coli with the use of a gene of Arthrobacter nicotianae. It was revealed that glucose isomerase activity and stability of biocatalysts prepared on the basis of the recombinant E. coli strain was 3-5 times greater compared with the biocatalysts prepared with the use of the donor strain A. nicotianae. Under conditions of continuous hydrolysis of 3 M fructose at 62-65 degrees C in a fixed bed reactor, time of half-inactivation of the biocatalysts prepared from the recombinant strain and A. nicotianae was -60 and -25 days, respectively.     

Heme-iron oxygenases: powerful industrial biocatalysts?

Are cytochrome P450 enzymes powerful industrial biocatalysts? Next to market demands, well-defined enzyme functionalities and process parameters allow generalizations on the basis of process windows. These can provide useful guidelines for the design of improved biocatalysts. Oxygenase-catalyzed reactions are of special interest for selective C-H bond oxidation. The versatile class of cytochrome P450 mono-oxygenases attracts particular attention, and impressive advances have been achieved with respect to mechanistic insight, enzyme activity, stability, and specificity. Recent major achievements include significant increases in productivities, yields, and rates of catalytic turnover as well as modification of substrate specificity and efficient multistep reactions in whole-cell biocatalysts. For some biocatalysts, these parameters are already of an industrially useful magnitude.     

Whole organism biocatalysis

The use of biocatalysts for the industrial synthesis of chemicals has been attracting much attention as an environment-friendly synthetic method. Microbial cells play a leading role in 'chemo-enzymatic synthesis' because of their great diversity. Several microbes with unique catalytic abilities have been found through intensive screening and put to practical use. Besides, advanced molecular biological techniques are powerful tools for developing more satisfactory biocatalysts.     

Immobilization of a recombinant strain producing glucose isomerase inside SiO<Subscript>2</Subscript>-xerogel and properties of prepared biocatalysts

An original method of immobilization of non-growing microorganism cells inside xerogel of silicium dioxide containing insoluble hydroxyl compounds of cobalt(II) has been developed. A recombinant strain producing glucose isomerase has been constructed on the basis of Escherichia coli with the use of a gene of Arthrobacter nicotianae. It was revealed that glucose isomerase activity and stability of biocatalysts prepared on the basis of the recombinant E. coli strain was 3–5 times greater compared with the biocatalysts prepared with the use of the donor strain A. nicotianae. Under conditions of continuous hydrolysis of 3 M fructose at 62–65°C in a fixed bed reactor, time of half-inactivation of the biocatalysts prepared from the recombinant strain and A. nicotianae was ∼60 and ∼25 days, respectively.     

Immobilized Yeast Membranes as Biocatalysts for Sucrose Inversion

Yeast membranes were obtained by autolysis of various strains with relatively high invertase activity. Heterogeneous biocatalysts for sucrose inversion were made of the yeast membranes and granulated carbon-containing supports made of common natural materials: expanded clay aggregate (ECA), sapropel, and lignin. The properties of these biocatalysts were studied. It was shown that the biocatalyst activity and stability of the immobilized yeast membranes increased with reference to the initial ECA, independent of the morphology of the carbon layer synthesized on the support surface. Heterogeneous biocatalysts prepared by adsorption of yeast membranes on sapropel had the greatest activity and stability, whereas lignin-based biocatalysts were relatively unstable.__________Translated from Prikladnaya Biokhimiya i Mikrobiologiya, Vol. 41, No. 4, 2005, pp. 454–459.Original Russian Text Copyright © 2005 by Kovalenko, Perminova, Plaksin, Komova, Chuenko, Rudina.     

Immobilized glucoamylase: A biocatalyst of dextrin hydrolysis

Heterogeneous biocatalysts of starch saccharification based on glucoamylase and carbon-containing carriers were obtained, and their biocatalytic properties in the enzymatic hydrolysis of corn dextrins were studied. It was shown that the morphology of the surface carbon layer of carriers markedly affected the properties of biocatalysts. Glucoamylase immobilized by adsorption on the surface of carriers covered with a layer of catalytic filamentous or pyrolytic carbon had the maximum enzymatic activity and stability, whereas biocatalysts prepared on the basis of carriers that had no carbon layer or were covered with graphite-like surface carbon had a low activity and stability.     

新型微生物脂肪酶资源开发

微生物脂肪酶是一类重要的工业生物催化剂, 广泛应用于工农业生产的诸多领域。筛选、挖掘和开发出具有新型催化活性或高稳定性的微生物脂肪酶一直是脂肪酶研究的重点。本文从极端微生物、宏基因组技术、基因组数据库挖掘、定向进化技术、固定化技术和化学修饰技术等方面介绍了当前新型脂肪酶开发的途径和方法。     

Immobilized glucoamylase: A biocatalyst of dextrin hydrolysis

Heterogeneous biocatalysts of starch conversion based on glucoamylase and carbon-containing carriers were obtained, and their biocatalytic properties in enzymatic hydrolysis of corn dextrins were studied. It was shown that the morphology of the surface carbon layer of carriers markedly affected the properties of biocatalysts. Glucoamylase that was immobilized by adsorption on the surface of carriers covered with a layer of catalytic fibrous or pyrolytic carbon had the maximum enzymatic activity and stability, whereas the biocatalysts prepared on the basis of carriers that had no carbon layer or were covered with graphite-like surface carbon had a low activity and stability.     

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.     

Genome mining approach for the discovery of novel cytochrome P450 biocatalysts

Cytochrome P450 enzymes (P450s) are able to regioselectively and stereoselectively introduce oxygen into organic compounds under mild reaction conditions. These monooxygenases in particular easily catalyze the insertion of oxygen into less reactive carbon–hydrogen bonds. Hence, P450s are of considerable interest as oxidation biocatalysts. To date, although several P450s have been discovered through screening of microorganisms and have been further genetically engineered, the substrate range of these biocatalysts is still limited to fulfill the requirements for a large number of oxidation processes. On the other hand, the recent rapid expansion in the number of reported microbial genome sequences has revealed the presence of an unexpectedly vast number of P450 genes. This large pool of naturally evolved P450s has attracted much attention as a resource for new oxidation biocatalysts. In this review, we focus on aspects of the genome mining approach that are relevant for the discovery of novel P450 biocatalysts. This approach opens up possibilities for exploitation of the catalytic potential of P450s for the preparation of a large choice of oxidation biocatalysts with a variety of substrate specificities.