Publication:
Cloning, expression and characterization of a thermostable esterase HydS14 from actinomadura sp. strain S14 in pichia pastoris

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Issued Date

2015

Resource Type

Article

File Type

application/pdf

ISSN

16616596

DOI

10.3390/ijms160613579

Other identifier(s)

2-s2.0-84935005175

Rights Holder(s)

Scopus

Bibliographic Citation

International Journal of Molecular Sciences. Vol 16, No.6 (2015), p.13579-13594

Suggested Citation

Sriyapai P., Kawai F., Siripoke S., Chansiri K., Sriyapai T. Cloning, expression and characterization of a thermostable esterase HydS14 from actinomadura sp. strain S14 in pichia pastoris. International Journal of Molecular Sciences. Vol 16, No.6 (2015), p.13579-13594. doi:10.3390/ijms160613579 Retrieved from: https://hdl.handle.net/20.500.14740/6130

Title

Cloning, expression and characterization of a thermostable esterase HydS14 from actinomadura sp. strain S14 in pichia pastoris

Author(s)

Sriyapai P.
 Kawai F.
 Siripoke S.
 Chansiri K.
 Sriyapai T.

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.

Subject(s)

Actinomadura
 Amino acid substitution
 Article
 Controlled study
 Enzyme activity
 Enzyme specificity
 Gene
 Gene cluster
 Gene expression
 Genetic conservation
 Genetic trait
 HydS14 gene
 Komagataella pastoris
 Molecular cloning
 Nonhuman
 Phylogeny
 Polyacrylamide gel electrophoresis
 Protein motif
 Sequence alignment
 Zymography
 Actinobacteria
 Amino acid sequence
 Chemistry
 Enzyme stability
 Enzymology
 Genetics
 Heat
 Metabolism
 Molecular cloning
 Molecular genetics
 Nucleotide sequence
 Pichia
 Protein denaturation
 Actinobacteria (class)
 Actinomadura
 Actinomadura sp.
 Pichia pastoris
 Bacterial protein
 Esterase
 Actinobacteria
 Amino Acid Sequence
 Bacterial Proteins
 Base Sequence
 Cloning, Molecular
 Enzyme Stability
 Esterases
 Hot Temperature
 Molecular Sequence Data
 Pichia
 Protein Denaturation
 Substrate Specificity

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URI

https://hdl.handle.net/20.500.14740/6130

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Scopus: Year 1983-2021

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