title: Loops Adjacent to Catalytic Region and Molecular Stability of Man1312.
abstract: 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.
authors.affiliation: College of Bioscience and Biotechnology, Hunan Agricultural University, Changsha, 410128, China.
authors.forename: Haiyan
authors.initials: H
authors.lastname: Zhou
authors.affiliation: College of Bioscience and Biotechnology, Hunan Agricultural University, Changsha, 410128, China.
authors.forename: Jie
authors.initials: J
authors.lastname: Yong
authors.affiliation: College of Bioscience and Biotechnology, Hunan Agricultural University, Changsha, 410128, China.
authors.forename: Han
authors.initials: H
authors.lastname: Gao
authors.affiliation: College of Bioscience and Biotechnology, Hunan Agricultural University, Changsha, 410128, China.
authors.forename: Zhihui
authors.initials: Z
authors.lastname: Yuan
authors.affiliation: College of Bioscience and Biotechnology, Hunan Agricultural University, Changsha, 410128, China.
authors.forename: Wenjiao
authors.initials: W
authors.lastname: Yang
authors.affiliation: Key Lab of Agricultural Biochemistry and Biotransformation, Hunan Agricultural University, Changsha, 410128, China.
authors.forename: Yun
authors.initials: Y
authors.lastname: Tian
authors.affiliation: College of Bioscience and Biotechnology, Hunan Agricultural University, Changsha, 410128, China. sws001@163.com.
authors.forename: Yongyao
authors.initials: Y
authors.lastname: Wu
mesh_terms: D019943:Amino Acid Substitution; D001412:Bacillus subtilis / Q000201:enzymology*; D002152:Calorimetry, Differential Scanning; D020134:Catalytic Domain; D002942:Circular Dichroism; D004795:Enzyme Stability; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D016296:Mutagenesis; D050505:Mutant Proteins / Q000737:chemistry; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D017384:Sequence Deletion; D013329:Structure-Activity Relationship; D013696:Temperature; D044902:beta-Mannosidase / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D050505:Mutant Proteins; D044902:beta-Mannosidase
keywords: Loop; Mannanase; Molecular stability; Rational design