Yujuan Han1, Peixin Gao1, Wengong Yu1, Xinzhi Lu2,3. 1. Key Laboratory of Glycoscience & Glycotechnology of Shandong Province; Key Laboratory of Marine Drugs, Chinese Ministry of Education; Laboratory for Marine Drugs and Bioproducts, Qingdao National Laboratory for Marine Science and Technology; School of Medicine and Pharmacy, Ocean University of China, 5 Yushan Road, Qingdao, 266003, China. 2. Key Laboratory of Glycoscience & Glycotechnology of Shandong Province; Key Laboratory of Marine Drugs, Chinese Ministry of Education; Laboratory for Marine Drugs and Bioproducts, Qingdao National Laboratory for Marine Science and Technology; School of Medicine and Pharmacy, Ocean University of China, 5 Yushan Road, Qingdao, 266003, China. luxinzhi@ouc.edu.cn. 3. Department of Glycobiology, School of Medicine and Pharmacy, Ocean University of China, 5 Yushan Road, Qingdao, 266003, China. luxinzhi@ouc.edu.cn.
Abstract
OBJECTIVE: To determine the effects of carbohydrate-binding modules (CBMs) on the thermostability and catalytic efficiency of chitosanase CsnA. RESULTS: Three CBMs (BgCBM5, PfCBM32-2 and AoCBM35) were engineered at the C-terminus of chitosanase CsnA to create hybrid enzymes CsnA-CBM5, CsnA-CBM32 and CsnA-CBM35. K m values of all the hybrid enzymes were lower than that of the wild type (WT) enzyme; however, only CsnA-CBM5 had an elevated specific activity and catalytic efficiency. The fusion of BgCBM5 enhanced the thermostability of the enzyme, which exhibited a 8.9 °C higher T50 and a 2.9 °C higher Tm than the WT. Secondary structural analysis indicated that appending BgCBM5 at the C-terminus considerably changed the secondary structure content. CONCLUSIONS: The fusion of BgCBM5 improved the thermal stability of CsnA, and the obtained hybrid enzyme (CsnA-CBM5) is a useful candidate for industrial application.
OBJECTIVE: To determine the effects of carbohydrate-binding modules (CBMs) on the thermostability and catalytic efficiency of chitosanase CsnA. RESULTS: Three CBMs (BgCBM5, PfCBM32-2 and AoCBM35) were engineered at the C-terminus of chitosanase CsnA to create hybrid enzymes CsnA-CBM5, CsnA-CBM32 and CsnA-CBM35. K m values of all the hybrid enzymes were lower than that of the wild type (WT) enzyme; however, only CsnA-CBM5 had an elevated specific activity and catalytic efficiency. The fusion of BgCBM5 enhanced the thermostability of the enzyme, which exhibited a 8.9 °C higher T50 and a 2.9 °C higher Tm than the WT. Secondary structural analysis indicated that appending BgCBM5 at the C-terminus considerably changed the secondary structure content. CONCLUSIONS: The fusion of BgCBM5 improved the thermal stability of CsnA, and the obtained hybrid enzyme (CsnA-CBM5) is a useful candidate for industrial application.