Optimization Of Fermentation Conditions Of Two Engineering Bacteria That Express Thermophilic Enzymes From Thermotoga Maritima

With the continuous development of biotechnology,more and more researchers are committed to the development and use of extremophilic microbial resources.Many microbes and their enzymes in extreme environments have significant application value and research significance in industrial production.Thermotoga maritima belongs to extreme thermophilic microorganisms and is derived from deep-sea volcanoes.Because of its very special growth environment,it is difficult to directly use it for industrial large-scale fermentation.The current mainstream method is to use genetic engineering methods to clone the thermophilic enzyme gene and transfer it into an appropriate expression system to make it highly efficient,and to obtain industrially desirable heat-resistant enzymes.The subjects of this study were endo-?-1,4-glucanase?TM1525?and arabino-?-1,4-galactanase?TM1201?extracted from Thermotoga maritima.The high-efficiency expression conditions of the two proteases,the enzyme activity of the two enzymes were examined,and the fermentation conditions of the two enzymes were optimized using orthogonal experiments and response surface methodology.The main research contents and results of this paper are as follows:1.Two recombinant expression plasmids constructed in this laboratory were used to transform Ecoli.BL21,and two engineering bacteria were obtained,named as TM1525 engineered bacteria and TM1201 engineered bacteria.2.Using IPTG to induce expression,SDS-PAGE electrophoresis analysis,Western blot and other methods to analyze the expression,and optimize the expression conditions of engineering bacteria.3.The physicochemical properties of the two enzymes were analyzed using bioinformatics software.Ni-NTA chromatography column was used for purification.Two enzymes with higher purity were obtained and the enzyme activity was detected.4.Through single factor experiments,the effects of OD₆₀₀ value,culture time,temperature,IPTG amount and pH of the culture solution on the expression of the target enzyme were investigated.Determine the main factors and use this as a basis to determine the factors and levels of orthogonal experiments.5.Using the Plackett-Burman experiment,the steepest climbing test,and the Box-Behnken experiment,the response surface regression equation was obtained to further optimize the fermentation conditions.The optimal fermentation scheme for TM1525 engineering bacteria was:initial OD600 was 0.8,culture medium pH was 7.0,culture time was 15h,and enzyme production reached approximately 361.513mg/L,which was approximately 1.25 times higher than before optimization;The optimal fermentation protocol for TM1201 engineering bacteria was:setting IPTG amount 0.3 mM,culture temperature 18°C,inoculation amount 3%,and enzyme yield up to about 379.074 mg/L compared to about 1.28 times before optimization.