Bacterial dehalogenases: biochemistry, genetics, and biotechnological applications.

This review is a survey of bacterial dehalogenases that catalyze the cleavage of halogen substituents from haloaromatics, haloalkanes, haloalcohols, and haloalkanoic acids. Concerning the enzymatic cleavage of the carbon-halogen bond, seven mechanisms of dehalogenation are known, namely, reductive, oxygenolytic, hydrolytic, and thiolytic dehalogenation; intramolecular nucleophilic displacement; dehydrohalogenation; and hydration. Spontaneous dehalogenation reactions may occur as a result of chemical decomposition of unstable primary products of an unassociated enzyme reaction, and fortuitous dehalogenation can result from the action of broad-specificity enzymes converting halogenated analogs of their natural substrate. Reductive dehalogenation either is catalyzed by a specific dehalogenase or may be mediated by free or enzyme-bound transition metal cofactors (porphyrins, corrins). Desulfomonile tiedjei DCB-1 couples energy conservation to a reductive dechlorination reaction. The biochemistry and genetics of oxygenolytic and hydrolytic haloaromatic dehalogenases are discussed. Concerning the haloalkanes, oxygenases, glutathione S-transferases, halidohydrolases, and dehydrohalogenases are involved in the dehalogenation of different haloalkane compounds. The epoxide-forming halohydrin hydrogen halide lyases form a distinct class of dehalogenases. The dehalogenation of alpha-halosubstituted alkanoic acids is catalyzed by halidohydrolases, which, according to their substrate and inhibitor specificity and mode of product formation, are placed into distinct mechanistic groups. beta-Halosubstituted alkanoic acids are dehalogenated by halidohydrolases acting on the coenzyme A ester of the beta-haloalkanoic acid. Microbial systems offer a versatile potential for biotechnological applications. Because of their enantiomer selectivity, some dehalogenases are used as industrial biocatalysts for the synthesis of chiral compounds. The application of dehalogenases or bacterial strains in environmental protection technologies is discussed in detail.     

Taxol: biosynthesis, molecular genetics, and biotechnological applications

Over the past decade, Taxol and its closely related structural analogue Taxotere have emerged as very important antitumor agents. Their widespread use in the treatment of a variety of cancer types, their likely approval for the treatment of additional forms of cancer, and their use at earlier stages of intervention will lead to increased demand for these drugs in the future. Because of yield considerations, Taxol and Taxotere are currently derived via semisynthesis from the advanced taxoid 10-deacetylbaccatin III, which must be isolated from yew (Taxus) trees. Thus, efforts are underway to produce Taxol (and other advanced taxoids for use in semisynthesis) by alternate, biotechnological means. This article provides a current overview of research on taxoid biosynthesis and an assessment of bioengineering applications for taxoid production in yew cell culture.     

Microbial degradation of furanic compounds: biochemistry,genetics, and impact

Microbial metabolism of furanic compounds, especially furfural and 5-hydroxymethylfurfural (HMF), is rapidly gaining interest in the scientific community. This interest can largely be attributed to the occurrence of toxic furanic aldehydes in lignocellulosic hydrolysates. However, these compounds are also widespread in nature and in human processed foods, and are produced in industry. Although several microorganisms are known to degrade furanic compounds, the variety of species is limited mostly to Gram-negative aerobic bacteria, with a few notable exceptions. Furanic aldehydes are highly toxic to microorganisms, which have evolved a wide variety of defense mechanisms, such as the oxidation and/or reduction to the furanic alcohol and acid forms. These oxidation/reduction reactions constitute the initial steps of the biological pathways for furfural and HMF degradation. Furfural degradation proceeds via 2-furoic acid, which is metabolized to the primary intermediate 2-oxoglutarate. HMF is converted, via 2,5-furandicarboxylic acid, into 2-furoic acid. The enzymes in these HMF/furfural degradation pathways are encoded by eight hmf genes, organized in two distinct clusters in Cupriavidus basilensis HMF14. The organization of the five genes of the furfural degradation cluster is highly conserved among microorganisms capable of degrading furfural, while the three genes constituting the initial HMF degradation route are organized in a highly diverse manner. The genetic and biochemical characterization of the microbial metabolism of furanic compounds holds great promises for industrial applications such as the biodetoxifcation of lignocellulosic hydrolysates and the production of value-added compounds such as 2,5-furandicarboxylic acid.     

Microalgal lipids biochemistry and biotechnological perspectives

In the last few years, there has been an intense interest in using microalgal lipids in food, chemical and pharmaceutical industries and cosmetology, while a noteworthy research has been performed focusing on all aspects of microalgal lipid production. This includes basic research on the pathways of solar energy conversion and on lipid biosynthesis and catabolism, and applied research dealing with the various biological and technical bottlenecks of the lipid production process. In here, we review the current knowledge in microalgal lipids with respect to their metabolism and various biotechnological applications, and we discuss potential future perspectives.     

A review of bacterial methyl halide degradation: biochemistry,genetics and molecular ecology

Methyl halide-degrading bacteria are a diverse group of organisms that are found in both terrestrial and marine environments. They potentially play an important role in mitigating ozone depletion resulting from methyl chloride and methyl bromide emissions. The first step in the pathway(s) of methyl halide degradation involves a methyltransferase and, recently, the presence of this pathway has been studied in a number of bacteria. This paper reviews the biochemistry and genetics of methyl halide utilization in the aerobic bacteria Methylobacterium chloromethanicum CM4T, Hyphomicrobium chloromethanicum CM2T, Aminobacter strain IMB-1 and Aminobacter strain CC495. These bacteria are able to use methyl halides as a sole source of carbon and energy, are all members of the alpha-Proteobacteria and were isolated from a variety of polluted and pristine terrestrial environments. An understanding of the genetics of these bacteria identified a unique gene (cmuA) involved in the degradation of methyl halides, which codes for a protein (CmuA) with unique methyltransferase and corrinoid functions. This unique functional gene, cmuA, is being used to develop molecular ecology techniques to examine the diversity and distribution of methyl halide-utilizing bacteria in the environment and hopefully to understand their role in methyl halide degradation in different environments. These techniques will also enable the detection of potentially novel methyl halide-degrading bacteria.     

Microbial glutaminase: biochemistry, molecular approaches and applications in the food industry

Glutaminase is widely distributed in microorganisms including bacteria, yeast and fungi. The enzyme mainly catalyzes the hydrolysis of γ-amido bond of -glutamine. In addition, some enzymes also catalyze γ-glutamyl transfer reaction. A highly savory amino acid, -glutamic acid and a taste-enhancing amino acid of infused green tea, theanine can be synthesized by employing hydrolytic or transfer reaction catalyzed by glutaminase. Therefore, glutaminase is one of the most important flavor-enhancing enzymes in food industries. In this review, subsequent to a discussion on the definition of glutaminase, the enzymatic properties, applications of glutaminase in the food industry, and occurrence and distribution of the enzyme are described. We then illustrate the gene cloning, primary structure, and 3D-structure of glutaminase. Finally, to facilitate the future applications of glutaminase in food fermentations, the mechanisms of action of salt-tolerant glutaminase are briefly discussed.     

Aminomutases: mechanistic diversity, biotechnological applications and future perspectives

Aminomutases carry out the chemically challenging exchange of a hydrogen atom and an amine substituent present on neighboring carbon atoms. In recent years, aminomutases have been intensively investigated for their biophysical, structural and mechanistic characteristics. The reactions catalyzed by these enzymes have considerable potential for biotechnological applications. Here, we present an overview of this diverse group of enzymes, with a focus on enzymatic mechanisms and recent developments in their use in applied biocatalysis.     

Nonconventional yeasts: their genetics and biotechnological applications

To date, more than 500 species of yeasts have been described. Most of the genetic and biochemical studies have, however, been carried out with Saccharomyces cerevisiae. Although a considerable amount of knowledge has been accumulated on fundamental processes and biotechnological applications of this industrially important yeast, the large variety of other yeast genera and species may offer various advantages for experimental study as well as for product formation in biotechnology. The genetic investigation of these so-called unconventional yeasts is poorly developed and information about corresponding data is dispersed. It is the aim of this review to summarize and discuss the main results of genetic studies and biotechnological applications of unconventional yeasts and to serve as a guide for scientists who wish to enter this field or are interested in only some aspects of these yeasts.     

3-Hydroxypropionaldehyde: applications and perspectives of biotechnological production

3-Hydroxypropionaldehyde (3-HPA) forms, together with HPA-hydrate and HPA-dimer, a dynamic, multi-component system (HPA system) used in food preservation, as a precursor for many modern chemicals such as acrolein, acrylic acid, and 1,3-propanediol (1,3-PDO), and for polymer production. 3-HPA can be obtained both through traditional chemistry and bacterial fermentation. To date, 3-HPA has been produced from petrochemical resources as an intermediate in 1,3-PDO production. In vivo, glycerol is converted in one enzymatic step into 3-HPA. The 3-HPA-producing Lactobacillus reuteri is used as a probiotic in the health care of humans and animals. The biotechnological production of 3-HPA from renewable resources is desirable both for use of 3-HPA in foods and for the production of bulk chemicals. The main challenge will be the efficient production and recovery of pure 3-HPA.     

Microbial formation, biotechnological production and applications of 1,2-propanediol

This short review covers metabolic pathways, genetics and metabolic engineering of 1,2-propanediol formation in microbes. 1,2-Propanediol production by bacteria and yeasts has been known for many years and two general pathways are recognized. One involves the metabolism of deoxyhexoses, where lactaldehyde is formed during the glycolytic reactions and is then reduced to 1,2-propanediol. The second pathway derives from the formation of methylglyoxal from dihydroxyacetonephosphate and its subsequent reduction to 1,2-propanediol. The enzymes involved in the reduction of methylglyoxal can generate isomers of lactaldehyde or acetol, which can be further reduced by specific reductases, giving chiral 1,2-propanediol as the product. The stereospecificity of the enzymes catalyzing the two reduction steps is important in deriving a complete pathway. Through genetic engineering, appropriate combinations of enzymes have been brought together in Escherichia coli and yeast to generate 1,2-propanediol from glucose. The optimization of these strains may yield microbial processes for the production of this widely used chemical. Received: 25 May 2000 / Received revision: 24 July 2000 / Accepted: 25 July 2000     

Biochemistry,genetics and physiology of microbial styrene degradation

The last few decades have seen a steady increase in the global production and utilisation of the alkenylbenzene, styrene. The compound is of major importance in the petrochemical and polymer-processing industries, which can contribute to the pollution of natural resources via the release of styrene-contaminated effluents and off-gases. This is a cause for some concern as human over-exposure to styrene, and/or its early catabolic intermediates, can have a range of destructive health effects. These features have prompted researchers to investigate routes of styrene degradation in microorganisms, given the potential application of these organisms in bioremediation/biodegradation strategies. This review aims to examine the recent advances which have been made in elucidating the underlying biochemistry, genetics and physiology of microbial styrene catabolism, identifying areas of interest for the future and highlighting the potential industrial importance of individual catabolic pathway enzymes.     

The genus Gluconobacter oxydans: comprehensive overview of biochemistry and biotechnological applications

The genus Gluconobacter comprises some of the most frequently used microorganisms when it comes to biotechnological applications. Not only has it been involved in "historical" production processes, such as vinegar production, but in the last decades many bioconversion routes for special and rare sugars involving Gluconobacter have been developed. Among the most recent are the biotransformations involved in the production of L-ribose and miglitol, both very promising pharmaceutical lead molecules. Most of these processes make use of Gluconobacter's membrane-bound polyol dehydrogenases. However, recently other enzymes have also caught the eye of industrial biotechnology. Among them are dextran dextrinase, capable of transglucosylating substrate molecules, and intracellular NAD-dependent polyol dehydrogenases, of interest for co-enzyme regeneration. As such, Gluconobacter is an important industrial microbial strain, but it also finds use in other fields of biotechnology, such as biosensor-technology. This review aims to give an overview of the myriad of applications for Gluconobacter, with a special focus on some recent developments.     

Penicillium roqueforti: an overview of its genetics,physiology, metabolism and biotechnological applications

Besides research on the model fungal genera Saccharomyces, Neurospora and Aspergillus, that has provided important biological knowledge in the areas of genetics, cell biology and physiology, recent investigations on non-model fungi used for food production offer insight into the mechanisms involved in food production but also adaptation and domestication processes. In this context, Penicillium roqueforti has been the most extensively studied species. This species is best known worldwide for its technological use for blue-veined cheese production and ripening. Recently, several advances related to taxonomy, population genetics, physiology and metabolism have been documented and provided deeper knowledge about this species. The methodological approaches used to study this species can be applied to other still largely understudied fungi associated with food production worldwide (e.g. P. camemberti, P. nalgiovense, P. salamii, Bisifusarium domesticum, Mucor spp.).     

Dirigent proteins: molecular characteristics and potential biotechnological applications

Dirigent proteins (DIRs) are thought to play important roles in plant secondary metabolism. They lack catalytic activity but direct the outcome of bimolecular coupling reactions toward regio- and stereospecific product formation. Functionally described DIRs confer specificity to the oxidative coupling of coniferyl alcohol resulting in the preferred production of either (+)- or (?)-pinoresinol, which are the first intermediates in the enantiocomplementary pathways for lignan biosynthesis. DIRs are extracellular glycoproteins with high β-strand content and have been found in all land plants investigated so far. Their ability to capture and orientate radicals represents a unique naturally evolved concept for the control of radical dimerization reactions. Although oxidative coupling is commonly used in biological systems, its wider application in chemical synthesis is often limited by insufficient selectivity. This minireview gives an overview of functionally described DIRs and their molecular characteristics and wants to inspire further research for their use in biotechnological applications.     

Catabolic pathways and biotechnological applications of microbial caffeine degradation

Catabolism of caffeine (1,3,7-trimethylxanthine) in microorganisms commences via two possible mechanisms: demethylation and oxidation. Through the demethylation route, the major metabolite formed in fungi is theophylline (1,3-dimethylxanthine), whereas theobromine (3,7-dimethylxanthine) is the major metabolite in bacteria. In certain bacterial species, caffeine has also been oxidized directly to trimethyl uric acid in a single step. The conversion of caffeine to its metabolites is primarily brought about by N-demethylases (such as caffeine demethylase, theobromine demethylase and heteroxanthinedemethylase), caffeine oxidase and xanthine oxidase that are produced by several caffeine-degrading bacterial species such as Pseudomonas putida and species within the genera Alcaligenes, Rhodococcus and Klebsiella. Development of biodecaffeination techniques using these enzymes or using whole cells offers an attractive alternative to the present existing chemical and physical methods removal of caffeine, which are costly, toxic and non-specific to caffeine. This review mainly focuses on the biochemistry of microbial caffeine degradation, presenting recent advances and the potential biotechnological application of caffeine-degrading enzymes.     

Physiology,biochemistry and possible applications of microbial caffeine degradation

Caffeine, a purine alkaloid is a constituent of widely consumed beverages. The scientific evidence which has proved the harm of this alkaloid has paved the way for innumerable research in the area of caffeine degradation. In addition to this, the fact that the by-products of the coffee and tea industry pollute the environment has called for the need of decaffeinating coffee and tea industry’s by-products. Though physical and chemical methods for decaffeination are available, the lack of specificity for removal of caffeine in these techniques and their non-eco-friendly nature has opened the area of microbial and enzymatic degradation of caffeine. Another important application of microbial caffeine degradation apart from its advantages like specificity, eco-friendliness and cost-effectiveness is the fact that this process will enable the production of industrially and medically useful components of the caffeine degradation pathway like theobromine and theophylline. This is a comprehensive review which mainly focuses on caffeine degradation, large-scale degradation of the same and its applications in the industrial world.     

The lipases from Yarrowia lipolytica: genetics, production, regulation, biochemical characterization and biotechnological applications

Lipases are serine hydrolases that catalyze in nature the hydrolysis of ester bonds of long chain triacylglycerol into fatty acid and glycerol. However, in favorable thermodynamic conditions, they are also able to catalyze reactions of synthesis such as esterification or amidation. The non-conventional yeast Yarrowia lipolytica possesses 16 paralogs of genes coding for lipase. However, little information on all those paralogs has been yet obtained and only three isoenzymes, namely Lip2p, Lip7p and Lip8p have been partly characterized so far. Microarray data suggest that only a few of them could be expressed and that lipase synthesis seems to be dependent on the fatty acid or oil used as carbon source confirming the high adaptation of Y. lipolytica to hydrophobic substrate utilization. This review focuses on the biochemical characterization of those enzymes with special emphasis on the Lip2p lipase which is the isoenzyme mainly synthesized by Y. lipolytica. Crystallographic data highlight that this latter is a lipase sensu stricto with a lid covering the active site of the enzyme in its closed conformation. Recent findings on enzyme conditioning in dehydrated or liquid formulation, in enzyme immobilization by entrapment in natural polymers from either organic or mineral origins are also discussed together with long-term storage strategies. The development of various biotechnological applications in different fields such as cheese ripening, waste treatment, drug synthesis or human therapeutics is also presented.     

Fungal tyrosinases: new prospects in molecular characteristics, bioengineering and biotechnological applications

Tyrosinases are type-3 copper proteins involved in the initial step of melanin synthesis. These enzymes catalyse both the o-hydroxylation of monophenols and the subsequent oxidation of the resulting o-diphenols into reactive o-quinones, which evolve spontaneously to produce intermediates, which associate in dark brown pigments. In fungi, tyrosinases are generally associated with the formation and stability of spores, in defence and virulence mechanisms, and in browning and pigmentation. First characterized from the edible mushroom Agaricus bisporus because of undesirable enzymatic browning problems during postharvest storage, tyrosinases were found, more recently, in several other fungi with relevant insights into molecular and genetic characteristics and into reaction mechanisms, highlighting their very promising properties for biotechnological applications. The limit of these applications remains in the fact that native fungal tyrosinases are generally intracellular and produced in low quantity. This review compiles the recent data on biochemical and molecular properties of fungal tyrosinases, underlining their importance in the biotechnological use of these enzymes. Next, their most promising applications in food, pharmaceutical and environmental fields are presented and the bioengineering approaches used for the development of tyrosinase-overproducing fungal strains are discussed.     

Properties and biotechnological applications of halohydrin dehalogenases: current state and future perspectives

Halohydrin dehalogenases (HHDHs) are lyases that catalyze the cleavage of carbon–halogen bond of halohydrins. They also can catalyze the reverse reaction in the presence of nucleophiles such as cyanide, azide, and nitrite ions. HHDHs have been recognized as the ideal tools for the degradation of various halogenated environmental pollutants. Moreover, they can be used as biocatalysts for the kinetic resolution of halohydrins and epoxides, and for the preparation of various substituted alcohols. This review is mainly focused on the current status of research on HHDHs, highlighting the production, characterization, structures and mechanism, protein engineering, and biotechnological applications of HHDHs.     

Microbial interaction with and tolerance of radionuclides: underlying mechanisms and biotechnological applications

Radionuclides (RNs) generated by nuclear and civil industries are released in natural ecosystems and may have a hazardous impact on human health and the environment. RN-polluted environments harbour different microbial species that become highly tolerant of these elements through mechanisms including biosorption, biotransformation, biomineralization and intracellular accumulation. Such microbial–RN interaction processes hold biotechnological potential for the design of bioremediation strategies to deal with several contamination problems. This paper, with its multidisciplinary approach, provides a state-of-the-art review of most research endeavours aimed to elucidate how microbes deal with radionuclides and how they tolerate ionizing radiations. In addition, the most recent findings related to new biotechnological applications of microbes in the bioremediation of radionuclides and in the long-term disposal of nuclear wastes are described and discussed.