Construction Of Engineering Bacteria For Endo-?-1,4-Glucanase Gene From Thermotoga Maritima And Fermentation Condions Optimization

Endo-?-1,4-glucanase is a major member of the cellulase system and can hydrolyze soluble cellulose to reducing oligosaccharides.Among the many biological properties of enzymes,its thermal stability is very important.Because thermophilic bacteria cells that exhibit high tolerance to high temperature environments contain high temperature resistant enzyme systems,they have great application value and development potential.Although the strictly anaerobic thermophilic microorganism Thermotoga maritima,which grows in submarine volcanic environments,has the ability to produce endo-?-1,4-glucanase,it is not suitable for industrial scale due to difficult cultivation.It is of great significance to clone the endo-?-1,4-glucanase gene of the bacteria using molecular biology methods to construct Bacillus subtilis engineering bacteria capable of culturing at room temperature.1)The gene encoding endo-?-1,4-glucanase(TM1525)in Thermotoga maritima has an open reading frame of 825 bp and encodes 274 amino acids.Using the secreted pHT43 shuttle plasmid as a vector,a recombinant TM1525 cloned plasmid pHT43-TM1525 fused with a His-tagged protein was constructed and amplified by Escherichia coli DH5? to prepare a large number of recombinant plasmids.The recombinant plasmid was transferred into B.subtilis WB800 N for expression by electroporation.The hydrolysis circle experiment was used to prove the expression of recombinant protein and the activity of hydrolyzed cellulase.The polyacrylamide gel(SDS-PAGE)electrophoresis analysis of the fermentation supernatant and the bacterial cells precipitated showed that the target protein achieved extracellular secretion expression.2)The His-tagged recombinant protein was purified by Ni-NTA chromatography column,and the purified proteinase activity was studied by DNS method.The results showed a specific activity of 180.94 U / mg,the optimum pH was 7.0,and the optimum reaction temperature was80 ° C,and it has better thermal stability.After 60°C and 70°C for 2.5 h,it still has more than 80%residual enzyme activity.After incubation at 80°C for 2 h,it still has 60% residual enzyme activity.3)The production of recombinant protein was optimized by the statistical method ofResponse Surface Methodology(RSM).The experimental design selected three factors: induction temperature,induction time and isopropyl-?-D-thiogalactoside(IPTG)addition amount.The enzyme activity per ml of fermentation supernatant is the response value.Using the Box-Behnken experimental design principle,17 groups of experiments were performed,and the second-order polynomial model was obtained by using Design-Expert v8.0.6 software.According to the model predicted by RSM,the optimal fermentation process parameters were 40°C,0.27 mM IPTG induced 6.47 h,and the enzyme activity reached 58.21 U/ml.In order to verify the optimized results,three replicate experiments were performed,and the actual value obtained was 57.90 U/ml,which was not significantly different from the theoretical value,and the model was reliable.In summary,Bacillus subtilis,as a host strain,is not only easy to culture,but also has no pathogenicity.It also achieves extracellular expression of heterologous protein,and endo-?-1,4-glucanase has high thermal stability.It works at high temperature,is not easy to deactivate,and is easy to package and transport.It can be developed and used in large scale in the industry.It is widely used in the food industry,textile industry,environmental protection and other fields,and can be mass-produced to meet market demand.