Study On Highly Selective Synthesis Of 1,3-diacylglycerol Lipase Microorganisms In Non-aqueous Media

The purpose of this paper is to high selectively synthesize1,3-diacylglycerol innon-aqueous media by microbial lipase. Its content includes screening of lipase-producingmicrobial, optimization of the conditions of lipase-producing microbial, optimization of thereaction system of lipase-producing microbial, amplification test of lipase-producingstrain in small fermenter.An intracelluar lipase-producing strain (GZUF36) with high selective synthesis of1,3-diglyceide was screened by combination of Rhodamine B-olive oil agar plat,para-nitrophenyl palmitate (ρ-NPP) for ester synthetase activity and HPLC analysis ofproducts of glycerolysis of triglyceride. Then, GZUF36(CCTCC No. M2012538) wasidentified as Aspergillus niger. For cost saving, the intracelluar lipase produed by thestrain was directly used as whole-cell lipase without purification.Fermentation conditions were optimized for Aspergillus niger GZUF36(CCTCCNo. M2012538). Three key factors were identified by a design of Plackett-Burman onbase of a single-factor test. And the optimal concentrations of the main factors weregot by using steepest ascent test and Box-Behnken response surface design, increasingthe selectivity of lipase on1,3-diglyceride and the yield of diacylglycerol. The yieldof1,3-diglyceride was25.287%by optimal medium, which contained glucose1.030%, soybean meal1.785%and potassium chloride0.0441%. The yield of1,3-diglyceride was improved1.46times compared with before optimizationcondition. The proportion of1,3-diglyceride in the diglycerides was88.253%.Reaction system catalyzed by whole-cell lipase from Aspergillus niger GZUF36was optimized for selective synthesis of1,3–diglyceride. The factors included singleorganic medium, mixed organic media, the ratio of mixing organic media, wateractivity, substrate concentration, the molar ratio of substrate, the amount of enzymeand so on. The results showed that: when reaction system contained29:1of n-hexane:tert-butyl alcohol (V/V),0.427of water activity,0.02mol/L of triglyceride,2:1ofmolar ratio of triglycerides to glycerol and80mg/mL of enzyme dosage, the yield ofdiglyceride was highest (33.378%) and increased0.18times. The selectivity of lipase on1,3-diglyceride was88.75%.In order to obtain large amounts of whole-cell lipase, Aspergillus niger GZUF36was fermented in five litre fermentor. The fermentation conditions were as follows:initial pH6, the temperature30℃, speed180, DO1.7%, loaded liquid80%,inoculation2%and time six days. The results showed that: when Aspergillus nigerwas cultured for5days, the yield of diglycerides reached the highest yeld of25.850%,the selectivity of whole-cell lipase on1,3-diglyeride was88%.