Screening Goat Rumen-derived Protease Geneand Its Heterologous Expression And Verificationbased On Fosmid Library

Investigating effective proteases is advantageous for growing the current protease resource pool because they are the most often utilized enzyme preparation in animal husbandry.Proteases generated from microorganisms are easily extracted,have a welldefined genetic basis,and have significant economic advantages.Microorganisms can be used as a source of effective proteases.The genome of rumen microbes contains a wealth of unexplored information,and current study mostly focuses on rumen microbial cellulase in ruminants.Few studies have been conducted on rumen microbial protease genes,and conventional in vitro cultivation techniques make it challenging to get proteases made by non-culturable microbes.Therefore,this study avoided using traditional culture techniques and instead used the Fosmid library function screening method to screen the genes with protease activity from goat rumen microorganisms,realizing the high expression in Escherichia coli and Bacillus subtilis;the engineering bacteria without resistance markers were obtained using CRISPR/Cas9 technology,and the research results have reference significance for investigating gene resources with protein degradation.(1)The Tibetan goat rumen Fosmid library produced a positive clone Pro4-C5 with a potent skim milk powder degradation plate.Casein served as the substrate for the discovery of Pro4-C5 protease.The examination of enzymatic characteristics and the enzyme activity indicated that the ideal reaction temperature and p H were 40 and 7.5,respectively.(2)Six putative proteolytic enzyme genes,including gene0170,gene0196,gene0400,gene0585,gene0683,and gene0833,were discovered in Pro4-C5.They were all highly similar to known protein sequences(> 98%)when compared to the NCBI Blast P database,however no functional verification of these proteins was carried out.Other target genes translated amino acid sequences that,except for gene0683,were similar to those of known proteases.According to the sequencing study,genes 0170 and 0683 are amidase hydrolases and exhibit strictly preserved diamidase hydrolysis catalysis,metalloproteinase-catalyzed triplet His-Glu-His structure is present in gene0196.Genes 0400,0585,and 0833 feature triplet Ser-His-Asp structures that are catalyzed by serine proteases.(3)The 6 chosen functional genes were seamlessly cloned into the p ET-28(+)vector and then transferred to Escherichia coli BL21(DE3)to successfully induce expression.Genes 0170,0683,and 0833,whose molecular weights were 20,37,and 87 k Da,were successfully induced,according to an SDS-PAGE study.It was found by enzymic characteristic analysis that the protease activity expressed by gene0833 can reach 10.45U/m L under the ideal reaction circumstances(50 ℃,p H 7.5),which is higher than the protease activity produced by gene0170(65℃,p H 7.5)and gene0683(50℃,p H 7.5)under the ideal reaction conditions(P < 0.05).When casein was used as the substrate for the protease produced by gene0170 and gene0833,it resulted in more activity,and gene0683 had stronger activity with skimmed milk powder as substrate(P < 0.05).Moreover,gene0683 exhibits significant L-asparaginase activity up to 88.52 U/m L.(4)Codon optimized gene0683 was inserted into the Bacillus subtilis C6 genome using CRISPR/Cas9 gene editing technology.This resulted in a substantial 8.67% increase in protease activity and a significant 30.06% rise in L-asparaginase activity.The existence of gene0683 as a bifunctional gene was further confirmed.In summary,the gene0683 with both protease activity and L-asparaginase activity was found from the rumen metagenomic Fosmid library of goat,enriched the current protease resource library,and provided a theoretical basis for screening novel protease genes based on Fosmid library.