Purification and characterization of a novel β-1,3-1,4-glucanase (lichenase) from thermophilic Rhizomucor miehei with high specific activity and its gene sequence.
Production, purification, and characterization of a novel β-1,3-1,4-glucanase (lichenase) from thermophilic Rhizomucor miehei CAU432 were investigated. High-level extracellular β-1,3-1,4-glucanase production of 6230 U/mL was obtained when oat flour (3%, w/v) was used as a carbon source at 50 °C. The crude enzyme was purified to homogeneity with a specific activity of 28818 U/mg. The molecular weight of purified enzyme was estimated to be 35.4 kDa and 33.7 kDa by SDS-PAGE and gel filtration, respectively. The optimal pH and temperature of the enzyme were pH 5.5 and 60 °C, respectively. The K(m) values of purified β-1,3-1,4-glucanase for barley β-glucan and lichenan were 2.0 mM and 1.4 mM, respectively. Furthermore, the gene (RmLic16A) encoding the β-1,3-1,4-glucanase was cloned and its deduced amino acid sequence showed the highest identity (50%) to characterized β-1,3-1,4-glucanase from Paecilomyces thermophila. The high-level production and biochemical properties of the enzyme enable its potential industrial applications.
Department of Biotechnology, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing, China.
Yanbin
Y
Tang
Shaoqing
S
Yang
Qiaojuan
Q
Yan
Peng
P
Zhou
Jian
J
Cui
Zhengqiang
Z
Jiang
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry; D006026:Glycoside Hydrolases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D020103:Rhizomucor / Q000201:enzymology*; D013379:Substrate Specificity
D018076:DNA, Complementary; D006026:Glycoside Hydrolases; C028020:licheninase