A novel salt-tolerant endo-beta-1,4-glucanase Cel5A in Vibrio sp. G21 isolated from mangrove soil.
Although cellulases have been isolated from various microorganisms, no functional cellulase gene has been reported in the Vibrio genus until now. In this report, a novel endo-beta-1,4-glucanase gene, cel5A, 1,362 bp in length, was cloned from a newly isolated bacterium, Vibrio sp. G21. The deduced protein of cel5A contains a catalytic domain of glycosyl hydrolase family 5 (GH5), followed by a cellulose binding domain (CBM2). The GH5 domain shows the highest sequence similarity (69%) to the bifunctional beta 1,4-endoglucanase/cellobiohydrolase from Teredinibacter turnerae T7902. The mature Cel5A enzyme was overexpressed in Escherichia coli and purified to homogeneity. The optimal pH and temperature of the recombinant enzyme were determined to be 6.5-7.5 and 50 degrees C, respectively. Cel5A was stable over a wide range of pH and retained more than 90% of total activity even after treatment in pH5.5-10.5 for 1 h, indicating high alkali resistance. Moreover, the enzyme was activated after pretreatment with mild alkali, a novel characteristic that has not been previously reported in other cellulases. Cel5A also showed a high level of salt tolerance. Its activity rose to 1.6-fold in 0.5 M NaCl and remained elevated even in 4 M NaCl. Further experimentation demonstrated that the thermostability of Cel5A was improved in 0.4 M NaCl. In addition, Cel5A showed specific activity towards beta-1,4-linkage of amorphous region of lignocellulose, and the main final hydrolysis product of carboxymethylcellulose sodium and cellooligosaccharides was cellobiose. As an alkali-activated and salt-tolerant enzyme, Cel5A is an ideal candidate for further research and industrial applications.
Gao
Zhaoming
Z
The State Key Lab of Microbial Technology, Marine Biotechnology Research Center, Shandong University, Jinan 250100, People's Republic China.
Ruan
Lingwei
L
Chen
Xiulan
X
Zhang
Yuzhong
Y
Xu
Xun
X
D000595:Amino Acid Sequence; D032407:Avicennia / Q000382:microbiology*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002480:Cellulase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D004795:Enzyme Stability; D008969:Molecular Sequence Data; D018517:Plant Roots / Q000382:microbiology; D017386:Sequence Homology, Amino Acid; D012965:Sodium Chloride / Q000494:pharmacology*; D012988:Soil Microbiology; D013379:Substrate Specificity; D014733:Vibrio / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification
D001426:Bacterial Proteins; D012965:Sodium Chloride; C407024:endoglucanase Cel5A; D002480:Cellulase