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

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

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 70–90oC. 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).

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DOI:
Digital Object Identifier (open in new tab) 10.18388/abp.2020_5730
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Category:
Articles
Type:
artykuły w czasopismach
Published in:
Acta Biochimica Polonica no. 68, pages 411 - 417,
ISSN: 0001-527X
Language:
English
Publication year:
2021
Bibliographic description:
Wicka-Grochocka M., Cieśliński H., Wanarska M.: Cloning, expression in Komagataella phaffii, and biochemical characterization of recombinant sequence variants of Pseudomonas sp. S9 GDSL-esterase// Acta Biochimica Polonica -Vol. 68,iss. 3 (2021), s.411-417
DOI:
Digital Object Identifier (open in new tab) 10.18388/abp.2020_5730
Verified by:
Gdańsk University of Technology

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