Cloning, expression and characterization of a novel thermophilic polygalacturonase from Caldicellulosiruptor bescii DSM 6725.
We cloned the gene ACM61449 from anaerobic, thermophilic Caldicellulosiruptor bescii, and expressed it in Escherichia coli origami (DE3). After purification through thermal treatment and Ni-NTA agarose column extraction, we characterized the properties of the recombinant protein (CbPelA). The optimal temperature and pH of the protein were 72 °C and 5.2, respectively. CbPelA demonstrated high thermal-stability, with a half-life of 14 h at 70 °C. CbPelA also showed very high activity for polygalacturonic acid (PGA), and released monogalacturonic acid as its sole product. The Vmax and Km of CbPelA were 384.6 U·mg⁻¹ and 0.31 mg·mL⁻¹, respectively. CbPelA was also able to hydrolyze methylated pectin (48% and 10% relative activity on 20%-34% and 85% methylated pectin, respectively). The high thermo-activity and methylated pectin hydrolization activity of CbPelA suggest that it has potential applications in the food and textile industry.
Key Laboratory for Molecular Enzymology & Engineering of the Ministry of Education, School of Life Science, Jilin University, Changchun 130012, China. cyy09@mails.jlu.edu.cn.
Yanyan
Y
Chen
School of Pharmaceutical Sciences, Jilin University, Changchun 130021, China. sundj@jlu.edu.cn.
Dejun
D
Sun
School of Pharmaceutical Sciences, Jilin University, Changchun 130021, China. zhouyl@jlu.edu.cn.
Yulai
Y
Zhou
Key Laboratory for Molecular Enzymology & Engineering of the Ministry of Education, School of Life Science, Jilin University, Changchun 130012, China. llp126@126.com.
Liping
L
Liu
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. zhengbaisong@126.com.
Baisong
B
Zheng
Key Laboratory for Molecular Enzymology & Engineering of the Ministry of Education, School of Life Science, Jilin University, Changchun 130012, China. wangzhi@jlu.edu.cn.
Zhi
Z
Wang
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
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D016937:Gram-Positive Endospore-Forming Rods / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism; D006358:Hot Temperature; D010368:Pectins / Q000378:metabolism; D011096:Polygalacturonase / Q000235:genetics*; D016415:Sequence Alignment
D001426:Bacterial Proteins; D010368:Pectins; D011096:Polygalacturonase