A monovalent anion affected multi-functional cellulase EGX from the mollusca, Ampullaria crossean.
A cellulose hydrolytic enzyme was isolated from the stomach juice of Ampullaria crossean, a kind of herbivorous mollusca. The enzyme was purified 45.3-fold to homogenety by ammonium sulfate precipitation, DEAE-Sephadex A-50 column, Bio-gel P-100 gel filtration column, and phenyl-Sepharose CL-4B column chromatography. The enzyme was designated as cellulase EGX. The purified enzyme is a multi-functional enzyme with the activities of exo-beta-1,4-glucanase (14.84 U/mg for p-nitrophenyl beta-D-cellobioside), endo-beta-1,4-glucanase (40.3 U/mg for carboxymethyl cellulose), and endo-beta-1,4-xylanase (196 U/mg for soluble xylan from birchwood). The monovalent anions such as F(-), Cl(-), Br(-), I(-), and NO(3)(-) are essential for its exo-beta-1,4-glucanase activity but have no effect on the activity for xylan, while I(-) higher than 5mM would inhibit the exo-beta-1,4-glucanase activity. The monovalent anions Cl(-) and Br(-) activate its endo-beta-1,4-glucanase activity. Binding of Cl(-) enhances the thermostability of EGX, but does not affect its fluorescence emission spectrum. The molecular mass of EGX is 41.5 kDa, as determined by SDS-PAGE. The pI value is about pH 7.35. The xylan hydrolytic activity of EGX reaches to the maximum between pH 4.8 and 6.0 and the pNPC hydrolytic activity reaches the maximum between pH 4.8 and 5.6, while that for CMC hydrolytic activity is between pH 4.4 and 4.8. Preliminary results showed that the enzyme was secreted by the mollusca itself.
Wang
Ji
J
Key Laboratory of Proteomics, Institute of Biochemistry and Cell Biology, Shanghai Institute for Biological Sciences, Chinese Academy of Sciences, 320 Yue-Yang Road, 200031, Shanghai, China.
Ding
Ming
M
Li
Yan-Hong
YH
Chen
Qin-Xi
QX
Xu
Gen-Jun
GJ
Zhao
Fu-Kun
FK
D000645:Ammonium Sulfate / Q000737:chemistry; D000818:Animals; D000838:Anions / Q000737:chemistry*; D015153:Blotting, Western; D001965:Bromides / Q000737:chemistry; D002266:Carboxymethylcellulose Sodium / Q000378:metabolism; D002480:Cellulase / Q000737:chemistry / Q000378:metabolism; D044602:Cellulases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002712:Chlorides / Q000737:chemistry; D003637:DEAE-Dextran / Q000031:analogs & derivatives* / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D043364:Endo-1,4-beta Xylanases / Q000737:chemistry / Q000378:metabolism; D004789:Enzyme Activation; D004795:Enzyme Stability; D005459:Fluorides / Q000737:chemistry; D005750:Gastric Juice / Q000201:enzymology; D044643:Glucan 1,4-beta-Glucosidase / Q000737:chemistry / Q000378:metabolism; D005960:Glucosides / Q000378:metabolism; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007700:Kinetics; D008099:Liver / Q000737:chemistry / Q000378:metabolism; D008970:Molecular Weight; D008974:Mollusca / Q000201:enzymology*; D009566:Nitrates / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013213:Starch / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature; D014990:Xylans / Q000378:metabolism
D000838:Anions; D001965:Bromides; D002712:Chlorides; D005960:Glucosides; D009566:Nitrates; D014990:Xylans; C034170:4-nitrophenyl beta-cellobioside; C027184:DEAE-Sephadex A-50; D013213:Starch; D003637:DEAE-Dextran; D044602:Cellulases; D002480:Cellulase; D044643:Glucan 1,4-beta-Glucosidase; D043364:Endo-1,4-beta Xylanases; D002266:Carboxymethylcellulose Sodium; D005459:Fluorides; D000645:Ammonium Sulfate