The High-effiency Secretory Expression,Separation And Purification Of Benzonase Nuclease Nuc A

Benzonase nuclease(Nuc A)is a nonspecific nucleic acid hydrolase derived from Serratia marcescens.It has high activity of hydrolyzing nucleotides and can degrade most forms of nucleotides.So it has important application value in biological products industry.Because Serratia marcescens can cause human diseases,the current recombinant nuclease expression system mostly uses Escherichia coli and Pichia pastoris expression system.But the E.coli has insufficient exocrine capacity,it is easy to form inclusion bodies and P.pastoris expression system has excessive glycosylation modification affecting enzyme activity,so it failed to achieve higher level of expression.Therefore,Bacillus licheniformis 2709 was used as the host for expression in this study.In view of its advantages of good production performance,simple culture components,simple and controllable culture conditions,clear genetic background,low fermentation cost,stable expression products,etc.It is widely used in the industrial production of various enzyme preparations and antibiotics.Compared with E.coli,B.licheniformis has the advantages of strong extracellular protein secretion ability and beneficial to downstream separation and purification process.Compared with P.pastoris expression system,B.licheniformis has a shorter fermentation period and relatively simple post-translational modification system,and does not carry out excessive glycosylation modification to affect enzyme activity.In this study,we screened B.licheniformis 2709 endogenous expression elements to study the recombinant expression of nuclease in view of the better compatibility between the expression regulatory element and host bacteria.Firstly,the main extracellular proteins secreted by B.licheniformis 2709 were isolated and identified by mass spectrometry,and the location and sequence of expression regulatory elements were obtained by analyzing the host genome data.In addition,B.licheniformis 2709,as one of the expression systems of Bacillus,is an important strain for industrial production of amylase and alkaline protease.Meanwhile,expression vectors for nuclease can be constructed by using regulatory elements related to gene expression and secretion of these enzymes.Combined with the genomic data of host bacteria,the expression regulatory elements were identified as apr,ggt,chi and amy.Recombinant vectors were constructed by using promoter,signal peptide and other expression regulatory elements,and single copy integrated expression of genome was carried out by gene editing technology.The enzyme yield of mutant strains was detected,and the selected apr expression element could effectively regulate the nuclease gene expression,with the highest yield of 27000±727 U/m L,significantly higher than that of the control strain.The other three expression elements could promote the nuclease expression,but the expression levels were all lower than the apr-mediated integrated expression.Considering that the high expression capacity of constitutive promoters often leads to the overexpression of exogenous genes,thus hindering the normal growth of bacteria,inducible promoters can effectively control the spatiotemporal nature of gene expression.In addition,nuclease was recombinant expressed by plasmid free expression in this study.The result showed that the highest free expression yield of apr-mediated high copy plasmid was 21596±671 U/m L at 48 h.And results showed that the expression of apr components consistent in terms of the expression of different genes,speculated that the purpose gene expression and regulation has a similar mechanism,compared and integrated expression plasmid of nucleic acid enzyme gene expression form of free expression is more efficient,in the fermentation conditions,the integrated expression can make nucleic acid enzyme for the synthesis and accumulation of stable efficiently.In order to further improve the expression level,this study adopted the method of multi-copy integration to increase the concentration of nuclease genes in the genome.The results showed that when the genome carried two copies,the nuclease production increased 14.64%,up to 30954±330 U/m L compared with the single-copy integration.On this basis,the fermentation conditions were preliminarily optimized by single factor screening test.Considering that nuclease was secreted under the environment of nutrient shortage,the medium carbon source was replaced by simple carbon source,in order to shorten the fermentation period and improve the enzyme yield.The results showed that when corn meal was replaced with sucrose and glucose,the enzyme yield increased by 76.16%and 83.9%,respectively,and the expression level of Nuc A was greatly increased.In this study,the element apr that can achieve high expression regulation of Nuc A was successfully screened,and a multi-copy integrated strain BL apr-Nuc A-2 with high yield of Nuc A was constructed.The enzyme yield was improved through preliminary optimization of fermentation process,and the expressed product was preliminarily separated and purified.Finally,the specific activity was 1.69×10~7 U/mg,which realized the high level of recombinant expression of Nuc A,which provided a basis for the further industrial production and application of Nuc A,and also provided a reference for the expression of other toxic proteins in the system.