Characterization of a GH family 8 β-1,3-1,4-glucanase with distinctive broad substrate specificity from Paenibacillus sp. X4.
A β-1,3-1,4 glucanase gene of Paenibacillus sp. X4, bglc8H, was cloned and characterized. BGlc8H was predicted to be a protein of 409 amino acid residues, including a signal peptide of 31 amino acids. The mature enzyme was predicted to have 378 amino acid residues; its [corrected] molecular mass and pI were estimated as 41,561 Da and 7.61, respectively. BGlc8H belongs to glycoside hydrolase family 8 (GH8). Site-directed mutants of Glu95 and Asp156 of BGlc8H showed a near-complete loss of activity, indicating that they are catalytically-active residues. Unlike other GH8 members, BGlc8H had broad substrate specificity and hydrolyzed barley-β-D-glucan > chitosan > carboxymethyl-cellulose > and lichenan. BGlc8H had a lower ratio of lichenase/barley-β-D-glucanase activities compared to GH16 enzymes. BGlc8H was optimally active at pH 5 and 50 °C, except for barley-β-D-glucanase (40 °C) and chitosanase (pH 7) activities. BGlc8H hydrolyzed cello-oligosaccharides (G3-G6) to G3 and G2 but not to G1. Ca(2+) increased the activity and thermostability of BGlc8H for lichenan suggesting its use for the saccharification of cellulosic biomass.
Department of Agricultural Chemistry, Sunchon National University, Suncheon, 540-950, Republic of Korea.
Han Beur
HB
Na
Won Kyeong
WK
Jung
Yu Seok
YS
Jeong
Hee Jung
HJ
Kim
Sung Kyum
SK
Kim
Jungho
J
Kim
Han Dae
HD
Yun
Jung-Kul
JK
Lee
Hoon
H
Kim
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D003001:Cloning, Molecular; D016000:Cluster Analysis; D004252:DNA Mutational Analysis; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004275:DNA, Ribosomal / Q000737:chemistry / Q000235:genetics; D004795:Enzyme Stability; D015870:Gene Expression; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D056507:Paenibacillus / Q000145:classification / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D010802:Phylogeny; D017354:Point Mutation; D021382:Protein Sorting Signals; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D012988:Soil Microbiology; D013379:Substrate Specificity; D013696:Temperature
D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D021382:Protein Sorting Signals; D012336:RNA, Ribosomal, 16S; D006026:Glycoside Hydrolases; C028020:licheninase