Cellulose type soluble polysaccharide monooxygenase as well as coding gene and application thereof
By heterologously expressing the soluble polysaccharide monooxygenase CaAA9 from Cantharellus anzutake and combining it with cellulase to catalyze the degradation of cellulose substrates, the problem of low enzyme activity of cellulose-type soluble polysaccharide monooxygenases was solved, and the efficient hydrolysis effect of cellulase was achieved.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-11
- Publication Date
- 2026-03-13
Smart Images
Figure CN121653084A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of enzyme engineering technology, and in particular to a cellulose-type soluble polysaccharide monooxygenase, its encoding gene, and its applications. Background Technology
[0002] Cellulose is the most widely distributed and abundant polysaccharide in nature. It consists of hundreds to thousands of D-glucose units linked by β-(1→4) bonds. Cellulose has important applications in industrial manufacturing, papermaking, textiles, and food. However, due to its high crystallinity and insolubility, improving its degradation efficiency remains a significant challenge.
[0003] Soluble polysaccharide monooxygenases (LPMOs) are copper-dependent oxidoreductases that oxidatively degrade insoluble and soluble polysaccharides such as cellulose, hemicellulose, chitin, and starch. LPMOs can break glycosidic bonds through oxidation, making the polysaccharide substrate structure looser and easier for glycoside hydrolases to hydrolyze. Cellulose-type LPMOs can degrade cellulose substrates through C1 and / or C4 oxidation. However, the reported sources of fungal-derived cellulose-type LPMOs are limited, and their enzyme activity is low, greatly restricting their industrial application. Therefore, developing highly active cellulose-type LPMO enzymes for cellulose degradation has significant practical value for production. Summary of the Invention
[0004] The technical problem to be solved by the present invention is to provide a cellulose-type soluble polysaccharide monooxygenase.
[0005] Another technical problem to be solved by the present invention is to provide a gene encoding the above-mentioned cellulose-type soluble polysaccharide monooxygenase.
[0006] Another technical problem to be solved by the present invention is to provide the application of the above-mentioned cellulose-type soluble polysaccharide monooxygenase.
[0007] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows:
[0008] A cellulose-type soluble polysaccharide monooxygenase, the amino acid sequence of which is shown in SEQ ID NO.1 of the sequence listing.
[0009] Nucleotides encoding the aforementioned cellulose-type soluble polysaccharide monooxygenases.
[0010] The pPI CZα-A vector or Pichia pastoris strain X-33 expressing the above-mentioned cellulose-type soluble polysaccharide monooxygenase.
[0011] The above-mentioned expression method for cellulose-type soluble polysaccharide monooxygenase involves synthesizing the cellulose-type soluble polysaccharide monooxygenase into an expression vector, transforming it into Escherichia coli, screening positive clones on LB plates and culturing them, extracting plasmids, and electroporating the plasmids into Pichia pastoris after enzyme digestion.
[0012] The above-mentioned cellulose-type soluble polysaccharide monooxygenases are used to improve the degradation activity of cellulose substrates.
[0013] Preferably, in the application of the above-mentioned cellulose-type soluble polysaccharide monooxygenase, the cellulose-type soluble polysaccharide monooxygenase works together with cellulase to catalyze the degradation of cellulose substrates to produce glucose.
[0014] Preferably, the application of the above-mentioned cellulose-type soluble polysaccharide monooxygenase involves the following specific steps:
[0015] (1) The reaction mixture consists of cellulose substrate, cellulose-type soluble polysaccharide monooxygenase, and ascorbate.
[0016] (2) The pH value in the reaction system was adjusted by sodium dihydrogen phosphate buffer to degrade the cellulose substrate.
[0017] Preferably, in the application of the above-mentioned cellulose-type soluble polysaccharide monooxygenase, the reaction mixture in step (1) further includes cellulase.
[0018] Preferably, in the application of the above-mentioned cellulose-type soluble polysaccharide monooxygenase, the concentration ratio of the added cellulase to the cellulose-type soluble polysaccharide monooxygenase is 1:2 to 2:1.
[0019] Preferably, in the application of the above-mentioned cellulose-type soluble polysaccharide monooxygenase, the pH value of the reaction system is 4-7.
[0020] Preferably, the application of the above-mentioned cellulose-type soluble polysaccharide monooxygenase is carried out at a reaction temperature of 35-50℃.
[0021] Preferably, the application of the above-mentioned cellulose-type soluble polysaccharide monooxygenase has a reaction time of 24 hours.
[0022] Beneficial effects:
[0023] The aforementioned cellulose-type soluble polysaccharide monooxygenase was heterologously expressed in Pichia pastoris using a soluble polysaccharide monooxygenase derived from *Cantharellus anzutake*. The heterologously expressed soluble polysaccharide monooxygenase enhanced the hydrolytic efficiency of cellulase on cellulose substrates. The synergistic action of the *Cantharellus anzutake* polysaccharide cleavage monooxygenase and cellulase in catalyzing the degradation of cellulose substrates increased glucose yield, laying the foundation for efficient cellulose degradation. Attached Figure Description
[0024] Figure 1 This study aims to determine the oxidative activity of CaAA9 on cellulose substrates.
[0025] Figure 2 This is a comparison of glucose production from cellulose substrates by CaAA9 and cellulase. Detailed Implementation
[0026] 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 further described in detail below with reference to specific embodiments.
[0027] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods. Unless otherwise specified, the experimental methods used in the following examples are conventional methods.
[0028] Example 1
[0029] Cloning of the soluble polysaccharide monooxygenase gene from Cantharellus anzutake
[0030] A soluble polysaccharide monooxygenase gene from *Cantharellus anzutake* was artificially synthesized by Suzhou Genewiz Biotechnology Co., Ltd., named CaAA9, with the amino acid sequence shown in SEQ ID NO.1. The gene was synthesized into the pPI CZα-A vector, transformed into *E. coli* BL21 Gold(DE3), and positive clones were screened on LB agar plates containing bleomycin. These positive clones were then cultured in LB liquid medium containing bleomycin at 37°C for 16 h. Plasmids were extracted, and sequencing confirmed the presence of the CaAA9 gene, confirming successful vector construction.
[0031] Example 2
[0032] Expression of soluble polysaccharide monooxygenase CaAA9
[0033] The successfully constructed plasmid was digested with SacI and then electroporated into Pichia pastoris strain X-33. The resulting single colonies were inoculated into YPD medium (25 μg / ml bleomycin) and cultured at 30°C and 220 rpm for 20 h. After incubation in BMG medium (25 μg / ml bleomycin) at 30°C and 220 rpm for 20 h, they were then transferred to BMM medium (25 μg / ml bleomycin) and cultured at 20°C and 220 rpm for 5 days, with 1% methanol added daily. YPD: 10 g / L yeast extract, 20 g / L tryptone, 2% glucose. BMG: 13.4 g / L YNB, 10 g / L glycerol, 0.0004 g / L biotin, 100 mM pH 6.0 potassium phosphate. BMM: 13.4 g / L YNB, 0.0004 g / L Biotin, 8 g / L L-alanine, 100 mM pH 6.0 potassium phosphate.
[0034] Example 3
[0035] Purification of soluble polysaccharide monooxygenase CaAA9
[0036] After cultivation, the supernatant was collected by centrifugation. The supernatant was first filtered through a 0.22 μm pore size filter. The filtered crude protein enzyme solution was purified using a DEAE ion-exchange chromatography column. A gradient wash was performed from 0-100% in 50 mM Tris-HCl (pH 7.5) containing 0.5 M NaCl, and the eluted protein was collected. The purified enzyme was obtained by SDS-PAGE analysis.
[0037] The gradient elution program was as follows: the mobile phase consisted of 50 mM Tris-HCl (A) at pH 7.5, 50 mM Tris-HCl (B) at pH 7.5, and 0.5 M NaCl (B). The flow rate was 3 mL / min. Gradient elution was performed using an increasing amount of mobile phase B and a decreasing amount of mobile phase A, as follows (all percentages are volume percentages): 0-45 min, 30% B; 45-60 min, 50% B; 60-75 min, 100% B.
[0038] Example 4
[0039] Example 3: Determination of the degradation activity of the soluble polysaccharide monooxygenase CaAA9 on cellulose substrates under different conditions.
[0040] 1. Preparation of the reaction system. The reaction system consisted of 300 μL of cellulose substrate Avice L, purified recombinant soluble polysaccharide monooxygenase CaAA9, and sodium ascorbate. The concentrations of Avice L, CaAA9, and sodium ascorbate were 10 mg / mL, 2 μM, and 1 mM, respectively. The reaction mixture was replenished with pH 6.0, 20 mM sodium dihydrogen phosphate buffer and reacted at different temperatures (35, 40, 45, 50 °C) for 24 h. The degradation activity of CaAA9 on the cellulose substrate was measured at different temperatures. Following the above reaction system, the reaction mixture was reacted at 40 °C for 24 h in buffer systems with different pH values (4, 5, 6, 7), and the degradation activity of CaAA9 on the cellulose substrate was measured under different pH conditions.
[0041] 2. After completing step 1, centrifuge at 12000 rpm for 10 min and collect the supernatant; then filter the supernatant with a filter (pore size of 0.22 μm) and collect the filtrate.
[0042] 3. The filtrate collected in step 2 was detected by ion exchange chromatography. The peak area of the soluble product in the chromatogram represents the degradation ability of the recombinant soluble polysaccharide monooxygenase on the cellulose substrate.
[0043] The ion exchange chromatography parameters are as follows: column PA-1; eluent consisting of 0.1M NaOH aqueous solution (A) and 1M CH3COONa aqueous solution (B); flow rate 0.25 ml / min; gradient elution with increasing eluent B and decreasing eluent A, specifically as follows: 0-10 min, 0-100 mM CH3COONa; 10-25 min, 100-300 mM CH3COONa; 25-30 min, 300-1000 mM CH3COONa; 30-31 min, 1000-0 mM CH3COONa; 31-40 min, 0 mM CH3COONa.
[0044] The activity of soluble polysaccharide monooxygenase CaAA9 was determined under different temperature and pH conditions as follows: Figure 1 As shown.
[0045] Example 5
[0046] Example 3: Activity assay of the synergistic degradation of cellulose substrates by the soluble polysaccharide monooxygenase CaAA9 and cellulase.
[0047] The enzymatic reaction mixture contained 10 g / L cellulose substrate (Avice 1), 1 mM ASC (sodium ascorbate), and 20 mM sodium acetate buffer at pH 5.0. Different concentrations of cellulase and CaAA9 were added. The cellulase concentration was 31.2 mg / L, and the CaAA9 concentrations were 15.6 mg / L, 31.2 mg / L, and 62.4 mg / L (i.e., enzyme ratios of 2:1, 1:1, and 1:2, respectively). The reaction was carried out at 40 °C for 24 h. After the reaction, 20 mM H₂SO₄ was added to terminate the reaction. The mixture was centrifuged at 12000 rpm for 15 min, and the supernatant was collected by filtration through a 0.22 μm filter. The glucose yield was compared between adding cellulase alone and using a mixture of CaAA9 and cellulase. The glucose yield after the reaction was determined by high-performance liquid chromatography (HPLC). The HPLC detection method used an Aminex HPX-87H column (300 × 7.8 mm, 9 μm, Bio-Rad, USA) with 5 mM H₂SO₄ as the mobile phase and a flow rate of 0.5 mL / min. A differential detector was used to detect the glucose peak at 30 °C. The results are as follows: Figure 2 As shown, when the soluble polysaccharide monooxygenase CaAA9 is used in combination with cellulase, the cellulase's ability to degrade cellulose is significantly improved.
[0048] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention. Improvements and modifications such as strain modification based on the method of the present invention or based on the method are all considered to be within the scope of protection of the present invention.
Claims
1. A cellulose-type soluble polysaccharide monooxygenase, characterized in that: The amino acid sequence is shown in SEQ ID NO.1 of the sequence listing.
2. A nucleotide encoding the cellulose-type soluble polysaccharide monooxygenase of claim 1.
3. The pPICZα-A vector or Pichia pastoris strain X-33 expressing the cellulose-type soluble polysaccharide monooxygenase of claim 1 or 2.
4. A method for constructing the expression strain of the cellulose-type soluble polysaccharide monooxygenase according to claim 1 or 2, characterized in that: Cellulose-type soluble polysaccharide monooxygenase was synthesized into an expression vector, transformed into Escherichia coli, positive clones were screened on LB plates and cultured, plasmids were extracted, and the plasmids were electroporated into Pichia pastoris after enzyme digestion.
5. The use of the cellulose-type soluble polysaccharide monooxygenase of claim 1 or 2 in improving the degradation activity of cellulose substrates.
6. The application of the cellulose-type soluble polysaccharide monooxygenase according to claim 5, characterized in that: The cellulose-type soluble polysaccharide monooxygenase and cellulase work together to catalyze the degradation of cellulose substrates to produce glucose.
7. The application of the cellulose-type soluble polysaccharide monooxygenase according to claim 5, characterized in that: The reaction mixture consists of cellulose substrate, cellulose-type soluble polysaccharide monooxygenase, and ascorbate.
8. The application of the cellulose-type soluble polysaccharide monooxygenase according to claim 7, characterized in that: The reaction mixture in step (1) also includes cellulase.
9. The application of the cellulose-type soluble polysaccharide monooxygenase according to claim 6 or 8, characterized in that: The concentration ratio of cellulase to cellulose-type soluble polysaccharide monooxygenase is 1:2 to 2:
1.
10. The application of the cellulose-type soluble polysaccharide monooxygenase according to claim 7 or 8, characterized in that: The pH value of the reaction system is 4-7, and the reaction temperature is 35-50℃.