A novel thermoacidophilic endoglucanase, Ba-EGA, from a new cellulose-degrading bacterium, Bacillus sp.AC-1.
A newly discovered bacterium, strain AC1, containing cellulase was isolated from the gastric juice of the mollusca, Ampullaria crosseans. Analysis of the 16S rDNA sequence and carbon sources revealed that the bacterium belonged to the genus Bacillus. A novel endoglucanase (Ba-EGA) was purified from culture supernatants of the bacterium growing in CMC-Na (low viscosity) induction medium. The cellulase was purified about 150-fold by ammonium sulfate fractionation, ion exchange, hydrophobic, and gel filtration chromatography, with a specific activity of 35.0 IU/mg. The molecular mass of the enzyme was 67 kDa. N-terminal amino acid sequencing revealed a sequence of SDYNYVEVLQKSILF, which had high homology with endoglucanases from the Bacillus and Clostridium species. The maximal activity of the enzyme with the substrate of CM-cellulose is at pH 4.5-6.5 and 70 degrees C, respectively. The studies on pH and temperature stability showed that the Ba-EGA is stable enough between pH 7.5 and 10.5 at 30 degrees C for 2 h, and more than 80% of the activity still remains when incubation was prolonged to 1 h at 50 degrees C. The activity of the enzyme was significantly inhibited by Fe(2+), Cu(2+) (5.0 mM of each), and sodium dodecyl sulfate (SDS) (0.5%) and obviously activated by Tween 20 and Triton X-100 (0.25% each). Binding studies revealed that the Ba-EGA had cellulose-binding domain.
Li
Yan-Hong
YH
Key Laboratory of Proteomics, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Graduate School of the Chinese Academy of Sciences, China.
Ding
Ming
M
Wang
Ji
J
Xu
Gen-Jun
GJ
Zhao
Fukun
F
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology*; D002480:Cellulase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D002482:Cellulose / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D013379:Substrate Specificity; D013696:Temperature
D002482:Cellulose; D002480:Cellulase