Application Of The Lipase-displaying Aspergillus Niger To The Enzymatic Preparation Of Glycerylphosphorylcholine

L-alpha-glycerophosphorylcholine(GPC),comprised of choline,glycerol,and phosphate.It is the natural water-soluble phospholipid product that exists in human body,and is also well known as the precursor for producing acetylcholine and phosphatidylcholine(PC)in the body.As a modified product of phospholipids,it has very important value in the field of medicine and health care.Furthermore,it is helpful for all types of people,and is very effective for the treatment of various brain diseases in the elderly and has significant effect in the treatment of Alzheimer's disease and the elderly stroke.But now,in our country,the manufacturers of GPC were very few,and the preparation methods of GPC mainly used chemical method.However,there are many deficiencies in the chemical method,such as the low yield of GPC and the complex process.Therefore,how to achieve the enzymatic preparation of GPC,amplification of the preparation system and purification of GPC has became the focus of the current research.This study aimed at solving the problems of environmental pollution and high cost of preparation which were caused by the extensive use of organic solvents in the traditional methods of GPC preparation and purification,and had established a set of methods in the enzymatic preparation and green purification of GPC.In this research,the whole cell catalyst AN-CALB prepared in our laboratory was used to prepare GPC through alcoholysis reaction of PC25 in the non-aqueous phase system.Then,a 2ml GPC enzymatic preparation system was established in the study.And we studied the main influencing factors of enzymatic reaction which included substrate molar ratio,substrate concentration,amount of water,reaction medium,enzyme concentration and reaction temperature.As a result,we get the optimal results of these main influencing factors that the substrate molar ratio(ethanol?PC25)was 7?1,the substrate concentration(PC25)was 24 mg/mL,the amount of water was 60%([w water /w substrate]%),the reaction medium was isooctane,the enzyme concentration of AN-CALB was 20 mg/m L,and the reaction temperature was 60?.Then,we carried out the alcoholysis reaction of PC25 on the basis of optimal reaction conditions,and 98% conversion of PC and 94% yield of GPC was obtained after 8h.On the other hand,the mixed solvent(tert-butanol?methanol =4?3)was used to extract GPC after reaction,and then,the whole cell biocatalyst(AN-CALB)was recovered and was re-used in the next reaction.After 10 times used of enzyme,it still had a higher enzyme activity.And at the 10 th reaction,we obtained 96% conversion of PC and 85% yield of GPC,and the research results showed that the enzyme of AN-CALB had a good stability in the reaction.In order to study the possibility of our reaction's amplification,we gradually amplified the volume of the reaction to 1L and 15 L.The results of the 1L reaction system showed that 94% conversion of PC and 85% yield of GPC was obtained after 15 h,and 97% conversion of PC and 81% yield of GPC was obtained after 8h in the 15 L reaction system.The result of this experiment showed that the enzyme of AN-CALB still had higher catalytic activity in a large volume of reaction,and the reproducibility of the experimental results were very well in the large volume reactions.With the purpose of evaluating the efficiency of GPC extraction,we did 5 batches of reactions consecutively in reaction volume of 1L and 15 L.Although the GPC extraction rate was low in the first time,but it was higher than 80% in the next four experiments and the concentration of GPC was better than 0.3g/L.However,the purity of enzymatic reaction products were not high enough and required further purification.Therefore,silica gel column chromatography-activated carbon decolorization combinations for purifying GPC were established.The optimal condition for L-?-GPC purification by silica gel: the mobile phase flow rate,2 mL/min;loading concentration,2.8mg/mL;loading volume,200 mL;eluent,100% methanol;silica gel,100 g.The final GPC was decolorized with activated carbon at 60? for 1.5h and a colorless GPC aqueous solution was obtained.Using the method of freeze-drying to remove the water in the GPC aqueous solution,and GPC solid was obtained.Then,taking out a certain amount of GPC solid and dissolving in methanol to measure the purity of GPC by the method of HPLC-ELSD.Finally,GPC at 65.7% purity.