Efficient Nepsilon-lauroyl-L-lysine production by recombinant epsilon-lysine acylase from Streptomyces mobaraensis.
epsilon-Lysine acylase from Streptomyces mobaraensis (Sm-ELA), which specifically catalyzes hydrolysis of the epsilon-amide bond in various Nepsilon-acyl-L-lysines, was cloned and sequenced. The Sm-ELA gene consists of a 1617-bp open reading frame that encodes a 538-amino acid protein with a molecular mass of 55,816Da. An NCBI protein-protein BLAST search revealed that the enzyme belongs to the YtcJ-like metal-dependent amidohydrolase family, which is further characterized as the metallo-dependent hydrolase superfamily. The Sm-ELA gene was ligated into a pUC702 vector for expression in Streptomyces lividans TK24. Expression of recombinant Sm-ELA in S. lividans was approximately 300-fold higher than that in wild-type S. mobaraensis. The recombinant Sm-ELAs from the cell-free extract and culture supernatant were purified to homogeneity. The specific activities of the purified Sm-ELAs were 2500-2800U/mg, which were similar to that obtained for the wild-type Sm-ELA. Using the cell-free extract of the recombinant S. lividans cells, Nepsilon-lauroyl-L-lysine was synthesized from 500mM L-lysine hydrochloride and 50, 100, or 250mM lauric acid in an aqueous buffer solution at 37 degrees C. The yields were close to 100% after 6 and 9h of reaction for 50 and 100mM lauric acid, respectively, and 90% after 24h for 250mM lauric acid.
Koreishi
Mayuko
M
Division of Chemistry and Biochemistry, Graduate School of Natural Science and Technology, Okayama University, Tsushima-Naka, Okayama, Japan.
Kawasaki
Ryoko
R
Imanaka
Hiroyuki
H
Imamura
Koreyoshi
K
Takakura
Yasuaki
Y
Nakanishi
Kazuhiro
K
D000581:Amidohydrolases / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D002474:Cell-Free System; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D007850:Lauric Acids / Q000378:metabolism; D008239:Lysine / Q000031:analogs & derivatives / Q000378:metabolism*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013302:Streptomyces / Q000201:enzymology*; D048170:Streptomyces lividans / Q000235:genetics
D007850:Lauric Acids; D011994:Recombinant Proteins; C030358:lauric acid; D000581:Amidohydrolases; C019665:amidase; D008239:Lysine