Preparation Of β-glucosidase And Enzymatic Synthesis Of Nerol Glucoside

Commonly used flavor and fragrance materials generally have a small molecular weight,a low boiling point,and are prone to volatilization during storage,especially during high temperature heating,which results in a long time for the fragrance in the flavored product,poor consistency of the aroma before and after,and the like.The problem affects the flavoring effect.Therefore,how to improve the thermal stability of the fragrance and develop a new type of fragrance that can be applied to high temperature processing products is a problem that needs to be solved urgently.In this paper,the enzymatic synthesis of thermostable spice neroliol glycosides is designed to solve the above problems,and provides a theoretical basis for the development of other thermally stable fragrance precursors.In this paper,Aspergillus niger M6 was used as a fermentation bacterium,andβ-glucosidase was prepared by optimizing the culture medium and culture conditions,and the crude enzyme solution was separated and purified;then,sodium alginate was used as an immobilized carrier,and the embedded cross-linking method was used.β-glucosidase was immobilized,the effects of different immobilization conditions on the recovery of enzyme activity were studied,and the enzymatic properties of immobilized β-glucosidase were studied;in the aqueous system,immobilized β-glucosidase was used as a catalyst,glucose and nerol were used as substrates,and the conditions for the enzymatic synthesis of nerol glucoside were optimized.At the same time,the types and ratios of different organic solvents in the organic solvent system were studied for the synthesis of nerol glucoside.Impact,and the product was isolated and purified and analyzed.Research indicates(1)Aspergillus niger M6 was used as a fermentation bacterium,andβ-glucosidase activity was used as an indicator,and the optimal experimental conditions for the preparation of β-glucosidase were determined by the Box-benhnken center combination test design.The results showed that:The basic conditions are corn cob 3.07%,ammonium sulfate + peptone(1:1)compound nitrogen source 1%,KH2PO4 0.2%,Mg SO40.1%,initial p H 4.80,inoculation amount 10%,30°C condition,150 r/min shaker cultured for 7 days,a higher enzyme activity of 4.86 U/m L was obtained.The crude enzyme solution was separated and purified by ammonium sulfate fractionation and glucan gel chromatography,and the protein content and enzyme activity of the active component were quantitatively analyzed.It was found that the protein and activity of some enzymes existed in the process of separation and purification.The purification factor after ammonium sulfate precipitation was 1.87,and the recovery rate was 70.78%;the purification factor after sephadex chromatography was 3.06 and the recovery rate was27.98%.The isolated and purified enzyme solution was subjected to SDS-PAGE gel.After electrophoresis,the molecular weight of β-glucosidase was about 50 k Da.(2)Using sodium alginate as a carrier,β-glucosidase was immobilized by embedding and cross-linking method.Through single factor and orthogonal experiments,it was determined that the optimal immobilized enzyme preparation conditions were sodium alginate 2%.The enzyme amount was 120 U/g,the calcium chloride concentration was 2%,the glutaraldehyde concentration was 1%,the hardening time was2 hours,and the crosslinking time was 2 hours.The enzymatic properties of free enzyme and immobilized enzyme were studied.The optimum temperature of free enzyme and immobilized enzyme was 65°C,the optimum p H was 5.0 and 5.5,and Km was 0.0832mmol/L.and 0.7593 mmol/L,immobilized β-glucosidase has good thermal stability and p H stability.After repeated use for 6 times,the enzyme activity was reduced to 65% and the reusability was good.(3)By optimizing the conditions for enzymatic synthesis in aqueous systems,the following conclusions were reached: 2 m L system with a water content of 15%,a glucose content of 1.5 mmol,an enzyme amount of 64 U,and a citrate buffer solution of p H 5.0 In the middle,the maximum nerol glycoside yield was obtained after 5 days of reaction at 65°C,and the yield of nerol glucoside was 2600 μg.By optimizing the organicsolvents and the ratio of organic solvents in the organic phase system,it was found that the yield of products synthesized from the reaction system containing acetonitrile and acetone was higher than that of tert-butyl alcohol when nerol was 8:1.At 8:1,the yield of nerol glucoside was maximized.The reaction product was isolated and purified to obtain better results.The alcohol was removed by extraction to remove a large amount of nerol.The nerolitol glucoside standard and the separated and purified product were analyzed by liquid phase.Nerolol glycoside appeared at a retention time of 9-10 min.The material peak was considered to be a neo-alcohol glycoside.The product was isolated and purified by silica gel column chromatography.The chromatographic process was followed by TLC to collect a single component solution of nerol glucoside,which was then concentrated by evaporation to obtain nerol glucoside solid.powder.The structure was characterized by 1H NMR,13 C NMR and LC-MS,which proved that it was a natural equivalent structure.