Immobilization Of A Novel Invertase From Gongronella Sp.w5 For High Fructose Syrup Preparation

High fructose syrup（HFS）is composed of fructose and glucose,at present,it is mainly prepared by multi-enzyme hydrolysis method.The preparation of HFS by multi-enzymatic hydrolysis requires a variety of enzyme proteins to synergistically hydrolyze and convert,and needs to undergo multiple steps such as activated carbon adsorption,evaporation,and ion exchange,resulting in a large increase in the cost of HFS preparation.Invertase（EC 3.2.1.26）takes sucrose as a substrate and hydrolyze it into equimolar glucose and fructose,which can be used for the preparation of HFS.However,invertases can easily be inhibited by sucrose,the cost of invertase preparation,and the high price of sucrose and other factors limits the use of invertase for the preparation of HFS.In the early stage,the laboratory obtained an invertase GspInv derived from Gongronella sp.w5,which was resistant to sucrose and has higher specific activity,and has the potential to prepare HFS.In order to achieve low-cost preparation of GspInv and use it for HFS preparation,GspInv was fused with cellulose binding module CBM₂₄（HF912724.1）derived from Clostridium thermocellumand fixed on microcrystalline cellulose to explore the feasibility of immobilized invertase to hydrolyze high-concentration sucrose and molasses to prepare HFS.The following results were obtained:（1）The CBM₂₄-GspInv fusion protein was constructed and prepared,and the influence of the glycosylation site of CBM₂₄ on the activity of the fusion enzyme was studied.The results showed that the deglycosylated CBM₂₄DG-GspInv can be successfully heterologously expressed in Komagataella pastoris,and the purity of the target protein of the fermentation supernatant was more than 90%.The amount of CBM₂₄DG-GspInv that can adsorb to cellulose approximately 3.5 mg(Cellulose-CBM₂₄DG-GspInv).The glycosylation of CBM₂₄ affects the expression or secretion of invertase,further reducing the activity of invertase by more than90%.（2）We conducted a comparison and analysis of the enzymatic properties of fusion proteins and GspInv.The results showed that CBM₂₄DG-GspInv and cellulose-CBM₂₄DG-GspInv retained the excellent physicochemical properties of GspInv with high specific activity and high sucrose(IC₅₀=1.2 M,IC₅₀:the half maximal inhibitory concentration),glucose(IC₅₀>2 M)and fructose(IC₅₀=1.5 M)tolerance.（3）Using sucrose as a substrate,the potential of immobilized invertase cellulose-CBM₂₄DG-GspInv for HFS preparation was studied.The results showed that in a 35 ^oC packed bed reactor,1.0 M sucrose with a flow rate of 2.5 mL/min was hydrolyzed by cellulose-CBM₂₄DG-GspInv for 50 h,and still retained more than 90%of the initial hydrolysis efficiency.（4）Using pretreated molasses as a substrate,the potential research of immobilized invertase cellulose-CBM₂₄DG-GspInv for the preparation of HFS was launched.The pretreated molasses can be completely hydrolyzed by 5000 U/L CBM₂₄DG-GspInv.The Ca（OH）₂chemical separation method was used to recover fructose in molasses hydrolysate and the fructose recovery efficiency was 83.7%,the fructose concentration was 72 g/L.However,the pretreated molasses was continuously hydrolyzed by cellulose-CBM₂₄DG-GspInv in a packed bed reactor for 2 h,and the hydrolysis efficiency dropped to zero.