New enzymes for biotransformations

Several novel bioprocesses that have little or no counterpart in traditional methodology have recently been reported. The stereoselective and enantioselective hydrolysis of sec-alkyl sulfate esters by alkyl sulfatases proceeds with inversion of configuration and furnishes a homochiral product mixture. Haloalcohol dehalogenases were shown to accept various non-natural nucleophiles, such as azide, cyanide and nitrite for the asymmetric opening of epoxides giving rise to the corresponding azido-, cyano-, and nitro-alcohols as non-natural products. Asymmetric carbon-carbon bond formation via the acyloin- and benzoin-reaction was successfully catalyzed in water by novel lyases, such as benzoylformate decarboxylase and benzaldehyde lyase. New methods for the production of chiral nonracemic alpha-L-amino acids and amines were recently reported. Enantioselective stereoinversion of racemic alpha-aryl- and alpha-aryloxycarboxylic acids via epimerase-catalyzed inversion led to a single stereoisomeric product from the racemate.     

Industrial biotransformations for the production of d-amino acids

Optically pure d-amino acids are industrially manufactured by biotransformations of cheap starting materials produced by chemical synthesis or fermentation in combination with the development of enzyme catalysts suitable for the starting materials. dl-Alaninamide, an intermediate of the chemical synthesis of dl-alanine, was efficiently converted to d-alanine by stereoselective hydrolysis with a d-isomer specific amidohydrolase produced by Arthrobacter sp. NJ-26. The total utilization system of dl-alaninamide for the production of optically pure d- and l-alanine was constructed by stereospecific amidohydrolases. On the other hand, d-amino acids were also produced from corresponding l-isomers, which are efficiently manufactured by fermentation. d-Glutamic acid was produced from l-glutamic acid. l-Glutamate was converted to the dl-form by the recombinant glutamate racemase of Lactobacillus brevis ATCC8287. Then l-glutamate in a racemic mixture was selectively decarboxylated to γ-aminobutyrate by the l-glutamate decarboxylase of E. coli ATCC11246. As a result of successive enzymatic reactions, d-glutamate was efficiently produced from l-glutamate by a one-pot reaction. d-Proline was produced by the same strategy from l-proline using the recombinant proline racemase of Clostridium sticklandii ATCC12262. In this case, l-proline was degraded by Candida sp. PRD-234. The strategy from l-amino acids to d-amino acids could be applicable to the manufacture of many d-amino acids.     

Microbial systems for study of the biotransformations of drugs

The metabolic fate of drugs and other xenobiotics in mammalian organisms represents an area of intense contemporary interest. Traditionally, it is a difficult area of research becausethe biological systems which are used to study biotransformations are capable of yielding only minute quantities of metabolites. Recent developments in comparative biochemistry have made itpossible to link diverse metabolic systems through similarities in the pathways by which they alter foreign organic compounds. The potential thus exists for utilizing microbial metabolic systems to study and possibly predict the metabolic fate of a drug or other foreign compound in mammals. The ease with which microbial systems may be used to obtain large amounts of metabolites is an obvious Advantage. We havhe attemped to review the ways in which mammalian and microbialorganisms metabolize a variety of organic compounds. Attention has been focused on the similarities and differences in the mechanisms by which these living systems metabolize xenobiotics. Particular emphasis has been given to four types of reactions which are important in drug biotransformations: aromatic hydroxylationl; N- and O-dealkylations; and sulfur oxygenations.     

Microbial epoxide hydrolases for preparative biotransformations

Epoxide hydrolases from microbial sources are highly versatile biocatalysts for the asymmetric hydrolysis of epoxides on a preparative scale. Besides kinetic resolution, which furnishes the corresponding vicinal diol and remaining non-hydrolysed epoxide in nonracemic form, enantioconvergent processes are possible: these are highly attractive as they lead to the formation of a single enantiomeric diol from a racemic oxirane. The data accumulated over recent years reveal a common picture of the substrate structure selectivity pattern of microbial epoxide hydrolases and indicate that substrates of various structural types can be selectively hydrolysed with enzymes from certain microbial sources.     

Industrial carbohydrate biotransformations

Nearly all major industrial processes which involve carbohydrates, include biotechnological transformations. This is due to the complex nature of carbohydrates where stereo- and regioselectivity are highly complex and difficult to control. Enzymes and microorganisms work highly selectively and efficiently in water solution, and provide high yield in general. The article focuses on different types of reactions, including large-scale processes. Topics are hydrolytic reactions, including starch processing, oxidation and reduction transformations including organic acids, such as gluconic and ketogluconic acids and vitamin C synthesis, and isomerization and transfer reactions, which are established on a very large scale to produce glucose/fructose syrups and sucrose isomers. The article will further discuss some mechanistic aspects which are relevant for technology and present selected details of industrial-scale processing. Finally an outlook outlines perspectives of future processes.     

Immobilisation of whole bacterial cells for anaerobic biotransformations

Anaerobically grown cells of Escherichia coli were immobilised within a range of entrapment matrices and packed into a column under standard conditions, and the ability of the immobilised cells to reduce nitrite (0.5 mM) was measured at a range of flow rates using sodium formate (20 mM) as the electron donor for nitrite reduction. A flow-rate/activity plot was constructed for each flow-through reactor and RA_1/2 values (residence time corresponding to 50 % nitrite removal) calculated for each reactor type. Cells immobilised in flat and hollow-fibre membranes were the most effective (RA_1/2 = 0.35 h and 0.47 h respectively), with cells entrapped by dialysis membrane (1.53 h), alginate beads (1.93 h), Hypol foam (2.31 h) and polyacrylamide gel (50 % nitrite not removed at maximum residence time tested: 4.9 h) performing progressively less effectively. Cells grown as a biofilm on a range of support materials were also tested in comparable packed-bed reactors. Cell loss from these supports was extensive and contributed to poor performance of the reactors despite high initial biomass loadings (RA_1/2 values using raschig rings, coke and activated-carbon supports: 1.6 h, 2.3 h and 1.0 h respectively). Biofilms grown on Pharmacia microcarrier supports and used in packed and also fluidised beds were more stable and the performance of these reactors was superior to that of biofilm reactors using other supports, and comparable to that of the membrane reactors (RA_1/2 values for Cytoline 2, Cytopore 2 and Cytodex 3: 0.76 h, 0.56 h, 0.68 h respectively). Received: 12 August 1996 / Received revision: 14 November 1996 / Accepted: 15 November 1996     

A membrane bioreactor for biotransformations of hydrophobic molecules

The Membrane Bioreactor for Biotransformations (MBB) is based on the aqueous/organic two-phase system, and uses a tubular silicone rubber membrane to separate the two liquid phases. This avoids the key problem associated with direct contact two-phase processes, specifically, product emulsification. The baker's yeast mediated reduction of geraniol to citronellol was used as a model biotransformation to demonstrate MBB operation. Values for the overall mass transfer coefficient were determined for geraniol, (2.0 x 10(-5) ms-1), and for citronellol, (2.1 x 10(-5) ms-1) diffusion across the silicone rubber membrane. Using these values, and the specific activity of the biocatalyst (5 nmols-1g biomass-1), a suitable membrane surface area: biomass ratio was determined as 2.4 x 10(-3) m2g biomass-1. The bioreactor was operated at this surface area: biomass ratio and achieved a product accumulation rate 90-95% that of a conventional direct contact two-phase system. The slight reduction in product accumulation rate was shown not to be due to mass transfer limitations with respect to reactant delivery or product extraction. Copyright 1998 John Wiley & Sons, Inc.     

Solvent-tolerant bacteria for biotransformations in two-phase fermentation systems

Product removal from aqueous media poses a challenge in biotechnological whole-cell biotransformation processes in which substrates and/or products may have toxic effects. The assignment of an additional liquid solvent phase provides a solution, as it facilitates in situ product recovery from aqueous media. In such two-phase systems, toxic substrates and products are present in the aqueous phase in tolerable but still bioavailable amounts. As a matter of course, adequate organic solvents have to possess hydrophobicity properties akin to substrates and products of interest, which in turn involves intrinsic toxicity of the solvents used. The employment of bacteria being able to adapt to otherwise toxic solvents helps to overcome the problem. Adaptive mechanisms enabling such solvent tolerant bacteria to survive and grow in the presence of toxic solvents generally involve either modification of the membrane and cell surface properties, changes in the overall energy status, or the activation and/or induction of active transport systems for extruding solvents from membranes into the environment. It is anticipated that the biotechnological production of a number of important fine chemicals in amounts sufficient to compete economically with chemical syntheses will soon be possible by making use of solvent-tolerant microorganisms.     

Chemical diversity through biotransformations

Diversity constitutes an intrinsic property of biosynthesis. This inherent property can be exploited and successfully applied in organic synthesis. Recent advances have been made in many areas, including the use of multifunctional enzymes and catalytic promiscuity, the synthesis of diverse products from a single substrate, the use of different biotransformations to make one product, and the use of in vivo biotransformations.     

Oxidative biotransformations using oxygenases

Considerable progress has been made in manipulating oxidative biotransformations using oxygenases. Substrate acceptance, catalytic activity, regioselectivity and stereoselectivity have been improved significantly by substrate engineering, enzyme engineering or biocatalyst screening. Preparative biotransformations have been carried out to synthesize useful pharmaceutical intermediates or chiral synthons on the gram to several-hundred-gram scale, by use of whole cells of wild type or recombinant strains. The synthetic application of oxygenases in vitro has been shown to be possible by enzymatic or electrochemical regeneration of NADH or NADPH.     

Enantioselective biotransformations using rhodococci

The use of enzymes and whole cells in enantioselective biotransformation reactions is briefly reviewed. A Rhodococcus strain is shown to possess nitrile hydratase and amidase activity. The organism can be used for the enantioselective biotransformation of racemic -amino amides to (S) -amino acids with an enantiomeric excess (ee) of > 98%. Enantioselectivity is effectively time independent allowing easy quantitative conversion of racemic mixtures into enantiomerically pure -amino amides and -amino acids. The reaction is effective for a wide range of - substituents. The pH-dependence of the reaction indicates that the -amino amide is bound to the amidase enzyme in its neutral unprotonated form.     

Recent advances in oxygenase-catalyzed biotransformations

Oxygenases continue to be widely studied for selective biooxidation of organic compounds. Protein engineering has resulted in heme and flavin monooxygenases with widely altered substrate specificities, and attempts have been reported to scale-up reactions catalyzed by these enzymes. Cofactor regeneration is still a key issue in these developments. Protein engineering contributed to understanding of structure versus function in dioxygenases.     

Monitoring biotransformations in polyamide fibres

The enzymatic hydrolysis of polyamide fibres yields amino and carboxylic groups. These groups can be found in solution treatments as polyamide monomers and soluble oligomers. The amino groups can also be found at the surface of the fibres as end group chains. In this paper we report two methods to quantify the formation of these groups as a result of the enzymatic action. Soluble amino groups can be quantified with 2,4,6-trinitrobenzenesulfonic acid (TNBS), which yields a coloured complex which can be determined spectrophotometrically. The amino groups on the fibre surface can be quantified by reaction with a wool reactive dye and determination of colour intensities after a dyeing procedure below the glass transition temperature of polyamide.     

Mercury biotransformations and their potential for remediation of mercury contamination

Bacterially mediated ionic mercury reduction to volatile Hg⁰ was shown to play an important role in the geochemical cycling of mercury in a contaminated freshwater pond. This process, and the degradation of methylmercury, could be stimulated to reduce the concentration of methylmercury that is available for accumulation by biota. A study testing the utility of this approach is described.Abbreviations Hg^R inorganic mercury resistance - Org-Hg organomercury - Org-Hg^R organomercury resistance - SRB sulfate reducing bacteria - Methyl-B12 methylcobalamine