Study On The Production Of Sophorolipids Based On Corn Straw And Heterologous Expression Of Lactonase And Its Catalytic Properties In Vitro

As a glycolipid biosurfactant,sophorolipids?SL?are low in toxicity,degradable,and have good environmental compatibility.They are widely used in petroleum,environmental protection,medicine and food.Sophorolipids not only have good physicochemical properties such as emulsification,dispersion and solubilization,but also some special types of sophorolipids have antibacterial,anti-inflammatory and anti-tumor biological activities,making them more valuable in the field of medicine.However,sophorolipids are not as invested in large-scale industrial production and applications as their demonstrated advantages.Compared with chemical surfactants,the production cost of sophorolipids is relatively high,and the components in the sophorolipids products are relatively complicated,so that the physicochemical properties of the final products depend on the composition ratio of different types of sophorolipids.Therefore,these two factors have become the main obstacles limiting the application of sophorolipids.In order to solve the problem of the diversity of sophorolipids,lactonase,the key enzyme in the lactonic sophorolipid synthesis pathway of Candida bombicola ATCC 22214,was heterologously expressed in Pichia pastoris GS115 in this study.And it was applied to in vitro catalysis to convert acid sophorolipids into more pharmaceutically valuable lactonic sophorolipids.After optimizing the induction expression conditions of the enzyme,the enzyme was purified by ammonium sulfate precipitation and His-tag affinity chromatography to obtain a higher purity lactonase.The enzymatic properties of the purified protein were characterized,and the mode of the acid sophorolipids lactonization reaction and the optimum enzymatic reaction conditions were determined.In order to reduce the cost of sophorolipid production,corn straw were used as raw materials,pretreated with solid acid,and the best process for preparing the hydrolyzate was determined.Then,the hydrolysate was used as the substrate to produce the sophorolipid by fermentation with C.bombicola ATCC 22214.The effects of hydrophobic carbon source,nitrogen source,metal ion,initial pH and fermentation temperature on the fermentation of sophorolipids were investigated to realize the conversion of corn straw into high value-added sophorolipid.The research results are as follows:?1?The lactonase gene in C.bombicola ATCC 22214 was cloned and the recombinant Pichia pastoris GS115/pPICZ?B-CBLE was successfully constructed.The high-efficiency heterologous expression of lactonase was achieved after induced by methanol.?2?The optimal conditions for the expression of GS115/pPICZ?B-CBLE lactones were as follows:induction temperature was 16?,the cells amount of initial inoculation was 2.18×10⁸,methanol concentration was 0.50%?v/v?,induction time was 60 h,the purpose protein concentration reached 2.41 g/L.?3?The enzymatic properties of lactonase were characterized,and the following results were obtained:the optimum pH of the enzyme-catalyzed reaction was 5.0,and the lactonase had better pH stability in the range of pH 4.0⁷.0;The optimum temperature for the enzyme-catalyzed reaction was 25?,and the lactonase maintained good thermal stability at20?;Metal ions and EDTA had no significant inhibition or promotion of the enzymatic activity of the lactonase.?4?In vitro catalyzed reaction of sophorolipids produced by corn straw fermentation with lactonase expressed by the recombinant Pichia pastoris GS115/pPICZ?B-CBLE,when the concentration of the acid sophorolipid was 8 mM,the temperature was 25°C,and the pH was5.0,the maximum conversion of the acid sophorolipids by the lactonase was 16.90%.?5?Investigating the optimal process for the preparation of corn straw hydrolysate,the results show that detoxification of corn strawhydrolysate by activated carbon adsorption and decompression rotary distillation can effectively reduce the presence of inhibitory compounds,and is helpful for the production of fermentation products.The optimum process conditions were treatment with 2%?w/v?activated carbon at 80?for 20 min and then concentrated at80?.The production of lactonic and total sophorolipids was increased by 231.78%and106.91%,respectively.?6?Through the study on the effects of hydrophobic carbon source,nitrogen source,metal ion,fermentation pH and fermentation temperature on sophorolipid production,it is found that when the hydrophobic carbon source in the medium was 6%?v/v?of Oleic acid,nitrogen source was 0.45%?w/v?ammonium nitrate,metal ions were 0.05%?w/v?Mg²⁺and 0.075%?w/v?Fe²⁺,and the pH of the broth was 3.0.The yield of total sophorolipids was 29.09 g/L.