Immobilization Of Lecitase~? Ultra Ontoorganically-modified SBA-15 And Their Applications

Lecitase~? Ultra(LU),one of the phospholipases,become the highlights of research and application in recent years due to its low price and good catalytic performance.Nowadays LU is mainly used for vegetable oil degumming and phospholipid modification.The immobilization of LU can overcome the disadvantages of poor stability and non-recyclability of free enzyme.Mesoporous molecular sieve SBA-15 is an excellent carrier to load enzymes better for its large specific surface area and the abundant silanol groups in the pore channel,which is convenient for functional modification to improve its performance.This paper is detailed the enzymatic properties of LU immobilized in different SBA-15 modified with radicals,and the immobilized LU is applied to produce DAG by glycerolysis and degumming of soybean crude oil,and the catalytic effect and reusability of immobilized LU in two reaction systems were studied.The main research results are as follows:1.Optimized the reaction conditions of SBA-15 immobilized LU,the optimal conditions were:immobilization phosphate buffer pH=6,enzyme protein concentration 200.80 ?g/mL,adsorption time 30 minutes.With these conditions,the optimal enzyme activity was 2200.00± 101.84 U/g.2.Organic modification of SBA-15(R-SBA-15)with common silane coupling agents,and then loaded LU(R-SBA-15-LU).It was found that N-aminoethyl-y-aminopropyl,3-(methacryloyloxy)propyl,3-aminopropyl,and isocyanopropyl groups with moderate hydrophobicity are beneficial to the improvement of lipase activity.The immobilized LU,#12 of enzyme activity respectively were 3555.56±900.21,3444.44±346.41,47.778± 115.47 and 3111.11 ±443.89 U/g.Structural characterizations such as XPS,XRD,and FT-IR show that organic functionalization and LU enzymes have been successfully modified and immobilized on SBA-15;and functionalized modifications and LU immobilized will not change the ordered hexagonal structure of SB A-15.3.The immobilized LU is suitable for catalyzing glycerolysis in a solvent-free system because the tert-amyl alcohol solvent impairs its glycerolysis reaction activity.SBA-15-LU catalyzed glycerolysis at a temperature of 60? with reaction of 8 hours,the DAG content reached 52.43±1.64%,and the TAG conversion reached 89.89±1.14%.After 12 hours of the reaction,TAG conversion rate reached 92.19±0.89%.Compared with SBA-15-LU(DAG/MAG is 1.40±0.06),(?)has a certain selectivity for DAG,and the DAG/MAG is 2.86±0.99 and 2.89±0.19,respectively.The optimization results for the conditions of H2N(?)SBA-15-LU catalyzed glycerolysis were:certain amount of enzyme(6.75 wt%of the reaction substrate),temperature 30?,and reaction time 4h.Under these conditions,the TAG conversionand DAG content were 90.92± 0.08%and 55.2±0.12%,respectively.In addition,the reaction progress showed that the reaction reached equilibrium more quickly at 60? than 30?,but the TAG conversion was higher at 30?.SBA-15-LU catalyzed the glycerol hydrolysis to obtain a higher DAG content,but its reusability was poor,since the enzyme activity only was maintain 9.79±6.17%after five times of repeated use.However,the enzyme activity of(?)could maintain 83.91±22.25%of its original enzyme activity after repeated 5 times.4.The hydrolytic phospholipase activity of LU was studied.The results showed that the enzyme activities of free LU and SBA-15-LU were 3581.67± 75.35 U/mL and 1852.50±297.83 U/g,respectively.Organic functionally modified R-SBA-15-LU,such as N-aminoethyl-?-aminopropyl,3-aminopropyl,anilinemethyl,3-glycidyloxypropyl,3-ureapropyl and long carbon chains have higher phospholipase activity activities.For exemple,the enzymatic phospholipase activities of#12 and(?)are 3840.00±511.72,3825.00±435.97,3981.67 ±319.34,4551.17±164.04,3702.50±5.00 U/g.The immobilized LU was used in soybean oil degumming for crude oil with lower phosphorus content(the content of phosphorus was 121.15 mg/kg),The degumming effect and reusability for(?)and(?)are excellent.After five repeated uses,the relative activity up to 100%,and the phosphorus content of degummed oil was less than 10 mg/kg and reach the standard for further refining.5.(?),with both high phospholipase activity and lipase activity,have favourable effect in catalyzing the glycerolysis reaction,degumming reaction and reusability,becoming the immobilized enzyme with excellent performance in every aspects.