Eukaryotic Expression And Immobilization Of Fusarium Solani Cutinase

Poly(butylene succinate)(PBS)is a biodegradable plastics that could substitute traditional plastic and conducive to solve white pollution.However,PBS degrades slowly in nature,so the key of PBS quckly degradation of is to obtain high efficient PBS degrading enzyme.Firstly,a recombinant Pichia pastoris expression system were built to realize the efficient expression of cutinase from Fusarium solani(FSC)with PBS degrading ability.According to the encoded codon in P.pastoris,FSC gene codon was optimized and the gene was transducted into P.pastoris X33to realize its recombinant expression.Then,the enzyme producing conditions of the recombinant strain was optimized based on single factor experiments.The results showed 157 bases in the optimized FSC gene sequence were changed,the G+C reduced from 59.6%to 48.3%,and the homology was at77.34%.After the constructed recombinant expression vector p PICZ?-FSC was transferred into P.pastoris X33,a recombinant strain L1 with higher enzyme producing capability was obtained based on initial screening on resistant plate,SDS-PAGE,Western blot,and assay of enzyme activity.Then,the fermentation conditions were further confirmed as follow:initial p H was at6.0,the shaker rotation was 220r/min,the addition of methanol was 1%,the dose of inoculation was 8%,incubation time was 72 hours and incubation temperature was 30?.Under these conditions the PBS depolymerase activity of fermentation broth reached up to 110U/m L.The recombinant protein with His-tag was purified by magnetic bead.The relatively high purity recombinant cutinase with a molecular weight about24k D was obtained from the fermentation broth of recombinant strain L1 based on was subjected to a series of separation and purification,such as centrifugation,re-dissolution,dialysis,magnetic bead binding,impurity washing,elution and re dialysis.The cross-linked magnetic chitosan microspheres were prepared by using genipin as the cross-linking agent.Under the conditions of genipin concentration of 0.6g/L,cross-linking time of 8h,enzyme concentration of50?g/ml and immobilization time of 16h,the recombinant cutinase was immobilized on the carrier.The assay of the immobilized enzyme showed that the Michaelis constant(K_m)of the immobilized cutinase increased.The optimal temperature of the immobilized enzyme was 50?,which was 5?higher than that of free enzyme.The optimal p H was increased from 5.0 to 9.0 of free enzyme.These showed that cutinase had better p H and thermal stability after being immobilized by the carrier.The activity of the immobilized enzyme could still be maintained more than 50%after 10 times of repeated operation.The activity of the immobilized enzyme still contained 88%after 30 days storage at 4?.