Construction Of Two-Dimensional Lipase Nanoreactors And Its Application In Synthesis Of Lutein Ester

Lutein is one of the six carotenoids in human serum,which has the ability of antioxidant,strengthening human immune system and delaying age-related macular degeneration.It is an important supplement in human diet.However,due to its high degree of unsaturation,lutein is easily decomposed under light and heat conditions.Esterifying the hydroxyl groups at both ends of lutein by different fatty acids or anhydrides to form lutein diester can not only improve its stability and bioavailability,but also endow lutein with different biological activities and physiological functions.Esterification catalyzed by lipases has a lot advantages such as high efficiency,strong specificity and environmental friendliness.But lipase is high cost,sensitive to the reaction condition,and easily inactivated.As an essential participator in the immobilization process,supporting material deeply affects lipase activity.The two-dimensional nanomaterials have large specific surface area,rich active sites and good biocompatibility,which has a broad application prospect in the field of immobilized enzyme and functional lipid synthesis.This paper constructed two kinds of two-dimensional lipase nano-reactors based on graphitic carbon nitride nanosheets?g-C₃N₄-Ns?and graphene-like meso-macroporous carbons?GMCs?.Then fixed CALB by using chemical covalent method and physical adsorption method respectively according to the material characteristics,and applied two kinds of immobilized lipases to synthesis of lutein disuccinate.The surface properties and physicochemical properties of the two carriers and lipase nanoreactor were characterized by a series of physicochemical methods,and the effects of factors in immobilization conditions and esterification process on the esterification rate were investigated.Specific conclusions are as follows:1.Preparation and characterization of graphitic carbon nitride lipase nanoreactor?g-C₃N₄-Ns-PEI-GA@CALB?.Firstly,g-C₃N₄-Ns was prepared by using dicyandiamide in thermal stripping method,and then the surface of g-C₃N₄-Ns was modified with amino group by polyethylene imide?PEI?.Finally,glutaraldehyde?GA?was used as cross-linking agent to fix the CALB covalently.SEM,TEM,XRD,FT-IR,TGA,XPS and BET were used to characterize g-C₃N₄-Ns and g-C₃N₄-Ns-PEI-GA@CALB.The specific surface area was 118.858 m² g^-1.In the XRD pattern,13°and 27°represented the lattice repeating element triazine ring and the interlayer diffraction between layered structure of the g-C₃N₄-Ns planar structure respectively.FT-IR and XPS indicated that g-C₃N₄-Ns had the bond of C-N?-C?-C with triazine ring structure.Secondly,the fixed CALB could be obviously observed in the TEM image of g-C₃N₄-Ns-PEI-GA@CALB.FT-IR and XPS showed the formation of new NH-CO bonds.TGA had a new obvious loss.The reduction of BET specific surface area and pore volume were caused by the immobilization of CALB.Thirdly,the optimal immobilization conditions:initial lipase concentration 25%,fixation time 8h,optimal buffer pH 7,highest lipase activity at 45°C,and g-C₃N₄-Ns-PEI-GA@CALB had better acid-base tolerance and thermal stability than CALB.2.The synthesis of lutein succinate was catalyzed by g-C₃N₄-Ns-PEI-GA@CALB,and the effects of solvent,lipase,the amount of immobilized lipase,molar ratio of substrate,substrate concentration,esterification temperature and other factors on the esterification rate,as well as the esterification time process and the cycling of immobilized lipase were investigated.The optimal conditions were as follows:DMF was the solvent,lutein molality was 0.7 mol ml^-1,molality ratio of lutein and succinic anhydride was 1:20,the amount of g-C₃N₄-Ns-PEI-GA@CALB was 10 mg,the reaction was carried out in the shaking table of 160 rpm at 50°C in 72 h.The esterification rate was the highest 92%.The lipase activity was still 65%after 9 cycles.3.Preparation and characterization of graphene-like mesoporous carbon lipase nanoreactor?GMCs@CALB?.GMCs was prepared with dicyandiamine as template material and glucose as carbon material by carbonization method,and CALB was fixed by physical adsorption.Using the same methods to characterize GMCs and GMCs@CALB.The results as follows:first,GMCs had high specific surface area of 372.1643 m² g^-1.The shape of mesoporous and macroporous could be observed clearly in SEM and a silk-like layer fold morphology was shown in TEM.Graphene-like layer structure diffraction between the interplanar of GMCs was showed in XRD.Two peaks of N in XPS signals were from pyridine nitrogen and graphite nitrogen respectively,proving that GMCs possessed the graphene-like interface of a macroporous structure.Second,the CALB which was adsorbed on the surface of GMCs could be obviously observed in the SEM and TEM images of GMCs@CALB.In the figure of TGA,the degradation curve of CALB was distinct.In XPS figure,a new peak of C appeared at 286.5 eV,which is derived from the oxygen-containing group C=O on CALB.The curve characteristics,specific surface area and pore volume reduction of BET also proved the successful adsorption of CALB.Third,the optimal conditions for immobilization were:initial enzyme concentration of 40%,fixed time of 10 h,optimal buffer pH of7,highest enzyme activity at 45°C,and better acid-base tolerance and thermal stability of GMCs@CALB.4.The synthesis of lutein succinate catalyzed by GMCs@CALB,and the optimal conditions were as follows:DMF was the solvent,the molal concentration of lutein was 0.2 mol ml^-1,the molar ratio of lutein to succinic anhydride was 1:16,the addition of GMCs@CALB was 10 mg,reaction was carried out in the shaking table of 160 rpm at 45°C in 72 h.The esterification rate was the highest at 93%.The lipase activity was 50%after 10 cycles.