title: Improved catalytic efficiency of endo-beta-1,4-glucanase from Bacillus subtilis BME-15 by directed evolution.
abstract: Bacillus subtilis endo-beta-1,4-glucanase (Cel5A) hydrolyzes cellulose by cleavage of the internal bonds in the glucose chains, producing new ends randomly. Using directed evolution techniques of error-prone polymerase chain reaction (PCR) and DNA shuffling, several Cel5A variants with improved catalytic activity had been screened from the mutant library, which contained 71,000 colonies. Compared with the wild-type enzyme, the variants (M44-11, S75 and S78) showed 2.03 to 2.68-fold increased activities toward sodium carboxymethyl cellulose (CMC), while the M44-11 also exhibited a wider pH tolerance and higher thermostability. Structural models of M44-11, S75, S78, and WT proteins revealed that most of the substitutions were not located in the strictly conserved regions, except the mutation V255A of S75, which was closed to the nucleophile Glu257 in the catalytic center of the enzyme. Moreover, V74A and D272G of M44-11, which were not located in the substrate binding sites and the catalytic center, might result in improved stability and catalytic activity. These results provided useful references for directed evolution of the enzymes that belonged to the glycoside hydrolase family 5 (GH5).
authors.affiliation: State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, People's Republic of China.
authors.forename: Ling
authors.initials: L
authors.lastname: Lin
authors.forename: Xin
authors.initials: X
authors.lastname: Meng
authors.forename: Pengfu
authors.initials: P
authors.lastname: Liu
authors.forename: Yuzhi
authors.initials: Y
authors.lastname: Hong
authors.forename: Gaobing
authors.initials: G
authors.lastname: Wu
authors.forename: Xiaoluo
authors.initials: X
authors.lastname: Huang
authors.forename: Congcong
authors.initials: C
authors.lastname: Li
authors.forename: Junli
authors.initials: J
authors.lastname: Dong
authors.forename: Liang
authors.initials: L
authors.lastname: Xiao
authors.forename: Ziduo
authors.initials: Z
authors.lastname: Liu
mesh_terms: D001412:Bacillus subtilis / Q000737:chemistry / Q000201:enzymology / Q000235:genetics*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D003001:Cloning, Molecular; D019020:Directed Molecular Evolution / Q000379:methods*; D004795:Enzyme Stability; D044643:Glucan 1,4-beta-Glucosidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D044643:Glucan 1,4-beta-Glucosidase