Molecular cloning and expression of the gene encoding family 19 chitinase from Streptomyces sp. J-13-3.
The gene encoding chitinase from Streptomyces sp. (strain J-13-3) was cloned and its nucleotide structure was analyzed. The chitinase consisted of 298 amino acids containing a signal peptides (29 amino acids) and a mature protein (269 amino acids), and had calculated molecular mass of 31,081 Da. The calculated molecular mass (28,229 Da) of the mature protein was almost same as that of the native chitinase determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometer. Comparison of the encoded amino acid sequences with those of other chitinases showed that J-13-3 chitinase was a member of the glycosyl-hydrolase family 19 chitinases and the mature protein had a chitin binding domain (65 amino acids) containing AKWWTQ motif and a catalytic domain (204 amino acids). The J-13-3 strain had a single chitinase gene. The chitinase (298 amino acids) with C-terminal His tag was overexpressed in Escherichia coli BL21(DE3) cells. The recombinant chitinase purified from the cell extract had identical N-terminal amino acid sequence of the mature protein in spite of confirmation of the nucleotide sequence, suggesting that the signal peptide sequence is successfully cut off at the predicted site by signal peptidase from E. coli and will be a useful genetic tool in protein engineering for production of soluble recombinant protein. The optimum temperature and pH ranges of the purified chitinase were at 35-40 degrees C and 5.5-6.0, respectively. The purified chitinase hydrolyzed colloidal chitin and trimer to hexamer of N-acetylglucosamine and also inhibited the hyphal extension of Tricoderma reesei.
Okazaki
Katsuichiro
K
Department of Life Sciences, Faculty of Agriculture, Kagawa University, Japan. okazaki@ag.kagawa-u.ac.jp
Yamashita
Yousuke
Y
Noda
Minoru
M
Sueyoshi
Noriyuki
N
Kameshita
Isamu
I
Hayakawa
Shigeru
S
D000595:Amino Acid Sequence; D001483:Base Sequence; D015139:Blotting, Southern; D002688:Chitinases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D002855:Chromatography, Thin Layer; D003001:Cloning, Molecular; D017931:DNA Primers; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics*; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics*; D015723:Gene Library; D006868:Hydrolysis; D025301:Hyphae / Q000187:drug effects / Q000254:growth & development; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010455:Peptides / Q000737:chemistry; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics*; D014242:Trichoderma / Q000187:drug effects
D017931:DNA Primers; D004269:DNA, Bacterial; D010455:Peptides; D011994:Recombinant Proteins; D002688:Chitinases