Functional diversity of family 3 β-glucosidases from thermophilic cellulolytic fungus Humicola insolens Y1.
The fungus Humicola insolens is one of the most powerful decomposers of crystalline cellulose. However, studies on the β-glucosidases from this fungus remain insufficient, especially on glycosyl hydrolase family 3 enzymes. In the present study, we analyzed the functional diversity of three distant family 3 β-glucosidases from Humicola insolens strain Y1, which belonged to different evolutionary clades, by heterogeneous expression in Pichia pastoris strain GS115. The recombinant enzymes shared similar enzymatic properties including thermophilic and neutral optima (50-60 °C and pH 5.5-6.0) and high glucose tolerance, but differed in substrate specificities and kinetics. HiBgl3B was solely active towards aryl β-glucosides while HiBgl3A and HiBgl3C showed broad substrate specificities including both disaccharides and aryl β-glucosides. Of the three enzymes, HiBgl3C exhibited the highest specific activity (158.8 U/mg on pNPG and 56.4 U/mg on cellobiose) and catalytic efficiency and had the capacity to promote cellulose degradation. Substitutions of three key residues Ile48, Ile278 and Thr484 of HiBgl3B to the corresponding residues of HiBgl3A conferred the enzyme activity towards sophorose, and vice versa. This study reveals the functional diversity of GH3 β-glucosidases as well as the key residues in recognizing +1 subsite of different substrates.
College of Animal Science, Zhejiang University, Hangzhou 310058, People's Republic of China.|Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, People's Republic of China.
Wei
W
Xia
Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, People's Republic of China.
Yingguo
Y
Bai
Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, People's Republic of China.
Ying
Y
Cui
Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, People's Republic of China.
Xinxin
X
Xu
College of Animal Science, Zhejiang University, Hangzhou 310058, People's Republic of China.
Lichun
L
Qian
Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, People's Republic of China.
Pengjun
P
Shi
Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, People's Republic of China.
Wei
W
Zhang
Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, People's Republic of China.
Huiying
H
Luo
College of Animal Science, Zhejiang University, Hangzhou 310058, People's Republic of China.
Xiuan
X
Zhan
Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, People's Republic of China.
Bin
B
Yao
D000595:Amino Acid Sequence; D001203:Ascomycota / Q000201:enzymology*; D002482:Cellulose / Q000737:chemistry; D003001:Cloning, Molecular; D017124:Conserved Sequence; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D005960:Glucosides / Q000737:chemistry; D007700:Kinetics; D020125:Mutation, Missense; D010802:Phylogeny; D010843:Pichia; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity; D001617:beta-Glucosidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D005656:Fungal Proteins; D005960:Glucosides; C025193:4-nitrophenyl beta-D-glucoside; D002482:Cellulose; D001617:beta-Glucosidase