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Linoleic acid isomerase was identified as a multi-component enzyme system that consists of three enzymes that exist in both the membrane and soluble fractions of Lactobacillus plantarum. One enzyme (CLA-HY) is present in the membrane fraction, while two enzymes (CLA-DH and CLA-DC) exist in the soluble fraction. Three Escherichia coli transformants expressing CLA-HY, CLA-DH, and CLA-DC were constructed. Conjugated linoleic acid (CLA) and 10-hydroxy-12-octadecenoic acid were generated from linoleic acid only when all these three E. coli transformants were used as catalysts simultaneously. CLA-HY catalyzed the hydration reaction, a part of linoleic acid isomerization, to produce 10-hydroxy-12-octadecenoic acid. This multi-component enzyme system required oxidoreduction cofactors such as NADH and FAD. This is the first report to reveal enzymes genes and the elaborate machinery that synthesizes CLA, especially an important isomer of cis-9, trans-11-CLA, in lactic acid bacteria., (Copyright © 2011 Elsevier Inc. All rights reserved.)

A novel enzymatic production system of optically pure β-hydroxy α-amino acids was developed. Two enzymes were used for the system: an N-succinyl L-amino acid β-hydroxylase (SadA) belonging to the iron(II)/α-ketoglutarate-dependent dioxygenase superfamily and an N-succinyl L-amino acid desuccinylase (LasA). The genes encoding the two enzymes are part of a gene set responsible for the biosynthesis of peptidyl compounds found in the Burkholderia ambifaria AMMD genome. SadA stereoselectively hydroxylated several N-succinyl aliphatic L-amino acids and produced N-succinyl β-hydroxy L-amino acids, such as N-succinyl- L-β-hydroxyvaline, N-succinyl- L-threonine, (2 S,3 R)- N-succinyl- L-β-hydroxyisoleucine, and N-succinyl- L- threo-β-hydroxyleucine. LasA catalyzed the desuccinylation of various N-succinyl- L-amino acids. Surprisingly, LasA is the first amide bond-forming enzyme belonging to the amidohydrolase superfamily, and has succinylation activity towards the amino group of L-leucine. By combining SadA and LasA in a preparative scale production using N-succinyl- L-leucine as substrate, 2.3 mmol of L- threo-β-hydroxyleucine were successfully produced with 93% conversion and over 99% of diastereomeric excess. Consequently, the new production system described in this study has advantages in optical purity and reaction efficiency for application in the mass production of several β-hydroxy α-amino acids. [ABSTRACT FROM AUTHOR]

The article describes the cloning of the gene encoding α-methylserine hydroxymethyltransferase from Aminobacter sp. AJ110403 and Ensifer sp. AJ110404 and the purification and characterization of the recombinant enzymes. It notes that gene encoding α-methylserine hydroxymethyltransferase was also expressed in Escherichia coli. The study reveals that the purified enzymes were homodimers with a 46-kDa subunit and contained 1 mol/mol-sub-unit of pyridoxal 5'-phosphate.

The article studies the alpha-methylserine aldolase gene from the NBRC15150, NBRC15149 and AJ110406 strains of Variovorax paradoxus that was expressed and cloned in Escherichia coli. It includes an in-depth analysis of the characterization and purification of the recombinant enzyme, as well as a discussion on the issues' implications for biotechnology.

Describes the purification and characterization of hydantoin racemase (HRase) from Microbacterium liquefaciens AJ 3912 (MHR). Enzymatic characteristics of the purified MHR; Comparison of the N-terminal amino acid sequence of MHR with those of other HRases.

Investigates the coenzyme specificity of enzymes in the oxidative pentose phosphate pathway of Gluconobacter oxydans. Activities of glucose-6-phosphate dehydrogenase; Expression of genes in Escherichia coli; Reduction of NAD.

Abstract: Screening was carried out for microorganisms able to produce N-(l-α-l-aspartyl)-l-phenylalanine methyl ester [APM] from l-isoasparagine and l-phenylalanine methyl ester hydrochloride. Of the 422 strains examined, 44 strains belonging to the family Enterobacteriaceae were found to produce APM. The enzyme catalyzing APM production was purified and identified as dipeptidase E. [Copyright &y& Elsevier]

Abstract: Aspartase (l-aspartate ammonia-lyase, EC 4.3.1.1), which catalyzes the reversible deamination of l-aspartic acid to yield fumaric acid and ammonia, is highly selective towards l-aspartic acid. We screened for enzyme variants with altered substrate specificity by a directed evolution method. Random mutagenesis was performed on an Escherichia coli aspartase gene (aspA) by error-prone PCR to construct a mutant library. The mutant library was introduced to E. coli and the transformants were screened for production of fumaric acid-mono amide from l-aspartic acid-α-amide. Through the screening, one mutant, MA2100, catalyzing deamination of l-aspartic acid-α-amide was achieved. Gene analysis of the MA2100 mutant indicated that the mutated enzyme had a K327N mutation. The characteristics of the mutated enzyme were examined. The optimum pH values for the l-aspartic acid and l-aspartic acid-α-amide of the mutated enzyme were pH 8.5 and 6.0, respectively. The K m value and V max value for the l-aspartic acid of the mutated enzyme were 28.3mM and 0.26U/mg, respectively. The K m value and V max value for the l-aspartic acid-α-amide of the mutated enzyme were 1450mM and 0.47U/mg, respectively. This is the first report describing the alteration of the substrate specificity of aspartase, an industrially important enzyme. [Copyright &y& Elsevier]

We determined the enzymatic characteristics of an industrially important biocatalyst, a-ketoglutarate-dependent L-isoleucine dioxygenase (IDO), which was found to be the enzyme responsible for the generation of (2S,3R,4S)-4-hydroxyisoleucine in Bacillus thuringiensis 2e2. Depending on the amino acid used as the substrate, IDO catalyzed three different types of oxidation reactions: hydroxylation, dehydrogenation, and sulfoxidation. IDO stereoselectively hydroxylated several hydrophobic aliphatic L-amino acids, as well as L-isoleucine, and produced (S)-3-hydroxy-L-allo-isoleucine, 4-hydroxy-L-leucine, (S)-4-hydroxy-L-norvaline, 4-hydroxy-L-norleucine, and 5-hydroxy-L-norleucine. The IDO reaction product of L-isoleucine, (2S,3R,4S)-4-hydroxyisoleucine, was again reacted with IDO and dehydrogenated into (2S,3R)-2-amino-3-methyl-4-ketopentanoate, which is also a metabolite found in B. thuringiensis 2e2. Interestingly, IDO catalyzed the sulfoxidation of some sulfur-containing L-amino acids and generated L-methionine sulfoxide and L-ethionine sulfoxide. Consequently, the effective production of various modified amino acids would be possible using IDO as the biocatalyst. [ABSTRACT FROM AUTHOR]

Abstract: Through the screening of microorganisms capable of utilizing α-methylserine, three representative strains belonging to the bacterial genera Paracoccus, Aminobacter, and Ensifer were selected as potent producers of α-methylserine hydroxymethyltransferase, an enzyme that catalyzes the interconversion between α-methyl-l-serine and d-alanine via tetrahydrofolate. Among these strains, Paracoccus sp. AJ110402 was selected as the strain exhibiting the highest α-methylserine hydroxymethyltransferase activity. The enzyme was purified to homogeneity from a cell-free extract of this strain. The native enzyme is a homodimer with apparent molecular mass of 85kDa and contains 1mol of pyridoxal-5′-phosphate per mol of the subunit. The K m for α-methyl-l-serine and tetrahydrofolate was 0.54mM and 73μM, respectively. The gene from Paracoccus sp. AJ110402 encoding α-methylserine hydroxymethyltransferase was cloned and expressed in Escherichia coli. Sequence analysis revealed an open reading frame of 1278bp, encoding a polypeptide with a calculated molecular mass of 46.0kDa. Using E. coli cells as whole-cell catalysts, 9.7mmol of α-methyl-l-serine was stereoselectively obtained from 15mmol of d-alanine and 13.2mmol of formaldehyde. [Copyright &y& Elsevier]