Heterologous Expression And Soluble Research Of Pullulanase

Pullulanase(EC.3.2.1.41)is a starch debranching enzyme that is mainly used for the saccharification reaction in starch processing.At present,most of the pullulanase reported in China is not high in thermal stability,specific enzyme activity,or can not adapt to the environment of acidic and high temperature.Therefore,finding and obtaining a pullulanase with high specific activity and resistance to heat and acid appears to be more important.The aim of this study was to use bioinformatics as a tool to screen out a putative pullulanase gene for heterologous expression in order to obtain a pullulanase with excellent enzymatic properties.The results are as follows:(1)Screening of pullulanase gene: Guided by bioinformatics,an acidic bacterial strain Candidatus Koribacter versatilis(DSM 22529)capable of coding pullulanase was screened from the database.Using the purchased genome as a template,we cloned the pullulanase gene KV-pul A(accession number: WP₀11523131.1).(2)Heterologous expression analysis: The recombinant plasmids p ET21a-KV-pul A,p ET22b-KV-pul A,p PIC3.5K-KV-pul A,p PIC9K-KV-pul A were constructed and introduced into Escherichia coli BL21(DE3)or Pichia pastoris GS115 for expression.Neither the pel B signal peptide nor the ? secretory factor can efficiently transport the protein to the extracell and intracellularly expresses in Escherichia coli was less than in Pichia pastoris.we finally chosen the intracell of Escherichia coli as the expression way of the target protein.(3)Optimization of fermentation conditions: To get the best soluble expression of Escherichia coli BL21(DE3),we explored the appropriate inducing conditions of the enzyme and finally determined the optimal inducer concentration was 0.1 mmol/L,the initial OD600 value of induction was 0.8 and the induction time was 6 h.(4)Analysis of crude enzymatic properties: The optimum p H range of the crude enzyme was 5.4 to 5.8,the optimal temperature was 60 ?,the half-life time under60 ? was 204 min.(5)Research on renaturation of inclusion body: Using the methods dialysis renaturation and ultrafiltration renaturation after purification,we found that simple dialysis renaturation did not work and although purified denatured protein was obtained after purification,ultrafiltration did not make the protein renaturation.(6)Fusion expression research: Trx A,GST,SUMO,Ars C,Ppi B,TF,MBP,NusA increased the solubility of the target protein to 21%,51%,30%,34%,8.6%,99%,85%,98%,respectively.Particularly,NusA fusion tag remain a significantly activity.