title: Cloning, Expression and Characterization of a Thermostable Esterase HydS14 from Actinomadura sp. Strain S14 in Pichia pastoris.
abstract: 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 Km and Vmax 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 (Kcat/Km = 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.
authors.affiliation: Department of Biology, Faculty of Sciences, Srinakharinwirot University, Bangkok 10110, Thailand. peechapack@g.swu.ac.th.
authors.forename: Pichapak
authors.initials: P
authors.lastname: Sriyapai
authors.affiliation: Center for Nanomaterials and Devices, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan. fkawai@kit.ac.jp.
authors.forename: Fusako
authors.initials: F
authors.lastname: Kawai
authors.affiliation: Innovative Learning Center, Srinakharinwirot University, Bangkok 10110, Thailand. somjaisi@swu.ac.th.
authors.forename: Somjai
authors.initials: S
authors.lastname: Siripoke
authors.affiliation: Department of Biochemistry, Faculty of Medicine, Srinakharinwirot University, Bangkok 10110, Thailand. kosum@swu.ac.th.
authors.forename: Kosum
authors.initials: K
authors.lastname: Chansiri
authors.affiliation: Faculty of Environmental Culture and Ecotourism, Srinakharinwirot University, Bangkok 10110, Thailand. thayat@g.swu.ac.th.
authors.forename: Thayat
authors.initials: T
authors.lastname: Sriyapai
mesh_terms: D039903:Actinobacteria / Q000201:enzymology* / Q000235:genetics; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004950:Esterases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006358:Hot Temperature; D008969:Molecular Sequence Data; D010843:Pichia / Q000201:enzymology / Q000235:genetics; D011489:Protein Denaturation; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D004950:Esterases
keywords: Actinomadura sp.; esterase; lipolytic family; thermophile; α/β-hydrolase superfamily