Expression and characterization of the recombinant gene encoding chitinase from Aeromonas caviae.
The gene encoding a chitinase from Aeromonas caviae was cloned by PCR techniques. Its recombinant gene expression was performed using pET20b(+) in Escherichia coli BL21 (DE3). The recombinant chitinase with the extra 33 and 13 amino acids in its N- and C-termini, respectively, was purified to near homogeneity using His-Tag affinity chromatography. The recombinant chitinase was found to be present in both the culture medium and the cytoplasm. A single protein band on the native polyacrylamide gel was confirmed by both the activity staining and protein staining. The optimum pH and temperature of the recombinant chitinase were determined to be 6.25-6.5 and 42.5 degrees C, respectively. It was stable within the pH range of 5-7. Significant activity stimulation by Cu2+ and inhibition by Fe3+ and Hg2+ were observed. Detergents such as SDS and Triton X-100 strongly inhibited the enzyme activity. Substrates such as 4-methylumbelliferyl-N,N'-diacetylchitobioside and 4-methylumbelliferyl-N,N',N"-triacetylchitotriose were hydrolyzed by the recombinant chitinase; however, 4-methylumbelliferyl-N-acetylglucosaminide was not cleaved during the activity assay periods. When chitin power was suspended in buffer with the chitinase (pH 6.5 and 42.5 degrees C), N-acetylchitooligosaccharides [(GlcNAc)n, n = 1-4] were detected at 24 h.
Lin
C S
CS
Institute of Marine Food Science, National Taiwan Ocean University, Keelung, Taiwan, Republic of China.
Chen
H C
HC
Lin
F P
FP
D000333:Aeromonas / Q000201:enzymology* / Q000235:genetics*; D002686:Chitin / Q000737:chemistry; D002688:Chitinases / Q000235:genetics*; D003001:Cloning, Molecular; D004274:DNA, Recombinant; D015870:Gene Expression; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry
D004274:DNA, Recombinant; D011994:Recombinant Proteins; D002686:Chitin; D002688:Chitinases