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Random and rational mutagenesis of an α-amino acid ester acyl transferase from Sphingobacterium siyangensis AJ2458 (SAET) was conducted to examine the production of aspartame, an α- l -aspartyl- l -phenylalanine methyl ester. We previously reported aspartame production via combination of enzymatic and chemical methods. However, the productivity of the aspartame intermediate by SAET was approximately one-fifth that of l -alanyl- l -glutamine (Ala-Gln), whose production method has already been established. Here, to improve the enzymatic activity of SAET, we performed random mutagenesis in the gene encoding SAET and obtained 10 mutations that elevated the enzymatic activity (1.2- to 1.7-fold increase) relative to that of wild-type SAET. To further improve the activity, we performed mutagenesis to optimize the combination of the obtained mutations and finally selected one SAET variant with 10 amino acid substitutions (M35-4 SAET). An Escherichia coli strain overexpressing M35-4 SAET displayed a 5.7-fold higher activity than that of the wild-type SAET, which was almost equal to that of Ala-Gln by an E. coli strain overexpressing wild-type SAET. The V max value of M35-4 SAET was 2.0-fold greater, and its thermostability was higher than those of wild-type SAET. These results suggest that the obtained SAET variants contribute to improvement in aspartame production. [ABSTRACT FROM AUTHOR]

The purification and cloning of the gene encoding the [alpha]-methylserine aldolase from Ralstonia sp. isolated from soil is described. The newly identified enzyme with the ability to catalyze the transfer of a hydroxymethyl moiety to [alpha]-amino acids independent of tetrahydrofolate (THF) might be used for efficient production of [alpha]-methyl-L-serine.

Here, we report a novel industrial aspartame production route, involving the enzymatic production of α- l -aspartyl- l -phenylalanine β-methylester from l - aspartic acid dimethylester and l -phenylalanine by α-amino acid ester acyl transferase. The route also involves the chemical transformation of α- l -aspartyl- l -phenylalanine β-methylester to α- l -aspartyl- l -phenylalanine methylester hydrochloride (aspartame hydrochloride) in an aqueous solution with methanol and HCl, followed by HCl removal to form aspartame. [ABSTRACT FROM AUTHOR]

The establishment of renewable biofuel and chemical production is desirable because of global warming and the exhaustion of petroleum reserves. Sebacic acid (decanedioic acid), the material of 6,10-nylon, is produced from ricinoleic acid, a carbon-neutral material, but the process is not eco-friendly because of its energy requirements. Laccase-catalyzing oxidative cleavage of fatty acid was applied to the production of dicarboxylic acids using hydroxy and oxo fatty acids involved in the saturation metabolism of unsaturated fatty acids inLactobacillus plantarumas substrates. Hydroxy or oxo fatty acids with a functional group near the carbon–carbon double bond were cleaved at the carbon–carbon double bond, hydroxy group, or carbonyl group by laccase and transformed into dicarboxylic acids. After 8 h, 0.58 mM of sebacic acid was produced from 1.6 mM of 10-oxo-cis-12,cis-15-octadecadienoic acid (αKetoA) with a conversion rate of 35% (mol/mol). This laccase-catalyzed enzymatic process is a promising method to produce dicarboxylic acids from biomass-derived fatty acids. Oxidative cleavage of oxo unsaturated fatty acid into dicarboxylic acid by laccase. [ABSTRACT FROM PUBLISHER]

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 focuses on a study regarding the production of L-alanyl-L-glutamine (Ala-Gin) from L-glutamine (Gin) and L-alanine methyl ester hydrochloride (AlaOMe). It informs that researchers produced Ala-Gin by providing optimum growth condition to Escherichia coli cells which overexpressed amino acid ester acyltransferase from Sphingobacterium siyangensis AJ 2458 (SAET). It also mentions that 69.7 gram per litre (g/L) Ala-Gin has been produced in 40 minutes.

Microorganisms are promising as producers of various polyunsaturated fatty acids (PUFAs) and as catalysts transforming them into unique molecular species beyond common PUFAs. This article describes PUFA production through chemical mutant- and molecular-breeding of an oleaginous filamentous fungus Mortierella alpina 1S-4 and PUFA transformation by anaerobic bacteria. M. alpina 1S-4 and its mutants and transformants produce oils containing not only common n − 6 and n − 3 PUFAs but also rare PUFAs. Unique PUFA-transforming activities were found in anaerobic bacteria. They isomerized PUFA to conjugated fatty acids and further transformed to partially saturated fatty acids with hydroxyl fatty acids as intermediates. The functions of these unique PUFAs have been attracting much attention for improving our health and for developing new chemical materials. [ABSTRACT FROM AUTHOR]

Abstract: Extracellular ligninolytic oxidoreductases produced by Cerrena sp. strain Ra, a soil-isolated basidiomycete with high artificial dye-decolorizing activity, were purified and characterized. One thermostable laccase, one typical manganese peroxidase, and two versatile peroxidases (VPI and VPII) were found in the culture of this fungus. The different characteristics of each enzyme enable the strain express wide-range of oxidizing activity under various conditions. VPI decolorizing activity was observed toward various kinds of dye compounds, and the activity and specificity varied depending on the oxidizing mediators added to the reaction mixture. Optimized VPI/mediator-coupling decolorizing will be widely used for industrial wastewater treatment. [Copyright &y& Elsevier]

l-isoleucine-4-hydroxylase ( IDO) is a recently discovered member of the Pfam family PF10014 (the former DUF 2257 family) of uncharacterized conserved bacterial proteins. To uncover the range of biochemical activities carried out by PF10014 members, eight in silico-selected IDO homologues belonging to the PF10014 were cloned and expressed in Escherichia coli. l-methionine, l-leucine, l-isoleucine and l-threonine were found to be catalysed by the investigated enzymes, producing l-methionine sulfoxide, 4-hydroxyleucine, 4-hydroxyisoleucine and 4-hydroxythreonine, respectively. An investigation of enzyme kinetics suggested the existence of a novel subfamily of bacterial dioxygenases within the PF10014 family for which free l-amino acids could be accepted as in vivo substrates. A hypothesis regarding the physiological significance of hydroxylated l-amino acids is also discussed. [ABSTRACT FROM AUTHOR]

4-Hydroxyisoleucine (HIL) found in fenugreek seeds has insulinotropic and anti-obesity effects and is expected to be a novel orally active drug for insulin-independent diabetes. Here, we show that the newly isolated strain Bacillus thuringiensis 2e2 and the closely related strain B. thuringiensis ATCC 35646 operate a novel metabolic pathway for l-isoleucine ( l-Ile) via HIL and 2-amino-3-methyl-4-ketopentanoic acid (AMKP). The HIL synthesis was catalyzed stereoselectively by an α-ketoglutaric acid-dependent dioxygenase and to be useful for efficient production of a naturally occurring HIL isomer, (2 S,3 R,4 S)-HIL. The (2 S,3 R,4 S)-HIL was oxidized to (2 S,3 R) -AMKP by a NAD-dependent dehydrogenase. The metabolic pathway functions as an effective bypass pathway that compensates for the incomplete tricarboxylic acid (TCA) cycle in Bacillus species and also explains how AMKP, a vitamin B antimetabolite with antibiotic activity, is synthesized. These novel findings pave a new way for the commercial production of HIL and also for AMKP. [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 molecular cloning and sequencing of a DNA fragment from Microbacterium liquefaciens AJ 3912, containing the genes responsible for the conversion of 5-substituted-hydantoins to α-amino acids, in Escherichia coli. Deduced amino acid sequence encoded by the hyuA gene; Expression of the hyuA, hyuH and hyuC genes in E. coli; Relation between the presence of the genes and the detection of hydantoin racemase, hydantoinase and N-carbamoyl α-amino acid amido hydrolase enzymatic activities.

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.

Investigates xylitol production from D-arabitol by the membrane and soluble fractions of Gluconobacter oxydans. Detection of two proteins in the soluble fraction; Ability of the proteins to increase xylitol production; Expression of the cloned genes in Escherichia coli.

Describes the production of D-arabitol, a five-carbon polyhydroxy alcohol, by Metschnikowia reukaufii AJ14787. Culture conditions effective for the production of D-arabitol; Effects of pH on D-arabitol production by resting cells; Effects of temperature on D-arabitol production.

Investigates the cloning of the xylitol dehydrogenase gene from Gluconobacter oxydans and improved production of xylitol from D-arabitol. Purification and characterization of xylitol dehydrogenase from G. oxydans ATCC 621; Cloning of the xdh gene from G. oxydans ATCC 621.

The article discusses the enzymatic production of 3,4-dihydroxyphenyl-L-alanine (L-dopa) using Erwinia herbicola cells that carry the mutant transciptional regulator TyrR. It notes that L-dopa is used in the treating individuals with Parkinson's disease and is synthesized through enzymatic method that uses tyrosine phenol-lyase (Tp)l, a tyrosine-inducible enzyme. It notes that the highest productivity of L-dopa was obtained for the strain that was developed under tyrosine non-induced conditions.

Describes the construction of a vector plasmid for use in Gluconobacter oxydans. Acetobacter-Escherichia coli shuttle vector introduced; Cryptic plasmid sequencing the nucleotides; Open reading frame of the plasmid; Shuttle vectors of Gluconobacter-E. coli.