Characteristics and function of Alcaligenes sp. NBRC 14130 esterase catalysing the stereo-selective hydrolysis of ethyl chrysanthemate.
OBJECTIVE
Alcaligenes sp. NBRC 14130 was found as a strain hydrolysing a mixture of (+/-)-trans- and (+/-)-cis ethyl chrysanthemates to (1R,3R)-(+)-trans-chrysanthemic acid. The Alcaligenes cells also have hydrolytic activity for 6-aminohexanoate-cyclic dimer (6-AHCD, 1,8-diazacyclotetradecane-2,9-dione). The correlation of function on the enzyme from the Alcaligenes strain with hydrolysis activities for both ethyl chrysanthemate and 6-AHCD was demonstrated.


RESULTS
The esterase was purified to homogeneity. The purified esterase hydrolysed 20 mmol l(-1) ester including the four stereoisomers to the corresponding (+)-trans acid with a 37% molar conversion of ethyl (+)-trans chrysanthemate. The esterase showed high hydrolytic activity for various short-chain fatty acid esters, n-hexane amide and 6-AHCD. The amino acid sequence of the Alcaligenes esterase was identical to that of Arthrobacter 6-AHCD hydrolase (EC 3.5.2.12) and similar to that of fatty acid amide hydrolase (EC 3.5.1.4) from Rattus norvegicus, having both serine and lysine residues of the catalytic site and the consensus motif Gly-X-Ser-X-Gly.


CONCLUSIONS
The stereo-selective hydrolytic activity was found in Alcaligenes sp. NBRC14130 by screening of ethyl chrysanthemate-hydrolysing activity in micro-organisms, and the purified esterase also acted on fatty acid esters and amides.


CONCLUSIONS
This study has demonstrated that there are great differences in the enzymatic properties, amino acid sequence and catalytic motif of esterases in both Alcaligenes and Arthrobacter globiformis with excellent stereo-selectivity for (+)-trans-ethyl chrysanthemate, but the amino acid sequence of Alcaligenes esterase is identical to that of Arthrobacter 6-AHCD hydrolase.
Mitsukura
K
K
Department of Biomolecular Science, Faculty of Engineering, Gifu University, Gifu, Japan. mitukura@gifu-u.ac.jp
Shimizu
M
M
Matsushita
K
K
Yoshida
T
T
Nagasawa
T
T
D000421:Alcaligenes / Q000201:enzymology*; D000595:Amino Acid Sequence; D004950:Esterases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D006868:Hydrolysis; D006899:Mixed Function Oxygenases / Q000378:metabolism; D008969:Molecular Sequence Data; D011722:Pyrethrins / Q000378:metabolism*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D011722:Pyrethrins; C007051:chrysanthemic acid; C056674:ethyl chrysanthemate; D006899:Mixed Function Oxygenases; D004950:Esterases