Cloning, Expression and Characterization of a Thermostable Esterase HydS14 from Actinomadura sp. Strain S14 in Pichia pastoris

International Journal of Molecular Sciences

Pichapak Sriyapai³  Fusako Kawai¹  Somjai Siripoke²  Kosum Chansiri⁴  Thayat Sriyapai⁵ 

[1] Center for Nanomaterials and Devices, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan; E-Mail:;Innovative Learning Center, Srinakharinwirot University, Bangkok 10110, Thailand; E-Mail:;Department of Biology, Faculty of Sciences, Srinakharinwirot University, Bangkok 10110, Thailand; E-Mail:;Department of Biochemistry, Faculty of Medicine, Srinakharinwirot University, Bangkok 10110, Thailand; E-Mail:;Faculty of Environmental Culture and Ecotourism, Srinakharinwirot University, Bangkok 10110, Thailand

关键词: Actinomadura sp.;    esterase;    α/β-hydrolase superfamily;    lipolytic family;    thermophile;   

DOI  :  10.3390/ijms160613579

来源: mdpi

【 摘 要 】

A thermostable esterase gene (hydS14) was cloned from an Actinomadura sp. S14 gene library. The gene is 777 bp in length and encodes a polypeptide of 258 amino acid residues with no signal peptide, no N-glycosylation site and a predicted molecular mass of 26,604 Da. The encoded protein contains the pentapeptide motif (GYSLG) and catalytic triad (Ser88-Asp208-His235) of the esterase/lipase superfamily. The HydS14 sequence shows 46%–64% identity to 23 sequences from actinomycetes (23 α/β-hydrolases), has three conserved regions, and contains the novel motif (GY(F)SLG), which distinguishes it from other clusters in the α/β-hydrolase structural superfamily. A plasmid containing the coding region (pPICZαA-hydS14) was used to express HydS14 in Pichia pastoris under the control of the AOXI promoter. The recombinant HydS14 collected from the supernatant had a molecular mass of ~30 kDa, which agrees with its predicted molecular mass without N-glycosylation. HydS14 had an optimum temperature of approximately 70 °C and an optimum pH of 8.0. HydS14 was stable at 50 and 60 °C for 120 min, with residual activities of above 80% and above 90%, respectively, as well as 50% activity at pH 6.0–8.0 and pH 9.0, respectively. The enzyme showed higher activity with p-nitrophenyl-C2 and C4. The K_m and V_max values for p-nitrophenyl-C4 were 0.21 ± 0.02 mM and 37.07 ± 1.04 μmol/min/mg, respectively. The enzyme was active toward short-chain p-nitrophenyl ester (C2–C6), displaying optimal activity with p-nitrophenyl-C4 (K_cat/K_m = 11.74 mM⁻¹·S⁻¹). In summary, HydS14 is a thermostable esterase from Actinomadura sp. S14 that has been cloned and expressed for the first time in Pichia pastoris.

【 授权许可】

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