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Title: Cloning, expression and characterization of a thermostable esterase HydS14 from actinomadura sp. strain S14 in pichia pastoris
Authors: Sriyapai P.
Kawai F.
Siripoke S.
Chansiri K.
Sriyapai T.
Keywords: Actinomadura
amino acid substitution
controlled study
enzyme activity
enzyme specificity
gene cluster
gene expression
genetic conservation
genetic trait
hydS14 gene
Komagataella pastoris
molecular cloning
polyacrylamide gel electrophoresis
protein motif
sequence alignment
amino acid sequence
enzyme stability
molecular cloning
molecular genetics
nucleotide sequence
protein denaturation
Actinobacteria (class)
Actinomadura sp.
Pichia pastoris
bacterial protein
Amino Acid Sequence
Bacterial Proteins
Base Sequence
Cloning, Molecular
Enzyme Stability
Hot Temperature
Molecular Sequence Data
Protein Denaturation
Substrate Specificity
Issue Date: 2015
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. © 2015 by the authors; licensee MDPI, Basel, Switzerland.
ISSN: 16616596
Appears in Collections:SCOPUS 1983-2021

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