Polysaccharide cleavage monooxygenase as well as preparation method and application thereof

Abstract

The invention discloses polysaccharide cleavage monooxygenase as well as a preparation method and application thereof. A novel polysaccharide splitting monooxygenase is constructed, glycosidic bonds of crystalline cellulose are efficiently oxidized and broken by applying a biological method of the novel polysaccharide splitting monooxygenase, the crystallinity of the cellulose is reduced, and the nanocellulose is prepared. By adopting the method disclosed by the invention, the defects of serious environmental pollution, high equipment requirement, poor product quality and the like caused by a large amount of inorganic strong acid waste liquid generated when cellulose is treated by a traditional chemical method are overcome, the cost is saved, the environmental pollution is avoided, and the method is environment-friendly; meanwhile, nanofibers prepared by using the polysaccharide splitting monooxygenase have the advantages of high yield, natural property, no pollution, uniform size and the like, in addition, the method is simple and convenient to operate and easy to industrially popularize and apply, and a green and sustainable way is provided for developing novel functional materials. The polysaccharide lysing monooxygenase provided by the invention can be widely applied to the fields of chemical engineering, agriculture, feed additives, medicines and the like, and has great production potential and economic value.

Description

Technical Field

[0001] The present invention relates to the preparation and application of a polysaccharide cleavage monooxygenase, belonging to the technical field of functional gene cloning and expression, and specifically relates to a polysaccharide cleavage monooxygenase which can be widely used in the fields of agriculture, food, feed additives and medicine, and a new method for applying the polysaccharide cleavage monooxygenase in the preparation of nanocellulose. Background Art

[0002] Cellulose is the most abundant renewable resource on earth. Its development and utilization are of great significance to solving the current resource, environmental and energy crises. Its degradation product, nanocellulose (NFC, Nanofibrillated Cellulose), is a nanomaterial with great development prospects. Due to its good optical and mechanical properties, adjustable surface chemical properties, low toxicity, biodegradability and biocompatibility, it has broad application prospects and greater application value in the fields of bioengineering and medicine. For example, nanofibers can be used to prepare antibacterial composite materials, ideal cell culture scaffold materials, and applied to drug delivery, skin tissue repair and wound dressing materials. It has great potential in the fields of polymer composite materials and energy storage devices.

[0003] However, since the fiber has a highly crystalline structure, it needs to be pretreated to convert the crystalline region into an amorphous substrate to improve the degradation efficiency of cellulose. At present, the main method for preparing nanocellulose is to use high-energy radiation, mechanical pressure, electrical energy or thermal energy to melt, wear, evaporate or condense the material, chemical acid-base hydrolysis or cellulase hydrolysis to form nanofibers. Among them, ball milling, low-temperature crushing or high-pressure homogenization are the most commonly used physical methods. High-pressure homogenization has been widely used to separate cellulose nanofibers from plants, but the mechanical energy consumption is large, and the fiber agglomeration will block the gap in the homogenizer, which is not conducive to the smooth progress of production; the crystallinity of nanocellulose is improved after solid alkali cooking and hydrogen peroxide bleaching, and the suspension has good stability. However, the chemical method for preparing nanocellulose is easy to cause environmental pollution, which is not conducive to the sustainable development of nanofiber preparation technology. This method wastes a lot of water resources in the production process and produces a lot of waste liquid, causing serious environmental pollution.

[0004] Enzymatic hydrolysis to prepare nanofibers generally uses cellulase systems or xylanase to effectively hydrolyze only the amorphous region of cellulose to obtain nanoscale products. However, the crystalline region of cellulose has a tight structure that makes it difficult for enzymes to contact it, limiting its application. Therefore, finding a green and efficient method to reduce the crystallinity of cellulose to prepare nanocellulose is gaining more and more attention. Summary of the invention

[0005] The purpose of the present invention is to provide a polysaccharide cleavage monooxygenase and its application in cellulose degradation and nanofiber preparation; it aims to overcome the shortcomings of the prior art such as chemical reagents polluting the environment and poor product quality in traditional chemical methods for hydrolyzing cellulose, and provide an economical, efficient and environmentally friendly enzymatic method for reducing cellulose crystallinity and preparing nanofibers.

[0006] The inventive concept of the present invention is: Lytic Polysaccharide Monooxygenase (LPMO) is a new type of copper ion-dependent oxidase, which can break the glycosidic bonds of polysaccharide substrates by oxidation, open the dense crystal structure of polysaccharide substrates, reduce cellulose crystallinity and improve cellulose accessibility, thereby increasing the yield of nanofibers. The present invention uses biological methods to efficiently hydrolyze the hydrogen bonds inside crystalline cellulose, effectively destroying its dense lattice network structure. On this basis, combined with the auxiliary effect of ultrasound, an environmentally friendly and efficient production process is realized, which can greatly improve the yield and quality of nanofibers.

[0007] The gene and amino acid sequence described in the present invention have not been reported in any literature or patent.

[0008] The specific technical scheme of the present invention is:

[0009] The invention provides a polysaccharide-cleaving monooxygenase, which is derived from Arthrobotrys sp. and has an amino acid sequence as shown in SEQ ID NO:1.

[0010] On the other hand, the present invention provides a gene encoding the polysaccharide cleaving monooxygenase, the nucleotide sequence of which is shown in SEQ ID NO:2.

[0011] The present invention also provides an expression bacterium for expressing the above-mentioned polysaccharide cleavage monooxygenase;

[0012] The present invention claims the use of the above-mentioned polysaccharide-cleaving monooxygenase in the degradation of cellulose substrates.

[0013] As a specific solution, the above application includes application in degrading cellulose substrates such as PASC, filter paper and microcrystalline cellulose.

[0014] As another specific embodiment, the polysaccharide-cleaving monooxygenase is mixed with cellulase for use in synergistically degrading cellulose.

[0015] As another specific embodiment, the polysaccharide-cleaving monooxygenase of the present invention is used to prepare nanofibers;

[0016] As a preferred embodiment, the polysaccharide-cleaving monooxygenase and ultrasound are used in synergistic degradation to prepare nanocellulose.

[0017] Furthermore, the average particle size of the uniformly dispersed nanofibers obtained in the above application is 232 nm.

[0018] The polysaccharide-cleaving monooxygenase of the present invention has good temperature stability. In addition, compared with the nanofiber preparation methods currently reported in the literature, the glycosidic bonds in the crystalline cellulose can be efficiently oxidatively broken by biological methods to reduce the crystallinity of the crystalline cellulose, and ultrasound can be used to assist in the green and efficient preparation of high-yield and high-quality nanofibers.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The present invention not only significantly reduces costs, but also avoids environmental pollution, and meets environmentally friendly technical requirements. In addition, the nanofibers prepared using the polysaccharide cleavage monooxygenase in the method have the advantages of high yield, natural pollution-free and uniform size. More importantly, the method has a simple operation process and is easy to promote and apply in industry, providing a green and sustainable way to develop new functional materials. The polysaccharide cleavage monooxygenase provided by the present invention has shown wide application potential in multiple fields such as chemical industry, agriculture, feed additives and medicine, and has large production capacity and economic value. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 : Electrophoresis detection of the cxLPMO23E gene of the present invention; wherein, the first lane: polysaccharide cleavage monooxygenase cxLPMO23E gene; the second lane: nucleic acid Marker (Takara, 3584A);

[0022] Figure 2 : SDS-PAGE electrophoresis of the purified pure protein of the polysaccharide cleaving monooxygenase cxLPMO23E of the present invention; wherein, the first lane: M is a standard protein Marker (Takara, 3596A); the second lane: the expression supernatant of the recombinant bacteria pPICZαA-cxLPMO23E / X-33; the third lane: the purified cxLPMO23E;

[0023] Figure 3 : The effect of pH change on the enzyme activity of the polysaccharide cleaving monooxygenase cxLPMO23E of the present invention;

[0024] Figure 4 : The effect of temperature change on the activity of the polysaccharide cleaving monooxygenase cxLPMO23E of the present invention;

[0025] Figure 5 : XRD diagram of the polysaccharide-cleaving monooxygenase of the present invention, hydrolyzing microcrystalline cellulose; wherein, (A) microcrystalline cellulose XRD; (B) filter paper XRD;

[0026] Figure 6 :Synergistic effect of cxLPMO23E and cellulase

[0027] Figure 7 : TEM image of nanofibers prepared by the polysaccharide cleaving monooxygenase cxLPMO23E of the present invention; wherein, (A) control group; (B) experimental group;

[0028] Figure 8 : The particle size distribution of nanofibers prepared by the polysaccharide-cleaving monooxygenase of the present invention. DETAILED DESCRIPTION

[0029] The present invention is further described below by way of examples, which are only used to illustrate the present invention and are not used to limit the scope of the present invention. The experimental methods in the examples are conventional methods unless otherwise specified. Unless otherwise specified, the reagents used in the present invention are all commercially available.

[0030] Example 1 Cloning of polysaccharide-cleaving monooxygenase gene fragments

[0031] The RNA of Arthrobotryssp.CX1 was extracted according to the operation steps of the Fungal RNA Extraction Kit (Shanghai Shenggong, SK8659), and cDNA was synthesized according to the operation steps of the One-Step RT-PCR Kit (Code No.RR064A) of TaKaRa Biological Company. PCR amplification was performed using the synthesized cDNA as a template. The PCR reaction conditions were 95℃ pre-denaturation for 5min; 94℃ denaturation for 1min, 55℃ annealing for 30s; 72℃ extension for 10min, 30 cycles; 72℃ extension for 10min. The PCR product was detected by agarose gel electrophoresis, and the results were as follows Figure 1 As shown, there is a specific band near 6000 bp. The target fragment was recovered by gel excision, and the target gene was connected to the expression vector pPICZαA (Invitrogen, Catalog No. V195-20) by unrestricted cloning (RF cloning) and then sequenced.

[0032] Example 2 Polysaccharide cleavage monooxygenase gene sequence analysis

[0033] The sequencing results were analyzed for homology and domain using the Basic Local Alignment Search Tool (BLAST) and Conserved Domain Database (CDD) in the GenBank database; the obtained polysaccharide cleavage monooxygenase gene (named cxLPMO23E) had a coding region length of 5793 bp, and its nucleotide sequence is shown in SEQ ID NO.1. cxLPMO encodes 1930 amino acids and 1 stop codon, and its amino acid sequence is shown in SEQ ID NO.2. Conserved domain analysis showed that the cxLPMO23E protein sequence had a characteristic polysaccharide cleavage monooxygenase catalytic domain and 23 glycoside hydrolase binding modules CBM, indicating that cxLPMO23E is a new member of the polysaccharide cleavage monooxygenase family.

[0034] Example 3 Recombination of cxLPMO23E gene in Pichia pastoris

[0035] The recombinant plasmid pPICZαA-cxLPMO23E obtained in Example 1 was transferred into Pichia pastoris X-33 competent cells and spread on YPD plates containing 0.5 μg / ml zeocin for screening. Multiple single colonies were picked and cultured in YPD liquid culture medium containing 5 μL Zeocin at 30°C and 200 rpm for 24 hours. Colony PCR verification was performed using upstream and downstream primers, and the amplification product of the correct size was obtained, proving that the recombinant expression bacteria were correctly constructed. The recombinant bacteria were named pPICZαA-cxLPMO23E / X-33.

[0036] Example 4 Recombinant expression of polysaccharide cleavage monooxygenase cxLPMO23E

[0037] Streak the correctly identified recombinant bacteria pPICZαA-cxLPMO23E / X-33 in Example 3 on a YPD plate containing 0.5μg / ml zeocin; pick a single colony in 5mL of 0.5μg / ml zeocinYPD liquid and culture for 24h as a seed solution; inoculate into 50mL of BMGY culture at a ratio of 1:50 until OD600=2; transfer the above culture medium to a 50mL centrifuge tube in a sterile operating table; centrifuge at 1500g for 5min, discard the supernatant, resuspend with BMMY culture medium, add 1.5% methanol, replenish once every 12 hours, and culture for 96 hours. The fermentation broth was purified by a nickel column to obtain the pure protein cxLPMO23E. The expression of the protein was detected by polyacrylamide gel electrophoresis, and the results are as follows Figure 2 As shown, polysaccharide-cleaving monooxygenase was expressed under the induction of methanol, and its protein size was above 200 kDa.

[0038] Example 5 Study on the enzymatic properties of polysaccharide-cleaving monooxygenase

[0039] The cxLPMO23E polysaccharide-cleaving monooxygenase obtained in Example 4 was used to measure the biological properties of microcrystalline cellulose as a substrate, including the effects of the optimal pH, optimal temperature and temperature stability on the cxLPMO23E polysaccharide-cleaving monooxygenase.

[0040] (1) Optimal pH determination

[0041] Using phosphoric acid swollen cellulose (PASC) as a substrate, cxLPMO23E was reacted at 60°C, 160 rpm, for 4 hours under different pH conditions (3.0, 4.0, 5.0, 6.0, 7.0, 8.0), and the amount of reducing sugar produced was measured by the DNS method; the results are shown in Figure 3 As shown, the optimum pH of cxLPMO23E is 5.0.

[0042] (2) Determination of optimum temperature

[0043] Under the above optimum pH conditions, the same system was reacted at different temperatures (30℃, 40℃, 50℃, 60℃, 70℃, 80℃), and the amount of reducing sugar produced was measured by DNS method. The results are as follows Figure 4 As shown, the optimum temperature of cxLPMO23E is 60°C.

[0044] Example 6 Polysaccharide cleavage monooxygenase acting on different cellulose substrates

[0045] 10 mg of cxLPMO23E obtained in Example 4 was used for filter paper and microcrystalline cellulose, reacted at 60°C, pH 5.0, 160 rpm for 24 h, centrifuged, the supernatant was aspirated, and the substrate was repeatedly washed three times with deionized water, and the insoluble substrate was dried at room temperature; the control group was an untreated substrate; the crystallinity and morphology of different substrates reacted with cxLPMO23E and water were detected using an X-ray diffractometer and a scanning electron microscope. Figure 5 As shown in the figure, the crystal characteristic diffraction peaks at 2θ values ​​of 15°, 22° and 34° correspond to the 101, 002 and 004 crystal planes of cellulose type I, respectively. After the action of cxLPMO23E, the crystal characteristic diffraction peak intensities of the filter paper and microcrystalline cellulose at 2θ = 15°, 22° and 34° positions were reduced. The crystallinity was analyzed by jade software as shown in Table 1. The crystallinity of microcrystalline cellulose and filter paper treated with cxLPMO23E decreased by 54.68% and 40.52%, respectively, indicating that cxLPMO23E oxidatively breaks the glycosidic bonds of the cellulose substrate, opens the dense crystal structure of the cellulose substrate, and reduces the crystallinity of cellulose.

[0046] Table 1: Changes in crystallinity of different celluloses treated with the polysaccharide-cleaving monooxygenase of the present invention

[0047]

[0048] Example 7 Polysaccharide cleavage monooxygenase synergistically degrades cellulose fibers

[0049] 5mg microcrystalline cellulose powder, 0.02mg cxLPMO23E / BSA, 0.8U cellulase, 1mmol / LVc and 50mmol / L citrate buffer (pH 5.0), 60℃, 200rpm for 4h. After the reaction, centrifuge and take the supernatant to determine the amount of reducing sugar using the DNS method. In order to characterize the synergistic effect of cxLPMO23E and cellulase, the degree of synergy (DS) was calculated according to formula 1. When DS>1, it shows synergy, and DS<1 shows antagonism. Figure 6 As shown, cxLPMO23E and cellulase have a significant synergistic effect in degrading microcrystalline cellulose.

[0050] DS=A (cellulase+cxLPMO23E) / c+A cxLPMO23E Formula 1

[0051] A (cellulase+cxLPMO23E) :The amount of reducing sugar in the synergistic reaction of cellulase and cxLPMO23E

[0052] A cellulase : The amount of reducing sugar when cellulase acts on the substrate alone

[0053] A cxLPMO23E :The amount of reducing sugar when cxLPMO23E acts on substrate alone

[0054] Example 8 Preparation of Nanofibers by Polysaccharide Lysis Monooxygenase

[0055] Take 30mL of 10mg cxLPMO23E enzyme solution to act on 0.5g of microcrystalline cellulose powder in a 250mL conical flask and seal it with tin foil. React at 200rpm and 50℃ for 48h. After the enzymatic hydrolysis reaction, centrifuge at 13000rpm for 10min, wash the precipitate twice with 95% ethanol and ddH2O respectively, and then resuspend it in 15mL ddH2O. The resuspension was ultrasonically crushed at 300W, ultrasonic for 3s, and intermittent for 5s for 8min. The treated sample was centrifuged at 6500rpm for 5min, and the supernatant and precipitate were collected respectively. The supernatant was centrifuged at 13000rpm for 5min, and the supernatant and precipitate were collected again. The supernatant separated out was nanocellulose CNC; the control group only used water to replace the enzyme solution, and other experimental operations were consistent. The nanocellulose CNC aqueous solution of the experimental group and the control group was diluted to an appropriate concentration (the experimental group and the control group were diluted at the same multiple), and a drop of the sample was placed on a carbon support film (300 mesh, Xinxing Bairui). After drying at room temperature, the sample morphology was observed using a transmission electron microscope (TEM). Figure 7 As shown in the figure, the morphology of the control group is a bundle of fibers in which some nanofibers are aggregated. Although there are small rod-shaped fibers adhered to them, they are unevenly distributed. The morphology of the experimental group is uniformly distributed rod-shaped nanofibers, and the number of nanofibers is large. The particle size of nanocellulose CNC was measured by dynamic light scattering (DLS) of a nanoparticle size analyzer. Figure 8 As shown in the figure, the average particle size of the nanofibers obtained after treatment with cxLPMO23E is 232nm, and they are uniformly dispersed, while the average particle size of the nanofibers in the control group with only ultrasound is larger and non-uniform, with most of them distributed at 562nm and a small amount distributed at 141nm. This shows that after cellulose is treated with cxLPMO23E, more nanofibers with uniform particle size can be obtained by ultrasound.

[0056] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical solutions and inventive concepts of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A polysaccharide-cleaving monooxygenase, characterized in that include: (1) The amino acid sequence of the polysaccharide cleaving monooxygenase is SEQ ID NO: 1; or (2) An amino acid sequence having polysaccharide-cleaving monooxygenase activity formed by substituting, deleting or adding one or more amino acids to the amino acid sequence shown in SEQ ID NO:

1.

2. A gene, characterized in that The gene encodes the polysaccharide-cleaving monooxygenase according to claim 1.

3. The gene according to claim 2, characterized in that The nucleotide sequence of the gene is SEQ ID NO:

2.

4. A recombinant expression bacterium, characterized in that: The recombinant expression bacteria are used to express the polysaccharide cleavage monooxygenase according to claim 1.

5. The use of the polysaccharide cleaving monooxygenase according to claim 1, characterized in that: Application of the enzyme in the degradation of cellulose substrates.

6. The use according to claim 5, characterized in that: The enzyme is used in the degradation of cellulose substrates PASC, filter paper and microcrystalline cellulose.

7. The use of polysaccharide cleaving monooxygenase according to claim 5, characterized in that: The polysaccharide-cleaving monooxygenase is mixed with cellulase for use in synergistically degrading cellulose.

8. The use of the polysaccharide cleaving monooxygenase according to claim 5, characterized in that: Application of polysaccharide-cleaving monooxygenase in the preparation of nanocellulose.

9. The use according to claim 8, characterized in that: The average particle size of the uniformly dispersed nanofibers obtained was 232 nm.

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