Hidenobu Komeda

New Enzymatic Method of Chiral Amino Acid Synthesis by Dynamic Kinetic Resolution of Amino Acid Amides: Use of Stereoselective Amino Acid Amidases in the Presence of α-Amino-ε-Caprolactam Racemase

ABSTRACT d- and l-amino acids were produced from l- and d-amino acid amides by d-aminopeptidase from Ochrobactrum anthropi C1-38 and l-amino acid amidase from Pseudomonas azotoformans IAM 1603, respectively, in the presence of α-amino-ε-caprolactam racemase from Achromobacter obae as the catalyst by dynamic kinetic resolution of amino acid amides.

Enhancement of the thermostability and catalytic activity of d-stereospecific amino-acid amidase from Ochrobactrum anthropi SV3 by directed evolution

d-Amino-acid amidases, which catalyze the stereospecific hydrolysis of d-amino-acid amide to yield d-amino acid and ammonia, have attracted increasing attention as catalysts for stereospecific production of d-amino acids. We screened for the enzyme variants with improved thermostability generated by a directed evolution method with the goal of the application of evolved enzyme to the production of d-amino acids. Random mutagenesis by error-prone PCR and a filter-based screening was repeated twice, and as a result the most thermostable mutant BFB40 was obtained. Gene analysis of the BFB40 mutant indicated that the mutant enzyme had K278 M and E303 V mutations. To compare the enzyme characteristics with the wild-type enzyme, the mutant enzyme, BFB40, was purified from the Escherichia coli (E. coli) transformant. Both the thermostability and apparent optimum temperature of the BFB40 were shifted upward by 5 ◦ C compared with those of the wild-type enzyme. The apparent Km value for d-phenylalaninamide of BFB40 enzyme was almost the same with that of the wild-type enzyme, whereas Vmax value was enhanced about three-fold. Almost complete hydrolysis of d-phenylalaninamide was achieved in 2 h from 1.0 M of racemic phenylalaninamide–HCl using the cells of E. coli transformant expressing BFB40 enzyme, the conversion of which was 1.7-fold higher than the case using cells expressing wild-type enzyme after the same reaction time.

Purification and Characterization of A Novel (R)-Hydroxynitrile Lyase from Eriobotrya japonica (Loquat)

A hydroxynitrile lyase was isolated and purified to homogeneity from seeds of Eriobotrya japonica (loquat). The final yield, of 36% with 49-fold purification, was obtained by 30–80% (NH4)2SO4 fractionation and column chromatography on DEAE-Toyopearl and Concanavalin A Sepharose 4B, which suggested the presence of a carbohydrate side chain. The purified enzyme was a monomer with a molecular mass of 72 kDa as determined by gel filtration, and 62.3 kDa as determined by SDS-gel electrophoresis. The N-terminal sequence is reported. The enzyme was a flavoprotein containing FAD as a prosthetic group, and it exhibited a K m of 161 μM and a k cat⁄K m of 348 s−1 mM−1 for mandelonitrile. The optimum pH and temperature were pH 5.5 and 40 °C respectively. The enzyme showed excellent stability with regard to pH and temperature. Metal ions were not required for its activity, while activity was significantly inhibited by CuSO4, HgCl2, AgNO3, FeCl3, β-mercaptoethanol, iodoacetic acid, phenylmethylsulfonylfluoride, and diethylpyrocarbonate. The specificity constant (k cat⁄K m) of the enzyme was investigated for the first time using various aldehydes as substrates. The enzyme was active toward aromatic and aliphatic aldehydes, and showed a preference for smaller substrates over bulky one.

A DmpA‐homologous protein from Pseudomonas sp. is a dipeptidase specific for β‐alanyl dipeptides

We have determined the nucleotide sequence of a DNA fragment covering the flanking region of the R‐stereoselective amidase gene, ramA, from the Pseudomonas sp. MCI3434 genome and found an additional gene, bapA, coding for a protein showing sequence similarity to DmpA aminopeptidase from Ochrobactrum anthropi LMG7991 (43% identity). The DmpA (called l‐aminopeptidase d‐Ala‐esterase/amidase) hydrolyzes alanine‐p‐nitroanilide, alaninamide, and alanine methylester with a preference for the d‐configuration of the alanine, whereas the enzyme acts as an l‐stereoselective aminopeptidase on a tripeptide Ala‐(Gly)2, indicating a reverse stereoselectivity [Fanuel L, Goffin C, Cheggour A, Devreese B, Van Driessche G, Joris B, Van Beeumen J & Frère J‐M (1999) Biochem J341, 147–155]. A recombinant BapA exhibiting hydrolytic activity toward d‐alanine‐p‐nitroanilide was purified from the cell‐free extract of an Escherichia coli transformant overexpressing the bapA gene and characterized. The purified enzyme contained two polypeptides corresponding to residues 1–238 (α‐peptide) and 239–366 (β‐peptide) of the precursor as observed for DmpA. On gel‐filtration chromatography, BapA in the native form appeared to be a tetramer. It had maximal activity at 60 °C and pH 9.0–10.0, and was inactivated in the presence of p‐chloromercuribenzoate, N‐ethylmaleimide, dithiothreitol, Zn2+, Ag+, Cd2+ or Hg2+. The enzyme hydrolyzed d‐alanine‐p‐nitroanilide more efficiently than l‐alanine‐p‐nitroanilide the same as DmpA. Furthermore, BapA was found to hydrolyze peptide bonds of β‐alanyl dipeptides including β‐Ala‐l‐Ala, β‐Ala‐Gly, β‐Ala‐l‐His (carnosine), β‐Ala‐l‐Leu, and (β‐Ala)2 with high efficiency compared to d‐alanine‐p‐nitroanilide. β‐Alaninamide was also efficiently hydrolyzed, but the enzyme did not act on the peptides containing proteinogenic amino acids or their d‐counterparts for N‐terminal residues. Based on its unique substrate specificity, the enzyme should not be called l‐aminopeptidase d‐Ala‐esterase/amidase but β‐Ala‐Xaa dipeptidase.

Functional expression of a plant hydroxynitrile lyase in Escherichia coli by directed evolution: creation and characterization of highly in vivo soluble mutants

Low protein solubility of recombinantly expressed proteins in Escherichia coli is a major factor hindering their application and analysis. We generated highly in vivo soluble mutants of a hydroxynitrile lyase in E.coli using protein engineering. Structure-guided saturation mutagenesis caused high solubility of single Lys-Pro mutations at positions 176, 199 and 224 of this low soluble wild-type enzyme. The triple Lys-Pro mutant generated at these surface conserved residues showed up to 8-fold increase in specific activity in the cell-free extract. Random mutagenesis also created a mutant of His103Met with 18.5-fold increase. The main expression form was reversed from insoluble to the soluble fraction following both types of above-mentioned mutations in E.coli at 37°C. The findings challenge the rationale of producing recombinant proteins in this host at 37°C. Formerly wild type low soluble protein was then present as soluble protein by these mutations, which also elevated the total soluble protein fraction in E.coli. Saturation mutagenesis of His103 provided other highly soluble mutants with hydrophobic substitutions. These mutations caused only minor secondary structural changes as determined by circular dichroism and Fourier-transform infrared spectroscopy and affected catalytic efficiency slightly for the purified mutants (0.82-1.6-fold for benzaldehyde and 0.9-1.9-fold for mandelonitrile). The stability of the mutants was differed from that of the wild type at high temperatures and at pH >8. Exchanging the buried basic-polar residue His103 with hydrophobic amino acids is in line with the overall structure of the enzyme, i.e. having hydrophilic residues in solvent-exposed areas and hydrophobic residues in the core.

Characterization of a New (R)-Hydroxynitrile Lyase from the Japanese Apricot Prunus mume and cDNA Cloning and Secretory Expression of One of the Isozymes in Pichia pastoris

PmHNL, a hydroxynitrile lyase from Japanese apricot ume (Prunus mume) seed was purified to homogeneity by ammonium sulfate fractionation and chromatographic steps. The purified enzyme was a monomer with molecular mass of 58 kDa. It was a flavoprotein similar to other hydroxynitrile lyases of the Rosaceae family. It was active over a broad temperature, and pH range. The N-terminal amino acid sequence (20 amino acids) was identical with that of the enzyme from almond (Prunus dulcis). Based on the N-terminal sequence of the purified enzyme and the conserved amino acid sequences of the enzymes from Pr. dulcis, inverse PCR method was used for cloning of a putative PmHNL (PmHNL2) gene from a Pr. mume seedling. Then the cDNA for the enzyme was cloned. The deduced amino acid sequence was found to be highly similar (95%) to that of an enzyme from Pr. serotina, isozyme 2. The recombinant Pichia pastoris transformed with the PmHNL2 gene secreted an active enzyme in glycosylated form.

The Screening, Characterization, and Use of ω-Laurolactam Hydrolase: A New Enzymatic Synthesis of 12-Aminolauric Acid

Several ω-laurolactam degrading microorganisms were isolated from soil samples. These strains were capable of growing in a medium containing ω-laurolactam as sole source of carbon and nitrogen. Among them, five strains (T7, T31, U124, U224, and U238) were identified as Cupriavidus sp. T7, Acidovorax sp. T31, Cupriavidus sp. U124, Rhodococcus sp. U224, and Sphingomonas sp. U238, respectively. The ω-laurolactam hydrolyzing enzyme from Rhodococcus sp. U224 was purified to homogeneity, and its enzymatic properties were characterized. The enzyme acts on ω-octalactam and ω-laurolactam, but other lactam compounds, amides and amino acid amides, cannot be substrates. The enzyme gene was cloned, and the deduced amino acid sequence showed high homology with 6-aminohexanoate-cyclic-dimer hydrolase (EC 3.5.2.12) from Arthrobacter sp. KI72 and Pseudomonas sp. NK87. Enzymatic synthesis of 12-aminolauric acid was performed using partially purified ω-laurolactam hydrolase from Rhodococcus sp. U224.

Synthesis of D-phenylalanine oligopeptides catalyzed by alkaline D-peptidase from bacillus cereus DF4-B

Abstract Synthesis of d -phenylalanine oligopeptides from d -phenylalanine methylester has been demonstrated by use of alkaline d -peptidase (ADP) from Bacillus cereus. An expression plasmid pKADP was constructed by placing the PCR-amplified ADP gene (adp) under the tac promoter of pKK223-3. Oligomerization of d -phenylalanine methylester by use of the purified ADP from the transformant Escherichia coli was investigated under several conditions. d -Phenylalanine dimer, ( d -Phe)2, and trimer, ( d -Phe)3, were produced in 25.4% and 8.6% yield, respectively, when 50 mM of the substrate was incubated for 8 h with ADP (2.0 U/ml and 0.4 U/ml, respectively) in 100 mM triethylamine–HCl (pH 11.5). Addition of dimethylsulfoxide to the reaction mixture resulted in the production of tetramer, ( d -Phe)4 in 6.7% yield with the decrease of the ( d -Phe)2 and ( d -Phe)3 production. This is the first study on the synthesis of d -phenylalanine oligomers by use of a d -stereospecific endopeptidase.

High Yield Synthesis of 12-Aminolauric Acid by “Enzymatic Transcrystallization” of ω-Laurolactam Using ω-Laurolactam Hydrolase from Acidovorax sp. T31

The genes encoding ω-laurolactam hydrolases from Cupriavidus sp. T7, Acidovorax sp. T31, Cupriavidus sp. U124, and Sphingomonas sp. U238 were cloned and sequenced. Nucleotide and amino acid sequence analysis of the four genes indicated that the primary structures of these ω-laurolactam hydrolases are significantly similar to the 6-aminohexanoate-cyclic-dimer hydrolase (EC 3.5.2.12). These genes were expressed in Escherichia coli, and the ω-laurolactam hydrolysing activity of the recombinant enzymes was compared with that of 6-aminohexanoate-cyclic-dimer hydrolase from Arthrobacter sp. KI72. The enzyme from Acidovorax sp. T31 was most successfully expressed in E. coli. Cell-free extract of the recombinant strain was used for the synthesis of 12-aminolauric acid from ω-laurolactam by “enzymatic transcrystallization,” because crystalline ω-laurolactam added into the enzyme solution was converted to crystalline 12-aminolauric acid (≧97.3% yield). Under the optimum conditions, 208 g/l of 12-aminolauric acid was produced in 17 h. The resulting pure product was identical to authentic 12-aminolauric acid.

Genes for an alkaline D-stereospecific endopeptidase and its homolog are located in tandem on Bacillus cereus genome.

Alkaline D-peptidase (Adp) from Bacillus cereus DF4-B is a D-stereospecific endopeptidase acting on oligopeptides composed of D-phenylalanine and the primary structure deduced from its gene, adp, shows a similarity with D-stereospecific hydrolases from Ochrobactrum anthropi strains. We have isolated DNA fragments covering the flanking region of adp from DF4-B genome and found an additional gene, adp2, located upstream of adp. The deduced amino acid sequence of Adp2 showed 96% and 85% identity with those of Adp from B. cereus strains AH559 and DF4-B, respectively. The recombinant Adp2 expressed in Escherichia coli was purified to homogeneity and characterized. It had hydrolyzing activity toward (D-Phe)3, (D-Phe)4, and (D-Phe)6 but did not act on (L-Phe)4, D-Phe-NH2, and L-Phe-NH2, some characteristics that are closely related to those of Adp from strain DF4-B. These results indicate that highly homologous genes encoding D-stereospecific endopeptidases are arranged in a tandem manner on the genomic DNA of B. cereus DF4-B.