Purification and characterization of two endo-beta-1,4-glucanases from mollusca, Ampullaria crossean.
Two novel endo-beta-1,4-glucanases, EG45 and EG27, were isolated from the gastric juice of mollusca, Ampullaria crossean, by anion exchange, hydrophobic interaction, gel filtration and a second round of anion exchange chromatography. The purified proteins EG45 and EG27 appeared as a single band on sodium dodecylsulfate polyacrylamide gel electrophoresis with a molecular mass of 45 kDa and 27 kDa, respectively. The optimum pH for CMC activity was 5.5 for EG45 and 4.4-4.8 for EG27. The optimum temperature range for EG27 was broad, between 50 degrees and 60 degrees; for EG45 it was 50 degrees. The analysis on the stability of these two endo-beta-1,4-glucanases showed that EG27 was acceptably stable at pH 3.0-11.0 even when the incubation time was prolonged to 24 h at 30 degrees, whereas EG45 remained relatively stable at pH 5.0-8.0. About 85% of the activity of EG27 could be retained upon incubation at 60 degrees for 24 h. However, less than 10% residual activity of EG45 was detected at 50 degrees. Among different kinds of substrates, both enzymes showed a high preference for carboxymethyl cellulose. EG45, in particular, showed a carboxymethyl cellulose hydrolytic activity of 146.5 IU/mg protein. Both enzymes showed low activities to xylan (from oat spelt) and Sigmacell 101, and they were inactive to p-nitrophenyl-beta-D-cellobioside, salicin and starch.
Li
Yan-Hong
YH
Key Laboratory of Proteomics, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Graduate School of the Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai 200031, China.
Guo
Rui
R
Yin
Qiu-Yu
QY
Ding
Ming
M
Zhang
Si-Liang
SL
Xu
Gen-Jun
GJ
Zhao
Fu-Kun
FK
D000818:Animals; D002480:Cellulase / Q000302:isolation & purification* / Q000378:metabolism*; D004795:Enzyme Stability; D005750:Gastric Juice / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D008974:Mollusca / Q000201:enzymology*; D013379:Substrate Specificity; D013696:Temperature
D002480:Cellulase