Expression of the Fusarium oxysporum lactonase gene in Aspergillus oryzae: molecular properties of the recombinant enzyme and its application.
The lactonase gene of Fusarium oxysporum was expressed in Aspergillus oryzae for optical resolution of DL-pantoyl lactone. When the chromosomal gene encoding the full-length form of the lactonase, which has its own NH2-terminal signal peptide, was introduced in the host cells, the resulting transformant produced an enzyme of 46,600 Da, which corresponded to the wild-type enzyme. In contrast, A. oryzae transformed with the cDNA coding the mature enzyme produced a protein of 41,300 Da. Deglycosylation analysis with an endoglycosidase revealed that the difference in molecular mass arose from the different sugar contents of the recombinant enzymes. The mycelia of the transformant were used as a catalyst for asymmetric hydrolysis of DL-pantoyl lactone. The initial velocity of the asymmetric hydrolysis reaction catalyzed by the transformant was estimated to be 30 times higher than that by F. oxysporum. When the mycelia of the transformant were incubated with a 20% DL-pantoyl lactone solution for 4 h, 49.9% of the racemic mixture was converted to D-pantoic acid (>95% ee).
Honda
Kohsuke
K
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa Oiwake-cho, Sakyo-ku, Kyoto, 606-8502, Japan.
Tsuboi
Hirokazu
H
Minetoki
Toshitaka
T
Nose
Hanae
H
Sakamoto
Keiji
K
Kataoka
Michihiko
M
Shimizu
Sakayu
S
D000595:Amino Acid Sequence; D001236:Aspergillus oryzae / Q000235:genetics; D002265:Carboxylic Ester Hydrolases / Q000378:metabolism*; D005670:Fusarium / Q000201:enzymology / Q000235:genetics*; D015870:Gene Expression; D007783:Lactones / Q000378:metabolism*; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
D007783:Lactones; D011993:Recombinant Fusion Proteins; D002265:Carboxylic Ester Hydrolases; C019103:delta-lactonase