Bacillus Cereus MH19 Genome Analysis And Collagenase Separation And Purification And Recombinant Expression

Collagenase is a type of proteolytic enzyme that specifically acts on collagen and gelatin.It can hydrolyze the three-dimensional spiral structure of collagen without damaging other proteins and tissues.Microbial collagenase sources are mainly Clostridium,Bacillus and Vibrio strains.Commercialized collagenase is prepared from Clostridium histolyticum.The structural properties of the Clostridium collagenases Col G,Col H and Clostridium perfringens collagenase Col A have been fully characterized.These three collagenases are often used as model enzymes to study enzymatic properties.With the deepening of research,more and more collagenase-producing strains have been isolated and screened.The structure and properties of collagenase from different sources are very different,indicating that the structure and function of collagenase are diverse.Bacillus cereus has a rich enzyme production system and is an excellent strain for producing collagenase.This article focuses on collagenase research and identified a strain of collagenase-producing Bacillus ceueus MH19.The entire genome sequence was obtained by sequencing.The collagenase gene Col B was excavated from the genome for E.coli recombinant expression.Natural collagenase was obtained,and enzyme activity was measured and evaluated separately.The main research results are as follows:?1?Identify the collagenase-producing strains isolated from the slaughterhouse for subsequent whole-genome sequencing and collagenase gene screening.According to strain culture observation,microscopic examination,MYP culture and some physiological and biochemical tests,combined with 16S r DNA sequence comparison analysis,screening and identification of Bacillus cereus MH19.?2?The second-generation combined with the third-generation sequencing technology was used to sequencing the whole genome of Bacillus cereus MH19.The genome size was 5,534,899 bp and the GC content was 35.28%.It consisted of a circular chromosome of 5,247,580 bp and a circular plasmid of 287,319 bp.It is predicted that there are 5,335 chromosomal genes and 253 plasmid genes.Chromosome circle diagram and plasmid circle diagram are drawn.Functional annotation of the genome,GO functional annotation mainly includes metabolic process,cellular process and single tissue process,there are 1615,1465,1259 genes involved.COG annotated a total of 3879genes,accounting for 69.41%of the entire genome sequence.COG functional annotation mainly includes 10.19%basic function prediction,9.07%transcription,8.78%amino acid transport and metabolism,6.80%translation,ribosomal structure and biosynthesis,followed by 5.55%cell wall/membrane/envelope biosynthesis,5.68%signal transduction mechanism,6.30%carbohydrate transport and metabolism,5.24%coenzyme transport and metabolism and 5.61%inorganic ion transport and metabolism.KEGG annotated a total of 2920 genes,accounting for 52.25%of the entire genome sequence.Comparative genomic analysis of collinearity,shared and unique genes,gene families found that the specificity of Bacillus cereus MH19 is mainly reflected in environmental adaptability,that is,the ability to adapt to the environment has been strengthened.Its genome contains 18specific genes and the largest number of unique genes 944.Evolutionary selection pressure analysis showed that Bacillus cereus MH19 genome has 36 positive selection genes and multiple transcriptional regulators.These regulatory factors have a positive regulatory effect on the development of drug resistance,virulence gene synthesis,DNA replication and repair,and cell survival.?3?B.cereus MH19 genome collagenase gene mining.Through multiple annotations and comparisons of GO,COG,KEGG,NR,SWISSPROT,IPR,it was found that the B.cereus MH19 genome has 5 different collagenase genes with a molecular weight distribution range of 35.7-110 k D.Sequence alignment and phylogenetic tree analysis showed that BCC000504 and BCC003388 collagenase gene sequences were closer to typical collagenase genes Col G,Col A and Col H,followed by BCC003615.Domain and function analysis showed that the properties of collagenases BCC000504 and BCC003388 are similar to those of collagenases Col G,Col A and Col H,and that of BCC004271 and BCC004272 are similar.BCC003615 may differ greatly from the other collagenases.The structure of collagenase was analyzed and the three-dimensional structure of BCC000504 was modeled using the crystal structure of Clostridium histolyticum collagenase G as a template.The established tertiary structure model of BCC000504 collagenase was an acceptable protein model.The establishment of the tertiary structure model helps to understand the mechanism of collagenase from the molecular level.BCC000504 was named Col B and used for prokaryotic expression of E.coli.?4?The separation,purification and enzymatic properties of natural collagenase in B.cereus MH19 fermentation broth were studied.Using stepwise purification,collagenase BCC was obtained by ammonium sulfate precipitation,ion exchange column chromatography and gel chromatography,with a molecular weight of 110 k D,enzyme activity of 257.20 U/mg,purification multiple of 6.07,and a yield of 9%.The optimal temperature of BCC enzyme is 37?,and the optimal p H is 7.5.Ca²⁺promotes enzyme can inhibit the enzymatic activity of collagenase,indicating that BCC is a metalloprotease.BCC is specific to type I collagen and belongs to type I collagenase.?5?The prokaryotic expression of collagenase Col B in E.coli was studied.The Col B gene sequence was extracted from the B.cereus MH19 genome.After optimization and transformation,the expression plasmid p ET30a-Col B was constructed and transformed into E.coli BL21 to induce expression.After purification by Ni-IDA affinity chromatography column,the recombinant Col B with higher purity was obtained,SDS-PAGE and Western Blot analysis showed that the molecular weight of recombinant Col B was 110 k D.The results of collagenase spectroscopy showed that the recombinant Col B had collagenase activity and was able to hydrolyze type I collagen.The measured collagenase activity was 200.76U/mg.Both the recombinant collagenase Col B and the extracted natural collagenase have a molecular weight of 110 k D,which is specific for type I collagen.The two may be the same enzyme.