title: Fusing a carbohydrate-binding module into the Aspergillus usamii β-mannanase to improve its thermostability and cellulose-binding capacity by in silico design.
abstract: The AuMan5A, an acidophilic glycoside hydrolase (GH) family 5 β-mannanase derived from Aspergillus usamii YL-01-78, consists of an only catalytic domain (CD). To perfect enzymatic properties of the AuMan5A, a family 1 carbohydrate-binding module (CBM) of the Trichoderma reesei cellobiohydrolase I (TrCBH I), having the lowest binding free energy with cellobiose, was selected by in silico design, and fused into its C-terminus forming a fusion β-mannanase, designated as AuMan5A-CBM. Then, its encoding gene, Auman5A-cbm, was constructed as it was designed theoretically, and expressed in Pichia pastoris GS115. SDS-PAGE analysis displayed that both recombinant AuMan5A-CBM (reAuMan5A-CBM) and AuMan5A (reAuMan5A) were secreted into the cultured media with apparent molecular masses of 57.3 and 49.8 kDa, respectively. The temperature optimum of the reAuMan5A-CBM was 75°C, being 5°C higher than that of the reAuMan5A. They were stable at temperatures of 68 and 60°C, respectively. Compared with reAuMan5A, the reAuMan5A-CBM showed an obvious decrease in K m and a slight alteration in V max. In addition, the fusion of a CBM of the TrCBH I into the AuMan5A contributed to its cellulose-binding capacity.
authors.affiliation: School of Biotechnology and Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi, Jiangsu, China.
authors.forename: Cun-Duo
authors.initials: CD
authors.lastname: Tang
authors.forename: Jian-Fang
authors.initials: JF
authors.lastname: Li
authors.forename: Xi-Huan
authors.initials: XH
authors.lastname: Wei
authors.forename: Rou
authors.initials: R
authors.lastname: Min
authors.forename: Shu-Juan
authors.initials: SJ
authors.lastname: Gao
authors.forename: Jun-Qing
authors.initials: JQ
authors.lastname: Wang
authors.forename: Xin
authors.initials: X
authors.lastname: Yin
authors.forename: Min-Chen
authors.initials: MC
authors.lastname: Wu
mesh_terms: D000595:Amino Acid Sequence; D001230:Aspergillus / Q000737:chemistry*; D001483:Base Sequence; D002241:Carbohydrates / Q000737:chemistry; D002482:Cellulose / Q000737:chemistry* / Q000378:metabolism; D043366:Cellulose 1,4-beta-Cellobiosidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004789:Enzyme Activation; D004795:Enzyme Stability; D019143:Evolution, Molecular; D015870:Gene Expression; D062105:Molecular Docking Simulation; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D010802:Phylogeny; D011485:Protein Binding; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013696:Temperature; D044902:beta-Mannosidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D002241:Carbohydrates; D011993:Recombinant Fusion Proteins; D002482:Cellulose; D044902:beta-Mannosidase; D043366:Cellulose 1,4-beta-Cellobiosidase