Method for improving cellulase activity

Abstract

A method for improving cellulase activity belongs to the technical field of biology, a nonionic surfactant and an anionic surfactant are compounded and then added into hydrolysate containing a substrate and cellulase, the mass ratio of the nonionic surfactant to the anionic surfactant is 1: (0.5-3.0), and the addition amount is 0.1%-1.0% of the total mass of the hydrolysate. A nonionic surfactant with a specific HLB value and an anionic surfactant are compounded and added into cellulase hydrolysate, so that the affinity of enzyme and cellulose is remarkably improved, and the catalytic activity is enhanced. According to the method, the yield of glucose can be increased by 30%-50%, the effect is remarkable compared with that of a single surfactant, the cost is reduced, and the method is suitable for the field of lignocellulose biomass conversion.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for improving the catalytic activity of cellulase by using a composite surfactant, and is particularly suitable for the enzymatic hydrolysis and saccharification process of lignocellulosic biomass. Background Art

[0002] Lignocellulose is one of Earth's most abundant renewable resources, and its efficient enzymatic hydrolysis into fermentable sugars is a key step in the production of biofuels and bio-based chemicals. However, the insufficient affinity between cellulase and the cellulose substrate results in low catalytic efficiency, which has become a bottleneck restricting its industrial application. Surfactants can enhance cellulase activity by reducing surface tension, reducing nonspecific adsorption of the enzyme to lignin, modifying substrate structure, and stabilizing the enzyme protein structure. While the addition of a single surfactant can improve enzymatic hydrolysis to some extent, the improvement is limited and often comes with issues such as high cost or cationic sterilization (which is detrimental to subsequent fermentation). For example, the use of a single surfactant typically only increases enzyme activity by 10%–20%, which is difficult to meet the requirements of industrial production. Summary of the Invention

[0003] The purpose of the present invention is to solve the above-mentioned problems in the prior art and provide a method for improving the activity of cellulase. The method is efficient and economical. By innovatively combining non-ionic surfactants and anionic surfactants with specific HLB values, the affinity of cellulase to cellulose is significantly improved, thereby enhancing catalytic activity and reducing the cost of lignocellulose conversion.

[0004] In order to achieve the above object, the present invention adopts the following technical solutions:

[0005] A method for improving cellulase activity comprises compounding a nonionic surfactant and an anionic surfactant and adding the compounded mixture to a hydrolyzate containing a substrate and cellulase, wherein the mass ratio of the nonionic surfactant to the anionic surfactant is 1:0.5-3.0, and the total amount of the nonionic surfactant and the anionic surfactant added is 0.1%-1.0% of the total mass of the hydrolyzate, and hydrolysis is performed in a mechanically stirred or oscillating environment.

[0006] The HLB value of the nonionic surfactant ranges from 8 to 18; the HLB value of the anionic surfactant ranges from 14 to 40.

[0007] Preferably, the mass ratio of the nonionic surfactant to the anionic surfactant is 1:1-2, and the total addition amount of the nonionic surfactant and the anionic surfactant is 0.3%-0.8% of the total mass of the hydrolyzate.

[0008] The cellulase comprises at least one of Trichoderma, Aspergillus and Penicillium.

[0009] The added amount of the cellulase is 5-20 FPU / g substrate.

[0010] The added amount of the cellulase is 8-15 FPU / g substrate.

[0011] The substrate is a pretreated product of corn cobs, straws, bamboo, wood chips or sugarcane bagasse.

[0012] In the present invention, the hydrolysis temperature is 45-60° C., the pH value of the hydrolyzate is 4.0-5.5, and the hydrolysis time is 24-96 hours.

[0013] Preferably, the hydrolysis temperature is 50-55° C., the pH value of the hydrolyzate is 4.5-5.2, and the hydrolysis time is 48-72 hours.

[0014] The nonionic surfactant can be selected from polyoxyethylene sorbitan fatty acid esters (such as Tween 80, HLB is 15), polyethylene glycol (PEG), etc.; the anionic surfactant can be selected from sodium lauryl sulfate (HLB is 40), fatty alcohol polyoxyethylene ether sulfate (AES), etc.

[0015] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0016] 1. Significantly improved catalytic efficiency: In the lignocellulose enzymatic hydrolysis system, the addition of composite surfactants can increase the glucose yield by 30%~50%, which is significantly better than a single surfactant (such as Tween 80 or sodium lauryl sulfate, which only increases by 10%~20% when used alone).

[0017] 2. The nonionic surfactant and anionic surfactant in this invention work synergistically to enhance cellulase activity. The nonionic surfactant adsorbs to the cellulose surface through hydrophobic interactions, reducing interfacial tension and enhancing the enzyme's diffusion capacity. The anionic surfactant binds to the negatively charged sites on the cellulose surface through electrostatic interactions, forming a stable interfacial film that further promotes enzyme adsorption. The synergistic effect of the two significantly increases the affinity between the enzyme and the substrate.

[0018] 3. In the present invention, the combination of a nonionic surfactant (HLB value 8-18) and an anionic surfactant (HLB value 14-40) can form a stable mixed micelle structure at the cellulose-water interface, optimize the enzyme microenvironment, and reduce enzyme inactivation caused by nonspecific adsorption.

[0019] 4. Reduce industrialization costs: Due to the reduction of enzyme dosage and the improvement of hydrolysis efficiency, the cost of lignocellulose conversion can be reduced by 20%~30%, which has significant economic value.

[0020] 5. Strong Universal Applicability: This method is applicable to a variety of cellulase preparations (such as Trichoderma reesei cellulase) and lignocellulosic substrates (such as corn straw, corncobs, sawdust, and sugarcane bagasse), showing broad application prospects. The composite surfactant, through its triple effects of "hydrophobic anchoring, electrostatic stabilization, and interface optimization," synergistically enhances the affinity between cellulase and substrate, addressing the limited effectiveness of traditional single surfactants and providing a new path for efficient enzymatic hydrolysis of lignocellulose. DETAILED DESCRIPTION

[0021] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention clearer and more understandable, the present invention is further described in detail below in conjunction with the embodiments.

[0022] Example 1

[0023] 1. Raw material pretreatment: The corn straw was crushed to a particle size of less than 1 mm, treated with 1% (w / v) dilute sulfuric acid at 160°C for 30 min, washed until neutral, and filtered to obtain a solid sample, which was the lignocellulosic substrate.

[0024] 2. Enzymatic hydrolysis system configuration: substrate concentration 5% (w / v), cellulase (derived from Trichoderma reesei of the genus Trichoderma) dosage 8 FPU / g substrate, pH 4.8, to obtain a hydrolyzate containing substrate and cellulase.

[0025] 3. Surfactant addition: Tween 80 (HLB 15) and sodium lauryl sulfate (HLB 40) were mixed in a 1:1 mass ratio and added at a rate of 0.5% of the total mass of the hydrolyzate. After the two surfactants were mixed, the critical micelle concentration (cmc) dropped to 0.14 mmol / L, the dynamic surface tension (DST) reached equilibrium within 1.42 seconds, and the surface tension reduction rate reached 14.47 mN·m -1 ·s -1 This shows that the composite system can significantly reduce interfacial tension at low concentrations and has excellent dynamic performance.

[0026] 4. Enzymatic Hydrolysis: After 48 hours of shaking reaction at 50°C, the reducing sugar concentration was measured. The results showed a glucose yield of 85% of the theoretical value. Free enzyme was separated from enzyme adsorbed on lignin by centrifugation. The adsorption capacity was calculated by measuring the concentration of free enzyme. The nonspecific adsorption capacity of enzyme to lignin was found to be approximately 0.5 FPU / g lignin.

[0027] Comparative Example 1

[0028] Compared with Example 1, the surfactant used in Comparative Example 1 was Tween 80 (HLB was 15), the addition amount was 0.5%, and the glucose yield was 70%.

[0029] Comparative Example 2

[0030] Compared with Example 1, the surfactant used in Comparative Example 2 was sodium lauryl sulfate (HLB of 40), the addition amount was 0.5%, and the glucose yield was 65%.

[0031] Comparative Example 3

[0032] Compared with Example 1, in Comparative Example 3, no surfactant was added, and the glucose yield was 60%. The free enzyme was separated from the enzyme adsorbed on lignin by centrifugation, and the adsorption amount was calculated by measuring the concentration of the free enzyme. It was found that the nonspecific adsorption amount of the enzyme to lignin was approximately 1.1 FPU / g lignin.

[0033] Compared with Example 3 without adding surfactant, the glucose yield of Example 1 is increased by 41.7%; compared with Example 1 with single Tween, the glucose yield is increased by 21.4%; compared with Example 2 with single sodium lauryl sulfate, the glucose yield is increased by 30.8%.

[0034] Example 2

[0035] 1. Raw material pretreatment: Bagasse is pretreated by steam explosion to obtain substrate.

[0036] 2. Enzymatic hydrolysis system configuration: substrate concentration 5% (w / v), cellulase (derived from Trichoderma viride) dosage 8 FPU / g substrate, pH 4.8, to obtain a hydrolyzate containing substrate and cellulase.

[0037] 3. Addition of surfactant: Polyethylene glycol 4000 (HLB is 14) and sodium polyoxyethylene fatty alcohol ether sulfate (HLB is 15) are compounded in a mass ratio of 1:2, and the addition amount is 0.8% of the total mass of the hydrolyzate.

[0038] 4. Enzymatic hydrolysis reaction: After 72 hours of oscillation reaction at 50°C, the glucose yield was 89% of the theoretical value.

[0039] Comparative Example 4

[0040] Compared with Example 2, the surfactant used in Comparative Example 4 was polyethylene glycol 4000 (HLB: 14), the addition amount was 0.8%, and the glucose yield was 75%.

[0041] Comparative Example 5

[0042] Compared with Example 2, the surfactant used in Comparative Example 5 was sodium fatty alcohol polyoxyethylene ether sulfate (HLB of 15), the addition amount was 0.8%, and the glucose yield was 70%.

[0043] Comparative Example 6

[0044] Compared with Example 2, in Comparative Example 6, no surfactant was added and the glucose yield was 62%.

[0045] Compared with Example 6 in which no surfactant was added, the glucose yield of Example 2 was increased by 43.5%; compared with Example 4 of single polyethylene glycol, the glucose yield was increased by 18.7%; compared with Example 5 of single fatty alcohol polyoxyethylene ether sodium sulfate, the glucose yield was increased by 27.1%.

[0046] In the present invention, nonionic surfactants (such as Tween 80) are adsorbed on the cellulose surface through hydrophobic interaction, and anionic surfactants (such as sodium lauryl sulfate) are combined through electrostatic interaction to form a mixed interfacial film, which promotes the adsorption and catalysis of cellulase.

[0047] The step-by-step mechanism of the composite surfactant of the present invention is described in detail as follows:

[0048] 1. Hydrophobic barrier on cellulose surface and surfactant adsorption

[0049] The surface of cellulose fibers is covered with lignin and hemicellulose, forming a hydrophobic barrier that hinders enzyme access.

[0050] Nonionic surfactant (Tween 80): The hydrophobic end (fatty acid chain) adsorbs to the hydrophobic region of cellulose / lignin, and the hydrophilic end (polyoxyethylene chain) faces the aqueous phase, reducing the interfacial tension and initially dispersing the cellulose.

[0051] Anionic surfactant (sodium dodecyl sulfate): The hydrophobic end is inserted into the hydrophobic structure of lignin, and the hydrophilic end is negatively charged, forming an electrostatic repulsion or stable hydration layer with the hydroxyl groups (partially negatively charged) on the surface of cellulose, while repelling the nonspecific adsorption of negatively charged enzyme proteins.

[0052] 2. Formation of mixed interface film and substrate structure optimization

[0053] The two surfactants form a mixed interfacial film on the cellulose surface: the non-ionic component is fixed by hydrophobic interaction, and the anionic component enhances the membrane stability through electrostatic interaction. The two surfactants synergistically destroy the lignin-cellulose complex and expose more cellulose hydroxyl groups and reducing ends.

[0054] The interfacial film reduces the crystallinity of cellulose and increases its porosity, making it easier for the carbohydrate binding module (CBM) of cellulase to recognize and bind to the cellulose surface.

[0055] 3. Inhibit nonspecific adsorption of enzymes and promote catalytic reactions

[0056] Untreated system: Lignin adsorbs enzyme molecules through hydrophobic interaction, resulting in ineffective binding.

[0057] Complex surfactant system: The acid radicals of the anionic surfactants reduce enzyme adsorption to lignin through electrostatic repulsion, while the hydrophilic layer of the nonionic surfactants creates steric hindrance, forcing the enzyme to preferentially bind to the active sites of cellulose. The catalytic domain of the cellulase efficiently hydrolyzes the β-1,4-glycosidic bonds of cellulose to produce glucose / cellobiose.

[0058] 4. Key synergy points

[0059] Hydrophobic-electrostatic dual effect: non-ionic surfactants are "anchored" to cellulose through hydrophobic action, and anionic surfactants stabilize the interfacial film through electrostatic action. The combination of the two significantly reduces interfacial tension (significantly lower than that of a single surfactant) and increases the enzyme diffusion rate.

[0060] Reduce enzyme loss: Inhibit nonspecific adsorption of enzymes to lignin (reduction of adsorption by 40% to 60%), increase the effective enzyme concentration, and improve catalytic efficiency by 30% to 50% (for example, in the example, glucose yield increased from 60% to 85%).

[0061] In summary, the synergistic effect of the composite surfactant of the present invention significantly improved the activity of cellulase, and the effect was better than that of a single surfactant.

Claims

1. A method for increasing cellulase activity, characterized in that: The nonionic surfactant and the anionic surfactant are compounded and added to the hydrolyzate containing the substrate and the cellulase, the mass ratio of the nonionic surfactant to the anionic surfactant is 1:0.5~3.0, and the total addition amount of the nonionic surfactant and the anionic surfactant is 0.1%~1.0% of the total mass of the hydrolyzate.

2. The method for increasing cellulase activity according to claim 1, wherein: The HLB value of the nonionic surfactant ranges from 8 to 18; the HLB value of the anionic surfactant ranges from 14 to 40.

3. The method for increasing cellulase activity according to claim 1, wherein: The mass ratio of the nonionic surfactant to the anionic surfactant is 1:1-2.

4. The method for increasing cellulase activity according to claim 1, wherein: The total amount of nonionic surfactant and anionic surfactant added is 0.3%~0.8% of the total mass of the hydrolyzate.

5. The method for increasing cellulase activity according to claim 1, wherein: The cellulase comprises at least one of Trichoderma, Aspergillus and Penicillium.

6. The method for increasing cellulase activity according to claim 1, wherein: The added amount of the cellulase is 5-20 FPU / g substrate.

7. The method for increasing cellulase activity according to claim 6, wherein: The added amount of the cellulase is 8-15 FPU / g substrate.

8. The method for increasing cellulase activity according to claim 1, wherein: The substrate is a pretreated product of corn cobs, straws, bamboo, wood chips or sugarcane bagasse.

9. The method for increasing cellulase activity according to claim 1, wherein: The hydrolysis temperature is 45-60°C, the pH value of the hydrolyzate is 4.0-5.5, and the hydrolysis time is 24-96 hours.

10. The method for increasing cellulase activity according to claim 9, wherein: The hydrolysis temperature is 50~55°C, the pH value of the hydrolyzate is 4.5~5.2, and the hydrolysis time is 48~72 hours.