Effect of K225 residue to the catalytic efficiency of Kex2 protease.

Abstract:

:The gene encoding S. cerevisiae Kex2 protease derivative Kex2-667 (encoding the N-terminal 20th to 667th amino acid residues of Kex2 protease, containing the propeptide, catalytic domain, P domain and Ser/Thr enrichment region) and its 225th amino acid residue mutant K225L were overexpressed in Pichia pastoris. Proteases were purified by dialysis and anion exchange chromatography (Q-FF). Their properties were further investigated. For catalysis efficiency, the value of Kcat/Km of Kex2-667-K225L was 3 folds higher than that of Kex2-667. Both were quite stable at 25 °C and 37 °C after 8 h of incubation at pH5.6, while Kex2-667 remained nearly 90% of the total activity while Kex2-667-K225L remained only 80%. The stability of Kex2-667-K225L was lower than that of Kex2-667 from pH4.0 to pH9.0. Due to the mutation site K225 was located at one of the calcium ion binding sites, it resulted in a tighter calcium ion binding region, which may be the reason why the catalytic efficiency of Kex2-667-K225L was improved while the stability was a little decreased.

journal_name

Protein Expr Purif

authors

Yang F,Liu L,Liu Y,Li S

doi

10.1016/j.pep.2020.105725

subject

Has Abstract

pub_date

2020-12-01 00:00:00

pages

105725

eissn

1046-5928

issn

1096-0279

pii

S1046-5928(20)30316-8

journal_volume

176

pub_type

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