Gene Construction,expression And Immobilization Of Inclusion Body Glucose Isomerase

Glucose isomerase（GI）,as the enzyme with the largest consumption and the most successful commercialization,is mainly immobilized by cross-linking method,embedding method and adsorption method.In order to overcome the problems of complex process and high cost of traditional immobilization methods,this study explored a new self-immobilization technology,that is,the self-immobilization fusion tag and the enzyme gene are linked through a linker peptide and then introduced into the expression host E.coli BL21,and then induced to express.Immobilized inclusion body type glucose isomerase is obtained by expressing.Comparing the self-immobilized enzyme with the inclusion body-type protease prepared by the traditional post-immobilization method,the differences in the enzymatic properties of the two inclusion body type enzyme were systematically studied.The main research contents and results of this study are as follows:（1）Using the pET-30a（+）vector,the self-immobilized tag 8PD0/1/2,the connecting peptide sequence L1/2/3 and the codon-optimized GI gene as the basic elements,the enzyme ligation method was used to construct 3 strains of inclusion body type glucose isomerase engineering bacteria and 12 self-immobilized inclusion body type glucose isomerase bacteria;after induction and expression,the recombinant proteins were purified by ultrasonic-assisted urea washing method,with enzyme activity as the first indicator and protein content as the second indicator,respectively,one optimal strain was screened from two types of engineering bacteria:pET-L1-GI and pET-8PD1-L1-GI.（2）Using the enzyme activity and protein content as indicators,the culture conditions of the inclusion body type glucose isomerase engineered strain pET-L1-GI were optimized,the optimal parameters are as follows:the induction temperature is31℃,the induction start time is 5 h,the induction duration is 7 h,the final concentration of the inducer isopropyl-β-D-thiogalactoside（IPTG）is 0.4 mmol/L,and the ultrasonic condition is ultrasonic for 4 h of induction in 1 min,the enzyme activity of the recombinant protein was 37.96 U/m L enzyme solution,and the yield was1723.79μg/m L enzyme solution.（3）Using enzyme activity and protein content as indicators,optimize the induction temperature,induction start time,induction duration,inducer concentration,amino acid species and ultrasound condition parameters of the self-immobilized inclusion body type glucose isomerase engineering bacteria pET-8PD1-L1-GI,the results are:under the condition of adding valine with a final concentration of 10mmol/L to the medium,IPTG with a final concentration of 0.6 mmol/L was added after 6 h of culture,and after induction at 28℃for 6 h,ultrasonication for 1.5 min,continued After induction for 1 h,the enzyme activity of the recombinant protein was12.01 U/m L enzyme solution,and the yield was 962.23μg/m L enzyme solution.（4）The inclusion body type glucose isomerase of pET-L1-GI has poor reusability,and it was post-fixed by chitosan-glutaraldehyde cross-linking method and the conditions were optimized.The results showed that the concentration of chitosan was0.1‰,glutaraldehyde concentration of 0.25%,cross-linking p H of 8.0 and cross-linking time of 2 h had the best immobilization rate（74.78%）and enzyme activity recovery rate（87.48%）.The basic enzymatic properties of the pET-L1-GI post-immobilized enzyme and the pET-8PD1-L1-GI self-immobilized enzyme were studied.The basic enzymatic properties of pET-L1-GI post-immobilized enzyme and pET-8PD1-L1-GI self-immobilized enzyme were studied.It shows that:compared with the traditional post-immobilized enzyme,the optimum temperature of the self-immobilized enzyme increased from 75℃to 80℃,and the optimum p H decreased from 10.0 to 9.0;the self-immobilized enzyme has a smaller temperature applicable range,a wider p H applicable range,and better temperature stability and p H stability;three metal ions,Mg²⁺,Co²⁺and Mn²⁺,can promote the activities of the two enzymes,among which Mg²⁺and Co²⁺have the most significant effects,Ca²⁺,Zn²⁺and Ba²⁺have inhibitory effects on the two enzyme activities,and Cu²⁺has a post-immobilized enzyme activity.The K_mvalues of the two enzymes are close to have a similar affinity for the substrate.In practical simulation applications,both enzymes showed better activity in the reaction with maltodextrin hydrolyzate as substrate compared with pitaya juice as substrate;compared with the post-immobilized enzyme,when juice as substrate,the self-immobilized enzyme has better catalytic performance,and the actual relative enzyme is 2.1 times（using maltodextrin hydrolyzate as substrate）and 3.8 times（using pitaya）respectively.).