Synthesis of phage-specific transfer RNA molecules by vibriophage phi 149

32P-Labelled tRNA was isolated from uninfected and phage phi 149-infected Vibrio cholerae cells. These tRNA preparations were then hybridised with DNA isolated from phage phi 149. Significant hybridisation was observed only with tRNA from phage phi 149-infected cells. This strongly suggests that infection of classical vibrio with phage phi 149 results in the synthesis of phage-specific tRNA molecules.     

Review: Biocatalytic transformations of ferulic acid: An abundant aromatic natural product

In this review we examine the fascinating array of microbial and enzymatic transformations of ferulic acid. Ferulic acid is an extremely abundant, preformed phenolic aromatic chemical found widely in nature. Ferulic acid is viewed as a commodity scale, renewable chemical feedstock for biocatalytic conversion to other useful aromatic chemicals. Most attention is focused on bioconversions of ferulic acid itself. Topics covered include cinnamoyl side-chain cleavage; nonoxidative decarboxylation; mechanistic details of styrene formation; purification and characterization of ferulic acid decarboxylase; conversion of ferulic acid to vanillin;O-demethylation; and reduction reactions. Biotransformations of vinylgualacol are discussed, and selected biotransformations of vanillic acid including oxidative and nonoxidative decarboxylation are surveyed. Finally, enzymatic oxidative dimerization and polymerization reactions are reviewed.     

Prediction of the Reaction Equilibrium of Biocatalytic Reactions in Aqueous-Organic Two-Phase Systems

Biocatalytic reactions can be carried out in aqueous-organic two-phase systems. Several models to describe the thermodynamically-determined equilibrium position in such systems have appeared in the literature. Some of these models are only valid for dilute systems, whereas others can also be used for nondilute systems. In this paper, these models are described and compared. It is explained in what way the equilibrium constants of each model can be used to predict the product concentration in different organic solvents.     

酶法生产L-高苯丙氨酸的研究进展

L-高苯丙氨酸(L-homophenylalanine,L-HPA)作为一种重要的非天然氨基酸,是合成治疗高血压的普利类药物等的关键中间体,具有广阔的市场前景。目前L-高苯丙氨酸的合成主要依赖于化学法,但化学合成L-高苯丙氨酸具有原料昂贵、步骤繁琐和污染严重等缺点,限制了广泛应用。因此,国内外研究者对L-高苯丙氨酸的酶法生产进行了深入的研究。本文就目前酶法合成L-高苯丙氨酸的工艺,包括脱氢酶法、转氨酶法、海因酶法和脱羧酶法的研究进展进行了综述,为酶法合成L-高苯丙氨酸提供一定的借鉴,为最终实现L-高苯丙氨酸的酶法工业化生产奠定基础。     

Mutant Escherichia coli penicillin acylase with enhanced stability at alkaline pH

Increased stability at alkaline pH should be a valuable attribute for the utilization of penicillin acylase in bioreactors employed to convert penicillins into 6-aminopenicillanic acid, a precursor of semisynthetic penicillins. In these systems, base is added for pH control, which results in local alkaline conditions that promote enzyme inactivation. Hydrolysis and synthesis reactions are also pH dependent. Here, we report work in which the gene coding for Escherichia coli penicillin acylase was subjected to oligonucleotide-directed random mutagenesis at regions coding for amino acids predicted to be at the surface of the enzyme. The resulting mutant library, cloned in E. coli, was screened by a filter paper assay of the colonies for the presence of penicillin acylase activity with enhanced stability at alkaline pH. Characterization of one of the selected clones revealed the presence of a mutation, Trp431-Arg, which would presumably alter the surface charge of the protein. In vitro experiments demonstrated a near twofold increase in the half-life of the mutant enzyme when stored at pH 8.5 as compared with the wild-type enzyme, with a comparable specific activity at several pH values. In general, the mutant displayed increased stability toward the basic side in the pH-stability profile. (c) 1995 John Wiley & Sons, Inc.     

Biocatalysis in natural products chemistry

The properties of enzymes and microbial cells as biocatalysts useful in natural products chemistry are discussed from the perspective of the chemical transformations they catalyse. Attention is focused on numerous reactions of value to natural products chemists, including the acyloin condensation, Baeyer-Villiger oxidation, regio- and enantioselective ester hydrolyses, oxidations of aromatic and non-aromatic substrates, oxidoreduction and O- and N-dealkylations. Compounds considered in this review include amino acids, alkaloids, antibiotics, coumarins, naphthoquinones, quassinoids, rotenoids and mono-, sesqui-, di- and triterpenoid substrates. The value of biocatalysis compared with traditional chemical catalysis is considered within the broad framework of natural products chemistry, and the potential for using immobilized enzyme and cell technology is presented.     

Solvent effects on biocatalysis in organic systems: equilibrium position and rates of lipase catalyzed esterification

Porcine pancreatic lipase immobilized on celite particles has been employed as a catalyst for the esterification of dodecanol and decanoic acid in a predominantly organic system. Solvent influence on the equilibrium position and on the catalyst activity has been studied using 20 solvents, including aliphatic and aromatic hydrocarbons, ethers, ketones, nitro- and halogenated hydrocarbons, and esters. The equilibrium constant for esterification correlates well with the solubility of water in the organic solvent, which in turn shows a good relationship with a function of Guttman's donor number and the electron pair acceptance index number of the solvent. This may be rationalized in terms of the requirements for solvation of water and of the reactants. The catalyst activity, measured as the initial rate of the esterification reaction, is best correlated as a function of both n-octanol-water partition coefficient (log P) and either the electron pair acceptance index or the polarizability.     

Studies on the effects of the antitumor agent camptothecin and derivatives on DNA. Camptothecin potentiated cleavage of DNA by bleomycin in vitro

Addition of sodium camptothecin (2a, Fig. 1) in comparable low concentrations to the glycopeptide antitumor antibiotic bleomycin (BLM) leads to enhanced rates of single-strand scission of PM2-covalently closed circular DNA, whereas sodium camptothecin alone has no effect. A similar enhancement of DNA scission by sodium camptothecin is produced with the 1 : 1 bleomycin-iron complex alone or in conjunction with NADPH as an additional reductant. The interpretation that camptothecin may substitute for the reducing requirement of the antibiotic is supported by its oxidation at 37°C by the 1 : 1 bleomycin iron complex, by iron salts or more efficiently by hydrogen peroxide to the known hemiacetal (3, Fig. 1).Electrochemical studies of 2a, its analogues and selected model compounds established that the α-pyridone ring D is most susceptible to a one-electron reduction at a reversible potential of ?0.95 ± 0.01 V. The reduced camptothecin is a transient species readily capable of donating an electron. This process may by compatible with a coupled reduction of the sequestered Fe(III) in the glycopeptide antibiotic necessary for the expression of antibiotic and antitumor properties. The results may provide a mechanistic rationale for the observed potentiation of the antitumor activity of bleomycin by camptothecin in vivo.     

Harnessing and engineering amide bond forming ligases for the synthesis of amides

The amide functional group is ubiquitous in nature and one of the most important motifs in pharmaceuticals, agrochemicals, and other valuable products. While coupling amides and carboxylic acids is a trivial synthetic transformation, it often requires protective group manipulation, along with stoichiometric quantities of expensive and deleterious coupling reagents. Nature has evolved a range of enzymes to construct amide bonds, the vast majority of which utilize adenosine triphosphate to activate the carboxylic acid substrate for amine coupling. Despite the fact that these enzymes operate under mild conditions, as well as possessing chemoselectivity and regioselectivity that obviates the need for protecting groups, their synthetic potential has been largely unexplored. In this review, we discuss recent research into the discovery, characterization, and development of amide bond forming enzymes, with an emphasis on stand-alone ligase enzymes that can generate amides directly from simple carboxylic acid and amine substrates.     

Optimization of factors influencing enzyme activity and product selectivity and the role of proton transfer in the catalytic mechanism of patchoulol synthase

The patchoulol synthase (PTS) from Pogostemon cablin is a versatile sesquiterpene synthase and produces more than 20 valuable sesquiterpenes by conversion of the natural substrate farnesyl pyrophosphate (FPP). PTS has the potential to be used as a biocatalyst for the production of valuable sesquiterpenes such as (−)-patchoulol. The objective of the present study is to develop an efficient biotransformation and to characterize the biocatalytic mechanism of the PTS in detail. For this purpose, soluble PTS was prepared using an optimized cultivation protocol and continuous downstream process with a purity of 98%. The PTS biotransformation was then optimized regarding buffer composition, pH-value, and temperature for biotransformation as well as functional and kinetic properties to improve productivity. For the bioconversion of FPP, the highest enzyme activity was reached with the 2-(N-morphlino)ethanesulfonic acid (MES) buffer containing 10% (v/v) glycerol and 10 mM MgCl₂ at pH 6.4 and 34°C. The PTS showed an unusual substrate inhibition for sesquiterpene synthases indicating an intermediate sesquiterpene formed in the active center. Deuteration experiments were used to gain further insights into the biocatalytic mechanism described in literature. Thus it could be shown that a second substrate binding site must be responsible for substrate inhibition and that further protonation and deprotonation steps are involved in the reaction mechanism.