Cross-Linked Enzyme Crystals Of Transglutaminase And Immobilization On Polypropylene Microporous Membrance

Microbial transglutaminase(MTGase)could catalyze the acyl-transfer reaction in which the ?-carboxyamide groups of glutamine residues in proteins,peptides and various primary amines act as acyl donors and primary amino groups including ?-amino groups of lysine residues,either as peptide-proteins bound or free lysine,act as the acyl acceptors.Through modifying to optimize protein's structure and physical properties,therefore,it is widely used in the food industry.In this study,the MTGase was cross-linked with glutaraldehyde.The morphology and enzymatic properties of cross-linked enzyme crystals were confirmed.Furthermore,MTGase was immobilized on a polypropylene microporous membrane to improve the enzyme characteristics,and the immobilized MTGase was applied to recovery soybean whey protein.The MTGase was purified by alcohol precipitation,ammonium sulfate fractionation precipitation,ultrafiltration,SephadexG-100gel chromatography,and the purity of the MTGase was examined by SDS-PAGE.The isolation and purification of MTGase was optimizated.The results showed that:purification multiple of MTGase was 9.81,The recovery of enzyme activity was 28.54%,specific activity was 100.97U/mg.MTGase activity reduced significantly by PMSF,NBS,IAc,BrAc,SUAN and NEM modified.But the activity of MTGase without change by DTT,Na2SO3,EDC and WRK,it testified that serine residues,tryptophan residues,histidine residues and homocysteine residues located on the enzyme activity center.The enzymatic properties of MTGase were preliminarily studied.The optimum temperature of free MTGase was determined to be approximately40? the optimal pH was 6.0.The free MTGase was stable between 30? and 40?,and was relatively stable at pH5.0-7.0.Some metal ions had a larger influence on the enzyme's ability,such as Fe3+?Zn2+ and Cu2+,and Fe3+ had serious effect on enzyme activity.Metal ions of Ca2+?Mg2+and Ba2+ had little effect on the activity of MTGase.The MTGase crystals obtained by the vapor diuffsion method ndependently using 2-methyl-2,4-pentnaediol as a preeipitant.The MTGase was cross-likned with glutaraldehyde,and the morphology of cross-linked enzyme crystals were confirmed by SEM,Polarizing microscope,FT-IR,Laser Raman Spectrum,NMR and XRD.The properties of corss-linked MTGase were also studied.The results indicated that the optimum temperature of corss-linked MTGase was determined to be approximately45?,the optimal pH was 6.0.Compared to free enzyme,the corss-linked MTGase enhanced thermal stability and pH stability,and the corss-linked MTGase was demonstrated significant resistance to Fe3+?Zn2+ and Cu2+.In addition,the corss-linked MTGase also maintain a high activity in aqueous-ogrnaie mixtures such as 50%terthaydrofuran and 50%dimethyl sulfoxideto.The corss-linked MTGase was immobilized on a polypropylene microporous membrane by UV-induced grafting.The conditions of graft polymerization were optimized by using response surface methodology,and the optimal conditions of immobilization were determined by experiments.The results indicate that the optimum graft polymerization conditions as followings:distance of ultraviolet was 10.1cm,consistency of methyl-methacrylate was 18.85%,and the time of ultraviolet was 21.4 minutes.The best grafting degree of the membrane was 34.62%.The conditions of immobilization was:the consistency of hexamethylendiamine was 20%,at 50? for 90min and consistency of glutaraldehyde was 2%,at 30? for 40 minutes,consistency of enzyme was 15mg/mL,at 4? for 24 hours.The morphology of immobilized MTGase was confirmed by SEM,FT-IR and XPS.The enzymatic properties of immobilized MTGase were also studied.The results showed that the optimum temperature of immobilized MTGase was approximately50?,the optimal pH was 6.0.Compared to free enzyme and corss-linked enzyme crystals,the immobilized MTGase enhanced thermal stability and pH stability,and the immobilized MTGase was also demonstrated significant resistance to Fe3+?Zn2+ and Cu2+.In addition,membrane immobilized MTGase has good storage stability and operation stability.Membrane immobilized MTGase was applied to recovery soybean whey protein.The optimum reaction conditions of soybean whey protein catalysis by membrane immobilized MTGase was determined.The composition of soybean whey wastewater is changed greatly after that was polymerized,and the molecular weight of soybean whey protein also changed.Under optimal conditions,membrane immobilized MTGase recovered 76%of proteins from soybean whey waste.It was found that the article will provide reference for further application of MTGase enzyme membrane reactor.