Separation And Purification Of Extracellular Lipase From Aspergillus Niger GZUF36 By Reverse Micellar Extraction And Investigated Its Enzymatic Properties,Immobilization Of Lipase

1,3-diacylglycerol is a healthy oil which can be converted into energy through the digestive system of body without accumulation,it can reduce the chance of obesity.It is the most effective way to prepare 1,3-diglycerides by lipase.Enzymatic method for preparing 1,3-diglycerides contains ester synthesis,glycerolysis,transesterification and triglyceride hydrolysis.Triglycerides direct hydrolyse is the most simple method for preparing 1,3-diglycerides,but it requires the lipase has the ability to specifically hydrolyze Sn-2 ester bond of triglyceride,and this type of lipase was reported few at home or abroad.In our previous study,we selected an Aspergillus niger strain GZUF36 with a high Sn-2-position selectivity of extracellular lipase.However,the strain showed a decline in producing extracellular Sn-2 lipase.So,in this paper,we rejuvenated Aspergillus niger GZUF36 to produce the Sn-2 lipase.And then we optimized the best conditions of purifying this lipase,investigated the enzymatic properties and conducted the enzyme immobilization.First,the culture medium?0.5% beef extract,0.5% peptone,1.0% olive oil,0.2% ammonium sulfate,0.5% dipotassium phosphate,0.02% magnesium sulfate,0.01% calcium chloride,0.5% sodium chloride?was successful in rejuvenating Aspergillus niger GZUF36 to produce Sn-2 lipase.Hydrolase activity of this extracellular lipase was 7.92±2.23 U/m L.The yield of 1,3-diacylglycerol and the positional selectivity were about 16.34±1.27% and 82.67±3.01%,respectively,by the liapse-catalyzed direct hydrolysis of triglycerides.The activity of the extracellular Sn-2 lipase and the yield of 1,3-diacylglycerol were lower than before,but the positional selectivity of lipase was restored to the previous level.So the recession of Aspergillus niger GZUF36 can still produce high Sn-2 selective lipase after rejuvenation.The activity of the extracellular Sn-2 lipase and the yield of 1,3-diacylglycerol can be improved by optimizing the culture medium and culture conditions of Aspergillus niger GZUF36.And then,the extracellular Sn-2 lipase was separated and purified by acetone precipitation and reverse micelle extraction.The optimum conditions of acetone precipitation were as follows: the volume ratio of acetone and fermentation broth was 2.5,temperature selected-18 ?,time chose one hour.Under the optimal conditions of acetone precipitation,the recovery rate of lipase activity was 73.2 ± 1.36% and the purification factor was 2.13 ± 0.072.Forward extraction of lipase was found to be maximum using aqueous phase of p H 9.0 Tris buffer contained 0.075 M NaCl and organic phase contained 0.125 M CTAB,which consisting of 75% isooctane,15% butanol and 10% hexanol.The organic phase and the aqueous phase were mixed at a volume ratio of 1: 1 and extracted at 30? for 30 min.In case of backward extraction,lipase was extracted from the organic phase to a fresh aqueous phase in Tris buffer containing 1.5 M KCl,10% anhydrous ethanol?pH 6.5?.The organic phase and the aqueous phase were mixed at a volume ratio of 1: 1 and extracted at 30 ? for 1.5 h.The activity recovery and purification factor of lipase were found to be 76.8% and 4.76±0.092,respectively.After purifying by acetone precipitation and reverse micellar extraction,the purity of lipase was 10.14 times than before.The purified enzyme was determined by SDS-PAGE and LC-MS / MS.The final results were retrieved in NCBI and found this purified enzyme belong to the family of lipase.Analysis of the extracellular Sn-2 lipase properties suggested that: the optimal temperature and pH were 35 ? and 6.5,respectively.Below 25 ?,the lipase has a good stability,but sensitive to high temperature.And the lipase was relatively stable at pH range 6.5-7 after incubated in different pH solution.But lipase incubated at pH <5 or pH> 8 for 12 hours,the enzyme activity of lipase loss highly.Mg²⁺?Ca²⁺ have a strong effect on the activity of lipase,but Zn²⁺?Mn²⁺?Al3+ have a certain inhibitory effect,Fe²⁺?Cu²⁺?Ni²⁺ have a strong inhibitory effect of lipase activity.The research to substrate specificity of lipase was found that corn oil,sesame oil,soybean oil have a strong effect on the activity of lipase,and rapeseed oil,olive oil,peanut oil have a certain promoting effect.This may be due to more linoleic acid were found in corn oil,sesame oil and soybean oil.Enzyme kinetic studies found that the lipase had Vmax and Km at 37.17 mM/min and 9.28 mM,respectively.Through single factor experiments and orthogonal experiments we found the optimum conditions to immobilized lipase by sodium alginate.The optimized conditions of preparing immobilized lipase were 3% of sodium alginate,the volume ratio of carrier to enzyme was 2:1 and 60 minutes immobilized time.Under these conditions,the immobilization efficiency of lipase was 63.67 ± 1.71%.The immobilized enzyme was subjected to repeated hydrolysis of olive oil for 6 times,and it was found that immobilized lipase had a better stability of 4 times,the enzyme activity was maintained 89.17 ± 0.61%.But this experiment repeated 6 times to retain only 73.91 ± 1.58% of the enzyme activity.This may be due to materials of immobilization were not suitable and repeated experimental operation caused the loss of enzymes or other reasons.