| Esterase is a enzyme catalyzing the hydrolysis of fatty acid esters with short acyl chain length. Esterase can catalyze esterification, transesterification and interesterification etc., and it shows high enantio-, regio- and stereo-selectivity. Compared with thermophilic and mesophilic esterase, cold active esterase exhibits much higher catalytic efficiency at low temperatures and lower thermostability. With regard to energy saving, it is also used for the synthesis of labile compounds at low temperatures, and it becomes a research focus of biocatalysis in recent years. Esterase is one of the most versatile class of biocatalysts, it have been broadly utilized in food industries, pharmaceutical industries, detergents industries and biodiesel industries etc..In this study, a esterase gene est S was cloned from Serratia sp. and expressed in Escherichia coli DE3(BL21), and then the protein were purified and characterzed. Finally, in order to improve the thermostability of Est S, a random mutant library was constructed. The result of this study were as follows:The esterase gene est S consists of 909 bp and encodes a polypeptide of 302 amino acid residues with a theoretical molecular weight of 32.5 KDa and an isoelectric point of 5.05. No signal peptide was predicted by Signl P 4.1 Server. Est S displayed maximum activity towards p-nitrophenyl acetate(C2) and nearly no activity towards p-nitrophenyl palmitate(C16). Est S showed the highest activity at 10°C and retained ~92% of its original activity at 0°C, indicating its cold activity. Est S was unstable above 50°C and showed no activity after incubation at 55°C for 20 min. Thermal inactivation analysis showed that the T1/2 value of Est S was 50 min at 50°C with residual activity 41.23% after 1 h incubation. Est S exhibited the optimal activity at p H 8.5, and it remained stable between p H 5.5 and 9.5. Meanwhile, Est S was salt-tolerant, as it retained more than 95% of its initial activity in the present of 5 M Na Cl and 80% activity after incubation in 4 M Na Cl at 4°C for 24 h. 1 mmol/L Mg2+, Ba2+and Mn2+ could slightly activate the activity of Est S and the same concentration of Zn2+ and Cu2+ could slightly inhibit its activity. Est S was slightly inhibited by low concentration of serine protein specific inhibitor PMSF, but it showed a drastic decrease in the activity in the presence of 10% acetonitrile, N-butyl alcohol and SDS. The Km, Vmax, Kcat and Kcat/Km value of Est S for p-nitrophenyl acetate were 74.02 μM, 7.351 μM·min-1, 2.339×103 s-1 and 31.60 s-1·μM-1, respectively.A random mutant library was constructed by error-prone PCR, and the library was finally screened by lysing the induced mutant with T7 phage and assaying the activity of the lysate after incubation at 50°C for 40 min. A variant 1-D5 was gained from about 8000 mutant, and multiple sequence alignment showed that 3 amino acid of Est S altered(A43V, R116 W, D147N). 1-D5 showed maximal activity at 10°C and p H 8.5, but 1-D5 retained 88.24% of its original activity at 30°C, about 10% higher than Est S. 1-D5 showed enhanced T1/2 of 70 min at 50°C and retained 63.29% of activity after incubation at 50°C for 60 min, which were about 22% higher than the wild type(WT), indicating the thermostability of 1-D5 improved. The Km, Kcat and Kcat/Km value of 1-D5 for pnitrophenyl acetate were 87.59 μM,2.501×103 s-1 and 28.55 s-1·μM-1, respectively. |
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