Fermentation Optimization Of CALB Displaying Pichia Yeast As Whole Cell Lipase Catalyst And Its Application On The Modification Of Natural Vitamin C

As a natural antioxidant, vitamin C has attracted considerable attention. However, the highly hydrophilic property limits its application. With the maturity of the two stage fermentation technology of vitamin C production, there is overcapacity in vitamin C production allover the world. Therefore, it is of great significance to modify vitamin C, so as to expand its usage, to increase its added value, and to alleviate overcapacity crisis as well. It's well known that biological methods have more advantages over the chemical methods in vitamin C modification. Yet the high cost and complicated produce of lipases remains the main obstacle for a commercially feasible enzymatic modification of vitamin C, thus vitamin C modification is limited to laboratory, unable to carry out at large industrial scale. The aim of this research is to produce an novel whole cell lipase catalyst with high efficiency and low cost by fermentation and to explore its application in the enzymatic synthesis of L-Ascorbic acid esters. To enhance the catalytical activity of the whole cell lipase catalyst, optimization of the fermentation and lyophilization were studied as well.In the fermentation optimization of the recombined Pichia yeast which surface displays Candida Antarctica lipase B (CALB), glycerol (carbon source) content, liquid volume and rotating speed were considerd in the cells enrichment period. In induce phase of enzyme prodution, the content of methanol, the adding pattern of it, the induce time, liquid volume and rotating speed were taken into consideration. As a result, the optimized condition for cells enrichment was:4% glycerol in BMGY, with 25mL/250mL liquid volume,250 r/min rotating speed. Cells density (OD600) increased to 55.4 from 24.68 after optimization The optimized condition for enzyme induce was:1% initial methanol content in BMMY, adding 0.75% methanol per 24h, inducing 144h with 12.5mL/250mL liquid volume and 250 r/min rotating speed. Enzyme activity per fermentation unit rose to 30.5 U/mL from 11.7 U/mL by optimization. In the enzyme property assay of CALB displayed on Pichia yeast. The optimum pH is 8, the optimum temperature range is 40℃in the catalytical hydrolysis of p-nitrophenol palmitate. Meanwhile, the thermal stability studies showed that Pichia displaying CALB has good thermal stability, after 4h at 40℃the the residual enzyme activity was still over 88%; after 6h at 50℃the residual enzyme activity kept over 50%.Synthesis of fatty acid ester of L-ascorbic acid must be carried out in non-aqueous phase. So CALB displayed on Pichia yeast must be dried before use. Here its freeze-dried conditions were optimized. The Optimized freeze-drying conditions were as follows:pH "memory" at pH 7.5 phosphate buffer; bioimprinted with oleic acid, freeze-dried with 5% skim milk/5% trehalose (1:1) as cryoprotector, The enzyme activity increased 5 times after freez drying.Synthesis of ascorbyl palmitate catalyzed by yeast whole cell ROL was successfully conducted. The process was strongly influenced by type of organic solvent used, mole ratio and concentration of substrates, molecular sieve, reaction temperature and the shaking speed. Under the optimum condition (palmitic acid/ L-ascorbic acid:7/1 (0.5 mmol L-ascorbic acid) at 45℃and 200 r/min with 0.5g of whole cell ROL in 5ml tetrahydrofuran), a maximum conversion (95.5%) of L-ascorbic acid was achieved after 36h transformation.