Preparation And Properties Of Cross-linked Enzyme Aggregates From Extracellular Lipase Of Aspergillus Niger GZUF36

Lipase is a commercially valuable serine hydrolase that can be used in food,pharmaceutical,chemical and other industries.However,most natural lipases have disadvantages such as poor stability,easy inactivation,and non-recyclability,which limits the application of lipase.Therefore,cross-linking enzyme aggregates from extracellular lipase of Aspergillus niger GZUF36 were prepared to obtain immobilized lipase with high enzyme activity,high stability and multiple recycling in this study.In addition,In addition,the enzymatic properties,secondary structure,morphology and selectivity of Aspergillus niger GZUF36 extracellular lipase aggregates(CLEAs-ANL)were studied.It is intended to provide a reference and theoretical basis for the subsequent study of the synthesis of functional oils and the large-scale application of industrial oils.The specific results are as follows:(1)It was confirmed when the volume of t-butanol and crude enzyme solution was 4:1,the amount of cross-linking agent was 30 ?l,the crosslinking time was 1.5hours,and the number of centrifugation was 6000 r/min,it were the best condition for preparing CLEAs-ANL.Under this condition,the CLEAs-ANL relative enzyme activity was 100.3±1.1% and the enzyme activity was 13.8±0.51 u/ml.The effects of different additives such as BSA,BSA hydrolysate,oleic acid,heptane,Tween-80,and SDS on CLEAs-ANL activity were investigated.The results showed that BAS,BSA hydrolysate and Tween-80 could all improve the activity of CLEAs-ANL,and the recovery rate of CLEAs-ANL enzyme activity could be increased by 11.3%,6.5% and4%,respectively.While oleic acid,SDS and n-heptane had a negative impact on CLEAs-ANL activity.They were not suitable for use as an additive in the preparation of CLEAs-ANL.(2)The hemolysin CZUF36 extracellular lipase could be partially purified by acetone precipitation and reverse micelle extraction.The electrophoresis results showed that the band appearing between 31 and 43 was the target band.The partiallypurified lipase was prepared into CLEAs and its enzymatic properties were investigated.The results showed that the optimum temperature for CLEAs-ANL was40?,which was 5? higher than the free enzyme.In terms of optimum pH,the optimum pH of the free enzyme was 6.5 and the difference of CLEAs-ANL was not significant at pH 4.0-6.0.This showed that the lipase shows better acid resistance after immobilization.CLEAs-ANL not only showed higher stability than free enzyme in terms of temperature stability and pH stability,but also generally more tolerant in different organic solvents than free enzyme.In terms of storage stability,the relative enzyme activities of free enzyme and CLEAs-ANL were 26.3±1.6% and 82.5±4.1%,respectively,after 30 days of storage.It indicated that the storage stability of the lipase was improved after crosslinking.In addition,the relative enzyme activity was34.9% when the CLEAs-ANL was recycled after 8 times,indicating that the operation stability was good.Finally,the kinetic parameters showed that the Vmax and Km of the free enzyme were 15.9 and 44.35,respectively,while the Vmax and Km 226 of CLEAs-ANLwere 11.95 and 226,respectively.It indicated that the substrate affinity and the maximum reaction rate were decreased after the lipase was immobilized.(3)The results of FTIR showed that the ?-fold and ?-turn of CLEAs-ANL increased by 31.54% and 2.88%,respectively,while the ?-helix and random curl decreased by 5.05% and 29.32%,respectively.This indicated that the lipase was increased in rigidity and reduced in flexibility after crosslinking.SEM results showed that the CLEAs-ANLsurface structure was more compact and uniform relative to the free enzyme.At 15;000X magnification,clusters and holes were observed on the surface of CLEAs-ANL.It indicated that the substrate molecule was more easily contacted with the enzyme catalytic site,which facilitated the reaction.The particle size of CLEAs-ANL is about 50 ?m,which was in line with the particle size range of general CLEAs.Finally,by selective analysis by TLC and HPLC,both the free enzyme and CLEAs showed a sn-1,3 positional selectivity in the hydrolysis reaction.It was indicated that the cross-linking process does not affect the positional selectivity of the black-strain enzyme GZUF36 in the hydrolysis reaction.Compared with the product of ppl,the free enzyme and CLEAs-ANL showed better sn-1,3 positionalselectivity than ppl in the hydrolysis reaction.However,the reaction product of the free enzyme glycerol solution in the glycerol hydrolysis reaction was not detected.The glycerol reaction product of CLEAs-ANL not only had the formation of1,3-diglyceride,but also the presence of some 1,2 diglyceride.This indicated that the selectivity of CLEAs-ANL in the glycerol hydrolysis reaction had changed.The reason may be that organic solvents have an effect on the selectivity of CLEAs-ANL.