Open AccessEnhancement of the catalytic efficiency and thermostability of S tenotrophomonas sp. keratinase KerSMD by domain exchange with KerSMF
Author(s) -
Fang Zhen,
Zhang Juan,
Liu Baihong,
Du Guocheng,
Chen Jian
Publication year - 2016
Publication title -
microbial biotechnology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.287
H-Index - 74
ISSN - 1751-7915
DOI - 10.1111/1751-7915.12300
Subject(s) - thermostability , chemistry , mutant , mesophile , protein precursor , protease , biochemistry , enzyme , peptide , specific activity , keratinase , biology , bacteria , gene , genetics
Summary In this study, we enhanced the catalytic efficiency and thermostability of keratinase KerSMD by replacing its N / C ‐terminal domains with those from a homologous protease, KerSMF , to degrade feather waste. Replacement of the N ‐terminal domain generated a mutant protein with more than twofold increased catalytic activity towards casein. Replacement of the C ‐terminal domain obviously improved keratinolytic activity and increased the k cat / K m value on a synthetic peptide, succinyl‐ A la‐ A la‐ P ro‐ P he‐ p‐ nitroanilide, by 54.5%. Replacement of both the N ‐ and C ‐terminal domains generated a more stable mutant protein, with a T m value of 64.60 ± 0.65°C and a half‐life of 244.6 ± 2 min at 60°C, while deletion of the C ‐terminal domain from KerSMD or KerSMF resulted in mutant proteins exhibiting high activity under mesophilic conditions. These findings indicate that the pre‐peptidase C ‐terminal domain and N ‐propeptide are not only important for substrate specificity, correct folding and thermostability but also support the ability of the enzyme to convert feather waste into feed additives.