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β-Peptides and their derivates are usually stable to proteolysis and have an increased half-life compared with α-peptides. Recently, β-aminopeptidases were described as a new enzyme class that enabled the enzymatic degradation and formation of β-peptides. As an alternative to the existing chemical synthesis routes, the aim of the present work was to develop a whole-cell biocatalyst for the synthesis and production of β-peptides using this enzymatic activity. For the optimization of the reaction system we chose the commercially relevant β,α-dipeptide L-carnosine (β-alanine-L-histidine) as model product. We were able to show that different recombinant yeast and bacteria strains, which overexpress a β-peptidase, could be used directly as whole-cell biocatalysts for the synthesis of L-carnosine. By optimizing relevant reaction conditions for the best-performing recombinant Escherichia coli strain, such as pH and substrate concentrations, we obtained high l-carnosine yields of up to 71%. Long-time as well as biocatalyst recycling experiments indicated a high stability of the developed biocatalyst for at least five repeated batches. Application of the recombinant E. coli in a fed-batch process enabled the accumulation of l-carnosine to a concentration of 3.7 g l(-1)., (© 2009 The Authors. Journal compilation © 2009 Society for Applied Microbiology and Blackwell Publishing Ltd.)

Biodegradable polyesters have the potential to replace nondegradable, persistent polymers in numerous applications and thereby alleviate plastic accumulation in the environment. Herein, we present an analytical approach to study enzymatic hydrolysis of polyesters, the key step in their overall biodegradation process. The approach is based on embedding fluorescein dilaurate (FDL), a fluorogenic ester substrate, into the polyester matrix and on monitoring the enzymatic cohydrolysis of FDL to fluorescein during enzymatic hydrolysis of the polyester. We validated the approach against established techniques using FDL-containing poly(butylene adipate) films and Fusarium solani cutinase (FsC). Implemented on a microplate reader platform, the FDL-based approach enabled sensitive and high-throughput analysis of the enzymatic hydrolysis of eight aliphatic polyesters by two fungal esterases (FsC and Rhizopus oryzae lipase) at different temperatures. While hydrolysis rates for both enzymes increased with decreasing differences between the polyester melting temperatures and the experimental temperatures, this trend was more pronounced for the lipase than the cutinase. These trends in rates could be ascribed to a combination of temperature-dependent polyester chain flexibility and accessibility of the enzyme active site. The work highlights the capability of the FDL-based approach to be utilized in both screening and mechanistic studies of enzymatic polyester hydrolysis. [ABSTRACT FROM AUTHOR]

Although the production and use of technical hexachlorocyclohexane ( HCH) and lindane (the purified insecticidal isomer γ- HCH) are prohibited in most countries, residual concentrations still constitute an immense environmental burden. Many studies describe the mineralization of γ- HCH by bacterial strains under aerobic conditions. However, the metabolic fate of the other HCH isomers is not well known. In this study, we investigated the transformation of α-, β-, γ-, δ-, ε- HCH, and a heptachlorocyclohexane isomer in the presence of varying ratios of the two enzymes that initiate γ- HCH degradation, a dehydrochlorinase ( LinA) and a haloalkane dehalogenase ( LinB). Each substrate yielded a unique metabolic profile that was strongly dependent on the enzyme ratio. Comparison of these results to those of in vivo experiments with different bacterial isolates showed that HCH transformation in the tested strains was highly optimized towards productive metabolism of γ- HCH and that under these conditions other HCH-isomers were metabolized to mixtures of dehydrochlorinated and hydroxylated side-products. In view of these results, bioremediation efforts need very careful planning and toxicities of accumulating metabolites need to be evaluated. [ABSTRACT FROM AUTHOR]

β-Peptides and their derivates are usually stable to proteolysis and have an increased half-life compared with α-peptides. Recently, β-aminopeptidases were described as a new enzyme class that enabled the enzymatic degradation and formation of β-peptides. As an alternative to the existing chemical synthesis routes, the aim of the present work was to develop a whole-cell biocatalyst for the synthesis and production of β-peptides using this enzymatic activity. For the optimization of the reaction system we chose the commercially relevant β, α-dipeptidel-carnosine ( β-alanine-l-histidine) as model product. We were able to show that different recombinant yeast and bacteria strains, which overexpress a β-peptidase, could be used directly as whole-cell biocatalysts for the synthesis ofl-carnosine. By optimizing relevant reaction conditions for the best-performing recombinant Escherichia coli strain, such as pH and substrate concentrations, we obtained highl-carnosine yields of up to 71%. Long-time as well as biocatalyst recycling experiments indicated a high stability of the developed biocatalyst for at least five repeated batches. Application of the recombinant E. coli in a fed-batch process enabled the accumulation ofl-carnosine to a concentration of 3.7 g l−1. [ABSTRACT FROM AUTHOR]

Partial microbial degradation of xenobiotic compounds in wastewater treatment plants (WWTPs) results in the formation of transformation products, which have been shown to be released and detectable in surface waters. Rule-based systems to predict the structures of microbial transformation products often fail to discriminate between alternate transformation pathways because structural influences on enzyme-catalyzed reactions in complex environmental systems are not well understood. The amide functional group is one such common substructure of xenobiotic compounds that may be transformed through alternate transformation pathways. The objective of this work was to generate a self-consistent set of biotransformation data for amide-containing compounds and to develop a metabolic logic that describes the preferred biotransformation pathways of these compounds as a function of structural and electronic descriptors. We generated transformation products of 30 amide-containing compounds in sludge-seeded bioreactors and identified them by means of HPLC-linear ion trap-orbitrap mass spectrometry. Observed biotransformation reactions included amide hydrolysis and N-dealkylation, hydroxylation, oxidation, ester hydrolysis, dehalogenation, nitro reduction, and glutathione conjugation. Structure-based interpretation of the results allowed for identification of preferences in biotransformation pathways of amides: primary amides hydrolyzed rapidly; secondary amides hydrolyzed at rates influenced by steric effects; tertiary amides were N-dealkylated unless specific structural moieties were present that supported other more readily enzyme-catalyzed reactions. The results allowed for the derivation of a metabolic logic that could be used to refine rule-based biotransformation pathway prediction systems to more specifically predict biotransformations of amide-containing compounds. [ABSTRACT FROM AUTHOR]

δ-Hexachlorocyclohexane (δ-HCH), one of the prevalent isomers of technical HCH, was enantioselectively dehydrochlorinated by the dehydrochlorinases LinA1 and LinA2 from Sphingobium indicum B90A to the very same δ-pentachlorocyclohexene enantiomer. Racemic δ-pentachlorocyclohexene, however, was transformed with opposite enantioselectivities by the two enzymes. A transformation pathway based on an anti-1,2-elimination, followed by a syn-1,4-elimination and a subsequent syn-1,2-elimination is postulated. [ABSTRACT FROM AUTHOR]

Review: ca. 100 refs. [ABSTRACT FROM AUTHOR]

Abstract: Several nitrilases were screened for the hydrolysis of the nitrile group of cis-1,2-dihydroxy-3-cyanocyclohexa-3,5-diene and trans-3-[(5S,6R)-5,6-dihydroxycyclohexa-1,3-dienyl]-acrylonitrile to the corresponding acids. Nitrilase from Rhodococcus sp. was able to convert both compounds with the activity of 0.3mU/mg protein and 0.05mU/mg protein, respectively. Nitrilase AtNIT1 from Arabidopsis thaliana converted only the latter but with a higher initial specific activity of 1.7mU/mg protein. Biotransformation of trans-3-[(5S,6R)-5,6-dihydroxycyclohexa-1,3-dienyl]-acrylonitrile was performed with AtNIT1 in the form of isolated enzyme and immobilized enzyme, and with recombinant cells containing AtNIT1. Biotransformations with isolated AtNIT1 resulted in 116mg of product in 10.6h with a yield of 77%. Forty-three percent of the enzymatic activity could be recovered after the biotransformation. Immobilization of AtNIT1 saturated with 3-phenylpropionitrile resulted in 3.5% of the free enzyme activity. Biotransformations with Escherischia coli JM101 (pQE10-AtNIT1) in shake-flasks produced 243mg of product in 23h with a yield of 48%. Maximum and average specific activities of 0.5U/g cell dry weight and 0.17U/g cell dry weight were achieved, respectively. [Copyright &y& Elsevier]

A novel bacterial strain that was capable of growing on the β-tripeptide H-βhVal-βhAla-βhLeu-OH as the sole carbon and nitrogen source was isolated from an enrichment culture. On the basis of physiological characterization, partial 16S rRNA sequencing, and fatty acid analysis, strain 3-2W4 was identified as a member of the family Sphingomonadaceae. Growth on the β-tripeptide and the β-dipeptide H-βhAla-βhLeu-OH was observed, and emerging metabolites were characterized. Small amounts of a persisting metabolite, the N-acetylated β-dipeptide, were identified in both media. According to dissolved organic carbon measurements, 74 to 80% of the available carbon was dissimilated. The β-peptide-degrading enzyme was purified from the crude cell extract of cells from strain 3-2W4 grown on complex medium. The enzyme was composed of two subunits, and the N-terminal sequences of both were determined. With this information, it was possible to identify the complete nucleotide sequence and to deduce the primary structure of the gene bapA. The gene encoded a β-peptidyl aminopeptidase (BapA) of 402 amino acids that was synthesized as preprotein with a signal sequence of 29 amino acids. The enzyme was cleaved into two subunits (residues 30 to 278 and 279 to 402). It belonged to the N-terminal nucleophile (Ntn) hydrolase superfamily. [ABSTRACT FROM AUTHOR]

Hexabromocyclododecanes (HBCDs) were used as flame-retardants until their ban in 2013. Among the 16 stereoisomers known, ε-HBCD has the highest symmetry. This makes ε-HBCD an interesting substrate to study the selectivity of biotransformations. We expressed three LinA dehydrohalogenase enzymes in E. coli bacteria, two wild-type, originating from Sphingobium indicum B90A bacteria and LinATM, a triple mutant of LinA2, with mutations of L96C, F113Y and T133 M. These enzymes are involved in the hexachlorocyclohexane (HCH) metabolism, specifically of the insecticide γ-HCH (Lindane). We studied the reactivity of those eight HBCD stereoisomers found in technical HBCD. Furthermore, we compared kinetics and selectivity of these LinA variants with respect to ε-HBCD. LC-MS data indicate that all enzymes converted ε-HBCD to pentabromocyclododecenes (PBCDens). Transformations followed Michaelis-Menten kinetics. Rate constants k cat and enzyme specificities k cat / K M indicate that ε-HBCD conversion was fastest and most specific with LinA2. Only one PBCDen stereoisomer was formed by LinA2, while LinA1 and LinATM produced mixtures of two PBCDE enantiomers at three times lower rates than LinA2. In analogy to the biotransformation of (−)β-HBCD, with selective conversion of dibromides in R-S-configuration, we assume that 1E,5S,6R,9S,10R-PBCDen is the ε-HBCD transformation product from LinA2. Implementing three amino acids of the LinA1 substrate-binding site into LinA2 resulted in a triple mutant with similar kinetics and product specificity like LinA1. Thus, point-directed mutagenesis is an interesting tool to modify the substrate- and product-specificity of LinA enzymes and enlarge their scope to metabolize other halogenated persistent organic pollutants regulated under the Stockholm Convention. Image 1 • Implementation of the LinA1 substrate-binding site to LinA2 by point-directed mutagenesis. • Expression of three LinA enzyme variants, LinA1, LinA2 and LinATM, in E. coli bacteria. • Three of eight HBCD stereoisomers are converted by the dehydrohalogenase LinA2. • All LinA variants follow Michaelis-Menten kinetics for the ε-HBCD transformation. • Stereoselective conversion of ε-HBCD to 1E,5S,6R,9S,10R-PBCDen by LinA2. [ABSTRACT FROM AUTHOR]