Cloning, Expression, and Characterization of a Thermophilic Endoglucanase, AcCel12B from Acidothermus cellulolyticus 11B.
The gene ABK52392 from the thermophilic bacterium Acidothermus cellulolyticus 11B was predicted to be endoglucanase and classified into glycoside hydrolase family 12. ABK52392 encodes a protein containing a catalytic domain and a carbohydrate binding module. ABK52392 was cloned and functionally expressed in Escherichia coli. After purification by Ni-NTA agarose affinity chromatography and Q-Sepharose® Fast Flow chromatography, the properties of the recombinant protein (AcCel12B) were characterized. AcCel12B exhibited optimal activity at pH 4.5 and 75 °C. The half-lives of AcCel12B at 60 and 70 °C were about 90 and 2 h, respectively, under acidic conditions. The specific hydrolytic activities of AcCel12B at 70 °C and pH 4.5 for sodium carboxymethylcellulose (CMC) and regenerated amorphous cellulose (RAC) were 118.3 and 104.0 U·mg(-1), respectively. The Km and Vmax of AcCel12B for CMC were 25.47 mg·mL(-1) and 131.75 U·mg(-1), respectively. The time course of hydrolysis for RAC was investigated by measuring reducing ends in the soluble and insoluble phases. The total hydrolysis rate rapidly decreased after the early stage of incubation and the generation of insoluble reducing ends decreased earlier than that of soluble reducing ends. High thermostability of the cellulase indicates its potential commercial significance and it could be exploited for industrial application in the future.
Key Laboratory for Molecular Enzymology & Engineering of the Ministry of Education, School of Life Science, Jilin University, Changchun 130012, China. wangjl09@mails.jlu.edu.cn.|Department of Biotechnology, Jilin Agricultural Science and Technology College, Jilin 132101, China. wangjl09@mails.jlu.edu.cn.
Junling
J
Wang
Key Laboratory for Molecular Enzymology & Engineering of the Ministry of Education, School of Life Science, Jilin University, Changchun 130012, China. gaogui@jlu.edu.cn.
Gui
G
Gao
Key Laboratory for Molecular Enzymology & Engineering of the Ministry of Education, School of Life Science, Jilin University, Changchun 130012, China. liyw11@mails.jlu.edu.cn.|State Key Laboratory of Supramolecular Structure and Materials, College of Chemistry, Jilin University, Changchun 130012, China. liyw11@mails.jlu.edu.cn.
Yuwei
Y
Li
Key Laboratory for Molecular Enzymology & Engineering of the Ministry of Education, School of Life Science, Jilin University, Changchun 130012, China. yanglz13@mails.jlu.edu.cn.
Liangzhen
L
Yang
Key Laboratory for Molecular Enzymology & Engineering of the Ministry of Education, School of Life Science, Jilin University, Changchun 130012, China. liangyl13@mails.jlu.edu.cn.
Yanli
Y
Liang
Key Laboratory for Molecular Enzymology & Engineering of the Ministry of Education, School of Life Science, Jilin University, Changchun 130012, China. hyjin13@mails.jlu.edu.cn.
Hanyong
H
Jin
Key Laboratory for Molecular Enzymology & Engineering of the Ministry of Education, School of Life Science, Jilin University, Changchun 130012, China. weiweihan@jlu.edu.cn.
Weiwei
W
Han
Key Laboratory for Molecular Enzymology & Engineering of the Ministry of Education, School of Life Science, Jilin University, Changchun 130012, China. yfeng2009@mail.sjtu.edu.cn.
Yan
Y
Feng
Key Laboratory for Molecular Enzymology & Engineering of the Ministry of Education, School of Life Science, Jilin University, Changchun 130012, China. zmzhang@jlu.edu.cn.
Zuoming
Z
Zhang
D039903:Actinobacteria / Q000201:enzymology* / Q000235:genetics* / Q000302:isolation & purification; D001483:Base Sequence; D002266:Carboxymethylcellulose Sodium / Q000378:metabolism*; D020134:Catalytic Domain; D002480:Cellulase / Q000737:chemistry / Q000235:genetics*; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006358:Hot Temperature; D006868:Hydrolysis; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013696:Temperature
D004269:DNA, Bacterial; D002480:Cellulase; D002266:Carboxymethylcellulose Sodium
AcCel12B; Acidothermus cellulolyticus 11B; endoglucanase; reaction slowdown; thermostability