[Expression of endoglucanase gene in Bacillus Megaterium and characterization of recombinant enzyme].
The signal peptide-encoding sequence, which was included in the gene (GenBank No. DQ782954) encoding for endoglucanase, was removed by PCR. The gene without the signal peptide was then ligated with the expression plasmid pHIS1525. The recombination plasmid pHIS1525 was transformed into Escherichia coli DH5a and the transformant was designated DH5a-pHIS1525-G7. The plasmid from the recombinant DH5a-pHIS1525-G7 was transformed into the proto-plasts of Bacillus megaterium strains WH320, and the genetically engineered bacterium, known as WH320-pHIS1525-G7, was acquired. The effective expression of the gene in the recombinant was confirmed by Congo-red dyeing and SDS poly-acrylamide gel electrophoresis (SDS-PAGE). WH320-pHIS1525-G7 was cultured in optimum condition. The activity of the endoglucanase was 899U, which was 11.22-fold higher than that of B.subtilis C-36. The properties of enzyme were deter-mined. The optimum temperature and pH value were 65 and pH 6.0, respectively. The enzyme maintained over 80% of the original enzyme activity between pH 4.5 and pH 10.0 after incubated at 50 for 30 min.
Chen
Hui
H
College of Biology and Science, Sichuan Agricultural University, Ya'an 625014, China. chenhui@sicau.edu.cn
Xu
Bing
B
Liao
Jun-Hua
JH
Guan
Xing-Ying
XY
Wu
Qi
Q
D001410:Bacillus megaterium / Q000201:enzymology* / Q000235:genetics*; D002480:Cellulase / Q000235:genetics* / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000235:genetics* / Q000378:metabolism*; D013696:Temperature
D011994:Recombinant Proteins; D002480:Cellulase