Application of N-acetylglucosamine incision enzyme in improvement of digestibility

Abstract

The invention discloses application of N-acetylglucosamine incision enzyme in improvement of digestibility, and relates to the technical field of biology. The N-acetylglucosamine incision enzyme disclosed by the invention can be used as a food additive to improve the digestibility of edible fungus mycelium protein and sporocarp.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to the application of N-acetylglucosaminyl endoside enzyme in improving digestibility. Background Art

[0002] Endo-beta-N-acetylglucosaminidase (ENGase) is widely distributed in various organisms, and mainly participates in the regulation of cells and life by degrading misfolded glycoproteins. ENGase is also an effective tool enzyme for sugar chain editing, which can specifically hydrolyze the β-1,4 glycosidic bond between free oligosaccharide chains and N-acetylglucosamine (GlcNAc) of the core pentasaccharide on glycopeptides or glycoproteins. Its hydrolysis products are oligosaccharide chains and a GlcNAc, or glycopeptides or glycoproteins with a GlcNAc.

[0003] N-acetylglucosaminidase (ENGase, EC 3.2.1.96) is a class of enzymes that can cleave the chitobiose core structure of high mannose and certain hybrid oligosaccharides in N-glycoproteins, removing N-linked high mannose in glycoproteins. Bacterial or fungal ENGase is a tool for N-glycan structure analysis and editing, which can be used for biological activity and can act on the entire process of glycoprotein synthesis and degradation. Protein glycosylation is a complex post-translational modification involving the attachment of sugar chains at specific sites on proteins. Based on the difference in sugar chain types, glycoproteins are divided into N-linked glycoproteins, O-linked glycoproteins, etc.; in addition, protein glycosylation is also affected by fluctuations in host cell types and fermentation conditions (such as culture medium, pH value, temperature, etc.). Therefore, glycoproteins are usually heterogeneous in glycosylation patterns, including glycosylation sites, glycosylation degrees, and specific structures of sugar chains, making sugar chain analysis and structural characterization extremely challenging. In order to obtain sugar chain structural information to the greatest extent, the current main strategy for sugar chain analysis is to first release the sugar chains from the protein and then conduct detailed analysis and characterization. In this strategy, efficient, accurate, and stable deglycosylation methods are crucial.

[0004] In 1971, Muramatsu first observed the hydrolytic activity of ENGase (Endo D) in a culture medium containing Diplococcus pneumoniae. In 1974, Tarentino first purified and characterized ENGase from Streptomyces griseus. The molecular weight of the oligosaccharide product of this endonuclease is relatively large, so it was named the endonuclease of high molecular weight product, namely Endo H. In 1988, Kadowaki et al. observed the hydrolytic activity of ENGase (Endo M) in a culture medium containing soil samples of Mucor hiemalis, proving that the enzyme also exists in fungi.

[0005] Although ENGase is widely distributed in various organisms, including bacteria, fungi, plants, animals and humans, it is particularly distributed in fungi. For example, ENGase is present in the genomes of Ogataea minuta, Candida parapolymorpha, Pichia anomala, and Zygosaccharomyces rouxii. However, it is not found in the genomes of Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris, and Candida boidinii. Among filamentous fungi and macrofungi, Trichoderma reesei, Cordyceps militaris, Aspergillus oryzae, and Flammulina filiformis also have ENGase, but Fusarium venenatum, Lentinula edodes, and Pleurotus spp. do not. This fact indicates that fungi are divided into two major categories, ENGase-positive and ENGase-negative fungi, based on the presence or absence of ENGase. The function of ENGase may be related to the differentiation and adaptation of fungi. In the Carbohydrate Active EnZyme (CAZy) database, the ENGases identified so far are distributed in glycoside hydrolases (GH) family 18 or 85. GH18 ENGase has only hydrolytic activity; GH85 ENGase has both hydrolytic activity and synthetic activity (transglycosylation activity). Although GH18 ENGase only has hydrolytic activity, during the crystal structure analysis and site-directed mutagenesis studies, it was also found that they have the ability to transglycosylate. Summary of the invention

[0006] The purpose of the present invention is to provide the application of N-acetylglucosamine endoside enzyme in improving digestibility. The present invention firstly finds that N-acetylglucosamine endoside enzyme can be used as a food additive to greatly improve the digestibility of fungal mycelium protein and fruiting body.

[0007] The present invention is achieved in that: The fungal cell wall is composed of polysaccharides and glycoproteins. It is a unique cell structure of fungi and is not only a physical barrier to protect fungal cells. The modification of the glycoproteins in the outermost layer of the cell wall is relatively complex, mainly including N-glycosylation. Through genetic and biochemical analysis of the cell wall glycoproteins and polysaccharide structures, it was found that mannose (Man) and N-acetylglucosamine (GlcNAc) are the main sugar chain structures of the outermost cell wall. Genes such as pmi1, srb1, uap1 and ugm1 in the synthesis pathway are necessary for cell wall synthesis; O-glycosylation is also involved in cell wall synthesis. In addition to N- and O-glycosylation modifications, there is also GPI modification. The folding of the glucan transferases Gel1 and Gel2 responsible for the synthesis of cell wall polysaccharides requires glycosylation modification. When the glycosylation process is blocked, Gel1 and Gel2 cannot fold correctly, resulting in cell wall synthesis defects and abnormal polar growth. The GPI protein Mp1, which is specifically located in the cell wall, triggers the extension of the O-sugar chain under the action of the mannosyltransferase Mnn9, and the extended mannan is covalently cross-linked with the cell wall glucan. These glycosylated proteins can resist the hydrolysis of various proteases, and the sugar chain gives the glycoprotein a certain viscosity and lubricity. For edible fungi as food, this layer of glycoprotein has become an obstacle to the digestion of edible fungi by the human digestive tract. Among them, edible fungi such as Flammulina velutipes and Shiitake mushrooms are the most typical, and are called "See You Tomorrow".

[0008] Therefore, the art needs to use genetic engineering and gene editing engineering technology to improve the digestibility of edible fungi by improving the cell wall of edible fungi, and provide people with a more diversified high-quality food supply chain. Although there are cases of using N-acetylglucosamine endonuclease to change the activity of glycoproteins, it does not mean that N-acetylglucosamine endonuclease can hydrolyze glycoproteins in the cell wall of edible fungi. First, glycoproteins and chitin and other components in the cell wall of edible fungi are combined together, which is not exactly the same as free glycoproteins. Secondly, whether the digestibility of the gastrointestinal tract can be improved by high expression of N-acetylglucosamine endonuclease or use as a food / feed additive is also not reported. Therefore, the present invention creatively proposes for the first time that N-acetylglucosamine endonuclease is used to improve the digestibility of edible fungal mycelium or fruiting body.

[0009] In a first aspect, the present invention provides an N-acetylglucosamine endoside enzyme, the amino acid sequence of which is shown in any one of SEQ ID NOs: 1 to 4, or has more than 90% identity thereto.

[0010] The present invention first discovered that the N-acetylglucosamine endoside enzyme described in SEQ ID NO: 1 to 4 can improve the digestibility of edible fungal mycelium and fruiting body, and has a wide range of uses. For example, it can be used to cultivate new edible fungal varieties, or as a feed additive and food additive to improve the digestibility of edible fungi; it provides an enzyme preparation with good safety for the food, feed and other industries.

[0011] In another aspect, the present invention provides an isolated nucleic acid molecule encoding the N-acetylglucosaminidase described above.

[0012] Alternatively, in some embodiments of the present invention, the nucleic acid molecule contains a coding region with a nucleotide sequence as shown in any one of SEQ ID NOs: 5 to 8.

[0013] Optionally, in some embodiments of the present invention, the nucleic acid molecule further comprises a promoter region located upstream of the coding region.

[0014] Optionally, in some embodiments of the present invention, the nucleotide sequence of the promoter region is as shown in any one of SEQ ID NOs: 9 to 12.

[0015] The promoter region sequence provided by the present invention can efficiently drive the coding region to express N-acetylglucosamine endo-nuclease, and transferring it into edible fungi can increase the expression amount of N-acetylglucosamine endo-nuclease and improve the digestibility of edible fungal mycelium and fruiting body.

[0016] The nucleic acid molecule provided by the present invention can encode and highly express the above-mentioned N-acetylglucosamine endoside enzyme, and the nucleic acid molecule can be used to transform edible fungi by genetic engineering technology, so that the mycelium and fruiting body of the transformed edible fungi have higher digestibility.

[0017] In another aspect, the present invention provides a vector comprising the above-mentioned nucleic acid molecule.

[0018] The term "vector" means that the vector can be a self-replicating vector, i.e., exists as an extrachromosomal entity, and its replication is independent of the vector of chromosomal replication, such as a plasmid, an extrachromosomal element, a minichromosome or an artificial chromosome. The vector can include any means for ensuring self-replication. Alternatively, the vector can be a vector that is integrated into the genome and replicated together with the chromosome into which it has been integrated when introduced into the host cell. In addition, a single vector or plasmid or two or more vectors or plasmids, or a transposon, that together contain the total DNA to be introduced into the host cell genome can be used.

[0019] In another aspect, the present invention provides a recombinant cell comprising the above-mentioned nucleic acid molecule or vector.

[0020] The recombinant cell can be a unicellular microorganism or a non-unicellular microorganism. Unicellular microorganisms such as Gram-positive bacteria include, but are not limited to, Bacillus cells, such as, alkaliphilic Bacillus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus megaterium, Bacillus subtilis, Bacillus licheniformis, Bacillus coagulans, Bacillus stearothermophilus and Bacillus thuringiensis, etc.; or Streptomyces cells, such as Streptomyces licheniformis; or Gram-negative bacteria, such as Escherichia coli and Pseudomonas. In preferred aspects, the bacterial host is Bacillus subtilis, Escherichia coli, Bacillus licheniformis, Bacillus stearothermophilus and Escherichia coli cells.

[0021] On the other hand, the present invention provides the use of the above-mentioned N-acetylglucosamine endoside enzyme, the above-mentioned nucleic acid molecule, or the above-mentioned vector in cultivating edible fungi varieties with high digestibility or as a food additive to improve the digestibility of mycelium protein.

[0022] On the other hand, the present invention provides the use of N-acetylglucosamine endonuclease as a component of a food additive in improving the digestibility of edible fungal mycelium or fruiting body, wherein the food additive is mixed with N-acetylglucosamine endonuclease, and the amino acid sequence of the N-acetylglucosamine endonuclease is shown in any one of SEQ ID NOs: 1 to 4.

[0023] On the other hand, the present invention provides a food additive for improving the digestibility of edible fungal food, wherein the food additive is mixed with N-acetylglucosamine endonuclease, and the amino acid sequence of the N-acetylglucosamine endonuclease is shown in any one of SEQ ID NOs: 1 to 4; and the food contains mycelium or fruiting body from edible fungi.

[0024] On the other hand, the present invention provides a method for improving the digestibility of edible fungus mycelium or fruiting body, the method comprising: causing the edible fungus to overexpress N-acetylglucosamine endoside enzyme in the cell, wherein the amino acid sequence of the N-acetylglucosamine endoside enzyme is shown in any one of SEQ ID NOs: 1 to 4.

[0025] Optionally, in some embodiments of the present invention, the method comprises: allowing the edible fungus to contain an exogenous nucleic acid molecule in the cell to overexpress the N-acetylglucosaminyl endo-nuclease.

[0026] Optionally, in some embodiments of the present invention, the exogenous nucleic acid molecule contains a coding region sequence as shown in any one of SEQ ID NOs: 5 to 8.

[0027] On the other hand, the present invention provides another method for improving the digestibility of edible fungi mycelium or fruiting body, the method comprising: modifying the endogenous promoter sequence in the genome of the edible fungus for driving the expression of N-acetylglucosamine endonuclease by gene editing technology so that it drives the overexpression of N-acetylglucosamine endonuclease.

[0028] Optionally, in some embodiments of the present invention, the edible fungus is selected from Aspergillus oryzae ( Aspergillus oryzae ), Cordyceps militaris ( Cordyceps militaris )、Trichoderma reesei( Trichoderma reesei ) and Flammulina velutipes ( Flammulina filiformis ).

[0029] Optionally, in some embodiments of the present invention, the edible fungus is selected from Cordyceps militaris ( Cordyceps militaris ); the nucleotide sequence of the endogenous promoter before modification is SEQ ID NO: 10; the nucleotide sequence of the endogenous promoter after modification is SEQ ID NO: 27.

[0030] It should be noted that gene editing technology (such as CRISPR) is a routine technology for those skilled in the art. It is easy to achieve modification under the premise of knowing the nucleic acid sequence to be modified and the modified sequence.

[0031] The Cordyceps militaris fungus having the promoter of SEQ ID NO: 27 overexpresses N-acetylglucosamine endoside enzyme, so that the digestibility of its mycelium and fruiting body is improved.

[0032] In another aspect, the present invention provides a recombinant edible fungus comprising an exogenous nucleic acid molecule to overexpress the N-acetylglucosaminidase.

[0033] The recombinant edible fungus provided by the invention expresses the N-acetylglucosamine endoside enzyme, and its mycelium and fruiting body have a higher digestibility.

[0034] In another aspect, the present invention provides a method for culturing mycelium or fruiting body of an edible fungus, comprising: culturing the above-mentioned recombinant edible fungus to obtain mycelium or fruiting body. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0036] Figure 1 The digestibility changes of mycelial protein of Cordyceps militaris transformants; A. The digestibility changes of mycelial protein of Cordyceps militaris starting strain and transformants; B. The digestibility changes of fruiting bodies of Cordyceps militaris starting strain and transformants.

[0037] Figure 2 This is a graph showing the changes in the digestibility of protein from the mycelium of Cordyceps militaris editor; A. The changes in the digestibility of protein from the starting strain of Cordyceps militaris and the mycelium of the editor; B. The changes in the digestibility of protein from the starting strain of Cordyceps militaris and the fruiting body of the editor.

[0038] Figure 3 The digestibility changes of mycelial proteins from different sources after pretreatment with recombinant N-acetylglucosamine endoside enzyme; A. The digestibility changes of Aspergillus oryzae mycelial protein under different treatment conditions; B. The digestibility changes of Trichoderma reesei mycelial protein under different treatment conditions; C. The digestibility changes of Cordyceps militaris mycelial protein under different treatment conditions; D. The digestibility changes of Flammulina velutipes mycelial protein under different treatment conditions; E. The digestibility changes of Lentinus edodes mycelial protein under different treatment conditions; F. The digestibility changes of Pleurotus eryngii mycelial protein under different treatment conditions; G. The digestibility changes of Pleurotus ostreatus mycelial protein under different treatment conditions. DETAILED DESCRIPTION

[0039] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0040] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0041] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0042] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0043] The present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually carried out according to conventional conditions such as those described in J. Sambrook et al., Molecular Cloning Experiment Guide, 3rd edition, Science Press, 2002, or according to the conditions recommended by the manufacturer.

[0044] Example 1

[0045] Overexpression of N-acetylglucosamine endoside improves the digestibility of Cordyceps militaris fruiting body and mycelium proteins.

[0046] 1. Construction of expression vector The ORF encoding gene of N-acetylglucosaminidase gene CmEndo (SEQ ID NO: 6, encoding the polypeptide shown in SEQ ID NO: 2) was cloned by PCR using Cordyceps militaris cDNA template. The forward primer used was: 5'ATGCGGTGGTCCTTGTTATCCGTC 3' (SEQ ID NO: 13); the reverse primer was 5'TCAGGCGTTGACCATGGCGTAGTAGTC 3' (SEQ ID NO: 14).

[0047] The PCR product was used to recover the enzyme-digested DNA fragment using the Axygen PCR product column recovery kit. The DNA fragment and the vector pHDt / sk (see Microbial Cell Factories, 2012, 11:21) recovered by XbaI digestion were dephosphorylated and then seamlessly connected using the Vazyme One Step Clone Kit - the linearized expression plasmid was connected to the PCR fragment, that is, the fusion gene was connected to the expression plasmid, and the expression modules of various fusion genes were constructed to obtain the recombinant expression vector pHDt / sk-CmEndo.

[0048] 2. Expression of N-acetylglucosaminylase in Cordyceps militaris The constructed plasmid pHDt / sk-CmEndo was transformed into Agrobacterium tumefaciens AGL1 (see Microbial Cell Factories, 2012, 11:21), and N-acetylglucosamine endoside was transformed into Cordyceps militaris (see Microbial Cell Factories, 2012, 11:21) under the mediation of Agrobacterium tumefaciens. The grown transformants were inoculated into SDB culture solution, and DNA was extracted as a template after 2 days to verify the positive transformants. The transformants of Cordyceps militaris and the starting strain were inoculated into SDB culture solution, and cultured at 28°C and 200rpm for 12 days to obtain mycelial protein. The transformants of Cordyceps militaris and the starting strain were inoculated into rice culture medium to obtain Cordyceps militaris fruiting bodies.

[0049] 3. Digestibility Determination The above-mentioned fruiting bodies and mycelium proteins are freeze-dried and then crushed into fungal protein powder. The following gastrointestinal digestive fluid is prepared: 1) Simulated gastric fluid: gastric electrolyte solution (Coolaber, SL66003) + pepsin (Macklin, P6322, >3000U / mg), prepared at 1mL: 1.3mg (4000U / mL), the concentration of the reaction system during use is 2000U / mL.

[0050] 2) Simulated intestinal fluid: intestinal electrolyte solution (Coolaber, Sl66101) + bile salts (Sigma, 48305, MW:422.55) + trypsin (Diamond, A003702, 237U / mg), prepared at a ratio of 1mL: 8.45mg: 0.844mg, i.e. 20mM bile salts and 200U / mL enzyme activity. During use, the concentrations of the reaction system were 10mM and 100U / mL, respectively.

[0051] The following method was used to simulate gastrointestinal digestion: 1) Add 2g of treated fungal powder to a 50mL centrifuge tube, rehydrate at 1g:2mL, add simulated gastric fluid at a volume ratio of 1:1, adjust the pH to 3.0 with 1M HCl, and add simulated gastric fluid until it accounts for 1 / 2 of the total system. Place the centrifuge tube in a 37℃ environment to simulate gastric digestion, take samples of 500μL each at 0, 3h, and 6h, and after gastric digestion, adjust the pH to 7.0 with 1M NaOH, boil in water for 10min to terminate the enzymatic action of pepsin, and store at -80℃ after cooling (samples are treated the same way).

[0052] 2) Intestinal digestion simulation The simulated gastric digestion products were mixed with simulated intestinal fluid in a volume ratio of 1:1. The pH of the digestion system was maintained at around 7.0 and the temperature was maintained at 37°C. 500 μL of samples were taken at 0, 3, and 6 h. After intestinal digestion, the samples were placed in a boiling water bath for 10 min to terminate the enzymatic action of trypsin and then cooled and placed at -80°C (the samples were treated the same way).

[0053] The results are as follows Figure 1 As shown: Both mycelial protein and fruiting body of the transformant had higher digestibility.

[0054] Example 2

[0055] Directed evolution of N-acetylglucosamine endoside promoter improves digestibility of Cordyceps militaris fruiting body and mycelium protein 1. Gene editing target selection By analyzing the promoter sequence of the Cordyceps militaris N-acetylglucosamine endonuclease gene CmEndo (SEQ ID NO: 10), we selected targets that may contain the carbon metabolism repressor factor CreA, and found three sequences that are suitable for designing gene editing targets and overlap with the conserved binding site of CreA, namely CCC TCAAGATCTGGGCCACCCCC (SEQ IDNO: 15), GGACCCCCGGTCTGATAACCG TGG (SEQ ID NO: 16), CCC ATGCATACCACCATAGTATC (SEQ IDNO: 17), the underlined nucleotide sequence is the PAM sequence, and the rest is the Spacer sequence. After the above sgRNA was synthesized by the company, the Cas9 protein purchased from novoprotein was used to incubate the synthesized ribonucleoprotein protein complex in vitro. Cordyceps militaris was edited by RNP-mediated gene editing technology (Microbial Biotechnology. 2021 Nov;14(6):2343-2355), and the N-acetylglucosamine endonuclease gene editor with increased expression was screened. Sequencing found that SEQ IDNO: 15 mutated to CCC TCAAGATCTGCCC (SEQ ID NO: 26); the endogenous CmEndo promoter after gene editing is SEQ ID NO: 27.

[0056] The Cordyceps militaris editor and the starting strain were inoculated into SDB culture medium and cultured at 28°C and 200rpm for 12 days to obtain mycelial protein. The Cordyceps militaris transformant and the starting strain were inoculated into rice culture medium to obtain Cordyceps militaris fruiting bodies.

[0057] 2. Digestibility Determination The above-mentioned fruiting bodies and mycelium proteins are freeze-dried and then crushed into fungal protein powder. The following gastrointestinal digestive fluid is prepared: The following method was used to simulate gastrointestinal digestion: 1) Add 2g of treated fungal powder to a 50mL centrifuge tube, rehydrate at 1g:2mL and add simulated gastric fluid (see Application Example 1) at a volume ratio of 1:1, adjust the pH to 3.0 with 1M HCl, and add simulated gastric fluid until it accounts for 1 / 2 of the total system. Place the centrifuge tube in a 37℃ environment to simulate gastric digestion, take samples of 500μL each at 0, 3h, and 6h, and after gastric digestion, adjust the pH to 7.0 with 1M NaOH, boil in water for 10min to terminate the enzymatic action of pepsin, and store at -80℃ after cooling (samples are treated in the same way).

[0058] 2) Intestinal digestion simulation The simulated gastric digestion product was mixed with the simulated intestinal fluid (see Example 1) in a volume ratio of 1:1. The pH of the digestion system was maintained at about 7.0 and the temperature was maintained at 37°C. 500 μL of samples were taken at 0, 3, and 6 hours. After the intestinal digestion was completed, the mixture was placed in a boiling water bath for 10 minutes to terminate the enzymatic action of trypsin, and then cooled and placed at -80°C (the samples were treated in the same way).

[0059] The results are as follows Figure 2 As shown: Both the mycelial protein and fruiting body of the editor have better digestibility.

[0060] Example 3

[0061] Recombinantly expressed N-acetylglucosidase endoside improves digestibility of edible fungal mycelial protein.

[0062] 1. Construction of recombinant expression vector in host Escherichia coli The corresponding ORF encoding genes of N-acetylglucosaminyl endosides AoEndo, TrEndo, CmEndo, and FfEndo (SEQ ID NOs: 5-8, encoding the polypeptides shown in SEQ ID NOs: 1-4, respectively) were cloned by PCR using cDNA templates of edible fungi such as Aspergillus oryzae, Trichoderma reesei, Cordyceps militaris, and Flammulina filiformis. The primer information of the four gene sequences is as follows: (1) Forward primer: 5' CCGGAATTCATGCCTCCCATACCTGCGCAGGTCGA 3' (SEQ ID NO: 18), with an EcoR I recognition site added to the 5' end: GAATTC; The reverse primer is: 5'CCGCTCGAGTTAGATGGACGCACCCAGCGAAG 3' (SEQ ID NO: 19), with an Xho I recognition site added to the 5' end: CTCGAG.

[0063] (2) The forward primer used is: 5' CCGGAATTCATGCGGTGGTCCTTGTTATCCGTC 3' (SEQ ID NO: 20), with an EcoR I recognition site GAATTC added to its 5' end; the reverse primer is 5'CCGCTCGAGTCAGGCGTTGACCATGGCGTAGTAG 3' (SEQID NO: 21), with an Xho I recognition site CTCGAG added to its 5' end.

[0064] (3) The forward primer used is: 5' CCGGAATTCATGAAGGCGTCCGTCTACTTGGCGTC 3' (SEQ ID NO: 22), with an EcoR I recognition site added to the 5' end: GAATTC; The reverse primer is: 5'CCGCTCGAGTTAAGCATTCACCATAGCATAATAG 3' (SEQ ID NO: 23), with an XhoI recognition site added to the 5' end: CTCGAG.

[0065] (4) The forward primer used is: 5' CCGGAATTCATGTCTGCCGTCGCCAATCGGCTCG 3' (SEQ ID NO: 24), with an EcoR I recognition site added to the 5' end: GAATTC; The reverse primer is: 5'CCGCTCGAGCTATGCTTCAACAGCGACATCCTTGAAG 3' (SEQ ID NO: 25), with an Xho I recognition site added to the 5' end: CTCGAG.

[0066] After the PCR product was purified, it was double-digested with Cla I and Xba I, and the digested DNA fragment was recovered using the Axygen PCR product column recovery kit. The DNA fragment and the vector pET28 recovered after the same double digestion were ligated with T4 DNA ligase at 16°C overnight to obtain the recombinant expression vectors pET28a-AoEndo, pET28a-TrEndo, pET28a-CmEndo, pET28a-FfEndo. The N and C termini of the expression product were provided with His tags (6×His-Tag) provided by the expression vector for subsequent purification.

[0067] 2 Expression of recombinant N-acetylglucosamine endoside and purification of the product Inoculate E. coli P1 clone into 5mL LB medium containing 100 µg / ml ampicillin, and culture overnight at 37℃, 200 rpm. Take 1 ml of culture solution into 100ml LB culture solution, and culture at 37℃, 200 rpm until OD600 is 0.6-0.8. After cooling, add IPTG to a final concentration of 25 µM, continue to culture at 16℃, 200 rpm for 16 hours, and collect the bacteria by centrifugation. Suspend the collected bacteria with lysis buffer (lysis buffer: NaH2PO4 50 mmol / L, NaCl 300 mmol / L, pH7.4), and the lysis solution after ultrasonic disruption of cells is the crude enzyme solution. Centrifuge the crude enzyme solution at 12000×g for 10 minutes, and collect the supernatant of the lysis solution, which is the required crude enzyme solution.

[0068] The supernatant of the lysate was purified using a Ni column (Ni-NTA Column) purchased from Qiagen. The wash buffer used during purification was: NaH2PO4 50 mmol / L, NaCI 300 mmol / L, pH7.0; the elution buffer with different imidazole concentrations (20, 40, 60, 100, 200, 500) was: NaH2PO4 50 mmol / L, NaCl 300 mmol / L, imidazole 20-500 mmol / L, pH 7.0. Among them, the purification effect was best when eluted with 40 mM imidazole. The supernatant was concentrated and dialyzed using a GE 10 Kd cutoff vivaspin6 ultrafiltration tube, and a replacement buffer of 20 mM pH7.4 NaH2PO4 was used to remove imidazole to obtain recombinantly expressed N-acetylglucosaminidase.

[0069] 3. Adding recombinant N-acetylglucosamine endoside enzyme to improve digestibility In a 50 mL centrifuge tube, 1 mg of recombinant protein N-acetylglucosamine endoside was dissolved in 4 mL buffer (20 mM Tris-HCl pH 7.5, 50 mM NaCl, 5 mM EDTA), 2 g of fungal mycelial protein was added, and pretreated at 30°C for 1 hour. Simulated gastric fluid (see Application Example 1) was added at a volume ratio of 1:1, and the pH was adjusted to 3.0 with 1M HCl, and the simulated gastric fluid was supplemented until it accounted for 1 / 2 of the total system. The centrifuge tube was placed in a 37°C environment to simulate the gastric digestion scene, and 500 μL of samples were taken at 0, 3h, and 6h. After the gastric digestion was completed, the pH was adjusted to 7.0 with 1M NaOH, and the enzymatic action of pepsin was terminated by boiling water bath for 10 minutes, and then stored at -80°C after cooling (the samples were treated in the same way).

[0070] 2) Intestinal digestion simulation The simulated gastric digestion product was mixed with the simulated intestinal fluid (see Application Example 1) in a volume ratio of 1:1. The pH of the digestion system was maintained at about 7.0 and the temperature was maintained at 37°C. 500 μL of samples were taken at 0, 3, and 6 hours. After the intestinal digestion was completed, the sample was placed in a boiling water bath for 10 minutes to terminate the enzymatic action of trypsin, and then cooled and placed at -80°C (the samples were treated in the same way).

[0071] The results are as follows Figure 3 As shown: Mycelial proteins of Aspergillus oryzae, Trichodermareesei, Cordyceps militaris, Flammulina filiformis, Lentinula edodes, Pleurotus eryngii or Pleurotus ostreatus all obtained better digestibility after pretreatment with recombinant N-acetylglucosamine endo-nuclease.

[0072] The sequence information involved in this application is as follows: SEQ ID NO: 1(AoEndo ENGase) MPPIPAQVDRCRVICYHQTLRPDRGQYVSALPLVQNNTGITHIIIAAFHLNAEPGHITLNDDPPHHSMYDELWTEVPLLKQSGVRVMGMLGGAAQGSFRCLDGDQDKFERYYLPLLAMIRRHQLEGLDLDVEEEMSLSGIIRLIDRLKSDLGDGFIITLAPVAAALLGIGNLSGFDYRELERQRASKISWYNTQFYNGWGPADDPRMYAAIVAQGWSPQRVVYGLLTNPGNGSQGYVPREIIGPILGQLVEQFPNFGGVMGWEYFNAKPGEREKPWQWAAEMSLSMHMKDLSTAVHHIFQTPTSLFSAPLLYLSECHLSFFSKARTENNSWLKEHSLSRSAPKTFPRSYGFLAAGCRGHELRDTQTRRLHLSSLGASI SEQ ID NO:2(CmEndo ENGase) MRWSLLSVVSLLAPLLRANPISNVQAGAKASATPTTTPGLPRLIVYFQTTHDQAGNPISMLPLINEKGIALTHLIVCSLHINRDAEIHLNDYPPGDPHFYTLWNETTHMKKAGVKVMGMIGGAAPGSFDSSTLDGDAVTFEEYYTQVRDVINTFGLQGMDLDVEQPMSQSGISRLISRLRKDFGRGFIITLAPVASALRNGGNLSGFNYKSLESSNGRDISFYNGQFYSGFGTMARTDDYVRIVSNGFTVSKVVAGQLTSSDNGYGYVPYSQLNATIISLRDKYGQIGGVMGWEYFNSQPGGTSEPWEWAQVVTEILRPNSIPDLRITMGDADHLTAVYDRTVGDASPNARPGSRAIDYYAMVNA SEQ ID NO:3(TrEndo ENGase) MKASVYLASLLATLSMAVPVKELQLRAEPTDLPRLIVYFQTTHDSSNRPISMLPLITEKGIALTHLIVCSFHINQGGVVHLNDFPPDDPHFYTLWNETITMKQAGVKVMGMVGGAAPGSFNTQTLDSPDSATFEHYYGQLRDAIVNFQLEGMDLDVEQPMSQQGIDRLIARLRADFGPDFLITLAPVASALEDSSNLSGFSYTALQQTQGNDIDWYNTQFYSGFGSMADTSDYDRIVANGFAPAKVVAGQLTTPEGAGWIPTSSLNNTIVSLVSEYGQIGGVMGWEYFNSLPGGTAEPWEWAQIVTEILRPGLVPELKITEDDAARLTGAYEESVKAAAADNKSFVKRPSINYYAMVNA SEQ ID NO:4(FfEndo ENGase) MSAVANRLVVYYQTHYKNGAFVSPLPLIGYVTHLLLAAFHLNMGNVGPDIVHLNDFMPSDAYFAPMWPEIAMMQGNGTKVLGMLGGAAPGTYECLVDELFDVYYPPLKDVITDYNLDGMDLDVEQWVSPETIMRIILTLKADFGDDFIITLAPVASALTEGGNLSGFNYIELEAEAGHLIDFYNAQFYSGFGTFFPEDQYVDIIEYEQGIDPSRITATVLTNPQNGYGYVSVGHVAESIGHLMERYGTGWGGVGGWEYFNSLPDANEPWYWAQTMHQAMKVTKENIERKQREEKERKEKEEALEGRSDRETGTVKPPRVRSRFFKDVAVEASEQID SEQ ID NO:5(AoEndo cds) SEQ ID NO:6(CmEndo cds) SEQ ID NO:7(TrEndo cds) SEQ ID NO:8(FfEndo cds) ATGTCTGCCGTCGCCAATCGGCTCGTCGTGTACTACCAGACGCACTACAAGAATGGCGCATTCGTCTCTCCCCTTCCCCTCATTGGCTACGTGACGCACCTCCTCCTCGCGGCGTTCCACCTGAACATGGGCAACGTCGGTCCGGACATTGTACATCTCAACGATTTCATGCCTAGCGACGCGTATTTCGCGCCCATGTGGCCGGAAATCGCAATGATGCAGGGGAACGGGACAAAGGTCCTAGGGATGTTGGGTGGTGCGGCACCGGGGACGTATGAGTGCCTCGTCGACGAGCTCTTCGACGTGTATTACCCCCCGTTGAAGGATGTGATCACCGACTACAACCTGGACGGCATGGACCTGGACGTCGAGCAGTGGGTATCCCCCGAGACAATCATGCGGATCATCTTAACCCTCAAAGCGGATTTTGGCGACGACTTCATCATTACTCTCGCGCCCGTCGCGTCTGCGCTGACGGAAGGCGGCAACCTCTCCGGATTCAACTACATCGAGCTCGAAGCCGAAGCGGGGCACCTCATCGATTTCTACAACGCACAGTTCTACTCTGGCTTTGGGACGTTCTTCCCCGAGGACCAGTATGTCGATATCATCGAGTATGAGCAGGGGATCGACCCGTCGCGAATTACGGCGACAGTGTTGACGAATCCACAGAATGGGTATGGGTATGTGAGTGTGGGGCATGTGGCGGAGAGTATTGGGCATTTGATGGAGAGATATGGGACGGGGTGGGGTGGGGTTGGGGGATGGGAGTATTTTAATTCGTTGCCGGATGCTAATGAGCCGTGGTACTGGGCGCAGACGATGCATCAGGCGATGAAGGTTACGAAGGAGAATATCGAGCGGAAGCAGAGGGAGGAGAAGGAACGGAAGGAGAAGGAGGAGGCGCTAGAAGGGAGGAGTGATAGGGAGACGGGCACGGTTAAGCCGCCGAGGGTGAGATCAAGGTTCTTCAAGGATGTCGCTGTTGAAGCATAG SEQ ID NO:9(AoEndo promoter) CCCACATCCGACAATTGGTCCTCATGGGGTAGCTTCGATTCTCCGAACAATTCGTCGTTCCGCTCCTCAAGACTCAACTGGCCGCTGGGGAATTGGTCAAAATCAAGCGTGTAATCTGCCAGGCGAGCGTCGAGCTCCTTCGCAGCGCGATCAAGCGATGATGCCCGGGAGGACGGCGGCGAGCCGGTCGGCGAATGCGCGTCGTCGATGTCATCGCCGGCGCGATGGGAGATTGGGCTTCGCGCACGCCGGGGGGATCCTTCAGGGCTATGGTTAACTTGCGGAGGGGAGGAAGGCATAAAGAGAGGCGTTGCGAGGGTGGACGCGCCGACTGTATCGTGAGAAGTTGTGTCCATGATGGTATTTGTCGCGGTCGGTAGGTGAGTGAATGCTAGGAATCAAGATCAATTGGCTAGGGCGACATTATTGATTTAGCAGAAGGGCTGACAAGGTCATGTAAATCGAGGTGGACTTTGATAACGATAACCAGCAGATAGAGTATGTCGATGTAACGGACGCGCCTGGACCGGCGATACTGGGAACCCGCCCGTGTGCCGGCCGGAATCCTCAGGTTTTCATATCAGGTGTTTATTATGCTCCATATTAGGTTTAGCGTAGAGGAAGAGCTTTCCTTGATATCGAACTGTACATACCCCGTATGTAATAGATACAAGTACCAGGATTACCGTACTGTAAAGAGACACCCCTTACCAAGACCATCACAGAGTCTTCTTGTAACCATCACCCATTCCGTCCTTTGTTGAATCCCCTCCTTATTTTATCTCCCCAAATATTGACCCTCTGAGGTGCGTTCGGACTGGCCTTCTGTCCAAACCGGTCGCGAAGCAATGGGAGAACCTGACGGCTAAGCTGCGGCCCCCCAGAGACCGTCTGTAATCTCACTACCCCGCTGTTGCTCCTCATCGAAATGCTGGACTTGGTCCTGACCAGTGCAGCAAATCCGCAGCCACCTCCAACCTCCTTTTAAAGCTAAGAAACG。 SEQ ID NO:10(CmEndo promoter) SEQ ID NO:11(TrEndo promoter) CCGTTTGCGGACGGAACGTACGGAGGCTGGTTTCCTTGGGGGCTTTCAGGTCAGGTTGCGAGTTGCCCCCCCCCCCCCCCCCCTCCTTGGCGCATTGCCGAGACGGGAAGGTTGCATGGATTAGAGACGTCCGAGGTATTCCCAAGGGAGAAGCTGGGATTTTTTTTTAACTTATCACAAAGCATGGCTAAAAAGGCTCTTTTATTGGGAATCTTGTTTATGCCACGGTACATGTGCTTTTCTTTTGCGGACTATTGATTTCTTTTTCTTCCCGGGAGGGGAAGGGGGGGGGGGGGGGGGGGGGGGGAGTAATACTTACATACATAGTAGTATGATTGAAGAGGCGCTCCTGGGTGGGTTTCCTATTCTTGGGGTTCTGGGTTTTGCAGAAGAGAGAGGGGAATGGAGGTTTCCTTGGCGAGCTTATAGGGCCGAATGAGTGCCTTGTATGGTGGACGTCTTATACAGGTATTGTATGGATGATGGTTGAATGCTGTTATGTTTGGGGGGGCAGAATGAAGGGAGTGGAATATACCTGCCTTGACTGGAGGGGGGAGTGAGATCACGGATATGGTGGGCACGGACGGGTTAGGTAGGTAGGTAGGTAGGTAGGTAGTAGATGCGGCGGCTTTTATGACTGGGGGAGTGGCATCAGCAGGGAGCAACGCATTATGCGGGAGTGATGATACTAGGTACTGCTACTACTACAAGTAGCTAACTGGTATGGGGGATGCAAGGAGGGCGAGGAGAAATCATGTGGAAGGTGCTTACAAGTATAAAGTGTCAGTTGTCCCGCTACTGATGTGTCTATAGAGATAGGGATATGTATCGTCCTGTAAGAGGCTTCACCCCGTCTCTTCTTTTCTGACTTGCTCCCTGCCCTTGCCCCCCCTCCTCCGACCCCCTCCGCCTCCCCCCTCCTTTGTTCACGATGAAGGCGTCCGTCTACTTGGCGTCCCTGCTGGCGACGCTGTCG SEQ ID NO:12(FfEndo promoter) SEQ ID NO: 13 ATGCGGTGGTCCTTGTTATCCGTC SEQ ID NO: 14 TCAGGCGTTGACCATGGCGTAGTAGTC SEQ ID NO: 15 CCCTCAAGATCTGGGCCACCCCC SEQ ID NO: 16 GGACCCCCGGTCTGATACCGTGG SEQ ID NO: 17 CCCATGCATACCACCATAGTATC SEQ ID NO: 18 CCGGAATTCATGCCTCCCATACCTGCGCAGGTCGA SEQ ID NO: 19 CCGCTCGAGTTAGATGGACGCACCCAGCGAAG SEQ ID NO: 20 CCGGAATTCATGCGGTGGTCCTTGTTATCCGTC SEQ ID NO: 21 CCGCTCGAGTCAGGCGTTGACCATGGCGTAGTAG SEQ ID NO: 22 CCGGAATTCATGAAGGCGTCCGTCTACTTGGCGTC SEQ ID NO: 23 CCGCTCGAGTTAAGCATTCACCATAGCATAATAG SEQ ID NO: 24 CCGGAATTCATGTCTGCCGTCGCCAATCGGCTCG SEQ ID NO: 25 CCGCTCGAGCTATGCTTCAACAGCGACATCCTTGAAG SEQ ID NO: 26 CCCTCAAGATCTGCCC SEQ ID NO:27: The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Application of N-acetylglucosamine endoside enzyme as a component of food additives in improving the digestibility of edible fungal mycelium or fruiting body, characterized in that: The food additive is mixed with N-acetylglucosamine endo-nuclease, and the amino acid sequence of the N-acetylglucosamine endo-nuclease is shown in any one of SEQ ID NOs: 1-4.

2. The use according to claim 1, characterized in that: The edible fungus is selected from Aspergillus oryzae ( Aspergillus oryzae ), Cordyceps militaris ( Cordyceps militaris )、Trichoderma reesei( Trichoderma reesei )、Flammulina velutipes( Flammulina filiformis ),mushroom( Lentinula edodes )、Pleurotus eryngii( Pleurotus eryngii ) Oyster mushroom ( Pleurotus ostreatus ).

3. A food additive for improving the digestibility of edible fungus food, characterized in that: The food additive is mixed with N-acetylglucosamine endo-nuclease, and the amino acid sequence of the N-acetylglucosamine endo-nuclease is shown in any one of SEQ ID NOs: 1 to 4; and the food contains mycelium or fruiting body from edible fungi.

4. A method for improving the digestibility of edible fungi mycelium or fruiting body, characterized in that: The method comprises: causing the edible fungus to overexpress N-acetylglucosamine endo-nuclease in cells, wherein the amino acid sequence of the N-acetylglucosamine endo-nuclease is shown in any one of SEQ ID NOs: 1 to 4.

5. The method according to claim 4, characterized in that The method comprises: allowing the edible fungus to contain an exogenous nucleic acid molecule in the cell to overexpress the N-acetylglucosamine endoenase.

6. The method according to claim 5, characterized in that The exogenous nucleic acid molecule contains a coding region sequence as shown in any one of SEQ ID NOs: 5 to 8.

7. The method according to any one of claims 4 to 6, characterized in that: The edible fungus is selected from Aspergillus oryzae ( Aspergillus oryzae ), Cordyceps militaris ( Cordyceps militaris )、Trichoderma reesei( Trichoderma reesei )、Flammulina velutipes( Flammulina filiformis ),mushroom( Lentinula edodes )、Pleurotus eryngii( Pleurotus eryngii ) Oyster mushroom ( Pleurotus ostreatus ).

8. A method for improving the digestibility of edible fungal mycelium or fruiting body, characterized in that: The method comprises: modifying the endogenous promoter sequence in the genome of the edible fungus for driving the expression of N-acetylglucosamine endonuclease by gene editing technology so as to drive the overexpression of N-acetylglucosamine endonuclease.

9. The method according to claim 8, characterized in that The edible fungus is selected from Aspergillus oryzae ( Aspergillus oryzae ), Cordyceps militaris ( Cordyceps militaris )、Trichoderma reesei( Trichoderma reesei ) and Flammulina velutipes ( Flammulina filiformis ).

10. The method according to claim 9, characterized in that The edible fungus is selected from Cordyceps militaris ( Cordyceps militaris ); the nucleotide sequence of the endogenous promoter before modification is SEQ ID NO: 10; the nucleotide sequence of the endogenous promoter after modification is SEQ ID NO: 27.

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