title: Replacing a piece of loop-structure in the substrate-binding groove of Aspergillus usamii β-mannanase, AuMan5A, to improve its enzymatic properties by rational design.
abstract: To perfect the enzymatic properties of AuMan5A, a mesophilic glycoside hydrolase (GH) family 5 β-mannanase from Aspergillus usamii, its loop-structure substitution was carried out by rational design and followed by megaprimer PCR. Based on the structural analysis and enzymatic property comparison of various β-mannanases, a piece of loop-structure with seven amino acids between two β-strands (βD and βE) in the substrate-binding groove, named "Loop DE," was speculated to be correlative to the thermostability and catalytic efficiency of GH family 5 β-mannanases. Therefore, three AuMan5A's mutants, AuMan5A-Af, AuMan5A-An, and AuMan5A-Th, were designed by substituting a Loop DE sequence ((316)KSPDGGN(322)) of AuMan5A with the corresponding sequences of other three family 5 β-mannanases, respectively. Then, the mutant-encoding genes, Auman5A-Af, Auman5A-An, and Auman5A-Th, were constructed as designed theoretically and then expressed in Pichia pastoris GS115. The expressed recombinant AuMan5A-Af (re-AuMan5A-Af) displayed the temperature optimum (T opt) of 75 °C, T m value of 76.6 °C and half-life (t 1/2) of 480 min at 70 °C, which were 10 and 12.1 °C higher and 48-fold longer than those of re-AuMan5A, respectively. Its catalytic efficiency (k cat/K m) was 12.7-fold that of re-AuMan5A. What is more, the site-directed mutagenesis of D320G in AuMan5A-Af was performed. The T opt and t 1/2 of expressed re-AuMan5A-Af(D320G) decreased to 70 °C and 40 min, respectively, while its k cat/K m was only 35 % of that of re-AuMan5A-Af. These results demonstrated that the mutation of G320 (in AuMan5A) into D320 (in AuMan5A-Af) through Loop DE substitution was mainly responsible for the thermostability and catalytic efficiency improvement of AuMan5A-Af.
authors.affiliation: State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, 1800 Lihu Road, Wuxi, 214122, China.
authors.forename: Yun Hai
authors.initials: YH
authors.lastname: Dong
authors.affiliation: State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, 1800 Lihu Road, Wuxi, 214122, China.
authors.forename: Jian Fang
authors.initials: JF
authors.lastname: Li
authors.affiliation: Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, 214122, China.
authors.forename: Die
authors.initials: D
authors.lastname: Hu
authors.affiliation: Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, 214122, China.
authors.forename: Xin
authors.initials: X
authors.lastname: Yin
authors.affiliation: State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, 1800 Lihu Road, Wuxi, 214122, China.
authors.forename: Chun Juan
authors.initials: CJ
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, 1800 Lihu Road, Wuxi, 214122, China.
authors.forename: Shi Han
authors.initials: SH
authors.lastname: Tang
authors.affiliation: Wuxi Medical School, Jiangnan University, 1800 Lihu Road, Wuxi, 214122, China. biowmc@126.com.
authors.forename: Min Chen
authors.initials: MC
authors.lastname: Wu
mesh_terms: D001230:Aspergillus / Q000201:enzymology*; D001665:Binding Sites; D004252:DNA Mutational Analysis; D004795:Enzyme Stability; D015870:Gene Expression; D007700:Kinetics; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013696:Temperature; D044902:beta-Mannosidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
chemical_list: D050505:Mutant Proteins; D011994:Recombinant Proteins; D044902:beta-Mannosidase
keywords: Catalytic efficiency; Loop-structure substitution; Rational design; Thermostability; β-Mannanase