阅读说明：本技术 一种生物质高固酶解过程中产物葡萄糖抑制的解决方法 (Solution method for product glucose inhibition in biomass high-solid enzymolysis process ) 是由 胡昌辉 王静 曾徐浩 罗家星 李勉 杨武龙 孟祥翔 甄妮 于 2020-12-24 设计创作，主要内容包括：本发明公开一种生物质高固酶解过程中产物葡萄糖抑制的解决方法,将粉碎后的木质纤维类生物质和PEG、硫酸镁混合溶液混合,获得高固酶解体系；再于高固酶解体系中加入纤维素酶液,进行酶解；酶解结束后,酶解液静置分离为两相,上相液体通过超滤膜超滤得高浓度葡萄糖溶液,下相液体离心,沉淀为不溶性废渣,上清为低浓度葡萄糖溶液和可回收纤维素酶。在高固酶解体系中,本发明方法可以在酶解过程中,将葡萄糖和反应相(木质纤维素类生物质和纤维素酶)分开,避免了高固酶解后期葡萄糖的积累抑制纤维素酶活性,提高整体酶解效率和转化率。(The invention discloses a solution for inhibiting glucose in a product in a biomass high-solid enzymolysis process, which comprises the steps of mixing crushed wood fiber biomass with a mixed solution of PEG and magnesium sulfate to obtain a high-solid enzymolysis system; adding cellulase liquid into the high-solid enzymolysis system for enzymolysis; after enzymolysis is finished, standing and separating the enzymolysis liquid into two phases, ultrafiltering the upper phase liquid through an ultrafiltration membrane to obtain a high-concentration glucose solution, centrifuging the lower phase liquid to precipitate insoluble waste residues, and collecting the supernatant which is the low-concentration glucose solution and the recyclable cellulase. In a high-solid enzymolysis system, the method can separate glucose from a reaction phase (lignocellulose biomass and cellulose) in the enzymolysis process, so that the accumulation of glucose in the later stage of high-solid enzymolysis is avoided, the activity of the cellulose is inhibited, and the overall enzymolysis efficiency and the conversion rate are improved.)

1. A solution for inhibiting glucose in a product in a biomass high-solid enzymolysis process is characterized in that a high-solid enzymolysis system is obtained by mixing crushed lignocellulose biomass with a mixed solution of PEG and magnesium sulfate; adding cellulase liquid into the high-solid enzymolysis system for enzymolysis; after enzymolysis is finished, standing and separating the enzymolysis liquid into two phases, ultrafiltering the upper phase liquid through an ultrafiltration membrane to obtain a high-concentration glucose solution, centrifuging the lower phase liquid to precipitate insoluble waste residues, and collecting the supernatant which is the low-concentration glucose solution and the recyclable cellulase.

2. The method for solving the problem of glucose inhibition of the product in the biomass high-solid enzymolysis process according to claim 1, wherein the mixed solution of PEG and magnesium sulfate is prepared by mixing a PEG solution and a magnesium sulfate solution according to a volume ratio of 1: 2-2: 1, the concentration of the PEG solution is 10wt% -25 wt%, the polymerization degree of PEG used in the PEG solution is 2000-6000, and the concentration of the magnesium sulfate solution is 10wt% -25 wt%.

3. The solution of glucose inhibition of products in biomass high-solid enzymatic hydrolysis process as claimed in claim 2, wherein the concentration of PEG solution is 20wt%, the polymerization degree of PEG used in PEG solution is 6000, and the concentration of magnesium sulfate solution is 15 wt%.

4. The method for solving the problem of glucose inhibition of the product in the biomass high-solids enzymolysis process according to claim 1, wherein the dry basis content of the lignocellulose biomass in the high-solids enzymolysis system is 15-20 wt%.

5. The solution for inhibiting the glucose in the product of the biomass high-solid enzymatic hydrolysis process according to claim 1, wherein the pH value of a high-solid enzymatic hydrolysis system is 5.5-6.0.

6. The solution of glucose inhibition of products in biomass high-solids enzymolysis process according to claim 1, wherein the lignocellulosic biomass comprises one or more of corncob, xylose residue and straw, and the pulverized lignocellulosic biomass is below 10 mesh.

7. The solution for inhibiting the product glucose in the biomass high-solids enzymolysis process according to claim 1, wherein the addition amount of the cellulase liquid is 4-6 wt% of the dry weight of the lignocellulose biomass, the concentration of the cellulase liquid and the enzyme protein is 195-200 mg/ml, and the activity is 1000 BHU-2/g.

8. The method for solving the problem of glucose inhibition of the product in the biomass high-solid enzymolysis process according to claim 1, wherein enzymolysis is carried out for 48-96 h at 45-50 ℃ and at a stirring speed of 50-200 rpm.

9. The solution for inhibiting the glucose in the product of the biomass high-solids enzymolysis process according to claim 1, wherein the enzymolysis solution is allowed to stand for 1-2 hours to separate into two phases.

10. The solution of glucose inhibition of a product in the biomass high-solid enzymolysis process according to claim 1, wherein the upper phase liquid is ultrafiltered by an ultrafiltration membrane with a molecular weight cut-off of 3kDa to 10kDa to obtain a high-concentration glucose solution.

Technical Field

The invention relates to the technical field of biomass enzymolysis, in particular to a solution for inhibiting glucose as a product in a biomass high-solid enzymolysis process.

Background

China is a big agricultural country, a large amount of lignocellulose biomass wastes such as straws, corncobs and the like are produced every year, and the current main method for treating the wastes is incineration. The main components of the lignocellulose biomass are cellulose, hemicellulose, lignin and a small amount of ash, wherein the cellulose component can be subjected to enzymolysis and saccharification by cellulase to obtain glucose.

At present, the main research direction of the process for producing glucose by cellulose enzymolysis in lignocellulose biomass is to improve the high solid content of an enzymolysis system, and the high solid content enzymolysis has the following advantages: the concentration of the product glucose is improved, the cost of the subsequent concentration process is reduced, and the method lays a foundation for the subsequent processes of monosaccharide fermentation and the like; secondly, the water consumption and the energy consumption in the enzymolysis process are reduced, and the time-space efficiency of the enzymolysis process is improved.

The development of high-solid enzymatic hydrolysis process also has many bottlenecks, and the most important problem is the problem of inhibition of the activity of the cellulase protein by the glucose product, namely product inhibition. In the high-solid enzymolysis process, particularly in the later stage of enzymolysis, the concentration of released monomer glucose is increased, the activity inhibition on cellulase is stronger, the overall enzymolysis efficiency is reduced, and the conversion efficiency of the enzymolysis cellulose is reduced. At present, some solutions are proposed by research in order to overcome the problem of product inhibition in the enzymatic hydrolysis process. Such as Simultaneous Saccharification and Fermentation (SSF), where the glucose is released enzymatically, i.e., fermented by the microorganisms to ethanol, thereby alleviating product inhibition. However, since the saccharification temperature is usually higher than the fermentation temperature, the simultaneous saccharification and fermentation process cannot take into account the optimal conditions for both enzymatic saccharification and fermentation, and both the enzymatic hydrolysis and fermentation efficiency are affected.

Disclosure of Invention

The invention aims to provide a solution for inhibiting glucose in a product in a biomass high-solid enzymolysis process, so as to overcome the defects of the prior art.

The invention adopts the following technical scheme:

a solution for inhibiting glucose in a product in a biomass high-solid enzymolysis process is characterized in that crushed lignocellulose biomass is mixed with a mixed solution of PEG and magnesium sulfate to obtain a high-solid enzymolysis system; adding cellulase liquid into the high-solid enzymolysis system for enzymolysis; after enzymolysis is finished, standing and separating the enzymolysis liquid into two phases, ultrafiltering the upper phase liquid through an ultrafiltration membrane to obtain a high-concentration glucose solution, centrifuging the lower phase liquid to precipitate insoluble waste residues, and collecting the supernatant which is the low-concentration glucose solution and the recyclable cellulase.

Furthermore, the PEG and magnesium sulfate mixed solution is prepared by mixing a PEG solution and a magnesium sulfate solution according to the volume ratio of 1: 2-2: 1, the concentration of the PEG solution is 10wt% -25 wt%, the polymerization degree of PEG used by the PEG solution is 2000-6000, and the concentration of the magnesium sulfate solution is 10wt% -25 wt%.

Further, the concentration of the PEG solution was 20wt%, the degree of polymerization of PEG used for the PEG solution was 6000, and the concentration of the magnesium sulfate solution was 15 wt%.

Further, the dry basis content of the lignocellulose biomass in the high-solid enzymolysis system is 15-20 wt%.

Further, the pH value of the high-solid enzymolysis system is 5.5-6.0.

Further, the lignocellulose biomass comprises one or more of corncobs, xylose residues and straws, and the crushed lignocellulose biomass is below 10 meshes.

Furthermore, the adding amount of the cellulase liquid is 4-6 wt% of the dry basis amount of the lignocellulose biomass, the concentration of the enzyme protein in the cellulase liquid is 195-200 mg/ml, and the activity is 1000 BHU-2/g.

Further, carrying out enzymolysis for 48-96 h at 45-50 ℃ and at a stirring speed of 50-200 rpm.

Further, standing the enzymolysis liquid for 1-2 h, and separating into two phases.

Further, the upper phase liquid is ultrafiltered by an ultrafiltration membrane with the molecular weight cutoff of 3-10 kDa to obtain a high-concentration glucose solution.

The invention has the beneficial effects that:

1. in a high-solid enzymolysis system, the method can separate glucose from a reaction phase (lignocellulose biomass and cellulose) in the enzymolysis process, so that the accumulation of glucose in the later stage of high-solid enzymolysis is avoided, the activity of the cellulose is inhibited, and the overall enzymolysis efficiency and the conversion rate are improved.

2. The method can effectively separate the glucose and the cellulase protein in the enzymolysis process, thereby simply recovering and reusing the cellulase protein.

Drawings

FIG. 1 is a schematic flow diagram of the process of the present invention;

FIG. 2 is a graph of glucose concentrations for different two-phase systems of example 1;

FIG. 3 is a graph of cellulase protein concentrations for different two-phase systems of example 1;

FIG. 4 is a graph showing the change in glucose amount in the enzymatic hydrolysate of example 2;

FIG. 5 is a comparison of the enzymatic effects of a single-phase system and a two-phase system.

Detailed Description

In order to make the technical problems, technical solutions and advantageous effects to be solved by the present invention more clearly apparent, the present invention is further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

A solution for product glucose inhibition in biomass high-solid enzymolysis process is shown in figure 1, and comprises the following steps:

1. and mixing the crushed wood fiber biomass with the PEG and magnesium sulfate mixed solution in a reactor to obtain a high-solid enzymolysis system. Wherein, the lignocellulose biomass comprises one or more of corncob, xylose residue and straw, but is not limited thereto, the crushed lignocellulose biomass is preferably below 10 meshes (in fig. 1, the corncob residue is taken as an example); the PEG and magnesium sulfate mixed solution is prepared by mixing a PEG solution and a magnesium sulfate solution according to the volume ratio of 1: 2-2: 1, the concentration of the PEG solution is 10wt% -25 wt%, the preferable weight is 20wt%, the polymerization degree of PEG used by the PEG solution can be 2000-6000, the preferable weight is 6000, and the concentration of the magnesium sulfate solution is 10wt% -25 wt%, the preferable weight is 15 wt%; the lignocellulose biomass dry basis content in the high-solid enzymolysis system is 15-20 wt%, the pH value of the high-solid enzymolysis system is 5.5-6.0, and the pH value is adjusted by using a hydrochloric acid solution and a sodium hydroxide solution.

2. And adding a cellulase solution into the high-solid enzymolysis system, and carrying out enzymolysis for 48-96 h at the temperature of 45-50 ℃ and the stirring speed of 50-200 rpm. Wherein the addition amount of the cellulase liquid is 4-6 wt% of the dry basis amount of the lignocellulose biomass, the concentration of the enzyme protein in the cellulase liquid is 195-200 mg/ml, and the activity is 1000 BHU-2/g.

3. After enzymolysis is finished, standing the enzymolysis liquid for 1-2 hours to separate the enzymolysis liquid into two clear phases, ultrafiltering the upper phase liquid through an ultrafiltration membrane with the molecular weight cutoff of 3-10 kDa to obtain a high-concentration glucose solution, centrifuging the lower phase liquid to precipitate insoluble waste residues, and obtaining the supernatant which is a low-concentration glucose solution and contains the recoverable cellulase.

The concentration of the cellulase liquid enzyme protein related to the following embodiment is 195-200 mg/ml, the activity is 1000BHU-2/g, and Novozymes Cellic CTec 2.

Example 1: effect of different two-phase systems on the distribution ratio of glucose and cellulase proteins

And (3) changing the polymerization degree of PEG, changing the concentration of the PEG solution and the concentration of the magnesium sulfate solution, and designing different two-phase systems.

Table 1: different two-phase system

Adding 100g of glucose and 4g of cellulase liquid mixture into different two-phase systems with the volume of 1L, stirring and mixing uniformly, standing for 1h, after the systems are separated, taking the upper phase liquid and the lower phase liquid with the volume of 0.46L and the lower phase liquid with the volume of 0.56L respectively, and measuring the concentrations of glucose and cellulase protein. The results are shown in FIGS. 2 and 3.

As a result: the proportion of the upper phase and the lower phase of glucose and cellulase protein in different two-phase systems is different, and generally speaking, the proportion of the glucose in the upper phase is more than that in the lower phase, and conversely, the proportion of the protein in the upper phase is less than that in the lower phase. Among them, the A3 system has the best separation effect of glucose and cellulase protein.

Example 2: the method of the invention prevents inhibition of glucose products during enzymatic hydrolysis

21. In the embodiment, the two-phase enzymolysis process is compared with the traditional direct enzymolysis process, cellulose is used as a raw material, and the effect of the two-phase enzymolysis process on the aspect of preventing the inhibition of the glucose product is analyzed.

22. Preparing a two-phase system, preparing 20wt% concentration PEG6000 solution and 15wt% magnesium sulfate solution, and mixing the two solutions in a ratio of 1: 1 volume of the mixture is mixed to prepare a two-phase system B0.

23. Two-phase enzymolysis: taking 15g of cellulose, adding 100g of two-phase system B0, adding 10g of glucose, maintaining the pH value of the system to be 5.5-6.0 by using 0.1mol/L sodium hydroxide solution and 0.1mol/L hydrochloric acid solution, adding 0.62g of cellulase solution, controlling the temperature to be 45-50 ℃, carrying out enzymolysis for 72 hours under the stirring condition (180-200 rpm), sampling every 12 hours, measuring the upper-phase glucose concentration and the lower-phase glucose concentration, and counting the whole glucose amount.

24. Single-phase enzymolysis: taking 15g of cellulose, adding 100g of water, adding 10g of glucose, maintaining the pH value of the system to be 5.5-6.0 by utilizing 0.1mol/L sodium hydroxide solution and 0.1mol/L hydrochloric acid solution, adding 0.62g of cellulase solution, controlling the temperature to be 45-50 ℃, carrying out enzymolysis for 72 hours under the stirring condition (180-200 rpm), and sampling every 12 hours to determine the enzymolysis glucose amount.

25. The glucose amount change curves of the two enzymolysis solutions are shown in FIG. 4. As a result: in the case of glucose, the two-phase enzymolysis method produces more glucose and has higher speed than single-phase enzymolysis.

Example 3: enzymatic glucose Release amount (15 wt% dry basis) of the method of the invention

31. Preparing a two-phase system, preparing 20wt% concentration PEG6000 solution and 15wt% magnesium sulfate solution, and mixing the two solutions in a ratio of 1: 1 volume of

Mixing to obtain a two-phase system B1.

32. Taking a corncob waste residue raw material containing 15.4g of dry basis content, adding 85g of a two-phase system B1, maintaining the pH of the system to be 5.5-6.0 by utilizing 0.1mol/L sodium hydroxide solution and 0.1mol/L hydrochloric acid solution, adding 0.62g of cellulase solution, controlling the temperature to be 45-50 ℃, and carrying out enzymolysis for 72 hours under the stirring condition (180-200 rpm).

33. After enzymolysis is finished, the enzymolysis liquid is kept stand for 1 hour, the enzymolysis liquid is separated into two clear phases, the volume of an upper phase is 48mL, the volume of a lower phase is 56mL, the glucose concentration measured by taking the upper phase is 133.5g/L, the glucose concentration of the lower phase is 45.7g/L, and the glucose conversion rate reaches 88.3%.

34. The upper phase liquid was ultrafiltered through an ultrafiltration membrane of 10kDa pore size to give 35mL of a glucose solution with a concentration of 170.3 g/L.

Example 4: enzymatic glucose Release amount (15 wt% dry basis) of the method of the invention

41. Preparing a two-phase system, preparing 20wt% concentration PEG6000 solution and 15wt% magnesium sulfate solution, and mixing the two solutions in a ratio of 1: 1 volume of the mixture is mixed to prepare a two-phase system B2.

42. Taking a corncob waste residue raw material containing 15.3g of dry basis content, adding 86g of a two-phase system B2, maintaining the pH of the system to be 5.5-6.0 by utilizing 0.1mol/L sodium hydroxide solution and 0.1mol/L hydrochloric acid solution, adding 0.63g of cellulase solution, controlling the temperature to be 45-50 ℃, and carrying out enzymolysis for 72 hours under the stirring condition (180-200 rpm).

43. After enzymolysis is finished, the enzymolysis liquid is kept stand for 1 hour, the enzymolysis liquid is separated into two clear phases, the volume of an upper phase is 49mL, the volume of a lower phase is 56mL, the glucose concentration measured by taking the upper phase is 131.5g/L, the glucose concentration of the lower phase is 44.7g/L, and the glucose conversion rate reaches 87.7%.

44. The upper phase liquid was ultrafiltered through an ultrafiltration membrane of 10kDa pore size to give 35mL of a glucose solution of 167.8g/L concentration.

Example 5: enzymatic glucose Release amount (20 wt% dry basis) of the method of the invention

51. Preparing a two-phase system, preparing 20wt% concentration PEG6000 solution and 15wt% magnesium sulfate solution, and mixing the two solutions in a ratio of 1: 1 volume of the mixture is mixed to prepare a two-phase system B3.

52. Taking a corncob waste residue raw material containing 20.3g of dry basis content, adding 80g of a two-phase system B3, maintaining the pH of the system to be 5.5-6.0 by utilizing 0.1mol/L sodium hydroxide solution and 0.1mol/L hydrochloric acid solution, adding 0.82g of cellulase solution, controlling the temperature to be 45-50 ℃, and carrying out enzymolysis for 72 hours under the stirring condition (180-200 rpm).

53. After enzymolysis is finished, standing the enzymolysis liquid for 1h, separating the enzymolysis liquid into two clear phases, wherein the volume of an upper phase is 48mL, the volume of a lower phase is 59mL, the glucose concentration measured by taking the upper phase is 161.1g/L, the glucose concentration of the lower phase is 55.5g/L, and the glucose conversion rate reaches 81.2%.

54. The upper phase liquid is ultrafiltered by an ultrafiltration membrane with the aperture of 10kDa to obtain 35mL of glucose solution with the concentration of 190.3 g/L.

Example 6: enzymatic glucose Release amount (20 wt% dry basis) of the method of the invention

61. Preparing a two-phase system, preparing 20wt% concentration PEG6000 solution and 15wt% magnesium sulfate solution, and mixing the two solutions in a ratio of 1: 1 volume of the mixture is mixed to prepare a two-phase system B4.

62. Taking a corncob waste residue raw material with the dry basis content of 20.1g, adding 81g of a two-phase system B4, maintaining the pH value of the system to be 5.5-6.0 by utilizing 0.1mol/L sodium hydroxide solution and 0.1mol/L hydrochloric acid solution, adding 0.82g of cellulase solution, controlling the temperature to be 45-50 ℃, and carrying out enzymolysis for 72 hours under the stirring condition (180-200 rpm).

63. After enzymolysis is finished, the enzymolysis liquid is kept stand for 1 hour, the enzymolysis liquid is separated into two clear phases, the volume of the upper phase is 49mL, the volume of the lower phase is 60mL, the glucose concentration measured by taking the upper phase is 160.5g/L, the glucose concentration of the lower phase is 54.8g/L, and the glucose conversion rate reaches 83.2%.

64. The upper phase liquid was ultrafiltered through an ultrafiltration membrane of 10kDa pore size to give 35mL of a glucose solution with a concentration of 188.3 g/L.

Example 7: the method of the invention and the single-phase enzymolysis method have the effect comparison

71. Data from examples 3-6 were collected and statistics were made for glucose conversion on a dry basis of 15wt% and 20wt% on a dry basis.

72. Taking 15g of a corncob waste residue raw material (15 wt% of dry basis) with dry basis content, adding water until the whole volume is 100mL, maintaining the pH of the system to be 5.5-6.0 by using 0.1mol/L sodium hydroxide solution and 0.1mol/L hydrochloric acid solution, adding 0.62g of cellulase solution, controlling the temperature to be 45-50 ℃, carrying out enzymolysis for 72h under a stirring condition (180-200 rpm), measuring the glucose concentration, and carrying out 3 batches in parallel.

73. Taking 20g of a corncob waste residue raw material (20 wt% of dry basis) with dry basis content, adding water until the whole volume is 100mL, maintaining the pH of the system to be 5.5-6.0 by using 0.1mol/L sodium hydroxide solution and 0.1mol/L hydrochloric acid solution, adding 0.81g of cellulase solution, controlling the temperature to be 45-50 ℃, carrying out enzymolysis for 72h under a stirring condition (180-200 rpm), measuring the glucose concentration, and carrying out 3 batches in parallel.

74. The glucose conversion was counted and the results are shown in FIG. 5. As a result: by utilizing a two-phase enzymolysis system, the conversion rate of glucose is improved by 7% compared with that of a normal enzymolysis system under the condition of 15wt% of dry basis, and the conversion rate of glucose is improved by 19% under the condition of 20wt% of dry basis. Therefore, under the condition of high-solid enzymolysis, the inhibition effect of glucose on cellulase can be effectively reduced by using a two-phase enzymolysis system, and the enzymolysis conversion rate is improved.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.