Preparation Of β-glucosidase@MOF High-temperature Resistant Catalysts And Research Its Enzymatic Hydrolysis Of Cellulose

As the most abundant biomass resource on the earth,the main components of lignocellulose are cellulose,hemicellulose and lignin.The cellulose can be converted into glucose by enzymatic hydrolysis,and then prepared as biofuels and chemicals by biological method.However,cellulase hydrolysis requires multi-enzyme synergistic catalysis,and the enzyme is easily inactivated by high temperature,and the low catalytic temperature（50°C）restricts the reaction speed,resulting in a long enzymatic hydrolysis time（48-72 h）.In order to improve the efficiency of enzymatic hydrolysis,cellulose was dissolved in ionic liquid and then rapidly hydrolyzed to glucose by usingβ-glucosidase（β-G）alone at high temperature in this paper.In view of the difficulty that the enzyme is easily inactivated in ionic liquid and high temperature,the three-dimensional structure ofβ-G is fixed to enhance the stability and maintaining catalytic activity in ionic liquids and high-temperature conditions.Then,to address the issue of reduced substrate and enzyme accessibility caused by MOF encapsulation,enzyme complexes with macroporous structures were constructed to promote cellulose diffusion and improve enzymatic hydrolysis efficiency.Finally,in order to shorten the mass transfer distance of cellulose substrates and improve the enzymatic hydrolysis reaction speed,two-dimensional（2D）enzyme complexes and 0D ZIF-8 nanospheres were prepared to reduce the thickness of MOF crystals and further improve enzymatic hydrolysis efficiency.（1）To increase the catalytic temperature ofβ-G in order to enhance the enzymatic efficiency,β-G was used to induce MOF nucleation.During the formation of MOF by Cu²⁺and para-aminobenzoic acid（PABA）,the enzyme was encapsulated in situ in the MOF structure to produce a floral porous enzyme complex ofβ-G@MOF（PABA）.Compared to the optimal enzyme activity of freeβ-G at 50℃in the buffer system,the encapsulatedβ-G could withstand a high temperature of 100℃,when the enzyme activity was 2 times higher than the free enzyme at 50℃.Moreover,β-G@MOF（PABA）could also maintain good activity in ionic liquid,with high temperature resistance up to 110℃,when its enzyme activity was 6.1 times that of free enzyme at 50℃.In addition,β-G@MOF（PABA）in the buffer system at 100℃,the V_maxand K_mwere 1.3 times and 0.64 times than free enzyme at 50℃.At 110℃,theβ-G@MOF（PABA）retained good activity after five consecutive cycles for 120 h.The research showed that the carboxyl and imidazole groups on the surface ofβ-G form a coordination bond with Cu²⁺,which induce MOF to encapsulated the enzyme in situ and form the enzyme complex with"inner flexibility and outer stiffness",thus improving the stability ofβ-G at Ionic liquids and high temperatures.（2）In view of the difficulty of large molecular weight cellulose to enter the MOF（PABA）pore,the enzyme complexβ-G@MOF-Fe was prepared by using the different coordination capacity of Fe²⁺and Fe³⁺.The macropore volume ofβ-G@MOF-Fe was increased by 3.4 times compared toβ-G@MOF（PABA）.After 64 h in the ionic liquid at 110℃,the cellulose conversion rate ofβ-G@MOF-Fe was 1.9 times that ofβ-G@MOF（PABA）.The thermotolerance ofβ-G@MOF-Fe in ionic liquids can be increased to 120℃,when the enzyme activity was 6.7 times higher than the free enzyme at 50℃.Moreover,the V_maxand K_m ofβ-G@MOF-Fe at 120℃were 1.2 and 0.79 times that ofβ-G@MOF（PABA）at 110℃.The study showed that Fe²⁺would preferentially coordinate with the imidazole group to form Fe²⁺-N bond.At this time,some Fe²⁺located at the edge of MOF fully contact with oxygen.Under the action of oxygen would be converted into Fe³⁺,while the bond of Fe³⁺and N was weak,and some Fe³⁺-N broke to form a defective structure,and then form a macroporous structure at the edge of MOFs.Moreover,due to the formation of(Fe²⁺-N)coordination in the competitive oxidation coordination process,the stability of the macroporous enzyme complex was enhanced.（3）In order to reduce the mass transfer path and further improve the enzymatic efficiency,cuprous chloride（Cu Cl）was used to induce the morphogenic evolution of 3D MOF crystals,and the 2D enzyme complexβ-G@ZIF-8-Cu with defective structures was prepared under mild conditions.In 50℃water system,the enzyme active retention rate of 2D quadrilateralβ-G@ZIF-8-Cu was 9.8 times higher than that of 3Dβ-G@ZIF-8.With the increase of temperature,2Dβ-G@ZIF-8-Cu showed better high temperature resistance,it was 1.8 times more active than freeβ-G at 50℃and 1.1 times that of floral porousβ-G@MOF（PABA）at90℃.Moreover,2Dβ-G@ZIF-8-Cu with excellent ionic liquid resistance at 70℃was 5.0 times that of 3Dβ-G@ZIF-8 under equivalent conditions.The 2D enzyme complex exhibited better mass transfer performance due to its ultra-thin structure,larger specific surface area,and smaller mass transfer resistance.The research showed that Cu Cl induces the coordination of the carboxyl group ofβ-G with copper to promote ZIF-8 crystal core formation.At the same time,some Cu Cl would coordinate with the organic ligand N to induce the absence of the original metal Zn and N coordination bond,leading to the defective structure of the crystal material,and then causing the evolution of morphogenesis in the composite material from 3D to 2D.（4）In order to further shorten the transmission distance and improve the enzymatic efficiency ofβ-G@MOF,the 0D ZIF-8 nanosphere single enzyme crystal material（β-G@ZIF-8-Cu₂O）with mesoporous structure was prepared by using the strategy of double competitive coordination of metal oxides.When the mass ratio of zinc acetate to Cu₂O was 1:2,the prepared enzyme complex was a single ZIF-8 wrapping a singleβ-G nanosphere with a diameter of about25 nm.At 50℃,the 0D ZIF-8 nanosphere single enzyme complexβ-G@ZIF-8-Cu₂O（1:2）had the highest enzyme activity retention rate,which was 1.82 times that ofβ-G@ZIF-8 and 1.2times that ofβ-G@MOF（PABA）under equivalent conditions.Moreover,with increasing temperature,the enzyme activity retention rate ofβ-G@ZIF-8-Cu₂O（1:2）at 85℃was 1.58times that ofβ-G@ZIF-8 under equal conditions,1.3 times that of 2D enzyme complex and2.23 times that of freeβ-G at 50℃.The special nanosphere morphology and the defective mesopore structure significantly improved the mass transfer efficiency of the substrate and thus enhanced the catalytic ability of the enzyme.The research showed that the addition of Cu₂O to the crystallization of ZIF-8 produces the double competitive coordination phenomenon of copper/zinc and oxygen/nitrogen,which changes the growth orientation of the whole crystal,leading to a single ZIF-8 growing around a singleβ-G,thus preparing ZIF-8 nanosphere single enzyme complexes with mesoporous structure.This paper addresses the problems of difficult synergistic action of multi-component enzymes,low enzymatic hydrolysis temperature,and long enzymatic hydrolysis time in the process of cellulase hydrolysis,resulting in low enzymatic hydrolysis efficiency.MOFs are used for encapsulationβ-G realizes its enzymatic hydrolysis of cellulose in ionic liquids,further constructing macroporesβ-G@MOFs-Fe,2Dβ-G@ZIF-8-Cu and the high mesoporous 0D ZIF-8 nanosphere single enzyme complex solved the problems of low enzyme heat resistance,poor substrate to enzyme accessibility,and long product transport pathways,ultimately achievingβ-G rapidly hydrolyzes cellulose at high temperature.