A novel alpha-amino-acid esterase from Bacillus mycoides capable of forming peptides of DD- and DL-configurations.
A novel alpha-amino-acid esterase possessing some properties favorable for the synthesis of D-amino acid-containing peptides has been purified from the culture broth of Bacillus mycoides. The enzyme consisted of 4 subunits of 39 kDa, had an isoelectric point of 7.0, and showed its maximum activity at around 47 degrees C and pH 7.6. The enzyme activity was strongly depressed by phenylmethanesulfonyl fluoride, but not by penicillin G or ampicillin, suggesting that the protein is a serine enzyme lacking penicillin-binding ability. The enzyme hydrolyzed a variety of D- and L-amino acid methyl esters with concomitant formation of homooligomers from D-Phe, D-Trp, D-Tyr, and D-Asp(OCH(3)) methyl esters, but it did not act on the D- or L-amino acid amides tested. Incubation of a mixture of Ac-D-Phe-OMe and D-/L-Leu-NH(2) with the enzyme yielded Ac-D-Phe-D-/L-Leu-NH(2) together with Ac-D-Phe-OH, the hydrolysate of the carboxyl component. To its credit, the enzyme failed to hydrolyze casein as well as peptides including diastereomers of diphenylalanine and dialanine, indicating that the enzyme would not cause secondary hydrolysis of once-formed peptides. These observations indicate the potential utility of the newly isolated enzyme for the synthesis of D-amino acid-containing peptides.
Sugihara
A
A
Osaka Municipal Technical Research Institute, 1-6-50 Morinomiya, Joto-ku, Osaka 536-8553, Japan. sugihara@omtri.city.osaka.jp
Shimada
Y
Y
Sugihara
S
S
Nagao
T
T
Watanabe
Y
Y
Tominaga
Y
Y
D001407:Bacillus / Q000201:enzymology*; D002265:Carboxylic Ester Hydrolases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002364:Caseins / Q000737:chemistry / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D013058:Mass Spectrometry; D008970:Molecular Weight; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism*; D010461:Peptones / Q000737:chemistry / Q000378:metabolism; D013237:Stereoisomerism; D013379:Substrate Specificity; D013696:Temperature
D002364:Caseins; D010455:Peptides; D010461:Peptones; D002265:Carboxylic Ester Hydrolases; C058058:alpha-amino acid esterase