Optimized Production Of D-psicose 3-epimerase And Immobilization Of Enzyme And Cell

At present,the living standards of human beings have generally improved,and due to excessive intake of fat and carbohydrates,coupled with reduced exercise,some diseases such as obesity,hyperglycemia and other diseases have also followed.The development of functional sweeteners has become a feasible measure to alleviate such problems.With low calorie and similar sweetness to sucrose,D-alllulose has been considered a potential functional sweetener.D-psicose 3-epimerase(DPEase,EC 5.1.3.30)catalyzes the synthesis of D-allulose from D-fructose.Free enzyme can not be reused and has poor stability,which limits its practical application.Therefore,the immobilization of enzyme and cell has received extensive attention.However,the activity of immobilized DPEase is too low,or the particle size is too small to be separated from the reaction solution.In this study,the conditions of Bacillus subtilis 1A751/p UB-P43dpe-dal to produce DPEase were optimized by using a 3 L fermentor.Amino-epoxide carrier Relizyme HFA403/M(HFA)and chitosan/aldehyde grafted hydrolyzed cellulose were used to immobilize enzyme and cell.Their characterization and enzymatic properties were studied and immobilized DPEase with high enzyme activity,high stability and ease of separation from the reaction solution was prepared.The main contents of this paper are as follows:Firstly,a 3 L fermenter was used to explore the conditions of Bacillus subtilis1A751/p UB-P43dpe-dal producing DPEase.The different initial and feeding conditions were systematically evaluated from the three aspects of bacterial growth,enzyme activity and glucose residue.The optimal conditions for producing DPEase were dissolved oxygen(DO)=30%,p H 6.0,temperature 37 °C and the initial glucose concentration of 15 g/L.Then carbon source was supplemented at a flow rate of 8 g/(L·h)at 5 h.Under this optimal strategy,the enzyme activity of the whole-cell reached 122.95 U/m L at 9 h.Secondly,the free DPEase was immobilized by the easy-to-separate HFA carrier,and the properties of free and immobilized enzyme were studied.The recovery of enzyme activity,the residual protein in the supernatant and the residual protein in the washing solution during the immobilization process was determined.The optimal immobilization conditions were as follows: ion exchange for 0～8 h,covalent binding for 8～20 h,0.01% glutaraldehyde crosslinking for 20～21 h and glycine blocking for 21～37 h,enzyme dosage 200 U/g carrier,and temperature of 20 °C and p H 7.5 for each step.Glycine-blocked DPEase(four-step immobilization,Blocked-E)activity was as high as 103.48 U/g support.The stability of immobilized and free enzyme was analyzed and results showed that Blocked-E has the best p H stability,thermal stability and reusability.Fourier transform infrared spectroscopy confirmed that the epoxy group of Blcoked-E was successfully blocked.Thirdly,hydrolyzed cellulose(HC)was prepared from peanut shells and HC was oxidized by sodium periodate to prepare aldehyde grafted hydrolyzed cellulose(HC-CHO).And the structure of these samples was characterized.Based on the content of aldehyde group,the optimal modification conditions were determined to be p H 5.0,temperature 40 °C and sodium periodate of 5 g/L.The results of scanning electron microscopy and fourier transform infrared spectroscopy were as follows: HC and HC-CHO have macroporous structure,which is conducive to immobilization.The hemicellulose and lignin are basically removed and the aldehyde group is successfully grafted to HC-CHO.Finally,the HC-CHO,chitosan(CS),glutaraldehyde and ultrasonically treated bacteria were cross-linked and flocculated.Then,immobilized cells were prepared by suction filtration,extrusion and drying,and properties were studied.Based on recovery of enzyme activity,the optimal immobilization conditions were determined to be ultrasonic treatment for 5 min,HC-CHO addition amount 5 g/L,CS final concentration 2.4 g/L and glutaraldehyde 0.25%.The activity of immobilized cell was as high as 259.20 U/g support.The residual enzyme activity was used as the measurement index to analyze the stability of immobilized cells constructed by CS(CS-E),by composite of CS and HC(CS-HC-E),and by composite of CS and HC-CHO(CS-HC-CHO-E).The results showed that the optimal temperature of the immobilized cells was 15 ℃ higher than that of the free enzyme.Among them,CS-HC-CHO-E was the most stable immobilized cell.Scanning electron microscopy showed that the pores of CS-HC-E and CS-HC-CHO-E are larger than CS-E,indicating that CS-HC-E and CS-HC-CHO-E are more conducive to mass transfer.