Purification and characterization of a novel endo-β-1,4-glucanases , AfEG22, from the giant snail, Achatina fulica frussac.
In this study, we confirmed that at least three endo-β-1,4-glucanases existed in the digestive juice of the giant snail, Achatina fulica ferussac, by Congo red staining assay. One of these enzymes, a novel endo-β-1,4-glucanase (AfEG22), was purified 29.5-fold by gel filtration, anion exchange, and hydrophobic interaction chromatography. The carboxymethyl cellulose (CMC) hydrolytic activity of the purified enzyme was 12.3 U/mg protein. The molecular mass of AfEG22 was 22081 Da determined by MALDI-TOF. N-terminal amino acid sequencing revealed a sequence of EQRCTNQGGILKYYNT, which did not have significant homology with any proteins in BLAST database. The optimal pH and temperature for hydrolytic activity toward CMC were pH 4.0 and 50°C, respectively. AfEG22 was stable between pH 3.0 and pH 12.0 when incubated at 4°C for 3 h or at 37°C for 1 h. The enzyme remained more than 80% activity between pH 4.5 and pH 7.0 after incubation at 50°C for 1 h. AfEG22 possessed excellent thermostability as more than 70% activity was remained after incubation at 60°C for 3 h. Substrate specific analysis revealed that AfEG22 was a typical endo-β-1,4-glucanase. This is the first time to report a novel endo-β-1,4-glucanase with high stability from the digestive juice of A. fulica.
Teng
Yigang
Y
Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, China.
Yin
Qiuyu
Q
ding
Ming
M
Zhao
Fukun
F
D000595:Amino Acid Sequence; D000818:Animals; D055162:Biocatalysis; D002266:Carboxymethylcellulose Sodium / Q000378:metabolism; D002480:Cellulase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D044644:Endo-1,3(4)-beta-Glucanase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008969:Molecular Sequence Data; D008970:Molecular Weight; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D012908:Snails / Q000201:enzymology* / Q000235:genetics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D013696:Temperature
D002480:Cellulase; D044644:Endo-1,3(4)-beta-Glucanase; D002266:Carboxymethylcellulose Sodium