Improved catalytic efficiency, thermophilicity, anti-salt and detergent tolerance of keratinase KerSMD by partially truncation of PPC domain.
The keratinase from Stenotrophomonas maltophilia (KerSMD) is known for its high activity and pH stability in keratin degradation. However, catalytic efficiency and detergent tolerability need to be improved in order to be used for industrial application. In this work, we obtained several keratinase variants with enhanced catalytic efficiency, thermophilicity, and anti-salt and detergent tolerability by partially truncating the PPC domain of KerSMD. The variants all showed improved catalytic efficiency to synthetic substrate AAPF, with the V355 variant having the highest kcat /Km value of 143.6 s(-1) mM(-1). The truncation of keratinase had little effect on alkaline stability but obviously decreased collagenase activity, developing its potential application in leather treatment. The variants V380, V370, and V355 were thermophilic, with a 1.7-fold enhancement of keratinlytic activity at 60 °C when compared to the wild type. The entire truncation of PPC domain obtained the variant V355 with improved tolerance to alkalinity, salt, chaotropic agents, and detergents. The V355 variant showed more than a 40% improvement in activity under 15% (w/v) NaCl or 4% (w/v) SDS solution, showing excellent stability under harsh washing and unhairing conditions. Our work investigated how protein engineering affects the function of PPC domain of KerSMD.
Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China.|Synergetic Innovation Center of Food Safety and Nutrition, Wuxi 214122, China.|School of Biotechnology, Jiangnan University, Wuxi 214122, China.
Zhen
Z
Fang
Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China.|School of Biotechnology, Jiangnan University, Wuxi 214122, China.
Juan
J
Zhang
School of Biotechnology, Jiangnan University, Wuxi 214122, China.|Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China.
Guocheng
G
Du
School of Biotechnology, Jiangnan University, Wuxi 214122, China.|National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, Wuxi 214122, China.
Jian
J
Chen
D000595:Amino Acid Sequence / Q000235:genetics; D002384:Catalysis; D003902:Detergents / Q000737:chemistry; D006358:Hot Temperature; D007633:Keratins / Q000378:metabolism; D010447:Peptide Hydrolases / Q000235:genetics* / Q000378:metabolism; D000072417:Protein Domains / Q000235:genetics; D015202:Protein Engineering / Q000379:methods*; D017433:Protein Structure, Secondary; D012965:Sodium Chloride / Q000737:chemistry; D020615:Stenotrophomonas maltophilia / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism
D003902:Detergents; D012965:Sodium Chloride; D007633:Keratins; D010447:Peptide Hydrolases; C035855:keratinase