Preparation Of Micro-Nano Fibrils Based On Enzyme Treatment And Mechanism Of Endoglucanase Recycling

Biomass resources are abundant,low-cost and environmentally friendly.Converting them into high value-added products is an important way to achieve green and sustainable development of resources Micro-nano fibrils derived from plant resources have excellent properties such as high specific surface area,high strength,low density,low expansion and biodegradability,and have shown great potential in many fields such as papermaking,food,medicine,cosmetics,etc.In order to meet the performance requirements of micro-nano fibrils in different fields,green and efficient deconstruction of plant resources to prepare micro-nano fibrils with different structural characteristics has become a research hotspot.In this study,enzyme treatment combined with mechanical grinding was employed to prepare micro-nano fibrils.The effect of enzyme treatment on the characteristics of micro-nano fibrils was studied.The deconstruction kinetics of pulp fibers and mechanism of enzyme recycling during enzyme treatment process were explored,which provides theoretical support for large-scale preparation of micro-nano fibrils.At first,the effect of disk milling as with a supermass-colloider(SMC)on the morphology,size,and rheological properties of micro-nano fibrils was explored.The feasibility of endoglucanase pretreatment to deconstruct fibers in neutral conditions was verified.The results showed with the decrease of the SMC disc gap and the increase of total refining pass,the viscosity of the micro-nano fibrils increased while the average diameter decreased.At the shear rate of 0.1 1/s,the viscosity of the micro-nano fibrils suspension prepared by SMC refining at the gap of-50 ?m for 10 times was 11.37 Pa?s(SMC-G5).Decreasing the SMC disc gap to-100 ?m,the viscosity of obtained micro-nano fibrils suspension(SMC-G10)was 91.98 Pa?s.The diameter distribution of SMC-G5 and SMC-G10 were in the range of 20-130 nm and 9-75 nm while the average diameter was 61 nm and 33 nm,respectively.With the addition of endoglucanase pretreatment in neutral condition,the diameter of micro-nano fibrils and entanglement between fibers were reduced.The fiber treated by endoglucanase in neutral condition(E12T50t1.5)was subjected to SMC grinding.The diameter distribution of obtained micro-nano fibrils(E50SMC-G10)was decreased to 14-58 nm while the average diameter was reduced to 30 nm,indicating that endoglucanase pretreatment improved the uniformity of micro-nano fibrils.Endoglucanase treatment facilitates the deconstruction of cellulosic fiber,but its efficiency is still limited.Therefore,the depolymerization kinetics of cellulosic fiber treated by endoglucanase combined with xylanase was explored.The results showed xylanase can assist endoglucanase to cleave cellulose chain and improve cellulose chains scission rate.The degree of polymerization(DP)of cellulosic fiber treated by endoglucanase pretreatment without and with supplement of xylanase for 240 min were 488(WOX-240)and 428(WX-240),respectively.The cellulose chains scission rate of cellulosic fiber treated by endoglucanase assisted by xylanase for 5 min was 3.2 times faster than the fiber treated only by endoglucanase.The content of intermolecular hydrogen bond O(6)H???O(3?)of untreated fiber was 59.03%.The content of O(6)H???O(3?)of fiber treated only by endoglucanase was 53.00% while the content of O(6)H???O(3?)of fiber treated by endoglucanase combined with xylanase was 34.28% at treatment time of 240 min,indicating that xylanase effectively assisted endoglucanase to open intermolecular hydrogen bonds.The SEM images of fiber treated by enzyme showed that xylanase effectively assisted in endoglucanase deconstructing fiber.The micro-nano fibrils prepared by SMC grinding was highly kinked and non-uniform.Therefore,endoglucanase post-milling treatment was adopted to produce micro-nano fibrils in order to reduce the entanglement between fibers and improve their uniformity.The results showed that endoglucanase post-milling treatment can effectively reduce the length of fiber.The DP of micro-nano fibrils prepared by SMC grinding at disk gap of-100 microns for 1 pass(SMC1P)was 520.After endoglucanase treatment at low endoglucanase dosage of 1 mg/g for24 h(P-SMC1P-1-24h),the DP decreased to 225.Besides,endoglucanase post-milling treatment can effectively reduce the diameter of SMC1 P from 55 nm to 25 nm(P-SMC1P-1-24h).When SMC1 P was treated by endoglucanase at 1mg/g dosage for 6 h,the DP of obtained micro-nano fibrils was 272 with high solid yield of 98.99%.But when the enzyme dosage was increased to 9 mg/g,although the DP of the obtained fiber(P-SMC1P-9-6h)was further reduced to193,the solid yield was greatly reduced to 89.95%.This was because fiber fragments were degraded into sugar by excess endoglucanase.All the micro-nano fibrils suspensions prepared by endoglucanase post-milling treatment were non-Newtonian fluids,which showed characteristics of shear thinning.When the concentration of the micro-nano fibrils suspension was 3 wt%,the viscosity of SMC1 P was 188.64 Pa?s at the shear rate of 0.11/s.SMC1 P after endoglucanase post-milling treatment for 3 h(P-SMC1P-1-3h),the viscosity was reduced to51.54 Pa?s,indicating endoglucanase post-milling treatment effectively reduced the viscosity of the micro-nano fibrils suspension.Endoglucanase pretreatment has great potential in disintegrating cellulosic fibers,but limited by its high price.In order to reduce endoglucanase expanses,endoglucanase was recycled.The results showed significant DP reduction can be achieved from 50.88% to 76.96%only by recycling liquid which included free endoglucanase.However,in the process with supplement of endoglucanase(SR process),no more pronounced reduction of DP was observed in all recycling rounds attributing to competitive adsorption between supplemented and recycled endoglucanase.Supplemented endoglucanase preferentially occupied active sites on cellulose surface,hydrolyzed amorphous region,and followed by degrading fibrils to saccharides together with recycled endoglucanase.At endoglucanase dosage of 54 mg/g in the process without supplement of endoglucanase(WR),the concentration of saccharides in the liquor of the third recycling was 1.27 mg/g.But in SR process,it was increased to 2.39 mg/g.The endoglucanase activity and protein recovery in WR process were 18.91-66.01% and 51.21-65.15%,respectively,while the endoglucanase activity and protein recovery in SR process were65.14-75.80% and 60.10-70.57%,respectively.The endoglucanase activity and protein recovery in SR process was higher than corresponding endoglucanase recycling in WR process.The saturated adsorption capacity of the fibers to fresh endoglucanase at dosage of 30 mg/g(FE-30)was 1.15 mg/g(L0-30).However,for the endoglucanase which was collected after treatment for fiber at one time,it was 0.54 mg/g.It indicated that the affinity of recycled endonuclease to fiber was reduced.By fitting Pseudo second-order kinetic model,the initial adsorption rate constants(h)was obtained.The initial adsorption rate constants of FE-30 and L0-30 were 0.82 g/(mg·min)and 0.26 g/(mg·min),respectively,demonstrating the adsorption rate of fiber to the recycled endoglucanase was relatively slow than fresh endoglucanase.This study can provide theoretical support to decrease endoglucanase cost during endoglucanase treatment process in industrial manufacture.