Preparation,modification And Application Of High Sn-1,3 Specific Lipase ROL

Diglycerides are widely found in various vegetable oils,including 1,2-diglycerides and 1,3-diglycerides.The oil rich in 1,3-diglycerides has various physiological functions including prevention of obesity,prevention of heart and brain diseases such as hyperlipemia,hypertension,and is therefore considered as "healthy oil".At the same time,it is an important raw material for the synthesis of some compounds such as the structural lipid 1,3-dioleoyl-2-palmitoylglyceride.Because of the low content of 1,3-diglycerides in natural oils,the preparation of oils rich in 1,3-diglycerides is important.In preparation of 1,3-diglycerides,there exists some defects such as the regioselectivity of lipase,the degree of reaction as well as separation and purification,resulting in a relatively low concentration of 1,3-diglyceride in products,which seriously affects the wide application of the product.In this work,we firstly studied the acyl migration of 1,3-diglyceride at different temperatures,and the temperature of the esterification reaction was initially selected to be 30OC.At this temperature,the regioselectivity of different lipases were assessed,and obtained the lipase(Rhizopus oryzae lipase)with highest sn-1,3-regioselectivity(>97%).In order to improve the dispersibility and recyclability of ROL in the reaction system of oleic acid with glycerol,lipase ROL was immobilized on nano-sized Fe3O4(15-20 nm).Fe3O4 was prepared and then modified with APTES and glutaraldehyde,and followed by immobilization of lipase ROL.With the help of "interfacial activation"of surfactant-sucrose ester SE-11,the activity of the immobilized lipase was greatly enhanced.The hydrolysis activities were 9.16-and 31.6 times of free enzyme,respectively,when p-NPB and p-NPP were used as substratesThe esterification reaction of oleic acid with glycerol catalyzed by immobilized ROL was optimized in a 1 L reactor.Under the optimal condition(30?,enzyme dosage was 8‰,molar ratio of oleic acid to glycerol was 2.8:1,vacuum degree was within 500 Pa,stirring speed was 400 rpm),the mono-glyceride could reach less than 3%after reaction for 8 h,and the content of 1,3-DAG reached about 76%.The immobilized enzyme retained about 75%of its initial activity after used for 55 batches.The excess oleic acid was removed by neutralization with NaOH or Na2CO3.The purity of 1,3-DAG was up to?95%(w/w)after purification with the recovery ratio?85%.In order to make the acquisition and modification of lipase ROL easier,heterologous expression of lipase ROL was achieved in Pichia pasioris GS115.The effect of leader sequence of ROL on its expression and regioselectivity was investigated.The activity in the fermentation broth reached 311 U/ml when the recombinant with leader sequence was induced for 96 h,which was about 2 times of that without leader sequence.And the former owns better sn-l,3-regioselectivity than the latter,the regioselectivities were>97%and?92%,respectively.Because the thermostability of ROL is poor,it is necessary to enhanced.Based on the results of multiple sequence alignments,two mutants with significantly improvement of thermostability were obtained,they were V209L and D262G(the half-lives at 55? were 4.38-and 4.2 times of the original one,respectively).Among them,mutant V209L leads to the decrease of the length of hydrogen bond,and the introduction of glycine at position 262 alleviates conformational strain and eliminates unfavorable steric interactions,thereby enhancing the thermostability of the protein.With the help of program DbD2,a new disulfide bond(E190C/E238C)was introduced into ROL,and the half-lives of this mutant were 72.4-and 8.33 times of the original one at 60 and 65?,respectivily.By combined mutations,a quadruple mutant V209L/D262G/E190C/E238C was obtained,whose half-life at 65? was 20 times of the original enzyme.The hydrolysis activity of quadruple mutant towards p-NPB maintained the same level to that of wild-type ROL.After enhancement of thermostability,the expression level of ROL was enhanced by increasing the gene copies of ROL in Pichia pasforis.After induction for 96 h,the activity of transformant with 3 copies reached 386 U/ml,which was about 25%higher than that with single copy.The thermostability-enhanced lipase was immobilized on nano-sized Fe3O4.With the help of "interfacial activation" of sucrose ester SE-11,the hydrolysis activity recovery of the immobilized enzyme reached 519.8%.The immobilized enzyme exhibits excellent performance in catalyzing the esterification reaction of different fatty acids with glycerin.Further,we attempted to investigate the regioselective mechanism of lipase ROL by molecular dynamics simulation On the basis of molecular simulation,when the lipase forms an enzyme-substrate complex with two diglycerides,the distance between hydroxy oxygen atom of the l45Ser of the catalytic triplet and the sn-1 carbonyl carbon atom of the 1,3-diglyceride was about 3-3.5 A,and when the complex is formed with 1,2-diglyceride,the distances between the hydroxy oxygen atom of the 145Ser and the sn-1/sn-2 carbonyl carbon atom of the 1,2-diglyceride is more than 5 A and 8 A,respectivily.At the same time,the distance between the carbonyl carbon atom of 1-MAG and the hydroxyl oxygen atom on the 145Ser of the catalytic triad was about 3 A,which is much smaller than the distance between carbonyl carbon atom of 2-MAG and the hydroxyl oxygen of 145Ser.The results indicate that the lipase ROL owns excellent 1,3-regioselectivity.