Soluble polysaccharide monooxygenase as well as coding gene and application thereof

Abstract

The invention provides soluble polysaccharide monooxygenase as well as a coding gene and application thereof, the amino acid sequence of the soluble polysaccharide monooxygenase is as shown in a sequence table SEQ ID NO.1, heterologous expression is realized in pichia pastoris, the soluble polysaccharide monooxygenase after heterologous expression can oxidize a cellulose substrate, and the soluble polysaccharide monooxygenase can be used for producing a cellulose substrate. And the hydrolysis efficiency of the cellulase on a cellulose substrate is improved. According to the present invention, the polysaccharide cleavage monooxygenase derived from the Autobasidium pullulans and the cellulase are mixed for use, and co-act to catalyze and degrade the cellulose substrate to produce the glucose, such that the degradation requirement of the cellulose substrate is easily achieved, the degradation activity of the cellulose substrate is improved, and the foundation is established for the efficient degradation of the cellulose substrate.

Description

Technical Field

[0001] This invention relates to the field of enzyme engineering technology, and in particular to a soluble polysaccharide monooxygenase, its encoding gene, and its applications. Background Technology

[0002] Cellulose is the most abundant biopolymer resource on Earth, primarily used as a raw material in industrial technologies such as fermentation and as a biodegradable material in the food industry. Due to the regular hydrogen bonds that can form between oxygen atoms and hydroxyl groups on adjacent chains, cellulose exhibits high crystallinity and insolubility. Traditional enzymatic hydrolysis methods mainly utilize mixed cellulases (exo- and endo-β-1,4-glucanases) to hydrolyze the glycosidic bonds of cellulose. Improving the hydrolysis efficiency of cellulose is crucial for the sustainable development of the bioeconomy.

[0003] Soluble polysaccharide monooxygenases (LPMOs) are copper-dependent oxidoreductases that oxidatively degrade polysaccharides such as cellulose, hemicellulose, chitin, and starch. LPMOs can act on crystalline polysaccharides, breaking glycosidic bonds through oxidation, making the polysaccharide structure looser and easier for glycoside hydrolases to hydrolyze. Cellulose-type LPMOs can degrade cellulose substrates through oxidation. However, currently reported LPMOs suffer from low enzyme activity, making their application in industrial production difficult. Therefore, developing highly active cellulose-type LPMOs for the degradation of cellulose and other polysaccharides has significant production application value. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a soluble polysaccharide monooxygenase.

[0005] Another technical problem to be solved by the present invention is to provide a gene encoding the above-mentioned soluble polysaccharide monooxygenase.

[0006] Another technical problem to be solved by the present invention is to provide the application of the above-mentioned soluble polysaccharide monooxygenase.

[0007] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows:

[0008] A 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 soluble polysaccharide monooxygenases.

[0010] The pPI CZα-A vector or Pichia pastoris strain X-33 expressed the above-mentioned soluble polysaccharide monooxygenase.

[0011] The above-mentioned expression method for soluble polysaccharide monooxygenase involves synthesizing the 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 soluble polysaccharide monooxygenases are used to improve the degradation activity of cellulose substrates.

[0013] Preferably, in the application of the above-mentioned soluble polysaccharide monooxygenase, the soluble polysaccharide monooxygenase works together with cellulase to catalyze the degradation of cellulose substrate to produce glucose.

[0014] Preferably, the application of the above-mentioned soluble polysaccharide monooxygenase involves the following specific steps:

[0015] (1) The reaction system consists of cellulose substrate, 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 soluble polysaccharide monooxygenase, the reaction mixture in step (1) further includes cellulase.

[0018] Preferably, in the application of the above-mentioned soluble polysaccharide monooxygenase, the concentration ratio of the cellulase and the cellulose-type soluble polysaccharide monooxygenase is 1:2 to 2:1.

[0019] Preferably, in the application of the above-mentioned soluble polysaccharide monooxygenase, the pH value of the reaction system is 4-7.

[0020] Preferably, the application of the above-mentioned soluble polysaccharide monooxygenase is carried out at a reaction temperature of 30-60℃.

[0021] Preferably, the application of the above-mentioned soluble polysaccharide monooxygenase involves a reaction time of 24 hours.

[0022] Beneficial effects:

[0023] The aforementioned soluble polysaccharide monooxygenase was heterologously expressed in Pichia pastoris using a soluble polysaccharide monooxygenase derived from Aureobasidium pullulans. The heterologously expressed soluble polysaccharide monooxygenase was able to oxidize cellulose substrates and improve the hydrolytic efficiency of cellulase on cellulose substrates. Combining the polysaccharide cleavage monooxygenase from Aureobasidium pullulans with cellulase allows for synergistic catalytic degradation of cellulose substrates to produce glucose, which is beneficial for the degradation of cellulose substrates and enhances its degradation activity, laying the foundation for efficient degradation of cellulose substrates. Attached Figure Description

[0024] Figure 1 This is a comparison of the oxidation activities of the soluble polysaccharide monooxygenase AuAA9 on cellulose substrates under different conditions.

[0025] Figure 2 This is a comparison of glucose production from cellulose substrates by the soluble polysaccharide monooxygenase AuAA9 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 monooxygenase gene derived from Aureobasidium pullulans soluble polysaccharide

[0030] The soluble polysaccharide monooxygenase gene derived from Aureobasidium pullulans was artificially synthesized by Suzhou Genewise Biotechnology Co., Ltd., and named AuAA9. The gene amino acid sequence is shown in SEQ ID NO.1. It was synthesized into the pPI CZα-A vector, transformed into Escherichia coli BL21 Gold(DE3), and positive clones were screened and cultured on LB plates containing bleomycin. Plasmids were extracted and sequenced to confirm successful vector construction.

[0031] Example 2

[0032] Expression of the soluble polysaccharide monooxygenase AuAA9

[0033] The successfully constructed plasmid was digested with SacI and then electroporated into Pichia pastoris strain X-33. The resulting single colonies were inoculated onto YPD medium (25 μg / ml bleomycin) using sterile toothpicks and cultured at 30°C and 220 rpm for 20 h. After incubation on BMG medium (25 μg / ml bleomycin) at 30°C and 220 rpm for 20 h, they were transferred to BMM medium (25 μg / ml bleomycin) and cultured at 20°C and 220 rpm for 5 days, with 1% methanol added daily. 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 the soluble polysaccharide monooxygenase AuAA9

[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 using 0-100% 50 mM Tris-HCl (pH 7.5) containing 0.5 M NaCl, and the eluted protein was collected using a 96-well deep-well plate. SDS-PAGE was used to determine the purity of the protein in each well, ultimately yielding pure enzyme.

[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 (A) at pH 7.5, and 0.5 M NaCl (B). The flow rate was 3 mL / min. Gradient elution was performed using an increasing gradient of mobile phase B and a decreasing gradient 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 AuAA9 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 AuAA9, and sodium ascorbate. The concentrations of Avice L, AuAA9, and sodium ascorbate were 10 mg / mL, 2 μM, and 1 mM, respectively. The reaction mixture was replenished with a pH 6.0, 20 mM sodium dihydrogen phosphate buffer and reacted at different temperatures (30, 40, 50, 60 °C) for 24 h. The degradation activity of AuAA9 on the cellulose substrate was measured at different temperatures. Following the above reaction system, the reaction compound was reacted at 40 °C for 24 h in buffer systems with different pH values ​​(4, 5, 6, 7), and the degradation activity of AuAA9 on the cellulose substrate was detected 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 AuAA9 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 AuAA9 under different temperature and pH conditions was measured as follows: Figure 1 As shown.

[0045] Example 5

[0046] Example 3: Activity assay of the soluble polysaccharide monooxygenase AuAA9 mixed with cellulase for degrading cellulose substrates.

[0047] The enzymatic reaction mixture contained 10 g / L Avice L, 1 mM ASC (sodium ascorbate), and 20 mM sodium acetate buffer at pH 5.0. The cellulase concentration was 31.2 mg / L. Different AuAA9 concentrations were added: 15.6 mg / L, 31.2 mg / L, and 62.4 mg / L. The cellulase to AuAA9 concentration ratios were 2:1, 1:1, and 1:2. The reaction was carried out at 50 °C for 24 h. After the reaction was completed, 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 before HPLC analysis. Glucose yield was determined by high-performance liquid chromatography (HPLC). For HPLC analysis, an Aminex HPX-87H column (300 × 7.8 mm, 9 μm, Bio-Rad, USA) was used with 5 mL H₂SO₄ as the mobile phase at a flow rate of 0.5 mL / min. A differential detector was used to monitor the glucose peak at 30 °C. The results are as follows: Figure 2 As shown, when the soluble polysaccharide monooxygenase AuAA9 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 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 soluble polysaccharide monooxygenase of claim 1.

3. The pPICZα-A vector or Pichia pastoris strain X-33 expressing the soluble polysaccharide monooxygenase of claim 1.

4. The expression method of the soluble polysaccharide monooxygenase according to claim 1 or 2, characterized in that: 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 soluble polysaccharide monooxygenase of claim 1 or 2 in enhancing the degradation activity of cellulose substrates.

6. The application of the soluble polysaccharide monooxygenase according to claim 5, characterized in that: The soluble polysaccharide monooxygenase and cellulase work together to catalyze the degradation of cellulose substrates to produce glucose.

7. The application of the soluble polysaccharide monooxygenase according to claim 5, characterized in that: The specific steps are as follows: the reaction system consists of cellulose substrate, soluble polysaccharide monooxygenase, and ascorbate.

8. The application of the soluble polysaccharide monooxygenase according to claim 7, characterized in that: The reaction mixture in step (1) also includes cellulase.

9. The application of the 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 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 30-60℃.

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