Masaru Sato

Synthesis of dl -tryptophan by modified broad specificity amino acid racemase from Pseudomonas putida IFO 12996

Broad specificity amino acid racemase (E.C. 5.1.1.10) from Pseudomonas putida IFO 12996 (BAR) is a unique racemase because of its broad substrate specificity. BAR has been considered as a possible catalyst which directly converts inexpensive l-amino acids to dl-amino acid racemates. The gene encoding BAR was cloned to utilize BAR for the synthesis of d-amino acids, especially d-Trp which is an important intermediate of pharmaceuticals. The substrate specificity of cloned BAR covered all of the standard amino acids; however, the activity toward Trp was low. Then, we performed random mutagenesis on bar to obtain mutant BAR derivatives with high activity for Trp. Five positive mutants were isolated after the two-step screening of the randomly mutated BAR. After the determination of the amino acid substitutions in these mutants, it was suggested that the substitutions at Y396 and I384 increased the Trp specific racemization activity and the racemization activity for overall amino acids, respectively. Among the positive mutants, I384M mutant BAR showed the highest activity for Trp. l-Trp (20xa0mM) was successfully racemized, and the proportion of d-Trp was reached 43% using I384M mutant BAR, while wild-type BAR racemized only 6% of initial l-Trp.

A New Method of Synthesis of Alkyl β-Glycosides Using Sucrose as Sugar Donor

Cellobiose phosphorylase from Clostridium thermocellum catalyzed the β-anomer-selective synthesis of alkyl glucosides from cellobiose. Synthesis of alkyl β-glucoside from inexpensive sucrose using cellobiose phosphorylase and sucrose phosphorylase from Pseudomonas saccharophilia was investigated. By combined use of these two phosphorylases, alkyl β-glucoside was anomer-selectively synthesized from sucrose and alkyl alcohol.

Substrate specificity of thermostable D-alanine-D-alanine ligase from Thermotoga maritima ATCC 43589.

D-Alanine-D-alanine ligase (Ddl) and its mutants maintain the biosynthesis of peptidoglycan, and the substrate specificity of Ddls partially affects the resistance mechanism of vancomycin-resistant enterococci. Through investigation of Ddls, Ddl from Thermotoga maritima ATCC 43589 showed novel characteristics, vis. thermostability up to 90 °C and broad substrate specificity toward 15 D-amino acids, particularly D-alanine, D-cysteine, and D-serine, in that order.

Identification of Rubisco anxiolytic-like peptides (rALPs) by comprehensive analysis of spinach green leaf protein digest

Rubisco, an enzyme for photosynthetic carbon dioxide fixation, is a major green leaf protein and known as the most abundant protein on the Earth. We found that Rubisco digested mimicking gastrointestinal enzymatic conditions exhibited anxiolytic-like effects after oral administration in mice. Based on a comprehensive peptide analysis of the digest using nanoLC-Orbitrap-MS and the structure-activity relationship of known anxiolytic-like peptides, we identified SYLPPLTT, SYLPPLT and YHIEPV [termed Rubisco anxiolytic-like peptide (rALP)-1, rALP-1(1-7) and rALP-2, respectively], which exhibited potent anxiolytic-like effects after oral administration. The anxiolytic-like effects of rALP-1/rALP-1(1-7) were blocked by a serotonin 5-HT1A receptor antagonist, whereas rALP-2-induced effects were inhibited by a δ-opioid receptor antagonist. In conclusion, novel Rubisco-derived anxiolytic-like peptides, rALP-1/rALP-1(1-7) and rALP-2, act via independent neural pathways.

Isolation of highly thermostable β-xylosidases from a hot spring soil microbial community using a metagenomic approach

Abstract The DNA extracted from a high-temperature environment in which micro-organisms are living will be a good source for the isolation of thermostable enzymes. Using a metagenomic approach, we aimed to isolate thermostable β-xylosidases that will be exploited for biofuel production from lignocellulosic biomass. DNA samples obtained from the soil near a spout of a hot spring (70°C, pH7.2) were subjected to sequencing, which generated a total of 84.2 Gbp with 967,925 contigs ofu2009>500u2009bp in length. Similarity search for β-xylosidase in the contigs revealed the presence of 168 candidate sequences, each of which may have arisen from more than one gene. Individual genes were amplified by PCR using sequence-specific primers. The resultant DNA fragments were cloned and introduced into Escherichia coli BL21 Star(DE3). Consequently, 269 proteins were successfully expressed in the E. coli cells and then examined for β-xylosidase activity. A total of 82 proteins exhibited β-xylosidase activity at 50°C, six of which retained the activity even at 90°C. Out of the six, three proteins were originated from a single candidate sequence, AR19M-311. An amino acid sequence comparison suggested the amino acid residues that appeared to be crucial for thermal stability of the enzymes.

Structure-Based Modification of D-Alanine-D-Alanine Ligase from Thermotoga maritima ATCC 43589 for Depsipeptide Synthesis

Depsipeptides are peptide-like polymers consisting of amino acids and hydroxy acids, and are expected to be new functional materials for drug-delivery systems and polymer science. In our previous study, D-alanyl-D-lactate, a type of depsipeptide, was enzymatically synthesized using D-alanine-D-alanine ligase from Thermotoga maritima ATCC 43589 (TmDdl) by Y207F substitution. Thereafter, in this study, further mutagenesis was introduced, based on structural comparison between TmDdl and a well-characterized D-alanine-D-alanine ligase from Escherichia coli. The S137A/Y207F mutant showed higher D-alanyl-D-lactate and lower D-alanyl-D-alanine synthesizing activity than the Y207F mutant. This suggests that substitution at the S137 residue contributes to product selectivity. Saturated mutagenesis on S137 revealed that the S137G/Y207F mutant showed the highest D-alanyl-D-lactate synthesizing activity. Moreover, the mutant showed broad substrate specificity toward D-amino acid and recognized D-lactate and D,L-isoserine as substrates. On the basis of these characteristics, various depsipeptides can be produced using S137G/Y207F-replaced TmDdl.