Fermentation Process Optimization And Immobilization On Haloalkane Dehalogenase

The production of a large number of halogenated compounds in the process of industrial and agricultural production has great harm to human health and the ecological environment.The use of haloalkane dehalogenase to degrade these compounds has the characteristics of mildness,non-pollution,non-toxicity and non-corrosiveness,and has gradually become a research hotspot in the treatment of environmental pollutants.Its wide application is currently mainly limited by the stability and yield of enzymes.In order to improve the yield and stability of haloalkane dehalogenase（Dha A）,the fermentation conditions were optimized at the shake flask level and the 5 L fermenter level,and the immobilization of haloalkane dehalogenase was studied.The main research contents and results are as follows:（1）The fermentation conditions were optimized at the shake flask level.Firstly,based on the TB medium,the components of culture medium were optimized by single factor experiments and response surface experiments;Secondly,the fermentation conditions were optimized on the basis of the optimal culture medium.The results showed that the optimal medium composition was glycerol 10 g·L^-1,yeast powder 23 g·L^-1,peptone 14 g·L^-1,Mg SO₄7H₂O 1.3 g·L^-1,Zn SO₄ 7H₂O 0.1 g·L^-1.The enzyme activity could reach 1182.94 U·L^-1,which was 94%higher than that of the initial medium;The optimal fermentation conditions were as follows:the loading volume was 40 m L/250 m L,the inoculum volume was 5%,IPTG with a final concentration of 0.4 mmol·L^-1 was added to induce 22 h at 20°C,when the OD₆₀₀ was 1.8at 37°C.The enzyme activity could reach 3585.83 U·L^-1,which was 5.87 times higher than the initial conditions.（2）Fermentation conditions were optimized at the level of 5 L fermenter.Firstly,the initial carbon source concentration,induction temperature,and induction p H were optimized in the batch fermentation experiments;Secondly,the inducer adding time,the feeding strategy in the induction period and the concentration of the inducer were optimized in the fed-batch fermentation experiments;Finally,the protein yield was verified on the optimal fermentation condition.The results showed that the optimal fermentation conditions of the batch fermentation were as follows:the initial carbon source concentration was 20 g·L^-1,the induction temperature was 25℃,and the induction p H was 7.0.Under these conditions,the activity of enzyme could be achieved at 8905.95 U·L^-1.The results of the optimal fermentation conditions of the fed-batch fermentation are as follows:the temperature was reduced to 25°C and induced by adding IPTG with a final concentration of 0.4 mmol·L^-1,when the bacterial concentration(OD₆₀₀)reached 45,and then exponential feeding was performed at a specific growth rate of0.15 h^-1.When the feeding rate reached 20 m L·h^-1,the constant feeding rate was carried out at this rate until the end of the fermentation.The enzyme activity could reach 35360.00 U·L^-1,which was 3.97 times higher than that of the batch fermentation,realizing high cell density fermentation of E.coli.Finally,the wet weight of the bacteria was 212.33 g·L^-1,and the dry weight of the bacteria was 51.40 g·L^-1.The concentration of total protein concentration was30.67 g·L^-1,and that of the purified protein was 11.93 g·L^-1.（3）Haloalkane dehalogenase was immobilized by metal-organic hybrid nanoflowers.Firstly,the immobilization conditions were optimized.Secondly,the morphology,functional groups and crystallization of the immobilized enzyme were characterized by SEM,FT-IR and XRD,respectively.Finally,the enzymatic properties of free and immobilized enzymes were determined.The results showed that the best immobilization conditions were as follows:Fe³⁺with a final concentration of 2.4 mmol·L^-1,phosphate concentration with a final concentration of 20 mmol·L^-1,protein concentration with a final concentration of 0.07 mg·m L^-1,and p H 8.0for 60 h.The recovery rate of enzyme activity could reach 138.54%.Compared with the free enzyme,the optimum temperature and optimum p H of the immobilized enzyme were not changed.However,the immobilized enzyme showed a higher activity than that of the free enzyme over the entire temperature and p H gradient.Meanwhile the p H stability and storage stability of the immobilized enzyme were improved.The relative enzyme activity of the immobilized enzyme was still above 40% after 6 recycling use.