Construction And Application Of Carrier-free Immobilized Enzyme-catalyzed Reaction Coupled With Separation In Aqueous Two-phase Microsystem

As a new type of food additive,oligosaccharides represented by xylan are widely used in the fields of intestinal health,immune modulation,blood glucose and lipid regulation.Currently,xylanase catalyzed hydrolysis of xylan is an effective method for the preparation of oligosaccharides.However,its practical application is limited by the disadvantages of enzyme sensitivity to environmental response,easy inactivation,poor stability and low reusability.Therefore,in this thesis,a new dual aqueous phase enzyme-catalyzed reaction coupled with a xylanase-polymer coupling as the catalyst and a low eutectic solvent as the medium,"low eutectic solvent+Na₂SO₄/PEG",was developed for the separation and coupling of pretreated mulberry xylan using W/W droplet microfluidic interfacial catalytic degradation coupled with S-Ca resin column chromatography to purify Xylan.The main elements are as follows:（1）Construction of a new system for the enzyme-catalyzed separation coupled with a low eutectic solvent+Na₂SO₄/PEG dual aqueous phase reaction.Eight low eutectic solvents（DESs）were prepared using betaine or choline chloride as hydrogen bond acceptors and glycerol,ethylene glycol,sorbitol,citric acid or malic acid as hydrogen bond donors,which were selected by the enzymatic hydrolysis activity of xylanase as an indicator.The results showed that 30%（w/w）Gly2B（glycerol-betaine,2:1）aqueous solution significantly increased the relative activity of xylanase by 53.26%（p<0.05）and the catalytic efficiency of the enzyme reached 933±104 m L/s/mg,which was 36%higher than that of the control（citric acid buffer）（p<0.05）.On this basis,a dual-phase system of“Gly2B+Na₂SO₄/PEG”was constructed,in which 93%of xylanase was enriched in Gly2B+Na₂SO₄ phase and more than75%of reducing sugars were enriched in PEG phase,and the yield of reducing sugars was79.17%after 1 h of reaction at 60℃and p H 6.0.The xylanase could be recycled by biphasic extraction,and the residual viability of the enzyme could be maintained at more than 60%after 7 times of reuse,which was 3.5 times higher than that of the homogeneous system.Therefore,the new system of"Gly2B+Na2SO4/PEG"can improve the catalytic performance of xylanase and achieve the reuse of catalyst.（2）Preparation of xylanase-polymer coupling catalysts based on hydrophobic polymers for two-phase catalytic systems.Three xylanase-polymer conjugates,XYL-p BMA,XYL-p（n PMA）and XYL-p HEMA,were constructed by atom transfer radical polymerisation（ATRP）using the Grating from technique by selecting methacrylate polymer monomers containing C2-C4 alkyl chains with different hydrophobicity and grafting them onto the lysine residues of xylanase.The three xylanase-polymer conjugates were characterized and their enzymatic properties were examined.The results showed that the p H and temperature ranges for the xylanase-polymer conjugates to maintain 60%relative activity were 3.0-8.0 and 40-80°C,respectively,which significantly widened the range compared to the free enzyme（4-7,40-60°C）,while their stability and catalytic efficiency were significantly improved,and increased with the hydrophobicity of the grafted polymer monomers（p<0.05）.In particular,XYL-p BMA maintained 67.61%residual activity after 1 h incubation at 90℃,which was significantly higher than that of the free enzyme（9.25%）;its catalytic efficiency was 1609.40m L/s/mg,which was 150%higher than that of the free enzyme（p<0.05）.Meanwhile,the enzymatic activity of XYL-p BMA was increased by 24%compared with the XYL-PNIPAAm coupling studied by our group previously.On the basis of this,XYL-p BMA was used as a two-phase aqueous interfacial catalyst,and the yield of reducing sugar could reach 89.16%at60℃and p H 6.0 for 1 h,which was 13%higher than that of the free enzyme（p<0.05）.Therefore,the xylanase-polymer coupling XYL-p BMA can be used as an interfacial catalyst to significantly improve the catalytic efficiency and stability.（3）Construction of a new W/W droplet microfluidic interfacial catalytic xylanase process with XYL-p BMA as the catalyst.A new W/W droplet microfluidic catalytic process for xylanase digestion was developed using Na₂SO₄+Gly2B as the disperse phase and PEG as the continuous phase,and the flow rate of the two phases was adjusted.The results showed that the W/W droplet size could be controlled at a flow rate of 2.0-3.5μL/min for the continuous phase and 1.5-3.0μL/min for the disperse phase,and was positively correlated with the flow rate ratio of the two phases.The reaction was carried out at p H 6.0,50°C and a substrate concentration of 1%at the W/W droplet microfluidic interface to catalyse the enzymatic digestion of xylan for 10 min,and the yield of reducing sugars reached 79.86%.The K⁰_m（app）of catalyst XYL-p BMA was 1.91 by kinetic calculations,which was 28%lower than the Km value of the conventional system;the temporal yield of its catalytic reaction reached 0.78μg/m L/min,which was 265%higher than that of the conventional system（p<0.05）.At the same time,the catalyst XYL-p BMA could be recycled through the dispersed phase and still maintained 60%relative activity after 10 uses.Compared with the XYL-PNIPAAm droplet microfluidic catalytic system constructed by the group,the space-time yield of the new system constructed in this thesis was significantly higher（p<0.05）and the reuse process was simpler.This indicates that the W/W droplet microfluidic interfacial catalytic system with XYL-p BMA as the catalyst is green,efficient and low-consuming for the enzymatic digestion of xylan.（4）W/W droplet microfluidic interfacial catalytic degradation of pretreated mulberry xylan coupled with S-Ca resin column chromatography purification of high purity xylan.Xylan was obtained by pretreating mulberry branches with H₂O₂ and Na OH in two steps,and xylan was prepared by enzymatic degradation of mulberry xylan through W/W droplet microfluidic interfacial catalytic system and purification of oligosaccharide products using S-Ca resin.The results showed that under the process conditions of p H 6.0,60℃and substrate concentration of 1%,the yield of reducing sugar from hydrolysis of mulberry xylan was77.63%with a spatiotemporal yield of 3.12±0.23 mg/m L/min,which was 478%higher than that of the conventional system using the W/W droplet microfluidic interfacial catalytic system（p<0.05）.In the first step,78%xylan was obtained with 10%ethanol as the eluent at40℃and a flow rate of 5 m L/min,and the yield was 63.32%.In the second step,the xylan fraction with 95%purity was purified with 15%ethanol as the eluent at 40℃and the flow rate of 5 m L min.Compared with the commonly used polyacrylamide gel chromatography,Na⁺resin chromatography and dextran gel G-10 chromatography,the constructed S-Ca resin two-dimensional liquid chromatographic separation method has the advantages of high product purity and low cost.Therefore,the constructed new process of W/W droplet microfluidic interfacial catalytic degradation of mulberry combined with S-Ca resin column chromatography can prepare high purity xylan efficiently.Therefore,in this paper,a new dual aqueous-phase catalytic reaction coupling system of Gly2B+Na₂SO₄/PEG with xylanase-polymer coupling as the catalyst and low eutectic solvent was constructed,and the W/W droplet microfluidic interfacial catalytic degradation of pretreated mulberry xylan combined with S-Ca resin two-dimensional liquid-phase chromatography was used.The separation of high purity xylan by chromatography improved the catalytic efficiency,stability and reusability of xylanase,and provided a new technology for the preparation of xylan by biocatalysis and a new idea for the high value utilization of natural lignocellulose.