Cloning And Expression Of The Protease Gene And Enzyme Analysis Of Bacillus Amyloliquefaciens And Its Mutant Strain

The main contents of this paper include several aspects:Through the comparison of the strain NCU116 and the strain NCU116-1,and the morphology of the colony and the morphology under the scanning electron microscope,it was found that the morphology of the colony and the morphology of the bacteria were not changed before and after the mutation.Colonies were round,smooth surface,the color is yellow,with irregular wavy edge circular protrusions.Observed by scanning electron microscope,the strain was rod-shaped,smooth surface,both ends with a rounded shape and it was single or multiple end to end arrangement.The results indicate that the physical and chemical mutagenesis may have great effect on the secreted enzyme genes for some strains,the DNA was mutated in certain degree,and had little change in the colony and cell morphology.The strains NCU116 and NCU116-1 were cultured by shaking flask culture,the extracellular enzymes were analyzed using the national standard method,CMC-Na method etc.The results showed that extracellular enzymes including protease,glucoamylase,pectinase,cellulase,amylase.The relationship between the extracellular enzymes activity and fermentation times was determined.Results show:The activities of neutral proteinase and amylase were the highest?4,536.5 U/m L and 4.8 U/mL respectively?when fermentation time was 44 h.The activity of glucoamylase was the highest?4070.9 U/m L?when fermentation time was 38 h.The activities of pectinase and cellulase were the highest?875.3 U/mL and 57.0 U/m L respectively?when fermentation time was 42 h.The activity of proteinase was the highest?9027.0 U/mL?with shaking flash fermentation for 44 h.The activities of glucoamylase,cellulase and amylase were the highest?10727.6 U/mL,6.3 U/m L and 29.2 U/m L,respectively?with shaking flash fermentation for 40 h.The activity of pectase was the highest?1644.2 U/mL?with shaking flash fermentation for 42 h.The enzymatic properties of Bacillus amyloliquefaciens NCU116 and NCU116-1 extracellular enzymes were analyzed using the national standard method,CMC-Na method etc.Results show:The proteinase had the maximum activity at 50 oC,but was unstable.Its optimum temperature and pH value were approximately 40-45 oC and 7.0 respectively.Mn²⁺ was an activator of neutral proteinase.The glucoamylase had the maximum activity at 45 oC,and was activated by Ca²⁺.Its optimum temperatures and pH value were 45-50 oC and 6.0 respectively.The pectinase had the maximum activity at 40 oC,and was activated by Ca²⁺.Its optimum temperatures and pH value were 35-40 oC and 7.0 respectively.The cellulase had the maximum activity at 35 oC,and was activated by Ca²⁺.Its optimum temperatures and pH value were 30-40 oC and 7.0 respectively.The proteinase had the maximum activity at 50 oC,but was unstable.Its optimum temperature and pH value were approximately 40-45 oC and 8.0 respectively.Mn²⁺ was an activator of neutral proteinase.The glucoamylase had the maximum activity at 35 oC,and was activated by Cu²⁺.Its optimum temperatures and pH value were 35 oC and 8.0 respectively.The pectinase had the maximum activity at 40 oC,and was activated by Ca²⁺.Its optimum temperatures and pH value were 35-40 oC and 6.0 respectively.The cellulase had the maximum activity at 50 oC,and was activated by Cu²⁺.Its optimum temperatures and pH value were 40-45 oC and 7.0 respectively.Bacillus amyloliquefaciens NCU116 was used as the research object,and its protease gene was cloned,expressed and purified.By gene cloning method,a length of 1494 bp protease gene was obtained.The gene of Bacillus amyloliquefaciens?Sequence ID: K02497.1?neutral protease gene had 99% homology with the protease gene cloned from the strain by blast sequence analysis.Protein molecular theory to protease gene encoding amount?Molecular weight?is 52.85 kDa,isoelectric point pl=8.25,positively charged amino acid residues?Arg+Lys?number was 53,negatively charged amino acid residues?Arg+Lys?number was 51.The coefficient of instability?The instability index?was 25.93,which indicated that the protein had good stability.The extinction coefficient of 280 nm is 59710M-1 cm-1.The fat coefficient was 71.33,and the total average hydrophilicity was-0.658.The protein is hydrophilic protein,containing a typical hydrophobic region.Consistent with the family of Peptidase_M4_C protein family domain,with LasB domain.Through the construction of heterologous expression system,the target gene was transferred into E.coli and the fusion protein was induced at 37oC.Finally,protein was purified by affinity chromatography and gel filtration chromatography and its concentration is 0.50mg/mL.