Screening Of Lipase And Its Application In Non-aqueous Phase In The Synthesis Of Vitamin C Palmitate

Lipase is an ester hydrolase that can catalyze the hydrolysis of naturalsubstrates such as oils and fats （triglycerides） to produce fatty acid andglycerol. In the organic phase, lipase can catalyze the reactions as follows:esters synthesis, transesterification, peptide synthesis, ester polymerizationand acylation. The various reactions catalyzed by lipase in the organicphase can synthesize many high-value products. However, not all lipasecan catalyze esterification reaction in non-aqueous phase, resulting in thatthe existing lipases that can efficiently catalyze ester synthesis reaction arevery limited, and exploring of a lipase with new high ester syntheticactivity is becoming the focus for researchers. Vitamin C as a water-solubleis applied as antioxidant in food processing to preserve the food value. Butits water solubility and instability constrain its applications in the oil andcosmetics industries. Therefore, it is of great significance to convert vitamin C into vitamin C fatty acid esters with lipase to improve its value.In this paper, screening of lipase-producing strain, strain identification,optimization of fermentation conditions and catalyzation of lipase inorganic phase were carried out.In this paper, we first established HPLC analysis of L-ascorbic acidpalmitate. Screening from laboratory lipase-producing strains, we get twothat can catalyze the esterification of vitamin C, recombinant C. antarcticaZJB09193and wild strain A113, and C. antarctica ZJB09193can highlycatalyze synthesis of vitamin C palmitate. Based on the morphology,physiological characteristics and18S rDNA, A113was confirmed asYarrowia lipolytica.Then, flask fermentation conditions of Yarrowia lipolytica forproducing lipase were optimized as follows: olive oil10g/L, soybean meal40g/L, MgSO₄7H₂O1g/L, K₂HPO₄5g/L, inoculum size5%, pH6.0,28℃and the highest enzyme activity was reached after55h of fermentation.Using of culture medium prescription which has been reported, the15L（5.5L basal medium） tank fermentation culture conditions of Candidaantarctic ZJB09193were optimized as follows: ventilation10L/min,stirring speed800r/min, the initial pH5.5, the inoculum size10%and thedosage of inducer3500mL, a high amount of enzyme could be reached.Finally, the application of CALB in the synthesis of vitamin Cpalmitate was studied. The optimum reaction conditions were determined as:10mL of acetone as solvent; reaction temperature,50℃; shaking speed,180rpm; Vc,0.1056g; the amount of immobilized CALB,1.2g; molarratio of Vc and palmitic acid,1:7; water content,2%; reaction time24h,the final conversion approximately reached86%. Kinetic studies showedthat Km was100.49mmol/L and Vm was3.96mol/min.