Makoto Yagasaki

Industrial biotransformations for the production of d-amino acids

Abstract 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.

A Novel L-Amino Acid Ligase from Bacillus subtilis NBRC3134, a Microorganism Producing Peptide-Antibiotic Rhizocticin

L-Amino acid ligase catalyzes the formation of an α-peptide bond from unprotected L-amino acids in an ATP-dependent manner, and this enzyme is very useful in efficient peptide production. We performed enzyme purification to obtain a novel L-amino acid ligase from Bacillus subtilis NBRC3134, a microorganism producing peptide-antibiotic rhizocticin. Rhizocticins are dipeptide or tripeptide antibiotics and commonly possess L-arginyl-L-2-amino-5-phosphono-3-cis-pentenoic acid. The purification was carried out by detecting L-arginine hydroxamate synthesis activity, and a target enzyme was finally purified 1,280-fold with 0.8% yield. The corresponding gene was then cloned and designated rizA. rizA was 1,242 bp and coded for 413 amino acid residues. Recombinant RizA was prepared, and it was found that the recombinant RizA synthesized dipeptides whose N-terminus was L-arginine in an ATP-dependent manner. RizA had strict substrate specificity toward L-arginine as the N-terminal substrate; on the other hand, the substrate specificity at the C-terminus was relaxed.

Dipeptide synthesis by l-amino acid ligase from Ralstonia solanacearum

Despite its utility, dipeptides have not been widely used due to the absence of an efficient manufacturing method. Recently, a novel method for effective production of dipeptides using l-amino acid alpha-ligase (Lal) is presented. Lal, which is only identified in Bacillus subtilis, catalyzes dipeptide synthesis from unprotected amino acids in an ATP-dependent manner. However, not all the dipeptide can be synthesized by Lal from B. subtilis (BsLal) due to its substrate specificity. Here, we attempted to find a novel Lal exhibiting different substrate specificity from BsLal. By in silico screening based on the amino acid sequence of BsLal, RSp1486a an unknown protein from Ralstonia solanacearum was found to show the Lal activity. RSp1486a exhibited different substrate specificity from BsLal, and preferably synthesized hetero-dipeptides where more bulky amino acid was placed at N terminus and less bulky amino acid was placed at C terminus in opposition to those synthesized by BsLal.

A Novel L-Amino Acid Ligase from Bacillus licheniformis

l-Amino acid alpha-ligase (EC 6.3.2.28) catalyzed formation of alpha-peptide bond in unprotected l-amino acids in an ATP-dependent manner. BL00235 gene in Bacillus licheniformis NBRC12200 coded as a new l-amino acid ligase. BL00235 substrate specificity was strict; only methionine or leucine was acceptable as dipeptide N-terminal residues.

Resistance acquisition of Thiobacillus thiooxidans upon cadmium and zinc ion addition and formation of cadmium ion-binding and zinc ion-binding proteins exhibiting metallothionein-like properties

Abstract Through subcultivations of Thiobacillus thiooxidans WU-79A in autotrophic media in which the concentrations of Cd2+ and Zn2+ were increased successively, Cd2+-resistant (CDR) and Zn2+-resistant strains (ZNR) were obtained. The growth of WU-79A was inhibited by the addition of 25 mM Cd2+ as well as Zn2+. However, CDR and ZNR could grow without any lag phase in media containing 200 mM Cd2+ and 250 mM Zn2+, respectively. CDR and ZNR were able to grow even in media containing up to 400 mM Cd2+ and 600 mM Zn2+, respectively, although they exhibited lag phases. CDR could grow in medium containing up to 250 mM Zn2+, as could ZNR in medium containing up to 200 mM Cd2+. Cd2+-binding and Zn2+-binding proteins were isolated from CDR and ZNR, respectively, by gel filtration and ion exchange chromatography. The molecular weights of both proteins were estimated to be approximately 13,000 by gel filtration. The fact that there was no strong absorption at 280 nm of the proteins suggested that they had few aromatic amino acids. Broad absorption bands which are typical of mercaptide (metal thiolate) complexes were detected. The properties of the proteins were spectrophotometrically similar to those of metallothionein.

Effect of multidrug-efflux transporter genes on dipeptide resistance and overproduction in Escherichia coli.

L-Alanyl-L-glutamine (Ala-Gln) is a clinically and nutritionally important dipeptide. We have already shown a novel method for the fermentative production of Ala-Gln using an Escherichia coli strain expressing L-amino acid alpha-ligase (Lal), which catalyzes the formation of dipeptides by combining two amino acids. In the course of Ala-Gln-producing strain development, it was revealed that Lal expression caused growth inhibition. We also found that the addition of some dipeptides, including Ala-Gln, inhibited the growth of a multiple peptidase-deficient strain. To further increase the productivity by overcoming the inhibitory effect of dipeptides, we focused on dipeptide transport systems. The four genes (bcr, norE, ydeE and yeeO) were selected from 34 genes encoding a multidrug-efflux transporter of E. coli as those conferring resistance to growth inhibitory dipeptides. Intracellular concentration of Ala-Gln was reduced by overexpressing these genes in a multiple peptidase-deficient strain. Furthermore, overexpression of each gene in the dipeptide-producing strains resulted in the increase of Ala-Gln and L-alanyl-L-branched chain amino acids titers. These results indicate that some multidrug-efflux transporters of E. coli can transport dipeptides and that enhancement of their activities is effective for fermentative production of dipeptides.

Identification and characterization of a novel L-amino acid ligase from Photorhabdus luminescens subsp. laumondii TT01.

L-amino acid ligase catalyzes dipeptide synthesis from unprotected L-amino acids in an ATP-dependent manner. We recently identified a new member of L-amino acid ligase, the plu1440 protein, from Photorhabdus luminescens subsp. laumondii TT01 by in silico analysis. This protein was found to synthesize dipeptides containing L-asparagine at the N-terminus, which is a novel substrate specificity.

Identification of Novel L-Amino Acid α-Ligases through Hidden Markov Model-Based Profile Analysis

L-Amino acid α-ligase (Lal), catalyzing the formation of α-dipeptides from unprotected L-amino acids in an ATP-dependent manner, is used in cost-effective fermentative production of dipeptides. We searched for novel Lals by in silico screening using Hidden Markov Model-based profile analysis, and identified five novel Lals that showed low similarity and different substrate specificity from known Lals.

The structure of L-amino-acid ligase from Bacillus licheniformis

L-Amino-acid ligases (LALs) are enzymes which catalyze the formation of dipeptides by linking two L-amino acids. Although many dipeptides are known and expected to have medical and nutritional benefits, their practical use has been limited owing to their low availability and high expense. LALs are potentially desirable tools for the efficient production of dipeptides; however, the molecular basis of substrate recognition by LAL has not yet been sufficiently elucidated for the design of ideal LALs for the desired dipeptides. This report presents the crystal structure of the LAL BL00235 derived from Bacillus licheniformis NBRC 12200 determined at 1.9 Å resolution using the multi-wavelength anomalous dispersion method. The overall structure of BL00235 is fairly similar to that of YwfE, the only LAL with a known structure, but the structure around the catalytic site contains some significant differences. Detailed structural comparison of BL00235 with YwfE sheds some light on the molecular basis of the substrate specificities.

Enzymatic production of d-glutamate from l-glutamate by a glutamate racemase

Abstract d -Glutamate was produced from l -glutamate by two successive cellular reactions with a glutamate racemase produced by Escherichia coli TM93 harboring a plasmid containing a glutamate racemase gene from Lactobacillus brevis ATCC 8287 and a glutamate decarboxylase produced by E. coli ATCC 11246. l -Glutamate was first racemized to dl -glutamate at pH 8.5 and l -glutamate was then decarboxylated at pH 4.2. Starting from 100 g/l of l -glutamate, 50 g/l of d -glutamate remained after 15 h reaction.