Liang Xiao1, Chuan Liu, Chichu Xie, Jun Cai, Dong Liu, Yuehua Chen. 1. Department of Microbiology, Nankai University, Key Laboratory of Molecular Microbiology and Technology, Ministry of Education, Tianjin 300071, China. xlnankai@gmail.com
Abstract
OBJECTIVE: To heterogeneously express the chitinase gene of Bacillus licheniformis strain MY75 in E. coli, and to characterize the recombinant chitinase ChiMY. METHODS: The extracelluar crude protein from B. licheniformis MY75 was analyzed by zymogram analysis. The partial amino acid sequence of the protein owned chitinase activity was given by time-of-flight mass spectrometry (TOF-MS). Then the corresponding chitinase gene chiMY was cloned and heterogeneously expressed in E. coli. The optimum temperature and pH of the ChiMY, and the effect of various metal ions on chitinase activity were studied. The antifungal activity and the synergistic effect on insecticidal activity were demonstrated by bioassays. RESULTS: A 55 kDa extracelluar protein produced by B. licheniformis MY75 exhibited chitinase activity in zymogram analysis. The chiMY gene was 1797 bp long and encoded a 599 amino acid protein. The molecular weight of the recombinant protein ChiMY over-expressed in E. coli was 67 kDa. The amino acid sequence of the 55 kDa extracelluar protein was proved identical to the 67 kDa ChiMY by the TOF-MS. The optimum temperature and pH were 50 degrees C and 7.0, respectively. The enzyme activity was improved by Li+, Na+ and Mg2+ and inhibited by Mn2+, Cr3+, Zn2+, and Ag+. Cu2+ and Fe3+ can inactivate the enzyme. The bioassays demonstrated the heterogeneously expressed ChiMY could inhibit the sporangia germination of G. saubinetii and A. niger, and reduce the LC50 (50% lethal concentration) of the crystal protein of Bacillus thuringiensis against S. exigua by approximately 27%. CONCLUSIONS: The B. licheniformis MY75 could produce a 55 kDa chitinase. The corresponding chitinase gene was over-expressed in E. coli. The molecular weight of heterogeneously expressed ChiMY showed significant different to the wild-type chitinase protein. This implicated the the protein processing of chitinase in the B. licheniformis MY75. The ChiMY also owned the antifungal activity and could improve the insecticidal activity of the Bacillus thuringiensis crystal protein against S. exigua. This is the first report about the chitinase from B. licheniformis in China.
OBJECTIVE: To heterogeneously express the chitinase gene of Bacillus licheniformis strain MY75 in E. coli, and to characterize the recombinant chitinase ChiMY. METHODS: The extracelluar crude protein from B. licheniformis MY75 was analyzed by zymogram analysis. The partial amino acid sequence of the protein owned chitinase activity was given by time-of-flight mass spectrometry (TOF-MS). Then the corresponding chitinase gene chiMY was cloned and heterogeneously expressed in E. coli. The optimum temperature and pH of the ChiMY, and the effect of various metal ions on chitinase activity were studied. The antifungal activity and the synergistic effect on insecticidal activity were demonstrated by bioassays. RESULTS: A 55 kDa extracelluar protein produced by B. licheniformis MY75 exhibited chitinase activity in zymogram analysis. The chiMY gene was 1797 bp long and encoded a 599 amino acid protein. The molecular weight of the recombinant protein ChiMY over-expressed in E. coli was 67 kDa. The amino acid sequence of the 55 kDa extracelluar protein was proved identical to the 67 kDa ChiMY by the TOF-MS. The optimum temperature and pH were 50 degrees C and 7.0, respectively. The enzyme activity was improved by Li+, Na+ and Mg2+ and inhibited by Mn2+, Cr3+, Zn2+, and Ag+. Cu2+ and Fe3+ can inactivate the enzyme. The bioassays demonstrated the heterogeneously expressed ChiMY could inhibit the sporangia germination of G. saubinetii and A. niger, and reduce the LC50 (50% lethal concentration) of the crystal protein of Bacillus thuringiensis against S. exigua by approximately 27%. CONCLUSIONS: The B. licheniformis MY75 could produce a 55 kDa chitinase. The corresponding chitinase gene was over-expressed in E. coli. The molecular weight of heterogeneously expressed ChiMY showed significant different to the wild-type chitinase protein. This implicated the the protein processing of chitinase in the B. licheniformis MY75. The ChiMY also owned the antifungal activity and could improve the insecticidal activity of the Bacillus thuringiensis crystal protein against S. exigua. This is the first report about the chitinase from B. licheniformis in China.