| D-psicose has a good sweetness,contains the other characteristics such as low heat value at the same time.It is a kind of high value of functional rare sugar that is widely used in food,medicine,health products and other fields.Enzymatic preparation of D-psicose has the advantages of high specific efficiency,mild reaction conditions,and is gradually used in large-scale industrial production.D-psicose 3-epimerase?DPEase?specifically catalyzes the epimerization of D-fructose C3 to produce D-psicose.The DPEase gene from Clostridium cellulolyticum H10 has good thermal stability and catalytic efficiency.In this study,DPEase from Clostridium cellulolyticum H10 was expressed in Bacillus subtilis CCTCC M 2016536 and its enzymatic properties were studied at first.Then shake-flask and 3-L fermentor experiments was optimized to increase the DPEase expression.Finally,the process optimization of D-psicose production by recombinant enzyme and immobilized recombinant cells were studied,which provided the foundation for industrial production.The main results are as follows:?1?We constructed two recombinant strains of B.subtilis/pHY300PLK-Pamy Q-dpe and B.subtilis/pHY300PLK-Phpa??-PamyQ-dpe.After the recombinant strains was cultured in shake flask for 48 h,the intracellular DPEase activity was 149.2 U·mL-1 and 147.5 U·mL-1.The target protein band with a molecular weight of 33 k Da indicated that the DPEase from Clostridium cellulolyticum H10 was successfully expressed in B.subtilis.Then the enzymatic properties of the recombinant DPEase were examined.The results showed that the optimum pH was 7.5,and the optimum temperature was 65?.Under the conditions of pH 7.5 and 0.1m M Co2+,the half-life of 55?and 60?were 3 h and 0.5 h,respectively.The stability of the reombinant DPEase was poor,and the whole cells had the best storage effect at 4?.There was still 82%residual enzyme activity after 15 days.?2?First,culture medium during shake-flask experiments was investigated.The optimized medium contains 20 g·L-1 soy peptone,15 g·L-1 corn steep powder,5 g·L-1 glycerol,1 mM Ca2+,K2HPO4 12.54 g·L-1,KH2PO4 2.31 g·L-1,the enzyme activity of DPEase reached314.3 U·mL-1.Then the different C/N ratios in the feeding medium,fermentation pH and temperature were tested for DPEase expression in a 3-L fermentor.The results showed that the optimal feed C/N for fermentation of 1:2,the pH 7.0,the temperature 33?,the enzyme activity reached 2246.3 U·mL-1,which was 15 times greater than that seen in the original shake-flask experiments.Finally,site-directed mutagenesis was performed on the PamyQmyQ promoter.It effectively relieved the effect of protein transcription inhibition due to high carbon source concentration.The DPEase activity increased from 495.4 U·mL-1 to 1030.6U·mL-1 by recombinant strain mutant B.subtilis/pHY300PLK-PamyQ/G212A-dpe when using a high concentration of carbon source in a 3-L fermentor.It shorten the fermentation period and reduced the cost.?3?The effects of different reaction conditions on the preparation of D-psicose by recombinant DPEase were examined.The optimum conditions were determined as follows:pH 7.5,reaction temperature 60?,the enzyme dosage added 30 U·g-1 fructose substrate,the reaction time 4 h,and the substrate concentration 300 g·L-1 fructose.The maximum conversion rate of D-psicose reached 28.17%.Then the production of D-psicose of the recombinant cells was optimized.When 300 g·L-1 fructose was used as a substrate,10 g·L-1whole cells were added and the reaction was performed at pH 7.5 and 65?,the conversion rate can reach 27.48%after 1 h.?4?The recombinant cells were immobilized by diatomite-sodium alginate?adsorption-occlusion method?.The optimized immobilized conditions were as follows:2%sodium alginate,50 g·L-1 cell concentration,2%CaCl2 and 1%diatomite.Under the optimum conditions,the recovery rate reached 64%.Compared with the free cells,the immobilized cells had the same optimal pH,the optimal temperature was 70?,5?higher than the free cells.The conversion rate was still 28%after 7 times repeated operations,it was also maintained 81%of the residual activity. |
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