Biochemical characterization of a novel thermostable beta-1,3-1,4-glucanase (lichenase) from Paecilomyces thermophila.
The purification and characterization of a novel extracellular beta-1,3-1,4-glucanase from the thermophilic fungus Paecilomyces thermophila J18 were studied. The strain produced the maximum level of extracellular beta-glucanase (135.6 U mL(-1)) when grown in a medium containing corncob (5%, w/v) at 50 degrees C for 4 days. The crude enzyme solution was purified by 122.5-fold with an apparent homogeneity and a recovery yield of 8.9%. The purified enzyme showed as a single protein band on SDS-PAGE with a molecular mass of 38.6 kDa. The molecular masses were 34.6 kDa and 31692.9 Da when detected by gel filtration and mass spectrometry, respectively, suggesting that it is a monomeric protein. The enzyme was a glycoprotein with a carbohydrate content of 19.0% (w/w). Its N-terminal sequence of 10 amino acid residues was determined as H2N-A(?)GYVSNIVVN. The purified enzyme was optimally active at pH 7.0 and 70 degrees C. It was stable within pH range 4.0-10.0 and up to 65 degrees C, respectively. Substrate specificity studies revealed that the enzyme is a true beta-1,3-1,4-D-glucanase. The K m values determined for barley beta-D-glucan and lichenan were 2.46 and 1.82 mg mL(-1), respectively. The enzyme hydrolyzed barley beta-D-glucan and lichenan to yield bisaccharide, trisaccharide, and tetrasaccharide as the main products. Circular dichroism studies indicated that the protein contains 28% alpha-helix, 24% beta-sheet, and 48% random coil. Circular dichroism spectroscopy is also used to investigate the thermostability of the purified enzyme. This is the first report on the purification and characterization of a beta-1,3-1,4-glucanase from Paecilomyces sp. These properties make the enzyme highly suitable for industrial applications.
Yang
Shaoqing
S
Department of Biotechnology, College of Food Science and Nutritional Engineering, College of Engineering, China Agricultural University, Beijing 100083, China.
Qiaojuan
Yan
Y
Jiang
Zhengqiang
Z
Fan
Guangsen
G
Wang
Lan
L
D004795:Enzyme Stability; D005656:Fungal Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D010143:Paecilomyces / Q000737:chemistry / Q000201:enzymology*; D017433:Protein Structure, Secondary; D013379:Substrate Specificity; D013696:Temperature
D005656:Fungal Proteins; D006026:Glycoside Hydrolases; C028020:licheninase