Aspects for the future of biotransformations

Biotransformations have gained extensive importance in practical use as a support for chemical synthesis or in the conversion of natural products. Biotransformations may present an enlargement, a sequential degradation or a specific modification of synthetic or natural compounds. The tools for biotransformations are principally mammalian, plant or microbial cells and their cell-free enzymes. In technical practice the biocatalysts are so far limited to the use of microorganisms and some cell-free enzymes of low cost. Although numerous microbial or enzymatical reactions were already developed for industrial processes, the capacities of biotransformations offer a broad field of inexhaustible possibilities for the future.

     

Hydrolytic enzyme activities of extracted humic substances during the vermicomposting of a lignocellulosic olive waste

Humic substances and three hydrolytic enzymes (beta-glucosidase, phosphatase and urease) were extracted by neutral sodium pyrophosphate from an olive waste (dry olive cake), alone or mixed with municipal biosolids, during a nine month vermicomposting process. Easily degradable compounds decreased during the vermicomposting process because of microbial consumption. When municipal biosolids were added to dry olive cake, microbial activity increased and the amounts of compounds extracted by pyrophosphate were three times lower than olive cake alone. In both instances, beta-glucosidase, phosphatase and urease activities of the organic extracts either increased or remained the same after a nine month period of vermicomposting, thus suggesting that the humus enzyme complexes resisted microbial and earthworm attack. It is known that humus immobilised enzymes also remain active in soil environments, reactivating the nutrient cycles in soil. The use as amendments of vermicomposted olive cake, alone or when mixed with biosolids, could be a good alternative to reactivate the C, P and N-cycles in degraded soils for regeneration purposes.     

Dioxygenase- and monooxygenase-catalysed synthesis of <Emphasis Type="Italic">cis</Emphasis>-dihydrodiols,catechols, epoxides and other oxygenated products

Oxidoreductases are an emerging class of biotechnologically relevant enzymes due to their regio- and stereo-specificity. The selective oxygenation of aromatic compounds by oxidoreductases has received much attention and a wide range of reactions have been documented using these enzymes from various microbial sources. This review gives an overview of various dioxygenase, monooxygenase and oxidase enzymes that have been manipulated for the synthesis of products such as cis-dihydrodiols, catechols, epoxides and other oxygenated products. The use of protein engineering and its advancement in the synthesis of recombinant enzymes is also discussed.     

Role of microbial enzymes in flavor development in foods

There are four main sources of enzymes in foods—these being the inherent enzymes, enzymes from microbial contaminants, enzymes elaborated by microorganisms added to foods, and specific enzymes added to foods. This study primarily deals with the latter two sources of enzymes in food. Although both plants and animals serve as sources of enzymes, they are not as economical or versatile sources as are enzymes obtained from microorganisms. In the meat industry, proteases are used to tenderize muscle and to obtain flavor precursors. In the preparation of cured meat products such as sausages, lipases, and proteases from bacterial cultures are utilized. Similarly, proteases and lipases are used in the dairy industry to develop flavor compounds. Proteases and amylases also have applications in the baking and milling industries where they are used to produce precursors for the nonenzymatic browning reactions. Carbohydrases such as amylase, amyloglucosidase, and glucose isomerase have found usage in the starch and syrup industry for the production of high dextrose and high fructose syrups. Other enzymes such as glucose oxidase, pectinase, and naringinase are of value to the wine and fruit juice industries. A better understanding of the mode of action of enzymes as well as the mechanisms of development of flavor compounds will further enhance the use of microbial enzymes to develop specific and desired flavors in foods.     

Microbial metabolism of quorum-sensing molecules acyl-homoserine lactones, γ-heptalactone and other lactones

The cell-to-cell communication of microorganisms is known to be via exertion of certain chemical compounds (signal molecules) and is referred to as quorum sensing (QS). QS phenomenon is widespread in microbial communities. Several Gram-positive and Gram-negative bacteria and fungi use lactone-containing compounds (e.g. acyl-homoserine lactones (AHLs), γ-heptalactone, butyrolactone-I) as signalling molecules. The ability of microorganisms to metabolise these compounds and the mechanisms they employ for this purpose are not clearly understood. Many studies, however, have focused on identifying AHL and other lactone-degrading enzymes produced by bacteria and fungi. Various strains that are able to utilise these signalling molecules as carbon and energy sources have also been isolated. In addition, several reports have provided evidence on the involvement of lactones and lactone-degrading enzymes in numerous biological functions. These studies, although focused on processes other than metabolism of lactone signalling molecules, still provide insights into further understanding of the mechanisms employed by various microorganisms to metabolise the QS compounds. In this review, we consider conceivable microbial strategies to metabolise AHL and other lactone-containing signalling molecules such as γ-heptalactones.     

Comparative efficiency of microbial enzyme preparations versus pancreatin for in vitro alimentary protein digestion

Utilisation of microbial enzymes may represent an alternative strategy to the use of conventional pancreatin obtained from pig pancreas for the treatment of severe pancreatic insufficiency. In this study, we focused on the capacity of two microbial preparations for their capacity to digest alimentary proteins (caseins and soya proteins) in comparison with pancreatin. These microbial enzymatic preparations were found to be able to generate small, medium-size and larger polypeptides from caseins and soya proteins but were inactivated at pH 3.0. As determined by Liquid Chromatography–Mass Spectrometry analysis, microbial enzymes generated very different peptides from caseins when compared with peptides generated through pancreatin action. These microbial preparations were characterised by relatively low trypsin- and low carboxypeptidase-like activities but high chymotrypsin-like activities and strong capacity for cleavage of caseins at the methionine sites. Although the efficiency of these microbial preparations to increase the rate of absorption of nitrogen-containing compounds in severe pancreatic insufficiency remains to be tested in vivo, our in vitro data indicate proteolytic capacities of such preparations for alimentary protein digestion.     

The University of Minnesota Biocatalysis/Biodegradation database: microorganisms, genomics and prediction

The University of Minnesota Biocatalysis/Biodegradation Database (http://www.labmed.umn.edu/umbbd/ ) begins its fifth year having met its initial goals. It contains approximately 100 pathways for microbial catabolic metabolism of primarily xenobiotic organic compounds, including information on approximately 650 reactions, 600 compounds and 400 enzymes, and containing approximately 250 microorganism entries. It includes information on most known microbial catabolic reaction types and the organic functional groups they transform. Having reached its first goals, it is ready to move beyond them. It is poised to grow in many different ways, including mirror sites; fold prediction for its sequenced enzymes; closer ties to genome and microbial strain databases; and the prediction of biodegradation pathways for compounds it does not contain.     

From rumen to industry

ABSTRACT: The rumen is one of the most complicated and most fascinating microbial ecosystems in nature. A wide variety of microbial species, including bacteria, fungi and protozoa act together to bioconvert (ligno)cellulosic plant material into compounds, which can be taken up and metabolized by the ruminant. Thus, the rumen perfectly resembles a solution to a current industrial problem: the biorefinery, which aims at the bioconversion of lignocellulosic material into fuels and chemicals. We suggest to intensify the studies of the ruminal microbial ecosystem from an industrial microbiologists point of view in order to make use of this rich source of organisms and enzymes.     

Dehalogenases: environmental defence mechanism and model of enzyme evolution

Studies on the fate of halogenated organic compounds in the environment from the 1950s onwards have led to the conclusion that the main method of degradation of these compounds is via microbial metabolism, and that this is mediated by enzymes which remove the halogen substituents. The enzymes are called dehalogenases. The study of these enzymes has yielded much useful information about the evolution of catabolic enzymes in general and represents an ideal tool for the investigation and illustration of many key concepts in enzymology including parallel evolution, convergent evolution, gene transfer, determination of reaction mechanisms and structure—activity relationships. This review summarises the key principles of microbial dehalogenation and illustrates the applications that these studies may have in teaching more generalised enzymological concepts.     

甾类化合物微生物转化与分解代谢机制研究进展

甾类化合物具有重要的生理医药作用,市场需求巨大。甾类化合物及其关键甾类药物通过微生物转化制备工艺较化学合成法具有区域立体选择性、减少合成步骤、缩短生产周期、提高收率以及生态友好等优点逐步被应用,然而甾类物质微生物分解代谢机制还有待进一步深入探索研究并确定。本文从甾类化合物结构种类与主要来源、生理功能、微生物转化与分解代谢机制的研究等方面进行了归纳,着重解析甾类化合物分解代谢过程关键酶系及其分子作用机制,为甾药化合物生产菌种改造与工程菌构建,以及微生物转化工业化生产工艺的开发提供参考。     

Microbes and microbial enzymes for cyanide degradation

Cyanide is an important industrial chemical produced on a grand scale each year. Although extremely toxic to mammalian life, cyanide is a natural product generated by fungi and bacteria, and as a result microbial systems have evolved for the degradation of cyanide to less toxic compounds. The enzymes which utilize cyanide as a substrate can be categorized into the following reaction types: substitution/addition, hydrolysis, oxidation, and reduction. Each of these categories is reviewed with respect to the known biochemistry and feasibility for use in treatment of cyanide containing wastes.     

Metagenomics,biotechnology with non-culturable microbes

Metagenomics as a new field of research has been developed over the past decade to elucidate the genomes of the non-cultured microbes with the goal to better understand global microbial ecology on the one side, and on the other side it has been driven by the increasing biotechnological demands for novel enzymes and biomolecules. Since it is well accepted that the majority of all microbes has not yet been cultured, the not-yet-cultivated microbes represent a shear unlimited and intriguing resource for the development of novel genes, enzymes and chemical compounds for use in biotechnology. However, with respect to biotechnology, metagenomics faces now two major challenges. Firstly, it has to identify truly novel biocatalysts to fulfil the needs of industrial processes and green chemistry. Secondly, the already available genes and enzymes need to be implemented in production processes to further prove the value of metagenome-derived sequences.     

Formation and physiological role of biosurfactants produced by hydrocarbon-utilizing microorganisms

Microbial growth on water-insoluble carbon sources such as hydrocarbons is accompanied by metabolic and structural alterations of the cell. The appearance of surface-active compounds (biosurfactants) in the culture medium or attached to the cell boundaries is often regarded as a prerequisite for initial interactions of hydrocarbons with the microbial cell. Under this point of view, biosurfactants produced by hydrocarbon-utilizing microorganisms, their structures and physico-chemical properties are reviewed. The production of such compounds is mostly connected with growth limitation in the late logarithmic and the stationary growth phase, in which specific enzymes are induced or derepressed. Addition of purified biosurfactants to microbial cultures resulted in inhibitory as well as in stimulatory effects on growth. Therefore, a more differentiated view of microbial production of surface-active compounds is proposed. Biosurfactants should not only be regarded as prerequisites of hydrocarbon uptake, but also as secondary metabolic products.     

Single molecule techniques for the study of membrane proteins

Lactone compounds are widely distributed in nature and play important roles in organisms. These compounds are synthesized and metabolized enzymatically in vivo; however, detailed investigation of these enzymes lags behind that of other common enzymes. In this paper, recent work on the enzymes involved in the metabolism of lactone compounds will be reviewed. In particular, fundamental and application studies on lactonases and Baeyer-Villiger monooxgenases of microbial origin are described.     

Hydrolysis of organophosphorus compounds by microbial enzymes

There are classes of microbial enzymes that have the ability to degrade harmful organophosphorus (OP) compounds that are present in some pesticides and nerve agents. To date, the most studied and potentially important OP-degrading enzymes are organophosphorus hydrolase (OPH) and organophosphorus acid anhydrolase (OPAA), which have both been characterized from a number of organisms. Here we provide an update of what is experimentally known about OPH and OPAA to include their structures, substrate specificity, and catalytic properties. Current and future potential applications of these enzymes in the hydrolysis of OP compounds are also addressed.     

Microbial transformation of alkaloids

Alkaloids continue to provide mankind with a plethora of medicines, poisons and potions. Because many valuable drugs are derived from such natural compounds, there is much interest in their transformation to provide new compounds or intermediates for the synthesis of new or improved drugs. This review aims to provide a survey of alkaloid transformations, and concerns microbial transformations and microbially expressed recombinant plant enzymes and their biotechnological applications.     

Microbial biosensors.

A microbial biosensor consists of a transducer in conjunction with immobilised viable or non-viable microbial cells. Non-viable cells obtained after permeabilisation or whole cells containing periplasmic enzymes have mostly been used as an economical substitute for enzymes. Viable cells make use of the respiratory and metabolic functions of the cell, the analyte to be monitored being either a substrate or an inhibitor of these processes. Bioluminescence-based microbial biosensors have also been developed using genetically engineered microorganisms constructed by fusing the lux gene with an inducible gene promoter for toxicity and bioavailability testing. In this review, some of the recent trends in microbial biosensors with reference to the advantages and limitations are been discussed. Some of the recent applications of microbial biosensors in environmental monitoring and for use in food, fermentation and allied fields have been reviewed. Prospective future microbial biosensor designs have also been identified.     

Use of delta-(alpha-aminoadipoyl) chromogenic amides in screening for aminoadipoyl amidohydrolases.

The synthesis of delta-(alpha-aminoadipoyl) aromatic amides and their use in screening for enzymes able to cleave delta-(alpha-aminoadipoyl) residues off the synthetic amides and cephalosporin C are described. A number of commercially available proteases and peptidases were not active with delta-(alpha-aminoadipoyl) chromogenic amides. Also, most tested microbial strains known to produce acylases did not hydrolyze these compounds. Only one microbial strain, Xanthomonas maltophila, had an appreciable activity toward the racemic form of chromogenic substrates. Activity measured in crude extracts from Xanthomonas cells indicated that this bacterium produces predominantly L-specific aminoadipoyl amidohydrolase and gamma-glutamyl hydrolase. A low level of cephalosporin C and glutaryl-cephalosporin acylase activities was also found.