Cloning, expression in Komagataella phaffii, and biochemical characterization of recombinant sequence variants of Pseudomonas sp. S9 GDSL-esterase

Monika Wicka-Grochocka; Hubert Cieśliński; Marta Wanarska

doi:10.18388/abp.2020_5730

Cloning, expression in Komagataella phaffii, and biochemical characterization of recombinant sequence variants of Pseudomonas sp. S9 GDSL-esterase

Monika Wicka-Grochocka Department of Molecular Biotechnology and Microbiology, Faculty of Chemistry, Gdańsk University of Technology, Gdańsk, Poland
Hubert Cieśliński Department of Molecular Biotechnology and Microbiology, Faculty of Chemistry, Gdańsk University of Technology, Gdańsk, Poland
Marta Wanarska Department of Molecular Biotechnology and Microbiology, Faculty of Chemistry, Gdańsk University of Technology, Gdańsk, Poland

DOI: https://doi.org/10.18388/abp.2020_5730

Abstract

Two recombinant Komagataella phaffii (formerly Pichia pastoris) yeast strains for production of two sequential variants of EstS9 esterase from psychrotolerant bacterium Pseudomonas sp. S9, i.e. αEstS9N (a two-domain enzyme consisting of a catalytic domain and an autotransporter domain) and αEstS9Δ (a single-domain esterase) were constructed. However, only one of recombinant K. phaffii strains, namely Komagataella phaffii X-33/pPICZαestS9Δ, allowed to successfully produce and secrete recombinant αEstS9Δ enzyme outside of the host cell. The purified αEstS9Δ esterase was active towards short-chain p-nitrophenyl esters (C2–C8), with optimal activity for the acetate (C2) ester. The single-domain αEstS9Δ esterase exhibits the highest activity at 60oC and pH 9.5. In addition, the enzyme retains 90% of its activity after 3 hour incubation at 70–90^oC. What should be also noted is that αEstS9Δ esterase produced in the K. phaffii expression system has a much higher specific activity (0.069 U/mg of protein) than the recombinant EstS9Δ esterase produced in an E. coli expression system (0.0025 U/mg of protein) (Wicka et al., 2016, Acta Biochim Pol 63: 117–125. https://doi.org/10.18388/abp.2015_1074).

Author Biographies

Monika Wicka-Grochocka, Department of Molecular Biotechnology and Microbiology, Faculty of Chemistry, Gdańsk University of Technology, Gdańsk, Poland

Department of Molecular Biotechnology and Microbiology

Hubert Cieśliński, Department of Molecular Biotechnology and Microbiology, Faculty of Chemistry, Gdańsk University of Technology, Gdańsk, Poland

Department of Molecular Biotechnology and Microbiology

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Published

2021-08-25

Issue

Vol. 68 No. 3 (2021): Issue dedicated to Professor Wacław Tadeusz Szybalski on the 100th anniversary of his birth

Section

Articles

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