Sukul_2018_Front.Microbiol_9_1925

A novel gene encoding for a lipolytic enzyme, designated ML-005, was recently identified using a functional metaproteomics approach. We heterologously expressed this protein in Escherichia coli and biochemically characterized it. ML-005 exhibited lipolytic activity toward short-chained substrates with the preferred substrate being p-nitrophenyl-butyrate, suggesting that ML-005 is an esterase. According to homology analysis and site-directed mutagenesis, the catalytic triad of the enzyme was identified as Ser-99, Asp-164, and His-191. Its optimal pH was determined to be at pH 8. Optimal activity was observed at 45 degrees C. It also exhibited temperature, pH and salt tolerance. Residual relative activity after incubating at 50-60 degrees C for 360 min was above 80% of its initial activity. It showed tolerance over a broad range of pH (5-12) and retained most of its initial activity. Furthermore, incubating ML-005 in 1 - 5M NaCl solution had negligible effect on its activity. DTT, EDTA, and ss-mercaptoethanol had no significant effect on ML-005's activity. However, addition of PMSF led to almost complete inactivation consistent with ML-005 being a serine hydrolase. ML-005 remains stable in the presence of a range of metal ions, but addition of Cu(2+) significantly reduces its relative activity. Organic solvents have an inhibitory effect on ML-005, but it retained 21% of activity in 10% methanol. SDS had the most pronounced inhibitory effect on ML-005 among all detergents tested and completely inactivated it. Furthermore, the Vmax of ML-005 was determined to be 59.8 muM/min along with a Km of 137.9 muM. The kcat of ML-005 is 26 s(-1) and kcat/Km is 1.88 x 10(5) M(-1) s(-1).