Molecular Modification,Expression Optimization And Stability Study On D-psicose 3-epimerase From Clostridium Cellulolyticum H10

D-psicose,as a kind of natural sugar substitute with low calorie,good taste and various physiological activities,has seen a surge in market demand.The content of D-psicose in nature is extremely rare,and it is mainly produced by D-psicose 3-epimerase(DPE)catalyzed by the isomerization reaction of D-fructose in industry.The cost of producing DPE accounts for a large proportion of the cost of producing D-psicose.However,the poor thermal stability and poor storage stability of the most existing DPE have limitted the further industrial production of D-psicose.In this study,DPE from Clostridium cellulolyticum H10(CcDPE)was taken as the research object.The mutants with improved thermal stability and specific activity were obtained through directed evolution,and the related enzymatic properties of these mutants were studied.Then the catabolite responsive element(CRE)region of the promoter P_amyE was modified to increase the fermentation yield further.Finally,by adding stabilizers,the storage problems of CcDPE was solved,which established a foundation for further industrial production of DPE.The main research conclusions of the study are as follows:1.Improving the thermal stability and specific activity of CcDPE by molecular modification.The mutant A107P with improved thermal stability was obtained with directed evolution.On the basis of A107P,the H56Q site found to improve thermal stability,is superimposed to construct mutant H56Q/A107P.After incubating at 55?for 6 h,the residual enzyme activities of wild-type,A107P and H56Q/A107P were 21.5%,47.5%,and 67.3%,respectively.Due to the special side chain structure of proline,it was speculated that the mutant on H207 had reduced the flexibility of loop,and strengthened the rigidity of the overall structure,and improved the thermostability of enzyme.The mutant L159M/H207L with increased specific vitality was obtained with directed evolution.Mutants L159M and H207L were constructed for single point analysis.The specific activities of wild type,L159M and H207L at 60?were 326.3,337.2 and 514.5 U·mg^-1,respectively.The specific activity of H207L was significantly increased to 1.6 times that of the wild type.Through the analysis of the three-dimensional simulation structure of the mutant,it was found that the hydrogen bond between the H207L mutant and the surrounding E34 and S240 disappeared,which affected the traction between the?2 and?8 folds.The changes might be the main causes of the increse in the specific activity and decrease in the optimum temperature of the mutant H207L.2.Optimizing the expression of CcDPE by molecularly modifying the CRE region of promoter P_amyE.Based on the Bacillus subtilis CCTCCM 2016536/p HY300PLK-P_amyE-ccdpe constructed in the early stage of the laboratory,the CRE region of the promoter was modified to alleviate the carbon catabolite repression(CCR)effect during the fermentation process.Experiments have shown that by destroying the palindrome and symmetry of the CRE region or changing the distance from the target gene,the CCR effect can be effectively alleviated.The fermentation enzyme activity of mutant?109 in shake flask fermentation process reached to297.7%of the wild type.B.substilus/p HY300PLK-?109-H207L was constructed,and the high-density fermentation was carried out in a 3-L fermenter.On the 73 h of the fermentation progresses,the enzyme activity was 4567.4 U·m L^-1,reaching 2.0 times of current highest level of CcDPE fermentation,which could reduce production costs further.3.Improving the storage stability of CcDPE by stabilizers.The poor storage stability of most DPE at room temperature has become a limiting factor in industrial applications.The effects of stabilizers such as carbohydrate,metal ions and polyols on the storage effect of CcDPE was investigated.The results showed that the combination of 20%glycerol and 0.5mmol·L^-1 Co²⁺prolonged the half-life of CcDPE from 4.5 d to 79.6 d at 25?,which also improved the residual enzyme activity of H207L from 9.7%to 65.8%after 15 d storaged at25?.The results provide technical support for further industrialization of CcDPE.