Screening And Purification Of A Neutral Protease Producing Strain Bacillus Sp. Study

The abuse of chemical pesticides and agricultural pesticides caused pesticides resistance and antibiotic resistance. The agricultural production was threatened pathogeny fungus and agriculture pest of resistance. The chitinase is indispensable enzyme for the plant pathogeny fungus and agriculture pest, and isn't necessary for human and mammals and plants. The chitinase as an anti-target is safe and feasible. This research have significance at both of theory and practice.This paper is about the search for chitinase inhibitor product strain, identification and classifying of the strain, optimization of fermentation of chitinase inbitor production by the strain, the purifying of the inhibitor, and study on the characteristics of the inhibitor.1. Screening of a chitinase inhibitor producing strainUsing the colloidal chitin plate culture method, the strain numbered CQBB218 with high chitinase inhibitor activity was found among 800 strains which were isolated from soil. The chitinase inhibitor activity of fermentative fluid is about 2.65 Uj/mL.2. Identification of the chitinase inhibitor producing strain CQBB218According to the characteristics of morphology, cultivation, physiology and 16S rDNA sequence, the strain CQBB218 was identified as Bacillus sp. The strain CQBB218 was named Bacillus sp. CQBB218.3. Optimization of fermentation of chitinase inbitor production by the strain CQBB218The primary optimization of cultivation conditions was selected through fermentation tests. The appropriate carbon source is starch, the appropriate nitrogen source is yeast extract and the appropriate metal ion is Mn²⁺. The highest chitinase inhibitor activity is 2.77 U_i/mL when the following conditions were meted: initial pH 8.0, incubation temperature 30°C, incubation time 72h, 40mL of the culture in100mL shake flasks, and the optimization culture medium of starch 0.2%, yeast extract 0.6%, MnCl₂ 0.1%.4. The purifying of the chitinase inhibitorThe inhibitor was purified by a method for extraction of the enzyme involving fractional ammonium sulphate precipitation, ion-exchange chromatography on DEAE Sepharose Fast Flow, gel filtration on Superdex G75. The molecular weight of the inhibitor is about 37 kDa by 15% SDS-PAGE. The inhibitor activity is 312 U_i, the purifying multiple is 25.89, activity reclaim rate is 3.76%.10 N-terminal amino acids of the protein were sequenced by PVDF transblorting and search of blast in GenBank suggests this protein is a kind of neutral protease.5. Study on the characters of the neutral protease of chitinase inhibitor activityThis neutral protease has chitinase inhibitor activity at pH5.5-9.5, and is unstable at high temperature. Bivalent metal ion is important to it retaining chitinase inhibitor activity and Protease K can hydrolyze it. The neutral protease has inhibitor activity to the chtinase of silkworm, but hasn't inhibitor activity to chitinase of bacteria.