Study Of Gene Construction And Expression Of Self-immobilized Lactase And Its Enzymatic Properties

Lactase as a common enzyme preparation in the food industry,it is widely used in dairy processing and functional polypeptides.However,there are problems such as poor stability,difficult separation and can not be reused in the use of free lactase.With the actual demand of producing low lactose dairy products,the immobilization technology of lactase was appeared,which could made up for the deficiency of free enzyme.Due to the limitation of technology and cost,the traditional immobilization methods include adsorption,embedding,crosslinking and covalent binding.With the emergence of self-immobilization fusion label,it is expected to become a new immobilization technology.The main purpose of this study is to construct a self-immobilized fusion label and express it with the enzyme gene,in order to construct an engineered bacterium capable of producing self-immobilized lactase.The main achievements are summarized as follows:(1)Based on our previous research on TunaAI tandem protein,two Tuna AI analogs,PVHIKFGD and PVFIKFGD,with successively enhanced hydrophobicity,were designed using the TunaAI amino acid sequence PTHIKWGD as the template.The 4-polymer genes of three tandem units PDn(n=0,1 and 2)were designed using the peptide expression master software and chemically synthesized.The 8,16 and32-polymer genes were successfully constructed on the p UC18 cloning vector using isotailase recursive construction method,and then 12 expression vectors p ET-m PDn(m=1,2,4 and 8)were constructed.The expression vectors were transferred into Escherichia coli BL21 for induction expression.By verifying the relationship between gene copy number and gene expression level,it was found that only BL21-p ET-8PD0and BL21-p ET-8PD1 in the 32 polymeric genes could efficiently express the target protein,which could be used as a self-immobilization fusion label.(2)Using the genomic DNA extracted from E.coli BL21 as a template,the lactase Lac Z gene was obtained by PCR amplification method.By linking the self-immobilized fusion tag with the lactase gene,the engineering bacteria BL21-PET-8PD0-Lac Z and BL21-PET-8PD1-Lac Z were successfully constructed.Tris-SDS-PAGE electrophoresis was performed on the two kinds of engineered bacteria to detect the expression products,it was found that only BL21-PET-8PD0-Lac Z could efficiently express the target protein.Using ultrasonic-assisted urea method to purify the target protein inclusion bodies,the yield of the obtained inclusion bodies was increased by 21.88%,the yield was 1.55 g/L in fermentation broth,and the enzyme activity was 1.01 U/mg inclusion bodies,which was better than the Ni²⁺column affinity method.(3)The fermentation medium of self-immobilized lactase engineered bacteria was optimized.By measuring the expression level of recombinant protein,the yield of inclusion bodies and enzyme activity indexes,the 2×YT medium was finally selected as the most suitable induction medium.On this basis,the influence of the strain's pre-induction culture time,induction duration,induction temperature and amino acid presence on the fermentation performance of the strain was studied.The study showed that the expression level of recombinant protein,the yield of inclusion bodies and enzyme activity are not directly corresponding to the positive correlation,it should be selected according to the actual situation.The optimal culture conditions were as follows:incubation time was 4 h before induction,induction duration was 8 h,induction temperature was 37?,and serine was added during induction,under this condition,the yield of the obtained inclusion bodies was 2.00 g/L in fermentation broth,and the enzyme activity was 6.65 U/mg inclusion bodies.(4)The research shows that the traditional denaturation-dialysis renaturation method cannot be used for renaturation of self-immobilized lactase.Ultrasound-assisted renaturation of self-immobilized lactase in PBS buffer solution with different concentrations of urea was explored.The results showed that the renaturation effect was the best when the concentration of urea was 1.5 mol/L,and the enzyme activity reached the maximum,which increased by 90.24%compared with the control group.Fourier transform infrared spectroscopy detection shows ultrasound did not change the secondary structure of the enzyme in a wide range,and the change of the content was less than 2.3%;ultrasound probably promotes the aggregation between proteins(quaterrestrial structure),thus affecting the enzyme activity.The enzymatic properties of the self-immobilized lactase were studied.The optimum reaction temperature of the enzyme was 40?,and the optimum reaction p H was 6.0-7.0.The enzyme had good thermal stability,strong acid and strong alkali would cause great loss of enzyme activity.The Km of michaelis constant of the self-immobilized lactase was low,so it had a good affinity with the substrate.Simulation practical application research shows that,the enzyme activity of the self-immobilized lactase could be maintained more than 90%within 10 days after storage at 4?,and the residual enzyme activity was51.3%after 30 days.The residual enzyme activity was 51.33%after repeated operation for 5 times,for 10 times its residual enzyme activity was 31.53%.A continuous lactase catalysis column was prepared by mixing the self-immobilized enzyme and pulp evenly.At room temperature and at a flow rate of 2 m L/min,the continuous catalysis could be achieved for more than 6 hours.It was added into pure milk to hydrolyze lactose,the lactase hydrolyze rate was 73.72%when hydrolyzed at37?for 3 h,which can meet the needs of industrial production.