Loops Adjacent to Catalytic Region and Molecular Stability of Man1312
Haiyan Zhou, Jie Yong, Han Gao, Zhihui Yuan, Wenjiao Yang, Yun Tian, Yongyao Wu
Applied Biochemistry and Biotechnology
Hemicelluloses are the second major polysaccharides in nature and can be converted to ethanol product by a variety of enzymes including mannanases. Mannanase is an important enzyme that hydrolyses mannose-containing polysaccharides which are abundant in plants. An optimized mannanase could help to improve conversion process and make the technology efficiently and competitively. In this work, the effects of loops adjacent to active region on enzymic properties of Man1312 were investigated. Loop 6 and 10 are two loops neighboring to Man1312 catalytic region, and deletion mutagenesis and residue substitution were performed on both loops. Deletion on sites S145, Q148, N244, and S255 and substitution on sites N146, S147, S156, and T157 gave significant increased stability to enzyme. The quadruplet mutant ManD4I4 combined all the mutations and had higher optimal temperature and T m value by 5 and 4 °C than Man1312, respectively. From our data, we are able to conclude the loops of enzymes are important to design mutagenesis and obtain improved properties, especially the loops neighboring to catalytic region from tertiary structure. In our experiment, residue deletion and substitution on loops neighboring to catalytic region made significant improvement on enzyme properties.
Circular dichroism, Secondary structure, Thermal stability, Biochemistry