Mutants of a cellulose disaccharide hydrolase from trichoderma reesei and uses thereof

Abstract

The application belongs to the technical field of biology and relates to a mutant of a cellulose disaccharide hydrolase from Trichoderma reesei and application thereof. The cellulose disaccharide hydrolase from Trichoderma reesei is TrCel7A, and the mutation sites of the mutant are T97A and / or K166A. The application successfully improves the catalytic activity of the cellulose disaccharide hydrolase through rational design of the cellulose disaccharide hydrolase from Trichoderma reesei.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a mutant of cellobiose hydrolase (CBH1) from Trichoderma reesei and its applications. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Cellulose degradation using cellulase (CBH) is one of the main methods for the high-value utilization of cellulose. The resistance of cellulose substrates to CBH catalytic degradation is a major challenge in cellulose degradation. To improve the catalytic activity of CBH, protein engineering techniques such as site-directed mutagenesis, random mutagenesis, loop modification, glycosylation removal, and substrate catalytic channel modification are currently employed. The inventors have found that although protein engineering can currently improve the catalytic activity of CBH1, its catalytic activity still requires further improvement. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the present invention aims to provide a mutant of cellobiose hydrolase from Trichoderma reesei and its application. Through rational design of cellobiose hydrolase from Trichoderma reesei, the present invention successfully improves the catalytic activity of cellobiose hydrolase.

[0005] To achieve the above objectives, the technical solution of the present invention is as follows:

[0006] On the one hand, a mutant of cellobiose hydrolase from Trichoderma reesei, wherein the cellobiose hydrolase from Trichoderma reesei is TrCel7A, and the mutation sites of the mutant are T97A and / or K166A.

[0007] On the other hand, the nucleic acid encoding the aforementioned mutant cellulosic disaccharide hydrolase from Trichoderma reesei.

[0008] Thirdly, an expression vector comprising nucleic acid encoding the aforementioned mutant of cellulosic disaccharide hydrolase from Trichoderma reesei.

[0009] In some embodiments, the backbone vector of the expression vector is selected from the pTCI vector, which is an integrative vector with hygromycin resistance.

[0010] Fourthly, a host cell capable of transforming or transfecting the aforementioned expression vector.

[0011] In some embodiments, the host cell is *Trichoderma reesei*.

[0012] The method for constructing the host cell includes: converting the constructed expression vector into a host cell.

[0013] Fifthly, a method for preparing a mutant of cellobiose hydrolase from Trichoderma reesei includes: culturing the above-mentioned host cells and inducing the host cells to express the mutant.

[0014] Sixthly, the application of the mutant, nucleic acid, expression vector, host cell, or product obtained by the above-mentioned preparation method in the catalytic hydrolysis of cellulose.

[0015] Seventhly, a biological agent comprising the aforementioned mutant, nucleic acid, expression vector, host cell, or product obtained by the preparation method.

[0016] Eighthly, a method for catalytically hydrolyzing microcrystalline cellulose, wherein the above-mentioned biological agent is added to a buffer solution containing microcrystalline cellulose, and catalytic hydrolysis is carried out under conditions of a temperature of 40-50°C and a pH of 4.5-5.8.

[0017] In some embodiments, the catalytic hydrolysis time is 12–72 h.

[0018] The beneficial effects of this invention are as follows:

[0019] This invention obtains the TrCel7A-T97A mutant by mutating threonine at position 97 of the TrCel7A protein to alanine, and obtains the TrCel7A-K166A mutant by mutating lysine at position 166 of the TrCel7A protein to alanine. Compared with the TrCel7A protein, the catalytic activity of cellobiose hydrolase in the TrCel7A-T97A and TrCel7A-K166A mutants is significantly improved. Catalytic activity studies show that, compared with TrCel7A, the catalytic activities of the TrCel7A-T97A mutant and the TrCel7A-K166A mutant are increased by 37% and 68%, respectively, under the condition of reaction at 45°C for 12 h. Attached Figure Description

[0020] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0021] Figure 1 This is a model diagram of the pTCI carrier in an embodiment of the present invention.

[0022] Figure 2The images shown are SDS-PAGE images of TrCel7A, TrCel7A-T97A, TrCel7A-S148A and TrCel7A-K166A after affinity purification in the embodiments of the present invention.

[0023] Figure 3 For the determination of the catalytic activity of the CBH1 mutant in this embodiment of the invention, the glucose concentration was measured based on the saccharification results of the extracellular enzyme solution of the Trichoderma reesei parent strain and the TrCel7A mutant strain using Avicel as a substrate. Detailed Implementation

[0024] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0025] Example 1: TrCel7A strain and vector

[0026] Escherichia coli Trans5α(F) was obtained from Beijing Transgen Biotech. - dlacZΔM15Δ(lacZYA-argF)U169 end Al recA1 hsdR17(r k - ,m k - The strain *Trichoderma reesei* (cat, No. CD201-01) was obtained from CCTCC (number: M2015804). The *TrCel7A* mutant strain was constructed using the pTCI vector, such as... Figure 1 As shown, the pTCI vector is an integration vector with a Cel7A promoter and hygromycin resistance, which is integrated into the genome of Trichoderma reesei through homologous recombination.

[0027] Example 2: Rational Design and Site-Directed Mutation of TrCel7A

[0028] Analysis of the three-dimensional crystal structure of TrCel7A and its molecular docking with substrates revealed that T97, S168, and K166 are potentially important sites affecting the hydrolytic activity of TrCel7A. Therefore, TrCel7A mutants TrCel7A-T97A and TrCel7A-K166A were obtained through rational design. The TrCel7A mutants TrCel7A-T97A and TrCel7A-K166A were obtained using the pTCI vector. The recombinant vectors pTCI-CBH1-WT, pTCI-CBH1-T97A, pTCI-CBH1-S18A, and pTCI-CBH1-K166A were constructed using the KOD-Plus mutagenesis kit from TOYOBO Co. (Osaka, Japan). The oligonucleotides used for plasmid construction in this example are shown in Table 1.

[0029] The amino acid sequence of TrCel7A is as follows:

[0030] QSACTLQSETHPPLTWQKCSSGGTCTQQTGSVVIDANWRWTHATNSSTNCYDGNTWSSTLCPDNETCAKNCCLDGAAYASTYGVTTSGNSLSIGFVTQSAQKNVGARLYLMASDTTYQEFTLLGN EFSFDVDVSQLPCGLNGALYFVSMDADGGVSKYPTNTAGAKYGTGYCDSQCPRDLKFINGQANVEGWEPSSNNANTGIGGHGSCCSEMDIWEANSISEALTPHPCTTVGQEICEGDGCGGTYSDNR YGGTCDPDGCDWNPYRLGNTSFYGPGSSFTLDTTKKLTVVTQFETSGAINRYYVQNGVTFQQPNAELGSYSGNELNDDYCTAEEAEFGGSSFSDKGGLTQFKKATSGGMVLVMSLWDDYYANMLW LDSTYPTNETSSTPGAVRGSCSTSSGVPAQVESQSPNAKVTFSNIKFGPIGSTGNPSGGNPPGGNPPGTTTTRRPATTTGSSPGPTQSHYGQCGGIGYSGPTVCASGTTCQVLNPYYSQCL, such as SEQ Shown as ID.NO.1.

[0031] The amino acid sequence of TrCel7A-T97A is as follows:

[0032] QSACTLQSETHPPLTWQKCSSGGTCTQQTGSVVIDANWRWTHATNSSTNCYDGNTWSSTLCPDNETCAKNCCLDGAAYASTYGVTTSGNSLSIGFVAQSAQKNVGARLYLMASDTTYQEFTLLGNEFSFDVDVSQLPCGLNGALYFVSMDADGGVSKYPTNTAGAKYGTGYCDSQCPRDLKFINGQANVEGWEPSSNNANTGIGGHGSCCSEMDIWEANSISEALTPHPCTTVGQEICEGDGCGGTYSDNRYGGTCDPDGCDWNPYRLGNTSFYGPGSSFTLDTTKKLTVVTQFETSGAINRYYVQNGVTFQQPNAELGSYSGNELNDDYCTAEEAEFGGSSFSDKGGLTQFKKATSGGMVLVMSLWDDYYANMLWLDSTYPTNETSSTPGAVRGSCSTSSGVPAQVESQSPNAKVTFSNIKFGPIGSTGNPSGGNPPGGNPPGTTTTRRPATTTGSSPGPTQSHYGQCGGIGYSGPTVCASGTTCQVLNPYYSQCL, as shown in SEQ ID.NO.2.

[0033] The amino acid sequence of TrCel7A-K166A is:

[0034] QSACTLQSETHPPLTWQKCSSGGTCTQQTGSVVIDANWRWTHATNSSTNCYDGNTWSSTLCPDNETCAKNCCLDGAAYASTYGVTTSGNSLSIGFVTQSAQKNVGARLYLMASDTTYQEFTLLGN EFSFDVDVSQLPCGLNGALYFVSMDADGGVSKYPTNTAGAAYGTGYCDSQCPRDLKFINGQANVEGWEPSSNNANTGIGGHGSCCSEMDIWEANSISEALTPHPCTTVGQEICEGDGCGGTYSDNR YGGTCDPDGCDWNPYRLGNTSFYGPGSSFTLDTTKKLTVVTQFETSGAINRYYVQNGVTFQQPNAELGSYSGNELNDDYCTAEEAEFGGSSFSDKGGLTQFKKATSGGMVLVMSLWDDYYANMLW LDSTYPTNETSSTPGAVRGSCSTSSGVPAQVESQSPNAKVTFSNIKFGPIGSTGNPSGGNPPGGNPPGTTTTRRPATTTGSSPGPTQSHYGQCGGIGYSGPTVCASGTTCQVLNPYYSQCL, such as SEQ Shown as ID.NO.3.

[0035] Table 1. Oligonucleotides used to construct plasmids in this embodiment.

[0036]

[0037]

[0038]

[0039] Example 3: Expression of cellobiase in Trichoderma reesei host fungus

[0040] The *Trichoderma reesei* strain was inoculated onto a bran solid medium (10% (w / v) bran, boiled with tap water, simmered for 30 min, filtered through gauze to obtain bran juice, dispensed, and sterilized at 121℃ for 30 min with 2% (w / v) agar powder). After culturing for 2-3 days, spores were activated. The spores were washed off the plates using a spore-washing solution (NaCl: 0.9 g, Tween-80: 50 μL, ddH2O to a final volume of 100 mL, autoclaved) in a clean bench. 50-100 μl of the spore suspension was then added to a bran plate lined with cellophane. The plate was evenly spread and incubated at 30℃ for approximately 20 hours. 0.1 g of lysin was dissolved in 20 mL of Solution I (sorbitol 1.2 M, KH2PO4 0.1 M). Pipette 2.5 ml of the mixture onto cellophane containing mycelia, then add another layer of cellophane, repeating this process 8-10 times. Incubate at 30°C for approximately 1.5-2 hours. After enzymatic digestion, remove the cellophane with tweezers and wash once. Filter the digest using a funnel with four layers of filter paper and collect it in a 50 ml centrifuge tube. Centrifuge at 3000 rpm for 10 min at 4°C, discard the supernatant, and resuspend the protoplasts in 4 ml of Solution II (1.2 M sorbitol, 0.01 M Tris-HCl, 0.05 M CaCl2). Centrifuge again at 3000 rpm for 10 min at 4°C, discard the supernatant, and resuspend the protoplasts in 0.6 ml of Solution III (25% PEG6000, 0.01 M Tris-HCl, 0.05 M CaCl2).

[0041] Protoplasmic transformation: Add 200 μl of protoplasts, 10 μl of purified plasmid, and 50 μl of PEG to a centrifuge tube, mix well using a shaker, and incubate on ice for 20 min. Then add 2 ml of PEG, mix gently, and incubate at 20°C for 5 min. Add 4 ml of Solution II and mix gently. Pipette 1 ml of the above solution into 4 ml of upper transformation medium containing 200 μg / ml hygromycin resistance (ammonium sulfate 10 g / L, potassium dihydrogen phosphate 15 g / L, magnesium sulfate heptahydrate 0.8 g / L, calcium chloride 0.8 g / L, glucose 20 g / L, sorbitol 364.4 g / L, agarose 24 g / L, pH 5.5) and mix well. Finally, pour in the lower transformation medium containing 200 μg / ml hygromycin resistance (sorbitol 92 g / L, glucose 200 g / L, potassium dihydrogen phosphate 15 g / L, agarose 20 g / L, pH 5.5). Place in a 30°C incubator until Trichoderma reesei transformants grow.

[0042] Obtaining the extracellular enzyme solution of TrCel7A: The above-mentioned Trichoderma reesei transformants were inoculated onto bran plates and cultured for 4-5 days. Then, they were inoculated into 50 mL of seed culture medium ((NH4)2SO4 2 g / L, KH2PO4 3 g / L, MgSO4 0.5 g / L, CaCO3 0 g / L). 5 g / L, peptone 10 g / L, microcrystalline cellulose 10 g / L), cultured at 30℃ and 200 rpm for 48 hours. 10 mL of the seed culture was then inoculated into 100 mL of enzyme-producing medium ((NH4)2SO4: 2 g / L, KH2PO4: 3 g / L, MgSO4: 0.5 g / L, CaCO3: 5 g / L, peptone: 10 g / L, Avicel: 10 g / L, urea: 1 g / L, sodium nitrate: 2 g / L, Tween-80: 3 g / L, trace elements: 0.1%), and cultured at 30℃ and 200 rpm for 7 days. The enzyme solution was then collected by centrifugation.

[0043] Example 4: Purification of cellobiose hydrolase

[0044] Using the AKATA protein purification system and gel filtration pre-packed columns (HiPrep) TM 16 / 60 Sephacryl TM The S-200HR was used to purify cellobiase. First, the pre-packed gel filter column was washed with ultrapure water for 2-3 column volumes. Then, it was washed again with PBS buffer for two more column volumes before loading 2 mL of sample each time. After loading, the protein was eluted with PBS buffer, and the eluted sample was collected when the peak appeared. After collection, SDS-PAGE was performed for verification. Figure 2 As shown.

[0045] Example 5: Determination of cellobiose hydrolase activity

[0046] Add 30 mg microcrystalline cellulose, 0.15 mg cellobiase, 84 μg β-glucosidase (Merck, Inc.), and 10 μL 10% sodium azide to centrifuge tubes. Add citrate buffer (pH 4.8) to bring the reaction volume to 1 mL. Incubate at 45°C using a rotary incubator. Take samples at 12 h, 24 h, 48 h, and 96 h and immerse them in boiling water for 10 minutes to terminate the reaction. Determine the glucose yield. Dilute the samples 3–15 times with distilled water and perform high-performance liquid chromatography (HPLC) analysis on a BioRad HPX-87H column. Elute the column with 5 mM H₂SO₄ at 45°C at a flow rate of 0.5 mL / min. Use Avicel as the substrate. Figure 3As shown, compared with TrCel7A, the catalytic activities of TrCel7A-T97A and TrCel7A-K166A increased by 37% and 68%, respectively, after reacting at 45℃ for 12 h. The catalytic activity of TrCel7A-S148A decreased by 37% compared with TrCel7A after reacting at 45℃ for 12 h, indicating that not all mutations at all sites increase the activity of cellobiose hydrolase. Compared with TrCel7A, the catalytic activities of TrCel7A-T97A and TrCel7A-K166A increased by 28% and 49%, respectively, after reacting at 45℃ for 72 h; compared with TrCel7A (40%), the conversion rates reached 51% (w / w) and 60% (w / w), respectively, after reacting at 45℃ for 72 h.

[0047] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A mutant of a cellobiose hydrolase from Trichoderma reesei, characterized in that, Cellulose disaccharide hydrolase from Trichoderma reesei is Tr Cel7A, whose amino acid sequence is shown in SEQ ID NO.1; the mutant is obtained by T97A or K166A mutation based on the amino acid sequence shown in SEQ ID NO.

1.

2. The nucleic acid encoding the mutant of the cellulosic disaccharide hydrolase from Trichoderma reesei as described in claim 1.

3. An expression carrier, characterized in that, It includes nucleic acids encoding a mutant of the cellulosic disaccharide hydrolase from Trichoderma reesei as described in claim 1.

4. The expression vector as described in claim 3, characterized in that, The backbone vector of the expression vector is selected from the pTCI vector.

5. A host cell characterized by, It can transform or transfect the expression vector described in claim 3 or 4.

6. The host cell as described in claim 5, characterized in that, The host cell is Trichoderma reesei.

7. A method for preparing a mutant of cellulosic disaccharide hydrolase from Trichoderma reesei, characterized in that, include: The host cells of claim 5 or 6 are cultured to induce the expression of the mutant in the host cells.

8. The use of the mutant of claim 1, the nucleic acid of claim 2, the expression vector of claim 3 or 4, the host cell of claim 5 or 6, or the product obtained by the preparation method of claim 7 in the catalytic hydrolysis of cellulose.

9. A biological agent, characterized in that, It includes the mutant of claim 1, the nucleic acid of claim 2, the expression vector of claim 3 or 4, the host cell of claim 5 or 6, or the product obtained by the preparation method of claim 7.

10. A method for catalytic hydrolysis of microcrystalline cellulose, characterized in that, The biological agent of claim 9 is added to a buffer solution containing microcrystalline cellulose and catalytically hydrolyzed at a temperature of 40-50 °C and a pH of 4.5-5.

8.

11. The method of claim 10, characterized in that, The catalytic hydrolysis time is 12~72 h.

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