Cloning and overexpression of a Paenibacillus beta-glucanase in Pichia pastoris: purification and characterization of the recombinant enzyme.
Isolation, expression, and characterization of a novel endo-beta-1,3(4)-D-glucanase with high specific activity and homology to Bacillus lichenases is described. One clone was screened from a genomic library of Paenibacillus sp. F-40, using lichenan-containing plates. The nucleotide sequence of the clone contains an ORF consisting of 717 nucleotides, encoding a beta-glucanase protein of 238 amino acids and 26 residues of a putative signal peptide at its N-terminus. The amino acid sequence showed the highest similarity of 87% to other beta-1,3-1,4-glucanases of Bacillus. The gene fragment Bg1 containing the mature glucanase protein was expressed in Pichia pastoris at high expression level in a 3-1 high-cell-density fermenter. The purified recombinant enzyme Bgl showed activity against barley beta-glucan, lichenan, and laminarin. The gene encodes an endo-beta-1,3(4)-D-glucanase (E. C. 3.2.1.6). When lichenan was used as substrate, the optimal pH was 6.5, and the optimal temperature was 60 degrees C. The K(m), V(max), and k(cat) values for lichenan are 2.96 mg/ml, 6,951 micromol/min x mg, and 3,131 s(-1), respectively. For barley beta-glucan the values are 3.73 mg/ml, 8,939 micromol/min x mg, and 4,026 s(-1), respectively. The recombinant Bg1 had resistance to pepsin and trypsin. Other features of recombinant Bg1 including temperature and pH stability, and sensitivity to some metal ions and chemical reagents were also characterized.
Department of Microbiology Engineering, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, PR China.
Peilong
P
Yang
Pengjun
P
Shi
Yaru
Y
Wang
Yingguo
Y
Bai
Kun
K
Meng
Huiying
H
Luo
Tiezheng
T
Yuan
Bin
B
Yao
D000595:Amino Acid Sequence; D001419:Bacteria / Q000201:enzymology* / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D005285:Fermentation; D015870:Gene Expression; D005798:Genes, Bacterial; D006026:Glycoside Hydrolases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D006031:Glycosylation; D006836:Hydro-Lyases; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D004269:DNA, Bacterial; D011994:Recombinant Proteins; D006026:Glycoside Hydrolases; C028020:licheninase; D006836:Hydro-Lyases