| Alkaline protease is widely used in detergent,food,pharmaceutical,leather,diagnosis,waste management and silver recovery and other industries,with important commercial value.In this paper,the mutagenesis and screening of strains producing alkaline protease,the production and fermentation optimization of microbial alkaline protease and cloning and expression of microbial alkaline protease gene were studied.In this study,the B.subtilis S1-4 strain was mutagenesis using Atmospheric and Room Temperature Plasma technology,and He was used as the discharge gas.The gas flow was 10L/min,the rf power was 120 W,and the mutagenesis time was 20 s,40 s,60 s and 80 s.Fourteen mutant strains with large hydrolytic circle were selected through casein plate preliminary screening,and then a high-yielding alkaline protease strain was obtained through laboratory flask rescreening.After morphological observation and comparative analysis of the16 S rDNA sequence,it was identified as B.subtilis S1-4-2-21,and its alkaline protease activity was up to 25.4 U/mL,about 2 times higher than that of the original strain,feather degradation rate increased by 4.33%.B.subtilis S1-4-2-21,the mutant with the highest enzyme activity,was resequenced and compared with the reference genome of the wild strain.SNP mutations mainly occurred between A-C and A-G bases,with non-homologous mutations accounting for 14%,and most SNP mutations occurred in intergene sequences.InDel mutation mainly occurs in Shift,CDS region sequence,which can cause Shift mutation.There are 2 internal migrations of chromosomes and 96 migrations between chromosomes.In order to study the effects of different medium components and fermentation conditions on the enzyme production of this strain,on the basis of single factor experiment,three factors including bran content,peptone content and initial pH value were selected as independent variables in this study,and alkaline protease enzyme activity was taken asdependent variable to carry out response surface test of three factors and three levels.After optimization of fermentation medium for: bran concentration of 49.46 g/L,peptone concentration of 25 g/L,the initial pH of 7,3% inoculated quantity,fermentation temperature of 40 ?,the fermentation time of 60 h,the alkaline protease enzyme activity is up to 128.98U/mL,and it is five times before optimization.In order to study the feather degradation mechanism of B.subtilis S1-4,the keratin gene BsuKER-68 of B.subtilis S1-4 was cloned in this study,and the full-length gene sequence2421 bp was obtained,which contained a 2271 bp open reading frame encoding 757 amino acids.DNAMAN,MEGA,TMPRED,SWISS-MODEL,SOPMA and other bioinformatics tools were used to analyze its evolutionary tree,transmembrane structure analysis,tertiary structure prediction,physical and chemical properties and secondary structure,etc.and it was found that its similarity with keratinase gene in NCBI was 99%.The relative molecular weight was 80.4 kD and the isoelectric point was 6.06.The gene protein is hydrophilic protein.The results of phylogenetic tree analysis were consistent with the strain information.The recombinant plasmid pSU03-BsuKER-68 was obtained by homologous recombination and transferred into WB600.As the product of the target gene is extracellular enzyme in WB600,its alkaline protease can be produced in the fermentation broth,and the maximum enzyme activity was 10.6 U/mL.In conclusion,B.subtilis with higher alkaline protease activity was obtained with ARTP mutagenesis technology in this study.The regions of SNP mutation,InDel mutation and SV mutation were obtained by genome resequencing analysis.Using B.subtilis S1-4 as the template,BsuKER-68 gene was successfully cloned and it was expressed in B.subtilis WB600.This study provided information for further screening strains producing high quality alkaline protease,and also laid a foundation for genetic engineering of alkaline protease and its industrial application in the future. |
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