Use of a coprinus cinereus mannanase

Abstract

The application discloses application of a Coprinus cinereus mannanase, and the Coprinus cinereus mannanase is used as a sugar chain analysis tool enzyme to hydrolyze beta-galactomannan, and the hydrolysis product oligosaccharide structure is analyzed and identified by combining a high-efficiency anion chromatography coupling mass spectrometry method, so that the fine structure of the beta-galactomannan is obtained; meanwhile, the beta-galactomannan hydrolyzed by the Coprinus cinereus mannanase is separated and purified by a molecular exclusion chromatography method to obtain mannobiose, the scavenging activity of the mannobiose to DPPH free radicals is 5-7 times that of the same concentration of mannose, and the DPPH free radical scavenging rate of the mannobiose is 3.5-8.0 times that of other cellobiose, laminarinose, lactose, gentiobiose and sucrose, so that the mannobiose can be used as high-quality functional food or dietary prebiotics.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to the application of an endoglucosanase of *Coprinus comatus* (CcMan5C). Background Technology

[0002] Mannan is a type of hemicellulose polysaccharide, primarily found in plant cork and specific tissues (such as fruits and seeds). Mannans are mainly classified into mannan, glucomannan, galactomannan, and galactoglucomannan. Galactomannan is a heteropolysaccharide composed of a D-mannan backbone polymerized by β-(1→4)-glycosidic bonds and individual D-galactose branches linked to the mannose groups on the mannan backbone by α-(1→6)-glycosidic bonds; it exists in plants and some fungi. The backbone of galactoglucomannan is composed of glucose and mannose polymerized by β-(1→4)-bonds, with branched side chains formed by galactoside glycosides linked to the mannan by α-(1→6)-glycosidic bonds. Galactomannan is used in food processing as a thickener, stabilizer, emulsifier, gelling agent, coffee whitening agent, and shelf-life extender. Locust bean gum glactomannan polysaccharides from seeds of Ceratoriasilique (LBG) is a commercially available β-galactomannan.

[0003] In the feed industry, mannans, as anti-nutritional factors, can reduce feed conversion efficiency and even lead to metabolic and digestive system diseases. Therefore, mannanase is often added to animal feed to hydrolyze anti-nutritional factors, thereby releasing the encapsulated proteins and producing beneficial mannan oligosaccharides.

[0004] Endo-β-1,4-mannanase (EC3.2.1.78) is a major β-mannan hydrolase capable of cleaving β-(1→4)-glycosidic bonds within the mannan backbone. According to the CAZy database (www.cazy.org), mannan hydrolases are found in the glycosidic hydrolase (GH) family, specifically GH5, 26, 45, 113, and 134. Mannanases are widely distributed in plants, mollusks, and microorganisms, exhibiting significant differences in enzymatic properties due to their diverse origins. Naturally expressed or recombinantly expressed microbial β-mannanases have been reported.

[0005] Currently reported or existing β-mannanases show that their ability to cleave the β-(1→4)-glycosidic bonds in the β-galactomannan backbone is related to the relative position of the mannose group with the α-(1→6)-branched galactosyl group. This results in the formation of only one type of mannooligosaccharide with a reduced α-(1→6)-branched galactosyl group at the end, or a non-reduced α-(1→6)-branched galactosyl group at the end, as well as mannooligosaccharides without either the α-(1→6)-branched galactosyl group at the end. Therefore, their application in galactomannan structural analysis and oligosaccharide preparation is limited. Summary of the Invention

[0006] Objective of the Invention: Addressing the problems existing in the prior art, this invention provides an endo-β-1,4-mannanase (CcMan5C) from *Coprinus pilosa* for the fine structural identification of β-galactomannan and its application in the hydrolysis of β-galactomannan to prepare mannobiose. This invention is the first to discover that *Coprinus pilosa* mannanase CcMan5C can endo-hydrolyze the hemiacetal hydroxyl group at the C1 position (reducing end) of the mannosyl group with α-(1→6)-branched galactosyl group on the galactomannan backbone via endo-hydrolysis. The β-(1→4)-mannoglycosidic bond formed by the C4 (non-reducing end) hydroxyl group and its corresponding hydroxyl group on the adjacent mannose group, as well as the β-(1→4)-mannoglycosidic bond without an α-(1→6)-branched galactosyl group within it, produces both reduced-terminal α-(1→6)-branched galactosyl groups and non-reducing-terminal α-(1→6)-branched galactosyl groups, as well as mannooligosaccharides without or without an α-(1→6)-branched galactosyl group at the terminal. Characterizing these various types of mannooligosaccharides with or without an α-(1→6)-branched galactosyl group can be used for the fine structure identification of β-galactomannan; simultaneously, the main final product, mannobiose, can be used in the preparation of mannobiose from the hydrolysis of β-galactomannan.

[0007] Technical solution: In order to achieve the above objectives, the present invention relates to the application of the gray-capped coprinus mannanase CcMan5C in the fine structure identification of β-galactomannan and in the preparation of mannobiose by hydrolysis of β-galactomannan.

[0008] The mannanase is Coprinus garicus mannanase CcMan5C, which can hydrolyze the β-galactomannan backbone in an endolytic manner. This hydrolyzes the β-(1→4)-mannosyl bonds formed by the C1-position (reducing end) hemiacetal hydroxyl group or the C4-position (non-reducing end) hydroxyl group of the mannosyl group with α-(1→6)-branched galactosyl group and the corresponding hydroxyl group of the adjacent mannosyl group, as well as the internal β-(1→4)-mannosyl bonds without α-(1→6)-branched galactosyl group. This produces mannooligosaccharides with α-(1→6)-branched galactosyl groups at the reducing or non-reducing ends, as well as mannooligosaccharides without α-(1→6)-branched galactosyl groups at the ends. The degree of polymerization (DP) of the oligosaccharides is 2-5, and essentially no mannose is produced.

[0009] As a preferred application, the gray-capped coprinus mannanase CcMan5C is used in the hydrolysis of carob gum β-galactomannan and the analysis and identification of its hydrolysis products.

[0010] In this study, *Coprinus comatus* mannanase was used as a glycan analysis tool enzyme to hydrolyze locust bean gum glactomannan polysaccharides (LBG). The oligosaccharide structure of the hydrolysis products was analyzed and identified using high-performance anion chromatography-mass spectrometry (HPLC-MS). The results showed that the number (m) of unbranched galactose residues between the two branched galactose residues in locust bean gum glactomannan varies, ranging from 1 to 5. Locust bean gum β-galactomannan possesses the following fine structure:

[0011]

[0012] The main product of the hydrolysis of carob gum β-galactomannan by the cymosinase described in the above-mentioned *Coprinus comatus* is mannobiose (accounting for about 50% of the oligosaccharide content of the hydrolysis product), which is obtained by separation and purification by size exclusion chromatography.

[0013] The mannobiose prepared by this invention has 5-7 times the DPPH free radical scavenging activity of mannan at the same concentration, and 3.5-8.0 times the DPPH free radical scavenging rate of other cellobiose, laminarin, lactose, gentiobiose and sucrose. It can be used as a high-quality functional food or dietary prebiotic.

[0014] Preferably, the mannanase is *Coprinus gracilis* mannanase CcMan5C, which hydrolyzes only β-(1→4)-mannan without hydrolyzing α-(1→2)-mannan or other types of dextran or xylan, and can hydrolyze β-(1→4)-mannoside bonds formed by the reducing or non-reducing ends of mannosyl groups with α-(1→6)-branched galactosyl groups, as well as internal β-(1→4)-mannoside bonds, in an endo-cleavage manner.

[0015] The mannanase CcMan5C described herein is a mature mannanase protein form (mCcMan5C), obtained by fermentation of a genetically engineered Pichia pastoris strain that heterologously recombinantly expresses mannanase. The cDNA fragment encoding the mature mannanase mCcMan5C gene is synthesized by RT-PCR using mRNA extracted from *Coprinus pastoris* as a template, or directly synthesized artificially based on the cDNA sequence of the encoding gene. The mCcMan5C cDNA fragment is then integrated into an expression plasmid and introduced into *Pichia pastoris* to obtain a heterologous recombinant expression strain of mature mannanase mCcMan5C.

[0016] Preferably, the heterologous recombinant expression of mannanase in *Coprinus gracilis* is obtained by introducing the mature mannanase mCcMan5C encoding gene from *Coprinus gracilis* as the host cell. The mature mannanase mCcMan5C encoding gene from *Coprinus gracilis* refers to the nucleotide sequence of mannanase CcMan5C cDNA from base 64 to 1329, that is, the mature mannanase cDNA (mCcMan5C cDNA) that does not contain the nucleotide sequence of base 1 to 63 base pairs encoding amino acids 1 to 21 and the stop codon.

[0017] The mannanase CcMan5C encoding gene cDNA is any one of the following genes in the NCBI database: CC1G_09314, EAU91632.1, XP_001830154.1, XM_001830102.1, or NW_003307542.1, or its homologous protein genes KAG2015513.1, KAG2015514.1, or KAG2015515.1; the Pichia pastoris strain is Pichia pastoris GS115 or other Pichia pastoris strains.

[0018] Furthermore, the heterologous recombinant expression plasmid of *Coprinus gracilis* mannanase used in the *Coprinus gracilis* mannanase heterologous recombinant expression strain is formed by mixing and ligating the cDNA fragment encoding the mature protein mCcMan5C of *Coprinus gracilis* CcMan5C and the plasmid pPICZαA after enzyme digestion, transforming it into competent cells, extracting the plasmid, and introducing the recombinant expression plasmid pPICZαA-mCcMan5C into *Pichia pastoris*.

[0019] The method for constructing the heterologous recombinant expression Pichia pastoris strain of *Coprinus comatus* mannanase includes the following steps:

[0020] (1) The mycelium or fruiting body of *Coprinus gracilistylus* grown on solid or liquid culture medium was frozen, ground into powder, and extracted to prepare total RNA samples. The mRNA was then reverse transcribed to synthesize cDNA.

[0021] (2) Design primers and use the cDNA synthesized in step (1) as a template to PCR amplify the mature CcMan5C cDNA fragment (mCcMan5C cDNA). The mCcMan5C cDNA fragment product and plasmid pPICZαA are digested with enzymes, mixed and ligated, and transformed into competent cells. The plasmid is extracted to obtain the recombinant expression plasmid pPICZαA-mCcMan5C.

[0022] (3) After linearizing the recombinant expression plasmid pPICZαA-mCcMan5C obtained above, it was electroporated into Pichia pastoris cells to obtain Pichia pastoris containing plasmid pPICZαA-mCcMan5C, which is the heterologous recombinant expression Pichia pastoris expressing mature mannanase mCcMan5C.

[0023] The primers used in step (2) are an upstream primer with an EcoRI restriction site (5'-AGAGAGGCTGAAGCTGAATTCGTAGGCCCTTGGGGCCAGT-3') and a downstream primer with a Not I restriction site (5'-TGTTCTAGAAAGCTGGCGGCCGCCCCGCGAGCCTTCATGG-3').

[0024] The preparation of the mannanase includes the following steps:

[0025] (1) The heterologous recombinant expression strain of mannanase, namely the heterologous recombinant expression strain of Pichia pastoris expressing mature mannanase mCcMan5C, was inoculated into BMGY medium and cultured overnight. The cells were collected by centrifugation, and the cells were resuspended and inoculated into BMMY medium for expansion culture. Methanol was added to induce expression, and the enzyme activity of the supernatant of the medium was measured at the same time. The culture was terminated when the enzyme activity no longer increased.

[0026] (2) Centrifuge the fermentation broth of the recombinant bacteria after the culture has stopped and collect the supernatant. Mix the supernatant with an equal volume of binding buffer and perform column chromatography. Collect the combined active recombinant protein components and dialyze them overnight with dialysis buffer to obtain purified mannanase preparation.

[0027] Preferably, in step (1), the heterologous recombinant expression strain of *Coprinus gracilistylus* mannanase is inoculated into BMGY medium and cultured overnight at 28°C and 180 rpm until OD500. 600 =2~6, collect the bacterial cells by centrifugation, suspend the bacterial cells in 1-2 mL of sterile water and inoculate them into BMMY medium at 0.5-1% volume, and expand the culture at 28℃ and 180 rpm. Add methanol every 24 h to the final concentration of 0.5% to induce expression. At the same time, take samples to measure the enzyme activity of the supernatant of the culture medium. When the enzyme activity no longer increases, the culture is terminated.

[0028] This invention is the first to propose the application of mannanase (recombinantly expressed mature mannanase mCcMan5C from *Coprinus rubrum*) in the hydrolysis of β-galactomannan and the analysis and identification of its hydrolysis products. Combined with high-performance anion chromatography-coupled mass spectrometry (HPAEC-PAD-MS), the oligosaccharide structure of the hydrolysis products was analyzed and identified, and the fine structure of carob gum β-galactomannan was obtained for the first time. Furthermore, the recombinantly expressed mature mannanase (mCcMan5C) from *Coprinus rubrum* described in this invention can hydrolyze carob gum galactomannan to prepare the main product, mannobiose.

[0029] This invention also proposes a recombinant expression strain comprising the mCcMan5C cDNA gene of mature mannanase mCcMan5C from *Coprinus gracilis*. The recombinant secretory expression plasmid pPICZαA-mCcMan5C is linearized by single digestion with the restriction endonuclease Pme I, and then electroporated into *Pichia pastoris* GS115 cells to obtain a *Pichia pastoris* strain expressing secretory β-1,4-mannanase (CcMan5C), which is the recombinant expression strain. The 5' end of the cDNA containing mature mannanase mCcMan5C is fused with the PAOX1 promoter and α-signal peptide sequence on pPICZαA, and the 3' end is fused with the coding sequences for the c-myc-tag, 6XHis-tag, and terminator, expressing and producing secretory mature β-1,4-mannanase mCcMan5C with a 6XHis-purification tag.

[0030] This invention provides a mature β-1,4-mannanase (mCcMan5C) recombinant expression plasmid of *Coprinus gracilis*, a recombinant expression strain of *Pichia pastoris*, and a recombinant expression of an endonuclease of *Coprinus gracilis*. The *Pichia pastoris* strain contains and is able to express the mCcMan5C gene, producing a highly active mature β-1,4-mannanase (mCcMan5C) with hyperglycosylated molecular weight larger than the natural enzyme protein.

[0031] The protein and mRNA sequences of the *Coprinus gracilis* mannanase CcMan5C described in this invention were obtained by searching the National Center for Bioinformatics (NCBI) database in the United States. The accession numbers for the *Coprinus gracilis* mannanase CcMan5C protein and mRNA sequences in the NCBI database are CC1G_09314, EAU91632.1, XP_001830154.1, XM_001830102.1, or NW_003307542.1, or its homologous protein accession numbers (KAG2015513.1, KAG2015514.1, or KAG2015515.1). The full-length CcMan5C cDNA consists of 1332 base pairs, encoding 443 amino acids. SignalP 5.0 Server software analysis confirmed that the N-terminal 1-21 amino acids (MKLSAGLVSLAIAVTSASAQA) of the protein are the signal peptide sequence. The mature protein mCcMan5C, which does not contain the signal peptide, has 422 amino acids and a theoretical molecular weight of 45.8 kDa.

[0032] This invention first used *Coprinopsis cinerea endo-1,4-beta-mannosidase* as a keyword to search the National Center for Bioinformatics (NCBI) database in the United States, finding the mannanase gene *CcMan5C* proposed in this invention. Then, based on the cDNA sequence of this gene, using a designed and synthesized upstream primer with an EcoRI restriction site (5'-AGAGAGGCTGAAGCTGAATTCGTAGGCCCTTGGGGCCAGT-3') and a downstream primer with a Not I restriction site (5'-TGTTCTAGAAAGCTGGCGGCCGCCCCGCGAGCCTTCATGG-3'), and using cDNA synthesized by reverse transcription from mRNA prepared from *Coprinopsis cinerea* as a template, the nucleotide sequence of 64-1329 base pairs of the *mCcMan5C* cDNA was amplified by PCR. This sequence contains 1-63 base pairs of nucleotides encoding amino acids 1-21 and a stop codon, representing the mature mannanase cDNA (mCcMan5C). The 5' end of the cDNA containing the mature mannanase mCcMan5C is fused with the PAOX1 promoter and α signal peptide sequence on pPICZαA, and the 3' end is fused with the coding sequences for the c-myc-tag, 6XHis-tag, and terminator, in order to express secretory mCcMan5C with the 6XHis-purified tag.

[0033] Although the mannanase-encoding gene sequence from *Coprinus gracilis* already exists in public, free gene banks and has been predicted by automated genome annotation to encode a certain mannanase, the function of the protein encoded by this gene has not been experimentally confirmed. In this invention, taking advantage of *Pichia pastoris* as the expression host, the mature mannanase mCcMan5 gene was introduced into *Pichia pastoris*, followed by fermentation and purification to obtain a highly active recombinant mature endonuclease β-1,4-mannanase (mCcMan5C). The purified form and catalytic properties of this mannanase are confirmed for the first time in this invention. The mannanase provided by this invention can be used in biotechnology applications.

[0034] This invention is the first to discover that mature endo-β-1,4-mannanase (mCcMan5C) of *Coprinus gracilis* can endo-hydrolyze β-(1→4)-mannoside bonds formed by reducing or non-reducing ends of mannosyl groups with α-(1→6)-branched galactosyl groups on the galactomannan backbone via endo-hydrolysis. This produces mannooligosaccharides with both reducing and non-reducing ends, as well as unbranched or terminally unbranched mannooligosaccharides. This invention utilizes recombinant expression of mature mannanase mCcMan5C for the hydrolysis of β-galactomannan. The specific products generated by the hydrolysis can be effectively used for the fine structure identification of β-galactomannan and to propose its fine structure. For example, the disaccharide product may include two isomers: Gal1-6Man and Man1-4Man. Figure 3 B, C); the trisaccharide product includes three isomers: Gal1-6Man1-4Man, Man1-4(Gal1-6)Man, and Man1-4Man1-4Man. Figure 3 D, 1, 2, 3); the tetrasaccharide product includes three isomers: Gal1-6Man1-4Man1-4Man, Man1-4Man1-4(Gal1-6)Man, and Man1-4Man1-4Man1-4Man. Figure 3 E, 1, 2, 3); the pentasaccharide products include four isomers: Gal1-6Man1-4Man1-4Man1-4Man, Man1-4Man1-4Man1-4(Gal1-6)Man, Man1-4(Gal1-6)Man1-4Man1-4Man and Man1-4Man1-4Man1-4Man1-4Man. Figure 3F, 1, 2, 3, 4), but the hydrolysis products basically do not contain the monosaccharide mannose. Because the mature endo-β-1-4-mannanase (mCcMan5C) of *Coprinus comatus* hydrolyzes carob gum galactomannan products, producing a series of mannooligosaccharides with different degrees of polymerization (DP2-DP5) with reduced or non-reduced ends bearing one branched galactose group, as well as mannooligosaccharides without or without branched galactose groups at the ends, the characterization of these various types of mannooligosaccharides with or without α-(1→6)-branched galactose groups revealed for the first time that the number (m) of unbranched mannose groups between two branched galactose groups in carob gum galactomannan varies, ranging from 1 to 5, rather than the three unbranched galactose groups reported in the prior art (Deaand Morrison, 1975, Adv Carbohydr Chem Biochem, 31:242–312.). Therefore, this invention reveals for the first time that carob gum β-galactomannan has the following fine structure:

[0035] In addition, it was discovered for the first time that since the main product of the final product formed by the hydrolysis of carob gum galactomannan by mature endo-β-1-4-mannanase (mCcMan5C) is mannobiose (accounting for about 50% of the hydrolyzed oligosaccharide product), it can be used in the preparation of mannobiose from the hydrolysis of β-galactomannan.

[0036] Previously reported or publicly available β-mannanases hydrolyzing galactomannan only produce one type of terminally galactosyl-substituted mannan oligosaccharide: either a reduced terminally branched galactosyl mannan oligosaccharide, or a non-reduced terminally branched galactosyl mannan oligosaccharide, as well as unbranched galactosyl-substituted mannan oligosaccharides and mannan oligosaccharides with different degrees of polymerization and non-terminally branched galactosyl groups. For example, a *Aspergillus niger* β-mannanase and a *Penicillium purpureum* mannanase hydrolyze galactomannan only produce a reduced terminally branched galactosyl mannan oligosaccharide, as well as non-terminally branched galactosyl mannan oligosaccharides, mannobioses, and mannotrioses with different degrees of polymerization and different degrees of polymerization (McCleary et al, 1983, Carbohydr Res, 119:191-219; Park et al, J Microbiol Biotechnol, 1992, 2:204-208). One Bacillus endo-β-1-4-mannanase, ManB-1601, hydrolyzes galactomannan to produce only one non-reducing terminally branched galactosyl mannobiose, α-(1→6)-galactosylmannose, as well as mannobiose and an unidentified pentasaccharide (Srivastava et al, 2017, J Agric Food Chem 65:2827-2838). Furthermore, none of these reported enzymes have been able to hydrolyze galactomannan to produce a dominant oligosaccharide hydrolysate.

[0037] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0038] (1) The Pichia pastoris engineered strain that heterologously recombinantly expresses mannanase CcMan5 constructed in this invention can be easily fermented and purified to obtain mature recombinantly expressed β-1-4-mannanase (mCcMan5C) for use in the field of biotechnology.

[0039] (2) The mature β-1,4-mannanase (mCcMan5C) recombinantly expressed in this invention has special catalytic properties and storage stability, which is beneficial for applications in the field of biotechnology.

[0040] (3) This invention is the first to discover that the mature β-1,4-mannanase (mCcMan5C) expressed by recombinant enzyme can hydrolyze the β-1-4-mannoside bonds formed by the reducing or non-reducing ends of mannosyl groups with α-(1→6)-branched galactosyl groups on the galactomannan backbone via endolysis, producing mannooligosaccharides with branched galactosyl groups at reducing or non-reducing ends, as well as mannooligosaccharides without or without branched galactosyl groups at the ends, but essentially not producing the monosaccharide mannose. This is the first application of this enzyme for the identification of the fine structure of β-galactomannan, and the first time that the fine structure of β-galactomannan has been revealed:

[0041]

[0042] The identification of this fine structure provides theoretical support and a basis for subsequent preparation and production of mCcMan5C-degraded carob gum β-galactomannan, which mainly produces mannobiose (the dominant sugar in the product).

[0043] (4) This invention is the first to discover that the main product in the final product formed by hydrolyzing carob gum galactomannan by the mature β-1,4-mannanase (mCcMan5C) expressed by recombinant expression is mannobiose, and for the first time it is used to hydrolyze β-galactomannan to prepare mannobiose with high yield and high purity.

[0044] (5) The present invention first discovered that the DPPH free radical scavenging activity of the prepared mannobiose is 5-7 times that of mannan of the same concentration, and 3.5-8.0 times that of other disaccharides such as cellobiose, laminarin, lactose, gentiobiose and sucrose. It can be used as a high-quality functional food or dietary prebiotic. Attached Figure Description

[0045] Figure 1 A) The expression vector pPICZαA-mCcMan5C constructed; B) SDS-PAGE analysis of the protein of mature mannanase mCcMan5C recombinantly expressed in *Pichia pastoris*, where M represents the molecular weight standard protein; 1) Fermentation supernatant of *Pichia pastoris* strain GS115 containing the empty vector plasmid pPICZαA; 2) Fermentation supernatant of *Pichia pastoris* strain GS115 containing the plasmid pPICZαA-mCcMan5C; 3) Purified mature mannanase mCcMan5C protein of *Pichia pastoris*; C) Two amino acid sequences (C1 and C2) of the trypsin-digested peptide fragment of the purified 66kD protein, and its position in the CcMan5C amino acid sequence displayed in the NCBI database (underlined) (C3); D) MALDI-TOF / TOF analysis of the trypsin-digested peptide fragment of the purified 64kD protein. The two amino acid sequences (D1 and D2) analyzed by MS and their positions in the CcMan5C amino acid sequence displayed in the NCBI database (D3), with the box lines indicating the CcMan5C signal peptide sequence.

[0046] Figure 2The hydrolytic activity of the mature mannanase mCcMan5C recombinantly expressed by Pichia pastoris for different polysaccharide substrates was evaluated (A); the hydrolysis rate of different concentrations of carob gum galactomannan was evaluated (B); the hydrolytic activity (C) and pH stability (D) at 50 °C and different pH conditions; the hydrolytic activity (E) and temperature stability (F) at pH 8.0 and different temperature conditions; the hydrolytic activity of the enzyme in the presence of different organic solvents (G) and surfactants (H); and the residual enzyme activity after storage at different storage temperatures for a certain period of time in liquid (I) or freeze-dried (J) states.

[0047] Figure 3 Figure A shows the HPAEC-PAD chromatogram of the product of carob gum galactomannan (LBG) after enzymatic hydrolysis at mCcMan5C for 24 h; Figure B shows the MS / MS mass spectrum and structure of disaccharide P1 in Figure A; Figure C shows the MS / MS mass spectrum and structure of disaccharide P2 in Figure A; Figure D shows the MS / MS mass spectrum and structure of trisaccharide P3 in Figure A; Figure E shows the MS / MS mass spectrum and structure of tetrasaccharide P4 in Figure A; and Figure F shows the MS / MS mass spectrum and structure of pentasaccharide P5 in Figure A.

[0048] Figure 4 A is the size exclusion chromatogram of mCcMan5C enzymatic hydrolysate of carob gum galactomannan (LBG); B is the primary mass spectrum of P1-P4 components in A; C is the HPAEC-PAD spectrum of the monosaccharide components after acid hydrolysis of the purified mannooligosaccharide; D is the MS / MS mass spectrum of the purified P2 disaccharide; E1 is the comparison of DPPH free radical scavenging rates of enzymatically hydrolyzed LBG (dLBG) and unhydrolyzed LBG obtained by size exclusion chromatography of 5 mg / mL and 10 mg / mL mannose (M) with polymerization degrees of DP2-DP5; E2 is the comparison of DPPH free radical scavenging rates of different disaccharides (5 mg / mL) such as sucrose, lactose, gentiobiose, cellobiose, laminarin, and mannobiose purified in this invention. Detailed Implementation

[0049] The present invention will be further described below with reference to the accompanying drawings and embodiments. Unless otherwise specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments used, unless otherwise specified, are all commercially available products.

[0050] Reagents and culture media used in the examples:

[0051] The binding buffer has a pH of 7.0 and consists of the following components in molar concentrations: 500 mM NaCl, 5 mM imidazole, and 20 mM K2HPO4.

[0052] The elution buffer has a pH of 7.0 and comprises the following components in molar concentrations: 500 mM NaCl, 500 mM imidazole, and 20 mM K2HPO4.

[0053] The dialysis buffer has a pH of 7.0 and comprises the following components at molar concentrations: 50 mM K2HPO4.

[0054] BMGY medium: 10g yeast extract, 20g tryptone, and 700mL double-distilled water were mixed and sterilized at 115℃ for 20 minutes. After cooling to room temperature, 100mL of 13.4% yeast nitrogen base and 2mL of 200μg / mL biotin were added separately, along with 100mL of 1M K2HPO4 / KH2PO4 (pH 6.0) and 100mL of 10% glycerol after sterilization at 115℃ for 20 minutes.

[0055] BMMY medium: 10g yeast extract, 20g tryptone, 700mL double-distilled water were mixed and sterilized at 115℃ for 20 minutes. After cooling to room temperature, 100mL of 13.4% yeast nitrogen base (filtered and sterilized), 2mL of 200μg / mL biotin, and 100mL of 5% methanol were added, along with 100mL of 1M K2HPO4 / KH2PO4 (pH 6.0) sterilized at 115℃ for 20 minutes.

[0056] Example 1

[0057] Construction of recombinant expression plasmid for endo-β-1,4-mannanase (CcMan5C) from *Coprinus gracilis* and recombinant expression strain of *Pichia pastoris*, purification and identification of the recombinant mannanase.

[0058] 1. A method for preparing recombinant expression plasmids, comprising the following steps:

[0059] Mycelia or fruiting bodies of *Coprinus gracilistylus* (strain ATCC 56838, purchased from the American Type Culture Collection, ATCC) grown on solid or liquid culture media were frozen in liquid nitrogen and then thoroughly ground into powder. Total RNA samples were prepared using the Total RNA Extractor (Trizol) kit from Sangon Biotech, following the manufacturer's instructions. Vazyme's product was used... The IIQ RT SuperMix for qPCR (+gDNAwiper) kit is used to degrade and remove genomic DNA from total RNA samples according to the instructions, and then reverse transcribe the mRNA to synthesize cDNA.

[0060] The upstream primer with the EcoRI restriction site SEQ ID NO. 3: (5'-AGAGAGGCTGAAGCTGAATTCGTAGGCCCTTGGGGCCAGT-3') and the downstream primer with the Not I restriction site SEQ ID NO.: 4: (5'-TGTTCTAGAAAGCTGGCGGCCGCCCCGCGAGCCTTCATGG-3') were designed. Using the cDNA synthesized in the above steps as a template, PCR amplification was performed using PrimeSTAR Mix (TAKARA, China) under the following conditions: ① 98℃, 5 min; ② 98℃, 20 sec; ③ 55℃, 20 sec; ④ 72℃, 1.5 min; ⑤ Repeat ②-④ for 30 cycles; ⑥ 72℃, 10 min, to obtain the cDNA fragment of mature CcMan5C protein (mCcMan5C cDNA).

[0061] The mCcMan5C cDNA fragment product and plasmid pPICZαA were treated with EcoR I and Not I restriction endonucleases, respectively, and then... The Entry One Step Cloning Kit (Vazyme, China) was used to mix and ligate the cells according to the instructions, and then transformed into E. coli DH5α. The plasmid was extracted using the SanPrep Column Plasmid DNA Mini-Extraction Kit (Sangon Biotech, China) to obtain the recombinant expression plasmid pPICZαA-mCcMan5C. Figure 1 Figure A shows the expression vector pPICZαA-mCcMan5C constructed in Example 1. In the figure, the mCcMan5C gene is inserted between the EcoRI and NotI restriction sites, replacing other multiple cloning sites.

[0062] The positive clone plasmid was sent to Sangon Biotech (Shanghai) for sequencing and identification. The nucleic acid sequence of mCcMan5C cDNA is shown in SEQ ID NO.1, which is consistent with the sequence registered in the NCBI database and will be used for subsequent experiments. The amino acid sequence of the protein it expresses is shown in SEQ ID NO.2.

[0063] 2. The method for preparing recombinant expression strains includes the following steps:

[0064] The recombinant secretory expression plasmid pPICZαA-mCcMan5C obtained above was linearized by single digestion with restriction endonuclease Pme I, and then electroporated into Pichia pastoris GS115 cells (Invitrogen, USA) according to the manufacturer's instructions. A Pichia pastoris strain expressing the endonuclease β-1,4-mannanase (CcMan5C) was obtained, which is the recombinant expression strain. The 5' end of the cDNA containing the mature mannanase mCcMan5C is fused with the PAOX1 promoter and α signal peptide sequence on pPICZαA, and the 3' end is fused with the coding sequences for the c-myc-tag, 6XHis-tag, and terminator, expressing and producing secretory mature β-1,4-mannanase mCcMan5C with a purified 6XHis-tag. Figure 1 A).

[0065] 3. The preparation method of mannanase includes the following steps:

[0066] The recombinant expression strain was inoculated into 10 mL of BMGY medium and cultured overnight at 28°C and 180 rpm until OD500. 600 =4. Centrifuge to collect bacterial cells, suspend the bacterial cells in 1 mL of sterile water and inoculate them into BMMY medium at 1% volume. Incubate at 28℃ and 180 rpm for expansion. Add methanol every 24 h to a final concentration of 0.5% to induce expression. At the same time, take samples to measure the enzyme activity of the supernatant of the medium. When the enzyme activity no longer increases, terminate the culture.

[0067] After stopping the culture of the recombinant bacteria, the fermentation broth was centrifuged to collect the supernatant. At 4°C, the supernatant was mixed with an equal volume of 2× binding buffer and loaded onto ProteinIso™ Ni-NTA resin (TransGen Biotech). Alternatively, ProBond can be used. TM Nickel-Chelating Resin (Invitrogen) or other similar affinity chromatography columns packed with Ni-affinity resin were used. After sample loading, the column was washed with 1× binding buffer until the UV absorbance (OD280) of the effluent was 0. Then, the recombinant protein on the column was eluted using a gradient of 5-100% elution buffer on a Bio-Rad low-pressure chromatography system. The combined active recombinant protein fractions were collected and dialyzed overnight with dialysis buffer using a 14 kDa cutoff dialysis bag (Biosharp, China) to obtain the purified mannanase preparation (i.e., the recombinantly expressed mature mannanase mCcMan5C of Coprinus comatus).

[0068] In this embodiment, the mature mannanase mCcMan5C expressed by recombinant Pichia pastoris was shown on polypropylene gel electrophoresis (SDS-PAGE) to have a molecular weight larger than the theoretical molecular weight of 48kD of mannanase from the protein of Coprinus pastoris. This was caused by the glycosylation modification of the recombinant mannanase by Pichia pastoris, and it had two molecular weight variants with different degrees of glycosylation, with molecular weights of 66kD and 64kD, respectively. Figure 1 Figure B shows the SDS-PAGE analysis results of the recombinant Pichia pastoris expression of mannanase mCcMan5C prepared in Example 1. M, molecular weight standard protein; 1, fermentation supernatant of Pichia pastoris strain GS115 containing empty vector plasmid pPICZαA; 2, fermentation supernatant of Pichia pastoris strain GS115 containing plasmid pPICZαA-mCcMan5C; 3, purified mature mannanase mCcMan5C protein of Pichia pastoris.

[0069] The mature mannanase mCcMan5C expressed by the recombinant Pichia pastoris strain was hydrolyzed with trypsin. The resulting peptide fragments were isolated and purified, and identified by MALDI-TOF / TOF MS analysis. The two variant proteins, 66kD and 64kD, were both mCcMan5C proteins expressed by Pichia pastoris strain GS115, with no difference in amino acid sequence. This is a common molecular weight difference caused by different degrees of glycosylation modification of recombinant Pichia pastoris proteins. Figure 1 C represents the two amino acid sequences (C1 and C2) of the purified 66kD protein trypsin-digested peptide fragment as analyzed by MALDI-TOF / TOF MS, and its position (underlined) in the CcMan5C amino acid sequence shown in the NCBI database (C3). Figure 1 D represents the two amino acid sequences (D1 and D2) of the trypsin-digested peptide fragment of the purified 64kD protein as analyzed by MALDI-TOF / TOF MS, and its position in the CcMan5C amino acid sequence displayed in the NCBI database (D3). The box line indicates the Man5C signal peptide sequence. This indicates that the mature mannanase mCcMan5C of *Coprinus comatus* can be obtained by expression in recombinant *Pichia pastoris* and purification by Ni-affinity resin chromatography.

[0070] Example 2

[0071] Enzyme catalytic properties

[0072] The present invention found that the recombinant expressed mature mannanase of *Coprinus gracilistylus* prepared in Example 1 has special catalytic properties, and is resistant to high temperature, alkali, surfactants, organic solvents and long-term storage.

[0073] The enzyme activity, substrate specificity, optimal reaction conditions, enzyme kinetics, enzyme stability, and tolerance to organic solvents and surfactants of the recombinant expressed and purified mature mannanase mCcMan5C prepared in Example 1 were analyzed. The determination methods and results are as follows:

[0074] 1. Analysis of mannanase activity and substrate specificity (reducing sugar method, also known as DNS method)

[0075] The standard reaction system, conditions, and operating procedures for mannanase enzyme activity assay are as follows: 100 μL of reaction solution contains 50 mM PBS buffer (pH 8.0), 2.5 mg / mL β-galactomannan or other polysaccharides, and 250 ng / mL recombinant expression purified β-mannanase mCcMan5C (the purified mannanase preparation obtained in Example 1). After incubating the reaction solution at 50°C and 800 rpm for 10 min, 100 μL of DNS reagent (3,5-dinitrosalicylic acid) is added, mixed well, and heated at 100°C for 10 min. After cooling to room temperature, the supernatant is centrifuged and the OD is measured. 520 nm. One unit of enzyme activity is defined as the amount of enzyme required to release 1.0 μmol of reducing sugar (based on mannose) per minute under the given reaction conditions.

[0076] Depend on Figure 2 As shown in Figure A, mCcMan5C exhibits hydrolytic activity only towards β-galactomannan from seeds of *Ceratoria silique* (LBG, containing β-D-Man-1,4-β-D-Man and α-D-Gal-1,6-β-D-Man glycosidic bonds, purchased from Sigma-Aldrich, USA), with a specific activity of 312.68 U / mg. However, it cannot degrade α-mannan from *Saccharomyces cerevisiae* (containing α-D-Man-1,2-α-D-Man and α-D-Man-1,6-α-D-Man glycosidic bonds, purchased from Sigma-Aldrich, USA). ), as well as microcrystalline cellulose Avicel (containing β-D-Glc-1,4-β-D-Glc glycosidic bonds, purchased from TCI Corporation, Japan), oat xylan (containing β-D-xyl-1,4-β-D-xyl glycosidic bonds, purchased from Sigma-Aldrich, USA) and palmate laminarin (containing β-D-Glc-1,3-β-D-Glc and β-D-Glc-1,6-β-D-Glc glycosidic bonds, purchased from Sigma-Aldrich, USA).

[0077] 2. pH analysis of the optimal reaction pH for mannanase

[0078] Replace the 50 mM PBS buffer (pH 8.0) in the reaction solution for the mannanase activity assay with buffers of different pH values: 50 mM NaAc-HAc buffer (pH 3.0–6.0), 50 mM PBS buffer (pH 6.0–8.0), or 50 mM Tris-HCl buffer (pH 8.0–9.0). Keep all other components, contents, reaction conditions, and operating procedures unchanged. Assess enzyme activity using the DNS method described above. Set the highest measured enzyme activity value as 100% and calculate the relative enzyme activity for each of the other pH reaction ranges.

[0079] like Figure 2 As shown in Figure C, the mature mannanase mCcMan5C prepared in Example 1 of this invention exhibits increased enzyme activity with increasing pH between pH 3.0 and 9.0, reaching its highest activity at pH 8.5-9.0, indicating that this enzyme prefers alkaline reaction conditions.

[0080] 3. pH stability analysis of mannanase

[0081] The aforementioned mannanase was prepared in 50 μL of 50 mM buffer at different pH values ​​(pH 3–9), incubated at room temperature for 1 h, and then the pH was adjusted to 8.0. It was then mixed with 50 μL of 50 mM PBS buffer (pH 8.0) to prepare 5 mg / mL β-galactomannan. The enzyme activity was then measured by the DNS method described above. The value measured by the unincubated storage enzyme was set as 100%, and the relative enzyme activity after incubation at different pH values ​​was calculated.

[0082] Depend on Figure 2 As shown in D, the mature mannanase mCcMan5C prepared in Example 1 of this invention can maintain more than 90% of its enzyme activity after incubation at pH 8-9 for 1 hour. This indicates that the mature mannanase mCcMan5C recombinantly expressed by *Coprinus comatus* of this invention has good pH stability.

[0083] 4. Analysis of the optimal reaction temperature of mannanase

[0084] The specified 50℃ temperature condition in the reaction system for mannanase activity determination was changed to 20℃, 30℃, 40℃, 50℃, 60℃, 70℃, or 80℃, respectively. Other reaction solution components and contents, reaction conditions, and operation steps remained unchanged. The enzyme activity was determined by the DNS method described above. The highest enzyme activity value was set as 100%, and the relative enzyme activity in other temperature reaction ranges was calculated.

[0085] The results are as follows Figure 2As shown in E, the mature mannanase mCcMan5C prepared in Example 1 of this invention showed that the enzyme activity gradually increased with the increase of reaction temperature in the range of 20℃ to 70℃, and the enzyme activity was the largest at 70℃. When the reaction temperature exceeded 70℃, the enzyme activity decreased sharply, indicating that the optimal temperature for the enzyme was 70℃.

[0086] 5. Temperature stability analysis of mannanase

[0087] First, 50 μL of the mannanase mCcMan5C solution prepared in 50 mM PBS buffer (pH 8.0) was incubated for 60 min at different temperatures from 20 to 80 °C. Then, it was mixed with 50 μL of 5 mg / mL β-galactomannan prepared in 50 mM PBS buffer (pH 8.0). The enzyme activity was then measured by the DNS method described above. The value measured by the unincubated storage enzyme was set as 100%, and the relative enzyme activity after incubation at different temperatures was calculated.

[0088] Depend on Figure 2 As can be seen from F, the enzyme is relatively stable between 20 and 50°C, but its activity decreases rapidly when the temperature exceeds 50°C. The mannanase prepared in Example 1 of this invention still retains more than 80% of its activity after incubation at 50°C for 1 hour. This indicates that the mature mannanase mCcMan5C recombinantly expressed by *Coprinus comatus* exhibits good temperature stability.

[0089] 6. Determination of the enzymatic kinetics of mannanase

[0090] Carob gum galactomannan solutions of 0.25 mg / mL, 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, 2 mg / mL, 2.5 mg / mL, 3 mg / mL, 3.5 mg / mL, 4 mg / mL, 4.5 mg / mL, and 5 mg / mL were prepared using 50 mM PBS buffer (pH 8.0). 50 μL of each of these carob gum galactomannan solutions replaced the 5 mg / mL β-galactomannan solution in the reaction system for the mannanase activity assay, while keeping other components, contents, reaction conditions, and operating procedures unchanged. Enzyme activity was measured using the DNS method described above, and Km curves were plotted using Origin software. Figure 2 B) Calculate Km and Vm. The calculated Km and Vmax values ​​for mature mannanase mCcMan5C are 1.23 mg / mL. -1 and 531.77 mol.min -1 .mg -1This indicates that within the mannan concentration range of 1-3 mg / mL, the catalytic activity of mature mannanase mCcMan5C of *Coprinus sarcodactylis* increases with increasing mannan concentration, but beyond 3 mg / mL, the catalytic activity of mannanase essentially no longer increases.

[0091] 7. Tolerance of mannanase to surfactants

[0092] Surfactants SDS, Triton X-100, or Tween-20 were added to the standard mannanase enzyme activity assay solution at final concentrations of 1% or 10%, respectively. Other components, contents, reaction conditions, and operating procedures remained unchanged. Enzyme activity was measured using the DNS method described above. The enzyme activity of the blank group without surfactant was taken as 100%, and the relative enzyme activity of each experimental group with added surfactant was calculated. Figure 2 As shown in Figure H, SDS inhibits the activity of mCcMan5C enzyme, but 1% or 10% Tween20 and Triton X-100 enhance the catalytic activity of the enzyme, increasing the enzyme activity by 26.2%–45.6% compared to the control. This indicates that mCcMan5C is not only resistant to surfactants, but its catalytic activity can also be enhanced by surfactants.

[0093] 8. Tolerance of mannanase to organic solvents

[0094] Organic solvents such as methanol, ethanol, isopropanol, or acetone were added to the standard mannanase enzyme activity assay solution at final concentrations of 10% or 20%, respectively. Other components, contents, reaction conditions, and operating procedures remained unchanged. Enzyme activity was measured using the DNS method described above. The enzyme activity of the blank group without the added solvent was taken as 100%, and the relative enzyme activity of each experimental group with added organic solvents was calculated. Figure 2 As shown in G, organic solvents such as 10% or 20% methanol, ethanol, isopropanol, and acetone had no significant inhibitory effect on the activity of CcMan5C. On the contrary, 10-20% isopropanol and acetone, as well as 20% methanol and ethanol, could enhance the catalytic activity of the enzyme, increasing the enzyme activity by 9.20% to 34.98% compared with the control. This indicates that mCcMan5C is not only resistant to organic solvents, but its catalytic activity can also be enhanced by higher concentrations of organic solvents.

[0095] 9. Storage stability of mannanase

[0096] The mature mannanase mCcMan5C solution samples obtained from the recombinant expression and purification of *Coprinus sarcodactylis* prepared in Example 1 were aliquoted and stored directly in refrigerators at 4℃, -20℃, and -80℃, respectively, or freeze-dried and then stored in refrigerators at 4℃, -20℃, and -80℃, respectively. Samples were retrieved after half a month, one month, three months, six months, and twelve months of storage, respectively, and the enzyme activity of the enzyme samples under different storage conditions was measured according to the standard mannanase enzyme activity assay reaction system described above. The initial enzyme activity of mCcMan5C measured at the beginning of storage was taken as 100%, and the relative enzyme activities of samples from other experimental groups with different storage conditions and storage times were calculated. Figure 2 As shown in Figure I, the activity of freeze-dried mannanase mCcMan5C decreased during the first three months of storage at -20°C or -80°C. However, after six months of storage, the enzyme activity of mCcMan5C increased by 40% and 60% compared to the initial activity, respectively. Even after 12 months of storage, the enzyme activity was still 28% higher than the initial activity. Figure 2 As shown in Figure J, the activity of purified dialysis-treated liquid mannanase mCcMan5C decreased compared to the control during the first three months of storage at -20°C or -80°C. However, after 6 months and even 12 months of storage, the activity of mCcMan5C remained almost unchanged compared to the initial activity. Furthermore, the activity of freeze-dried or liquid mannanase mCcMan5C samples stored at 4°C for 12 months consistently remained above 50% of the control. This indicates that mannanase mCcMan5C exhibits high storage stability.

[0097] Example 3

[0098] Mannanase is used for the hydrolysis of β-galactomannan and the analysis and identification of its hydrolysis products.

[0099] The composition and content of 200 μL carob gum β-galactomannan enzymatic hydrolysis solution were as follows: 50 mM PBS buffer (pH 8.0) containing a final concentration of 1.5 μg / mL mature carob gum mannanase mCcMan5C (prepared in Example 1) and a final concentration of 2.5 mg / mL carob gum β-galactomannan (LBG, purchased from Sigma-Aldrich, USA). The enzymatic hydrolysis solution was reacted at 50 °C for 24 h, then heated at 100 °C for 15 min, cooled to room temperature, centrifuged, and the supernatant was filtered through a 0.22 μm filter for later use.

[0100] The filtered enzymatic hydrolysate was loaded onto a CarboPac PA1 column (4×250mm, Dionex, Sunnyvale, CA) and analyzed by high performance anion liquid chromatography (HPLC) with pulsed amperometric detector (HPAEC-PAD) using a Professional IC Vario 940. The column temperature was 30℃, the flow rate was 1mL / min, and the quantitative loop was 20μL. Elution was performed according to the following procedure: 0–30 min: 100% A isogradient; 30–60 min: 100% A—60% A + 40% B; 60–65 min: 60% A + 40% B—100% B; 65–70 min: 100% B isogradient; 70–71 min: 100% B—100% A + 0% B; 71–80 min: 100% A isogradient; Mobile phase A: 12 mM NaOH, Mobile phase B: 12 ​​mM NaOH + 300 mM NaOAc. Figure 3 A is the HPAEC-PAD chromatogram of the LBG enzymatic hydrolysis products, showing five oligosaccharide peaks: P1, P2, P3, P4, and P5.

[0101] The oligosaccharide peaks in the enzymatic hydrolysate were analyzed and identified using high-performance anion exchange liquid chromatography combined with pulsed amperometric detector-mass spectrometry (HPAEC-PAD-Q-TOF-MS). During analysis, the sample solution was separated using a PA-1 analytical column, and the eluent was split via a three-way splitter. One portion of the eluent flowed directly into the pulsed amperometric detector (PAD), while the other portion was treated with a desalting column before being analyzed by the mass spectrometer. The Q-TOF-MS analytical parameters were as follows: nebulizer pressure: 40 psi; drying gas flow rate / temperature: 10 L / min, 350 °C; capillary voltage: 3500 V; scan mass range: 100 m / z to 3000 m / z; fragmentation voltage: 100 V; collision energy adjusted appropriately between 10 V and 50 V according to molecular weight; negative ion mode was used. Q-TOF-MS analysis showed that P1 and P2 were disaccharides, P3 was a trisaccharide, P4 was a tetrasaccharide, P5 was a pentasaccharide, and no obvious monosaccharide mannose was detected. Figure 3 B is the QTOF-MS / MS secondary mass spectrum of the product peak P1 disaccharide. In its MS / MS fragment ion peaks, in addition to the typical fragment ions B1 and C1 from glycosidic bond breakage, there are also transcyclic fragment ions. 0,2 A2, 0,3 A2, 0,4A2 originates from the second sugar ring from the non-reducing end, confirming that the glycosidic bonds are 1,6 linked. LBG is known to be a heteropolysaccharide composed of mannan as the main chain linked by β-(1→4)-glycosidic bonds and D-galactose as branched side chains linked by α-(1→6)-glycosidic bonds, with galactose and mannose in a ratio of approximately 1:4. Therefore, this P1 disaccharide is Gal1-6Man. Figure 3 C is the Q TOF-MS / MS mass spectrum of the P2 disaccharide. In the MS / MS fragment ion peaks, in addition to the typical fragment ions B1 and C1 from glycosidic bond breakage, there are also transcyclic fragment ions from the second sugar ring at the non-reduced end. 0.2 A2, 2.4 A2, 2.5 A2, therefore they are linked by (1→4)-glycosidic bonds, and based on the known LBG structure, the P2 disaccharide can be identified as Man1-4Man. Figure 3 D is the TOF-MS / MS mass spectrum of the P3 trisaccharide. The spectrum shows that, in addition to the typical fragment ions B1, C1, B2, and C2 from glycosidic bond breakage, there is also a diagnostic ion peak indicating transglycosylation of the 1,6-glycosidic bond. 0.3 Diagnostic ion peaks for the transglycolic ring breakage of A2 and 1,4-glycosidic bonds. 2.5 A2 indicates that the first glycosyl group at the non-reducing end can be linked to the second glycosyl group at the non-reducing end via either a (1→6)-glycosidic bond or a (1→4)-glycosidic bond. This suggests that the non-reducing end of the trisaccharide molecule is either a galactose group linked to a mannosyl group via a (1→6)-glycosidic bond, or a mannosyl group linked to another mannosyl group via a (1→4)-glycosidic bond. Additionally, a transring breakage ion peak is present due to the third sugar ring at the non-reducing end. 0.2 A3, 2.4 A3, 2.5 A3 indicates that the second and third glycosyl groups from the non-reducing end are connected by a -(1→4)-glycosidic bond; a special transcyclic cleavage ion peak was also observed, generated when a sugar ring is simultaneously attached to C4 and C6 of the third glycosyl group from the non-reducing end. 3.5 A3. Considering that the β-(1→4)-main chain of carob gum β-galactomannan contains an α-(1→6)-galactose branched side chain, and the ratio of mannose to galactose is approximately 4:1, it is impossible for two galactose molecules to exist simultaneously in one molecule of trisaccharide. Therefore, it is determined that the product of DP3 contains a mannobiose with an α-(1→6)-galactose branched side chain on the mannose group at the reducing end. Thus, the trisaccharide of DP3 has three isomers: Man1-4Man1-4Man(1), Gal1-6Man1-4Man(2), and Man1-4(Gal1-6)Man(3). Figure 3E is the QTOF-MS / MS spectrum of the P4 tetrasaccharide. The figure shows typical fragment ions B1, C1, B2, C2, B3, and C3, generated from glycosidic bond breakage, in the fragment ion peaks. These are diagnostic trans-glycan ring cleavage ions. 0.3 A2 and 2.5 A2 is present at the second glycosyl group from the non-reducing end, indicating that the first and second glycosyl groups from the non-reducing end are linked either by a (1→6)-glycosidic bond or by a (1→4)-glycosidic bond; transcyclic cleavage ion 0.2 A, 2.4 A, 2.5 A is generated at the third and fourth glycosyl groups from the non-reduced end, confirming that they are (1→4)-linked; in addition, transcyclic cleavage ions... 3.5 Given the presence of A4 and the approximately 4:1 ratio of mannose to galactose in LBG, it's impossible for two galactose molecules to coexist in a single oligosaccharide molecule; at most, only one can exist. Therefore, it can be concluded that the product of DP4 contains a structure with an α-(1→6)-branched galactose group at its reduced terminal mannose group. Consequently, the tetrasaccharide product P4 has three isomers: Man1-4Man1-4Man1-4Man(1), Gal1-6Man1-4Man1-4Man(2), and Man1-4Man1-4(Gal1-6)Man(3). Figure 3 F is the TOF-MS / MS mass spectrum of the P5 pentasaccharide. Among the fragment ion peaks are typical fragment ions B1, C1, B2, C3, B4, and C4, resulting from glycosidic bond breakage, and diagnostic trans-glycan ring cleavage ions. 0.3 A2 and 2.5 A2 is present at the second sugar ring from the non-reducing end, indicating the presence of both (1→6)-glycosidic bonds and (1→4)-glycosidic bonds. This suggests that the non-reducing end of the pentasaccharide molecule is either a galactosyl group linked to a mannose group via an α-(1→6)-glycosidic bond, or a mannose group linked to another mannose group via a β-(1→4)-glycosidic bond; transcyclic cleavage ion 0.2 A, 2.4 A, 2.5 A is generated at the third, fourth, and fifth sugar rings from the non-reduced end, confirming that they are linked by β-1,4 glycosidic bonds. Additionally, the transcyclic cleavage ion at the third sugar ring from the non-reduced end... 3,5 A3 and the fifth sugar ring transcyclic cleavage ion 3.5A5 also exists. Since the ratio of mannose to galactose in LBG is approximately 4:1, it is impossible for two galactose molecules to exist simultaneously in one oligosaccharide molecule. At most, only one galactose group can exist. Therefore, it is suggested that in the mannopentasaccharide molecule, there is an α-(1→6)-branched galactose group on the third sugar group (mannose group) or the fifth sugar group (reduced end manose group) from the non-reducing end. Thus, the pentasaccharide has four isomers, namely Man1-4Man1-4Man1-4Man1-4Man(1), Gal1-6Man1-4Man1-4Man1-4Man(2), Man1-4(Gal1-6)Man1-4Man1-4Man(3), and Man1-4Man1-4Man1-4(Gal1-6)Man(4).

[0102] Based on the structural analysis of the oligosaccharides in the hydrolysis products, the disaccharide structures are Gal1-6Man and Man1-4Man, the trisaccharide structures are Man1-4Man1-4Man, Gal1-6Man1-4Man and Man1-4(Gal1-6)Man, the tetrasaccharide structures are Man1-4Man1-4Man1-4Man, Gal1-6Man1-4Man1-4Man and Man1-4Man1-4(Gal1-6)Man, and the pentasaccharide structure is Man1-4Man1-4M. The genera are an1-4Man1-4Man, Gal1-6Man1-4Man1-4Man1-4Man, Man1-4(Gal1-6)Man1-4Man1-4Man, and Man1-4Man1-4Man1-4(Gal1-6)Man, but the monosaccharide mannose was not detected. This indicates that the number (m) of unbranched mannose units between the two branched galactose units in carob gum β-galactomannan varies, ranging from 1 to 5. Thus, carob gum β-galactomannan has the following fine structure:

[0103]

[0104] Instead of the previously reported arrangement of three unbranched galactose groups of mannose (Dea and Morrison, 1975, Adv. Carbohydr Chem Biochem, 31:242–312.):

[0105]

[0106] This embodiment is the first to discover an endo-β-(1→4)-mannanase (mCcMan5C) capable of endo-hydrolyzing β-(1→4)-mannoside bonds formed by reducing or non-reducing ends of mannosyl groups with α-(1→6)-branched galactosyl groups, as well as internal β-(1→4)-mannoside bonds, on the galactomannan backbone. This produces mannooligosaccharides with α-(1→6)-branched galactosyl groups at reducing or non-reducing ends, as well as mannooligosaccharides without branches or without α-(1→6)-branched galactosyl groups at the ends. Characterizing these various types of mannooligosaccharides with or without α-(1→6)-branched galactosyl groups is beneficial for the application of fine structure identification of β-galactomannan. This embodiment utilizes recombinant mannanase as a glycan analysis tool enzyme applied to the hydrolysis of carob gum β-galactomannan. The oligosaccharide structure of the hydrolysis product is analyzed and identified using high-performance anion chromatography-mass spectrometry (HPLC-MS / MS), leading to the derivation of the fine structure of carob gum β-galactomannan. Previously, there were no reports on using mannanase to hydrolyze β-galactomannan and using HPLC-MS / MS to analyze the oligosaccharide structure and derive the fine structure of β-galactomannan. In this invention, the oligosaccharide structure of the product generated from the hydrolysis of β-galactomannan by mannanase is analyzed using HPLC-MS / MS. Compared with previous methods using NMR or derivative methods combined with GC-MS, this approach has advantages such as requiring less sample and being able to accurately analyze the sequence of glycosyl groups and their glycosidic bonds.

[0107] Example 4

[0108] Mannanase is used in the preparation of mannobiose.

[0109] The composition and content of the 1.2 mL carob gum β-galactomannan enzymatic hydrolysis solution were as follows: 50 mM ammonium acetate (NH4Ac) buffer (pH 7.12) containing a final concentration of 30 μg / mL mature carob gum β-galactomannan enzyme mCcMan5C (prepared in Example 1) and a final concentration of 2.5 mg / mL carob gum β-galactomannan (LBG, purchased from Sigma-Aldrich, USA). The enzymatic hydrolysis solution was incubated at 50 °C and 800 rpm for 24 h, then heated at 100 °C for 15 min to inactivate the enzyme. After cooling to room temperature and centrifugation, the supernatant was filtered through a 0.22 μm filter and used for later use.

[0110] Isolation and purification of the enzymatic hydrolysis product, mannan oligosaccharides. 1.0 mL of the supernatant from the above enzymatic hydrolysis solution was loaded onto a size exclusion column (Superdex) installed on an Agilent 1100 system. TMA 30 XK26 triple column (pre-equilibrated with 0.15M NH4Ac, pH 7.12) was used as the mobile phase for elution at a flow rate of 0.5–2.0 mL / min. Elution was monitored using an Agilent 1100 series G1362 ARID refractive index detector. All eluted fractions were collected stepwise in one tube every 1–5 min to separate and purify the enzymatic hydrolysis product, β-mannooligosaccharide. Figure 4 As shown in Figure A, the molecular size exclusion chromatogram reveals five peaks with large areas and good separation: P1, P2, P3, P4, and P5. Figure 4 B was identified by primary mass spectrometry as the anionized molecules [MH] produced by peaks P1-P5. - [M+2H2O-H] - And [M+HSO4] - P1 was identified as a disaccharide (DP2), P2 as a trisaccharide (DP3), P3 as a tetrasaccharide (DP4), P4 as a pentasaccharide (DP5), and P5 was not a carbohydrate. After purification by size exclusion chromatography, 0.56 mg of disaccharide, 0.24 mg of trisaccharide, 0.14 mg of tetrasaccharide, and 0.24 mg of pentasaccharide were obtained from 5 mg LBG enzymatic hydrolysate, with a disaccharide yield of 11.2%, accounting for approximately 50% of the oligosaccharide content in the hydrolysis products. Figure 4 As shown in Figure C, after acid hydrolysis, purified MOS was analyzed using HPAEC-PAD monosaccharide composition. The molar ratio of mannose to galactose in LBG was 3.98:1. The molar ratios of mannose to galactose in the purified oligosaccharides DP2, DP3, DP4, and DP5 were 40.33:1, 7.17:1, 7.70:1, and 15.23:1, respectively. The manniobiose content in the DP2 disaccharide reached 95.15%. Figure 4 As shown in D, MS / MS mass spectrometry analysis of the P1 disaccharide revealed fragment ions B1 and C1 from glycosidic bond breakage, as well as transcyclic fragment ions from the second sugar ring at the non-reducing end. 0.2 A2, 2.4 A2, 2.5 A2, therefore they are linked by 1,4 glycosidic bonds, forming Man1-4Man.

[0111] The free radical scavenging ability of mannobiose or other hexosiobioses against DPPH was determined. 100 μL of 0.15 mM DPPPH ethanol solution was added to 100 μL of an aqueous solution of mannobiose (DP2 obtained above after purification) or other hexosiobioses at 5 mg / mL or 10 mg / mL. After incubation at 37 °C in the dark for 30 min, the absorbance (Ab) at 517 nm was measured. 100 μL of size exclusion chromatography eluent, thoroughly lyophilized and reconstituted in 50 mM ammonium acetate, was mixed with 100 μL of 0.15 mM DPPH and reacted to serve as a blank control. Other hexosiobioses were dissolved in 50 mM ammonium acetate, thoroughly lyophilized, and then reconstituted in ultrapure water as control samples. Another 100 μL solution of carob gum galactomannan (dLBG) enzymatically hydrolyzed with mCcMan5C was prepared. 100 μL of 50 mM (pH 8.0) Tris buffer containing 5 mg / mL LBG and 15 μg / mL mCcMan5C, or 10 mg / mL LBG and 30 μg / mL mCcMan5C, was added. The reaction was carried out at 37 °C and 800 rpm for 24 h, followed by inactivation at 100 °C and cooling to room temperature. A control of the same concentration of unhydrolyzed carob gum galactomannan (LBG) was used. 100 μL of 0.15 mM DPPH ethanol solution was added to test the effect of enzymatic degradation on the DPPH free radical scavenging ability of carob gum β-galactomannan. The free radical scavenging activity was calculated as the percentage of DPPH inhibition: Inhibition% = (Abscontrol - Abssample) / Abscontrol × 100.

[0112] like Figure 4 As shown in E1, the DPPH free radical scavenging activities of 5 mg / mL and 10 mg / mL LBG hydrolysates (dLBG) were 2.4 times and 2.7 times that of the unhydrolyzed LBG solution, respectively. This indicates that short-chain mannooligosaccharides have higher antioxidant activity compared to long-chain mannans. Furthermore, the mannobiose (DP2) prepared for the first time in this invention was found to have higher and more significant antioxidant activity than DP3, DP4, and DP5 mannooligosaccharides. Specifically, 5 mg / mL mannobiose exhibited 54% DPPH free radical scavenging activity, while 10 mg / mL mannobiose exhibited 73% DPPH free radical scavenging activity, representing 5-7 times the DPPH free radical scavenging activity of galactomannan at the same concentration. Further comparisons of the DPPH free radical scavenging activities of various disaccharides with mannobiose are shown below. Figure 4As shown in E2, the DPPH radical scavenging rates of 5 mg / mL cellobiose, laminabiose, lactose, gentiobiose, and sucrose were only 15.44%, 13.76%, 10.67%, 8.03%, and 6.59%, respectively. In contrast, the DPPH radical scavenging rate of mannobiose was 3.5-8.0 times higher than that of cellobiose, laminabiose, lactose, gentiobiose, and sucrose. This study reveals for the first time that mannobiose exhibits significantly higher DPPH radical scavenging activity than other hexosylbioses, making it suitable for use as a high-quality functional food or dietary prebiotic.

[0113] β-Mannanases can partially hydrolyze the β-(1→4)-glycosidic bonds of mannan to produce mannooligosaccharides through enzymatic, chemical, or physical methods, or a combination of these methods. These mannooligosaccharides can be used as functional foods or dietary prebiotics, exhibiting functions such as regulating gut microbiota and enhancing immunomodulatory activity. They also possess anticancer, antioxidant, anti-inflammatory, antifreeze, anti-stress, and antidiabetic effects. However, the function of purified mannobiose and its antioxidant activity with other hexose disaccharides have not been reported. This example is the first to discover that the main product in the final product of the hydrolysis of carob gum galactomannan by the endo-β-1,4-mannanase of *Coprinus comatus* (CcMan5C) is mannobiose (accounting for approximately 50% of the hydrolyzed oligosaccharide product), which can be applied to the preparation of mannobiose from the hydrolysis of β-galactomannan. Previously reported fungal β-mannanases have not produced a dominant hydrolyzed oligosaccharide from the hydrolysis of galactomannan. Example 1 of this invention proposes a method for obtaining mannobiose by separating and purifying carob gum galactomannan hydrolyzed by recombinant mature mannanase mCcMan5C using size exclusion chromatography. The mannobiose obtained has 5-7 times the DPPH free radical scavenging activity of mannan at the same concentration, and 3.5-8.0 times the DPPH free radical scavenging rate of other cellobiose, laminarin, lactose, gentiobiose, and sucrose. It can be used as a high-quality functional food or dietary prebiotic. sequence list <110> Nanjing Normal University Suzhou University <120> Application of a gray-capped coprinus mannanase <160> 4 <170> SIPOSequenceListing 1.0 <210> 1 <211> 1266 <212> DNA <213> Artificial Sequence <400> 1 gtaggccctt ggggccagtg tggtggtagt ggatggagtg gtgcaacgac ttgcgagagc 60 ggctatactt gccagaagca caacgaatgg tactctcaat gcgtccctgg tacctccagc 120 gcgcctcccc ctcccgtcac tccccagccg tcgaccactg ccgctcctcc cgtcgtgacc 180 ccgccacctc ccacctctgc gactggcttc gtcaagacca acggtacccg cttcgttttg 240 gacggaaagc cgtacactgt ggttggatcc aactcgtact gggtcggtct ttctggacac 300 agcagggaca atatgaaccg ggcctttgct gacattgcag ctgctggagg caccactgtc 360 cgaacctggg gtttcaacga agtcactgcg tatggcggta tcccctatta tcagatttgg 420 aacggaagga cgccgtccgt caacactggc gccaatggtc ttcaaaactt cgaccaggtc 480 atcgccgcgg ccaaggccaa cggcatcaag ctcatcgttg ccctgacgaa caactggtcc 540 gattatggcg gcatggacgt atatgtgaga caaattctca actccaacaa ccacgatttg 600 ttctacacgg acccggacgt caaggcggcc ttcaagaact atattagggc tttcgtcggg 660 agatacgtca atgaaacagg tatccttgga tgggagcttg cgaacgaacc aaggtgtcgt 720 ggaagcaccg gcaccacctc tggcaggtgc acaccggcca cgattacggc ttgggcacgg 780 gaaatgtctg ccttcatcaa gtctattgac cccaaccacc tggtggccct tggagacgaa 840 ggcttctaca accaacccgg tcacccggtc tacccatacc agggcgggga aggtattgac 900 ttcgacgtca accttcaaat cgataccctc gactttggta ctgtccatgc ctatcctgag 960 cactggggtc agcaaggcaa cgaagtgggc ttcggaaacg attggatcaa ggaccacgcc 1020 gaatcccaga agagatacgg caagcccgtc atccttgagg aatacggcgt aaccaccaac 1080 aagccagccg tttacactga atggctcagg accatccaaa cctccggcct tgctggggac 1140 ctctactggc aagcgggatc tcgactccca actggtagca ctcatgacga cggattcact 1200 gtctaccctg accagccagc ctaccagctc ttgcgaagcc acgctgccgc catgaaggct 1260 cgcggg 1266 <210> 2 <211> 422 <212> PRT <213> Artificial Sequence <400> 2 Val Gly Pro Trp Gly Gln Cys Gly Gly Ser Gly Trp Ser Gly Ala Thr 1 5 10 15 Thr Cys Glu Ser Gly Tyr Thr Cys Gln Lys His Asn Glu Trp Tyr Ser 20 25 30 Gln Cys Val Pro Gly Thr Ser Ser Ala Pro Pro Pro Pro Val Thr Pro 35 40 45 Gln Pro Ser Thr Thr Ala Ala Pro Pro Val Val Thr Pro Pro Pro Pro 50 55 60 Thr Ser Ala Thr Gly Phe Val Lys Thr Asn Gly Thr Arg Phe Val Leu 65 70 75 80 Asp Gly Lys Pro Tyr Thr Val Val Gly Ser Asn Ser Tyr Trp Val Gly 85 90 95 Leu Ser Gly His Ser Arg Asp Asn Met Asn Arg Ala Phe Ala Asp Ile 100 105 110 Ala Ala Ala Gly Gly Thr Thr Val Arg Thr Trp Gly Phe Asn Glu Val 115 120 125 Thr Ala Tyr Gly Gly Ile Pro Tyr Tyr Gln Ile Trp Asn Gly Arg Thr 130 135 140 Pro Ser Val Asn Thr Gly Ala Asn Gly Leu Gln Asn Phe Asp Gln Val 145 150 155 160 Ile Ala Ala Ala Lys Ala Asn Gly Ile Lys Leu Ile Val Ala Leu Thr 165 170 175 Asn Asn Trp Ser Asp Tyr Gly Gly Met Asp Val Tyr Val Arg Gln Ile 180 185 190 Leu Asn Ser Asn Asn His Asp Leu Phe Tyr Thr Asp Pro Asp Val Lys 195 200 205 Ala Ala Phe Lys Asn Tyr Ile Arg Ala Phe Val Gly Arg Tyr Val Asn 210 215 220 Glu Thr Gly Ile Leu Gly Trp Glu Leu Ala Asn Glu Pro Arg Cys Arg 225 230 235 240 Gly Ser Thr Gly Thr Thr Ser Gly Arg Cys Thr Pro Ala Thr Ile Thr 245 250 255 Ala Trp Ala Arg Glu Met Ser Ala Phe Ile Lys Ser Ile Asp Pro Asn 260 265 270 His Leu Val Ala Leu Gly Asp Glu Gly Phe Tyr Asn Gln Pro Gly His 275 280 285 Pro Val Tyr Pro Tyr Gln Gly Gly Glu Gly Ile Asp Phe Asp Val Asn 290 295 300 Leu Gln Ile Asp Thr Leu Asp Phe Gly Thr Val His Ala Tyr Pro Glu 305 310 315 320 His Trp Gly Gln Gln Gly Asn Glu Val Gly Phe Gly Asn Asp Trp Ile 325 330 335 Lys Asp His Ala Glu Ser Gln Lys Arg Tyr Gly Lys Pro Val Ile Leu 340 345 350 Glu Glu Tyr Gly Val Thr Thr Asn Lys Pro Ala Val Tyr Thr Glu Trp 355 360 365 Leu Arg Thr Ile Gln Thr Ser Gly Leu Ala Gly Asp Leu Tyr Trp Gln 370 375 380 Ala Gly Ser Arg Leu Pro Thr Gly Ser Thr His Asp Asp Gly Phe Thr 385 390 395 400 Val Tyr Pro Asp Gln Pro Ala Tyr Gln Leu Leu Arg Ser His Ala Ala 405 410 415 Ala Met Lys Ala Arg Gly 420 <210> 3 <211> 40 <212> DNA <213> Artificial Sequence <400> 3 agagaggctg aagctgaatt cgtaggccct tggggccagt 40 <210> 4 <211> 40 <212> DNA <213> Artificial Sequence <400> 4 tgttctagaa agctggcggc cgccccgcga gccttcatgg 40

Claims

1. Gray-covered parasol tree ( Coprinopsis cinerea The application of mannanase in the fine structure identification of carob gum β-galactomannan and / or in the preparation of mannobiose from carob gum β-galactomannan hydrolysis; the mannanase is *Coprinus comatus* mannanase CcMan5C, and the nucleotide sequence of the cDNA of *Coprinus comatus* mannanase CcMan5C is shown in SEQ ID NO.1; the fine structure identification of carob gum β-galactomannan involves applying *Coprinus comatus* mannanase as a glycan analysis tool to the hydrolysis of carob gum β-galactomannan, and combining high-performance anion chromatography-mass spectrometry to analyze and identify the oligosaccharide structure of the hydrolysis product. It was found that the number *m* of unbranched galactosyl groups between two branched galactosyl groups in carob gum β-galactomannan varies, ranging from 1 to 5. The fine structure of carob gum β-galactomannan is as follows: ，m=1-5。 2. Use according to claim 1, characterized in that, The mannanase is Coprinopsis cinerea mannanase CcMan5C, which can hydrolyze the β-(1→4)-mannosidic bond formed by the adjacent mannose hydroxyl group of the β-galactomannan backbone with the α-(1→6)-branch galactosyl group of the reducing end C1 hemiacetal hydroxyl or the non-reducing end C4 hydroxyl.

3. Use according to claim 1, characterized in that, The main product of the Coprinopsis cinerea mannanase hydrolysis of the β-galactomannan of locust bean gum is mannobiose, which is obtained by molecular exclusion chromatography separation and purification.

4. Use according to claim 1, characterized in that, The Coprinopsis cinerea mannanase is obtained by integrating a cDNA fragment of a mature mannanase mCcMan5C into an expression plasmid, introducing into Pichia pastoris, and then fermenting to obtain a recombinant expression Pichia pastoris strain expressing the mature mannanase mCcMan5C; the cDNA fragment of the gene encoding the mature mannanase mCcMan5C is synthesized by RT-PCR using mRNA extracted from Coprinopsis cinerea mycelium as a template, or directly synthesized artificially according to the cDNA sequence of the encoding gene.

5. Use according to claim 4, characterized in that, The Pichia pastoris is Pichia pastoris GS115.

6. Use according to claim 4, characterized in that, The construction method of the recombinant expression Pichia pastoris of the Coprinopsis cinerea mannanase comprises the following steps: (1) grinding the mycelium or fruiting body of Coprinopsis cinerea grown on solid or liquid medium into powder after freezing to extract and prepare a total RNA sample, and reverse transcribing mRNA into cDNA; (2) designing primers, using the cDNA synthesized in step (1) as a template to obtain a mature CcMan5C cDNA fragment by PCR amplification, and mixing and connecting the mCcMan5C cDNA fragment product and the plasmid pPICZαA after enzyme digestion, and then transforming into a competent cell to extract a plasmid to obtain a recombinant expression plasmid pPICZαA-mCcMan5C; (3) linearizing the recombinant expression plasmid pPICZαA-mCcMan5C obtained above and then electrotransferring into Pichia pastoris cells to obtain Pichia pastoris containing the plasmid pPICZαA-mCcMan5C, which is the recombinant expression Pichia pastoris expressing the mature mannanase mCcMan5C.

7. Use according to claim 6, characterised in that, The primers in step (2) are an upstream primer 5'-AGAGAGGCTGAAGCTGAATTCGTAGGCCCTTGGGGCCAGT-3' with an EcoR I enzyme digestion site and a downstream primer 5'-TGTTCTAGAAAGCTGGCGGCCGCCCCGCGAGCCTTCATGG-3' with a Not I enzyme digestion site.

8. The use according to claim 4, wherein the preparation of the mannanase comprises the following steps: (1) inoculating the recombinant expression Pichia pastoris expressing the mature mannanase mCcMan5C into a culture medium, culturing overnight, collecting the mycelium by centrifugation, suspending the mycelium, inoculating into a culture medium, and expanding the culture for inducing expression, while sampling to determine the enzyme activity of the supernatant of the culture medium, and terminating the culture when the enzyme activity no longer increases; (2) The supernatant of the centrifuged recombinant bacteria fermentation broth is collected, column chromatography is performed, the active recombinant protein component is collected, dialysis is performed overnight, and the purified mannanase is obtained.

9. The fine structure of the carob gum β-galactomannan of claim 1 is: ，m=1-5。

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