title: Enhancement of the catalytic efficiency and thermostability of Stenotrophomonas sp. keratinase KerSMD by domain exchange with KerSMF.
abstract: 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-Ala-Ala-Pro-Phe-p-nitroanilide, by 54.5%. Replacement of both the N- and C-terminal domains generated a more stable mutant protein, with a Tm 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.
authors.affiliation: Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi, 214122, China.|Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi, 214122, China.|School of Biotechnology, Jiangnan University, Wuxi, 214122, China.
authors.forename: Zhen
authors.initials: Z
authors.lastname: Fang
authors.affiliation: Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi, 214122, China.|School of Biotechnology, Jiangnan University, Wuxi, 214122, China.
authors.forename: Juan
authors.initials: J
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi, 214122, China.|Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi, 214122, China.|School of Biotechnology, Jiangnan University, Wuxi, 214122, China.
authors.forename: Baihong
authors.initials: B
authors.lastname: Liu
authors.affiliation: School of Biotechnology, Jiangnan University, Wuxi, 214122, China.|Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi, 214122, China.
authors.forename: Guocheng
authors.initials: G
authors.lastname: Du
authors.affiliation: School of Biotechnology, Jiangnan University, Wuxi, 214122, China.|National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, Wuxi, 214122, China.
authors.forename: Jian
authors.initials: J
authors.lastname: Chen
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D055162:Biocatalysis; D002364:Caseins / Q000378:metabolism; D004795:Enzyme Stability; D007700:Kinetics; D010447:Peptide Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000072417:Protein Domains; D020588:Stenotrophomonas / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D002364:Caseins; D010447:Peptide Hydrolases; C035855:keratinase