A method for promoting the efficiency of enzymatic hydrolysis of cellulose by cellic ctec3

Abstract

The application discloses a method for promoting enzymatic hydrolysis efficiency of cellulose by Cellic CTec3, and belongs to the technical field of biology.The cellulose is uniformly dispersed in a buffer, Cellic CTec3 and Knickkopf protein are added, and after being uniformly mixed, the mixture is reacted at 25-60 DEG C for 5-60 h, so that an enzymatic hydrolysis solution containing high-concentration reducing sugar is obtained; the addition of the Knickkopf protein can significantly reduce the dosage of the Cellic CTec3 and simultaneously improve the enzymatic hydrolysis efficiency, so that the production cost of cellulose ethanol can be greatly reduced, the industrialization process can be accelerated, and the method has a very good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for improving the efficiency of cellulose enzymatic hydrolysis by Cellic CTec3. Background Technology

[0002] With the global energy transition, the resource utilization of agricultural and forestry waste has become a key path to solving the energy crisis and environmental pollution. Among them, straw biomass is considered an ideal renewable resource due to its large yield and high content of cellulose (35-50%), hemicellulose (20-35%), and lignin (10-25%). Converting straw into fermentable sugars through enzymatic hydrolysis and saccharification technology, and then producing biofuels such as cellulosic ethanol or high-value chemicals, is the core approach to achieving its high-value utilization.

[0003] Cellic CTec3, launched globally by Novozymes in February 2012, is specifically designed for the commercial production of cellulosic ethanol and is the third-generation upgrade following CTec (2009) and CTec2 (2010). Its core components include highly active cellulases (such as exoglucanase and endoglucanase), a modified β-glucosidase, and a novel hemicellulase, which work together to degrade cellulose and hemicellulose into fermentable sugars. It also contains a cleaving polysaccharide monooxygenase (LPMO) that cleaves cellulose chains through oxidation, disrupting the lignin-carbohydrate complex and significantly improving the accessibility of crystalline cellulose. Compared to other commercially available enzyme preparations, the amount of Cellic CTec3 enzyme preparation required to produce the same yield of cellulosic ethanol is only one-fifth that of other enzyme preparations, improving the economics of bioethanol. Further improving the enzymatic hydrolysis efficiency based on Cellic CTec3 is expected to significantly reduce the production cost of cellulosic ethanol and accelerate the industrialization process.

[0004] Knickkopf (Knk) is a protein family found in many insect groups with known genomic information, but research on the function of Knk family proteins is still very limited, with reports only in Drosophila and the red flour beetle. In Drosophila, mutations in the gene encoding Knk prevent the normal formation of fibrous structures from chitin in the epidermis and trachea. The expression period of the Knk gene has a significant impact on the macroscopic morphology, microscopic structure, and osmotic resistance of Drosophila wings. RNA interference with Knk1 in the red flour beetle and the migratory locust leads to epidermal structural disorder, molting failure, and death. However, current research on insect Knk is mainly at the gene level, and its biochemical functions and mechanisms of action remain unknown. No studies have been reported on its use to improve the enzymatic digestion efficiency of Cellic CTec3. Summary of the Invention

[0005] Therefore, the object of the present invention is to provide a method for improving the efficiency of cellulose enzymatic hydrolysis by Cellic CTec3.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] This invention provides a method for improving the efficiency of Cellic CTec3 enzymatic hydrolysis of cellulose, comprising the following steps: adding cellulose to a buffer solution and dispersing it evenly, adding Cellic CTec3 and Knickkopf protein, mixing evenly, and reacting at 25~60℃ for 5~60h to obtain an enzymatic hydrolysate containing a high concentration of reducing sugars.

[0008] Based on the above technical solution, the amino acid sequence of the Knickkopf protein is further selected from: (a) the amino acid sequence shown in SEQ ID NO.1, or (b) an amino acid sequence that has at least 70% identity with the amino acid sequence shown in SEQ ID NO.1, preferably an amino acid sequence that has at least 80% identity.

[0009] Based on the above technical solution, the preparation method of the Knickkopf protein is as follows: a linearized plasmid vector containing the Knickkopf protein encoding gene is introduced into Pichia pastoris to induce recombinant protein expression, and the protein is obtained after ammonium sulfate precipitation and dialysis.

[0010] Based on the above technical solution, further, the mass concentration of Knickkopf protein in the reaction system is 0.01~0.5%, the mass concentration of Cellic CTec3 is 0.1~1%, and the mass concentration of cellulose is 10~30%.

[0011] Based on the above technical solution, the reaction temperature is further 40~60℃ and the reaction time is 5~48h.

[0012] Based on the above technical solution, the pH of the buffer solution is further specified as 4-8.

[0013] Based on the above technical solution, the buffer solution further includes phosphate buffer solution.

[0014] Based on the above technical solution, the cellulose further includes steam-exploded corn stalks.

[0015] Compared with the prior art, the present invention has the following beneficial effects: 1. In the enzymatic hydrolysis experiment of 20% concentration of steam-exploded straw, the reducing sugar yield of the experimental group with 1% Cellic CTec3 and the same mass concentration of Knickkopf protein was basically the same as that of the experimental group with 5% Cellic CTec3, indicating that Knickkopf can reduce the amount of Cellic CTec3 used.

[0016] 2. As the storage time increases, the enzymatic activity of Cellic CTec3 gradually decreases. In an enzymatic hydrolysis experiment using Cellic CTec3 stored for more than three months, when enzymatically hydrolyzing 20% ​​concentration of steam-exploded straw, the reducing sugar yield in the experimental group with only Cellic CTec3 added was significantly reduced. After adding an equal mass concentration of Knickkopf protein, the reducing sugar yield returned to normal levels, indicating that Knickkopf protein can extend the shelf life of Cellic CTec3. Attached Figure Description

[0017] To more clearly illustrate the embodiments of the present invention, the accompanying drawings involved in the embodiments will be briefly described below.

[0018] Figure 1 The graph shows the promoting effect of Knickkopf protein on the enzymatic hydrolysis of steam-exploded straw by Cellic CTec3. The horizontal axis represents the enzymatic hydrolysis reaction time in hours, and the vertical axis represents the reducing sugar content in the system as measured by the DNS method in g / L.

[0019] Figure 2 The graph shows the promoting effect of Knickkopf protein on the enzymatic hydrolysis of steam-exploded straw by Cellic CTec3 with reduced activity. The horizontal axis represents the enzymatic hydrolysis reaction time in hours, and the vertical axis represents the reducing sugar content in the system as measured by the DNS method in g / L. Detailed Implementation

[0020] The present invention will be described in detail below with reference to the embodiments. However, the implementation of the present invention is not limited thereto. Obviously, the embodiments described below are only some embodiments of the present invention. For those skilled in the art, other similar embodiments can be obtained without creative effort and all fall within the protection scope of the present invention.

[0021] Example 1 The amino acid sequence of Knickkopf (Knk) in this embodiment is shown below (SEQ ID NO.1): MALSSRVLCLRLQHHLLLVILTIFLLCPAASAQNEEEDGPYRGKYLGKLNSYHHQVSGDVYAVNEYTFLIVGFNYDGNGADTFFWSGASNRPGPQGFIVPDEYGKTNILDRYHNKDFTLTLPDRKKITEIKWLAVYDLSSQNNFGDVYIPEEFDPPMSQLGGTFSKRSHNVSSSSVEILDSKTIRIKDFTYDGRGKRTFFWTGVGPQPSSRGSKLPDERGYLDPIRQYNKETIELELPGDKTIFDIDWISVYDVADNENYGHVLFNDKLNVPPSLVKVTPFEFSLPNCRQLHKDMQVSWEVFGPQITFQLSGQVGGNDYMSFGISGSDVSSQMIGSDVVVAYIDDIRGYTVDYNITSLAPCVQVLGQNKGVCRDDVVGGLDSFQLNTYSRKDGINTISFRRTLKSSDDGDKEIFLDRSNYVIWAFGPLDSNNEPAFHTYYPKSDIVIDFNTTEPVNDCFAFTKRAETTNPPVWERTRITDATVRTFNAYLGPSGGLRGYQGLTNHVSSGLAWYINGYMIPELYLKRGLTYTFKVRGGNNPHSPEHYHPLVITDDPQGGYDRLSDAKQSEIRVLAGVEFTRRGRPKPTAAGPLCLSRYPQNSDRRLDDNFPTFKKFNRSLITECVEGEPALLEITPNITWPDTVYYNSFTHGNMGWKIHIVDSYTNLKSGSMGLSWSLCIILLPWLVLQN。

[0022] Knickkopf expression: pPIC9k was used as the expression vector for Knickkopf. The constructed plasmid vector containing the Knickkopf gene was introduced into Pichia pastoris GS115 cells for recombinant expression. The plasmid was synthesized by GenScript Biotech Co., Ltd. and linearized by treating with Sac I restriction endonuclease at 37°C for 1 h. Pichia pastoris GS115 competent cells were purchased from Shanghai Weidi Biotechnology Co., Ltd. 80 μL of Pichia pastoris GS115 competent cells and 10 μL of linearized plasmid were added to a pre-chilled electroporation cuvette, mixed, and incubated on ice for 10 min. The electroporation conditions were: voltage 1.5 kV, capacitance 25 μF, resistance 200 Ω, and pulse time approximately 5 msec. After electroporation, 1 mL of pre-cooled 1 M sterile sorbitol solution was quickly added and transferred to a 1.5 mL centrifuge tube. The tube was incubated at 30°C for 3 h. After centrifugation at 1500 rpm for 1 min, 800 μL of supernatant was discarded. The remaining bacterial culture was then plated on a histidine-deficient plate and incubated at 30°C for 3-5 days, observing colony growth. Larger colonies were picked and dissolved in 100 μL of sterile deionized water. 40 μL of the bacterial culture was added to 10 μL of 25 mM NaOH and boiled at 100°C for 15 min. After centrifugation, the supernatant was used as a PCR template. PCR was performed using the universal primers 5'-AOX and 3'-AOX of the pPIC9K plasmid. The PCR product was verified by agarose gel electrophoresis to confirm successful gene transformation. The Knickkopf positive strain was then inoculated into 20 mL of YPD medium (2% glucose, 1% yeast extract, 2% peptone) for activation and cultured at 30°C and 200 rpm for 24 h. The activated strain was then inoculated into 100 mL of BMGY medium (1% yeast extract, 20% peptone, 1% glycerol, 0.2% biotin, 1.34% yeast nitrogen base, 100 mM phosphate buffer, pH 6.0) and cultured at 30°C and 200 rpm for 24 h. The cells were then collected by centrifugation. The cells were resuspended in 1 L of BMMY medium (1% yeast extract, 2% peptone, 1% methanol, 1.34% yeast nitrogen base, 0.2% biotin, 100 mM potassium phosphate buffer, pH 6.0) and cultured at 30°C and 200 rpm, with 20 mL of methanol added every 24 h. After 3 days of culture, the supernatant was collected by centrifugation at 4°C. 350 g of [unspecified ingredient] was added to 1 L of the supernatant. Add ammonium sulfate, stir until dissolved, and let stand overnight at 4°C to precipitate.

[0023] The ammonium sulfate precipitate was centrifuged at 4°C. The precipitate was resuspended in buffer (100 mM potassium dihydrogen phosphate, pH 5) and filtered through a 0.22 μM filter membrane. The precipitate was then placed in a dialysis bag (purchased from Beijing Solarbio Science & Technology Co., Ltd., 45mm wide, molecular weight cutoff 3500 Da) and dialyzed in 100 mM potassium dihydrogen phosphate, pH 5 to remove residual ammonium sulfate. The total protein concentration was then concentrated to 5 mg / mL using a concentration tube (total protein concentration was determined using BCA).

[0024] Example 2 Prepare 50% straw: Weigh 20 g of steam-exploded corn straw, add 20 g of buffer (100 mM potassium dihydrogen phosphate, pH 5), stir well, and then adjust the pH to 5 with NaOH.

[0025] Preparation of 100 mg / mL Cellic CTec3: Weigh 1 g of Cellic CTec3 enzyme preparation (purchased from Novozymes, Denmark) and bring the volume to 10 mL using buffer (100 mM potassium dihydrogen phosphate, pH 5).

[0026] Three experimental groups were set up, each with a mass of 5 g: (1) 20% straw (1 g) + 1% Cellic CTec3 (10 mg): Weigh 2 g of 50% straw, add 0.1 mL of 100 mg / mL Cellic CTec3, and use a buffer to measure to 5 g; (2) 20% straw (1 g) + 1% Cellic CTec3 (10 mg) + 3.3 mg Knickkopf protein: Weigh 2 g of 50% straw, add 0.1 mL of 100 mg / mL Cellic CTec3, add 3.3 mg of Knickkopf protein, and use a buffer to quantify to 5 g (by BCA method, 10 mg Cellic CTec3 contains 3.3 mg of protein). (3) 20% straw (1 g) + 5% Cellic CTec3 (50 mg): Weigh 2 g of 50% straw, add 0.5 mL of 100 mg / mL Cellic CTec3, and use a buffer to measure to 5 g; The reaction was carried out at 50 °C and 200 rpm. Samples were taken at 0 h, 6 h, 12 h, 24 h and 48 h after the reaction. 100 μL of each sample was taken, centrifuged and the supernatant was collected. Chloroform was added at a ratio of 1:1 to remove protein. After shaking and centrifugation, the supernatant was collected and the reducing sugar yield was determined by the DNS method.

[0027] Experimental results are as follows Figure 1 As shown in the experimental results, after enzymatic hydrolysis of 20% steam-exploded straw for 36 h, the reducing sugar yield of the experimental group with 1% Cellic CTec3 and an equal mass concentration of Knickkopf protein was basically the same as that of the experimental group with 5% Cellic CTec3, indicating that Knickkopf can reduce the amount of Cellic CTec3 used.

[0028] Example 3 Preparation of 100 mg / mL Cellic CTec3 with reduced activity: Weigh 1 g of CellicCTec3 enzyme preparation that has been stored for more than 3 months, and make up to 10 mL with buffer (100 mM potassium dihydrogen phosphate, pH 5).

[0029] 1) 20% straw (1 g) + 1% Cellic CTec3 (10 mg): Weigh 2 g of 50% straw, add 0.1 mL of 100 mg / mL Cellic CTec3 with reduced activity, and use a buffer to quantify to 5 g; (2) 20% straw (1 g) + 1% Cellic CTec3 (10 mg) + 3.3 mg Knickkopf protein: Weigh 2 g of 50% straw, add 0.1 mL of Cellic CTec3 with reduced activity of 100 mg / mL, add 3.3 mg Knickkopf protein, and use buffer to quantify to 5 g; (3) 20% straw (1 g) + 3% Cellic CTec3 (30 mg): Weigh 2 g of 50% straw, add 0.3 mL of Cellic CTec3 with activity reduced by 100 mg / mL, and use a buffer to quantify to 5 g; The reaction was carried out at 50 °C and 200 rpm. Samples were taken at 0 h, 6 h, 12 h, 24 h and 48 h after the reaction. 100 μL of each sample was taken, centrifuged and the supernatant was collected. Chloroform was added at a ratio of 1:1 to remove protein. The reducing sugar yield was then determined by the DNS method.

[0030] Experimental results are as follows Figure 2As shown, after 48 hours of enzymatic hydrolysis of 20% concentration of steam-exploded corn straw with 1% Cellic CTec3 (normal, uninactivated), the reducing sugar yield was 26.5 g / L. However, when using Cellic CTec3 stored for more than three months, the reducing sugar yield was only 4.5 g / L, just 17% of the normal level. This indicates that the enzymatic activity of Cellic CTec3 gradually decreases with prolonged storage time. Adding Knickkopf protein at the same mass concentration as Cellic CTec3 increased the reducing sugar yield to 25.0 g / L, almost restoring it to the normal level. These results demonstrate that Knickkopf protein can extend the shelf life of Cellic CTec3.

[0031] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for improving the efficiency of enzymatic hydrolysis of cellulose by Cellic CTec3, characterized in that, The process includes the following steps: cellulose is added to a buffer solution and dispersed evenly; Cellic CTec3 and Knickkopf proteins are added and mixed evenly; and the mixture is reacted at 25-60°C for 5-60 hours to obtain an enzymatic hydrolysate containing a high concentration of reducing sugars.

2. The method for improving the efficiency of enzymatic hydrolysis of cellulose by Cellic CTec3 according to claim 1, characterized in that, The amino acid sequence of the Knickkopf protein is selected from: (a) the amino acid sequence shown in SEQ ID NO.1, or (b) an amino acid sequence that has at least 70% identity with the amino acid sequence shown in SEQ ID NO.

1.

3. The method for improving the efficiency of enzymatic hydrolysis of cellulose by Cellic CTec3 according to claim 2, characterized in that, The preparation method of the Knickkopf protein is as follows: a linearized plasmid vector containing the Knickkopf protein encoding gene is introduced into Pichia pastoris to induce recombinant protein expression, and the protein is obtained after ammonium sulfate precipitation and dialysis.

4. The method for improving the efficiency of enzymatic hydrolysis of cellulose by Cellic CTec3 according to claim 1, characterized in that, The mass concentration of Knickkopf protein in the reaction system was 0.01~0.5%, the mass concentration of Cellic CTec3 was 0.1~1%, and the mass concentration of cellulose was 10~30%.

5. The method for improving the efficiency of enzymatic hydrolysis of cellulose by Cellic CTec3 according to claim 1, characterized in that, The reaction temperature is 40~60℃, and the reaction time is 5~48h.

6. The method for improving the efficiency of enzymatic hydrolysis of cellulose by Cellic CTec3 according to claim 1, characterized in that, The pH of the buffer solution is 4-8.

7. The method for improving the efficiency of enzymatic hydrolysis of cellulose by Cellic CTec3 according to claim 1, characterized in that, The buffer solution includes phosphate buffer.

8. The method for improving the efficiency of enzymatic hydrolysis of cellulose by Cellic CTec3 according to claim 1, characterized in that, The cellulose mentioned includes steam-exploded corn stalks.

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