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    Surface residues serine 69 and arginine 194 of metagenome-derived lipase influence catalytic activity
    (Elsevier, 2020) Özgen, Fatma Feyza; Vardar-Yel, Nurcan; Roth, Owen Scott; Shahbaz, Lersa Sayyad; Vardar-Schara, Gonul
    A lipase gene was identified and isolated from a fosmid metagenomics library constructed from forest topsoil samples. Error prone PCR and saturation mutagenesis were used to generate seven lipase variants: S69 P/R194Q, S69 G, S69 P, S69E, S69 P/T99S/N190S/R194Q, R194S, and R194A; by testing five different fatty acids of p-nitrophenyl (p-NP) ester, positions S69 and R194 were found to influence the catalytic activity. Specifically, variant R194S was identified that had 5.0, 4.5, 2.1, 2.2, and 1.4-fold higher catalytic efficiency (v(max)/K-m) towards p-NP acetate, p-NP butyrate, p-NP octanoate, p-NP dodecanoate, and p-NP palmitate, respectively, compared to the metagenome-derived native lipase. Among the S69 variants, variants S69 P/R194Q and S69 P hydrolyzed p-NP acetate 1.8- and 1.5-fold faster than wild-type in terms of apparent v(max)/K-m values, respectively. The activity is lowered by introducing mutations S69E, R194A, and S69 P/T99S/N190S/R194Q. Here, two additional residues, T99 and N190, were also identified that may have important roles in catalysis. Present work confirms the advantages of combining directed evolution and saturation mutagenesis approaches of protein engineering and expands the knowledge on the architecture of the industrially important lipase enzyme and its relation to its catalytic activity.