Study On Characteristics And Expression Regulation Elements Of Chitinase From Bacillus Licheniformis MY75

Strain MY75 is a gram-positive, aerobic, endospore-forming bacterium that can secrete high levels of extracellular chitinase (4.645 U/ml) when chitin powder exists in media as an inducer.In our identification research, the strain MY75 was found belonged to the Bacillus subtilis group. The 16S rDNA sequence analysis can't be used to identify this strain, because the strains included in the Bacillus subtilis group exhibits very high sequence identity. The sequences of protein-coding genes, gyrA (codes for DNA gyrase subunit A) and rpoB (codes for DNA-directed RNA polymerase subunit beta) were used to the identification of the strain MY75 and this strain was identified as Bacillus' lichenifromis finally. After being cultured for 96 h, the chitinase production of strain MY75 reached the maximum at 4.645 U/mL.The extracellular crude proteins from the culture of strain MY75 were used in the antifugal activity assays. The germination of the spores from Gibberella saubinetii and Aspergillus niger mixed with the induced crude proteins was totally inhibited, whereas the spores mixed with the uninduced crude proteins germinated normally. This result demonstrated the chitinase produced by the strain MY75 played an important role in the stain's antifungal activity.The crude proteins from induced cultured of the strain MY75 were analyzed by zymogram assays. The protein band showed chitinase activity was send to the time-of-flight mass spectrometer (TOF MS) analysis. The partial amino acid sequence of the sample showed high identity with the Bacillus lichenifromis chitinase. Then the corresponding gene was cloned and sequenced. The open reading frame (ORF) of the gene is 1797 bp long and encodes 599 amino acids protein. It owns 99% identity to the Bacillus lichenifromis chiB gene in GenBank. The cloned gene was designated as chiMY75. Then the chiMY75 was subcloned to the expression vector pET28a, the recombined vector was transformed to the E.coli BL21(DE3)codon plus. A specific 67 kDa protein band was detected in the supernatant of the disrupted transformant cells. The characteristics of the recombinant chitinase ChiMY75 have been studied. The optimum temperature and pH of the enzyme were 50℃and 7.0 respectively. The enzyme activity was remarkably inhibited by Mn2+, Cr3+, Zn2+ and Ag+. And there was a slight increment in enzyme activity in the presence of Li+, Na+ and Mg2+. The Cu2+ and Fe3+ would inactivate the enzyme. Its antifungal activity against two types of pathogenic fungi also has been assayed. The expressed chitinase can totally inhibited the germination of the spores. In bioassays, the existence of the the ChiMY75 can enhance the toxicity of the Bacillus thuringiensis against Spodoptera exigua significantly.To study the regulator of the chitinase from Bacillus, the entire ORF with 432 bp upstream of the MY75 chitinase gene was cloned (designated as chiMY75full), the chitinase gene with 248 upstream from Bacillus thuringiensis strain 15A3 (designated as 15A3chiB) cloned previously was also used in this research. The sequences of the chiMY75full and 15A3chiB were analysed by online software, putative promoter regions were detected in their upstream sequence, including the-10 and-35 areas. Both of them could expressed in E. coli XL-Blue with their natural promoter. A 16bp direct repeat (DR) sequence was found existing in the upstream area of each gene respectively. The function of the DR region was revealed by the truncation. The results of the chitinase assays demonstrated the chitinase activity of the transformants with the truncation of the DR were significant higher than the original ones. The SDS-PAGE showed that the deletion of the DR caused the increase of the chitinase expression. The northern blot revealed that the amount of the mRNA increased according to the truncation of the DR region. So we suggested that the DR region should be the binding site of the repressor that regulates the transcription of the chitinase gene.In this paper, the strain MY75 was identified as Bacillus lichenifromis. The chitinase gene of this strain chiMY75 was cloned and expressed in the E. coli. The antifungal activity and the synergistic effect to the Bacillus thuringiensis toxicity of the ChiMY75 were proved. The putative promoter region and direct repeat sequence were found existing in the upstream of two kinds of chitinase, chiMY75full and 15A3chiB. The results of the DR truncation suggested that the DR sequence should be the negative control element of the bacillus chitinase, could regulate the expression of the chitinase gene on transcription level. Our research revealed the potential of the strain MY75 in biocontrol application and found the theoretical basis on the regulation mechanisms of the Bacillus chitinase gene.