Mannase mutant as well as coding gene and application thereof

Abstract

The invention discloses a mannase mutant as well as a coding gene and application thereof. The invention provides a mannase mutant which is any one of the following proteins: A1) the amino acid sequence of the protein is obtained by mutating the 38th Gln of the amino acid sequence of wild type mannase as shown in a sequence 3 into Lys, mutating the 224th Asp into Ala and mutating the 272th Ala into Thr, and the amino acid sequence of the protein is obtained by mutating the 38th Gln of the amino acid sequence of wild type mannase as shown in the sequence 3 into Lys, mutating the 224th Asp of the amino acid sequence of wild type mannase as shown in the sequence 3 into Thr; a2) a fusion protein obtained by connecting the N end or / and C end of the protein shown in A1) with a protein tag. The mannase mutant provided by the invention is derived from aspergillus niger, has good enzymatic properties and hydrolysis characteristics, can efficiently hydrolyze guar bean slices under a natural pH value condition to generate mannan oligosaccharide, and has great application potential in the field of food.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and relates to a mannanase mutant, a coding gene thereof and application. BACKGROUND

[0002] Mannans can be divided into four categories according to the different monomers and linkage modes, namely linear mannans, galactomannans, glucomannans and galactoglucomannans (Malgas et al. World Journal of Microbiology and Biotechnology, 2015, 31: 1167-1175). The main chains of these mannans are composed of mannose (and glucose) through 1,4-glycosidic bonds, and also contain galactose residue side chains connected by α-1,6-glycosidic bonds. These mannans, as structural polysaccharides and energy storage polysaccharides, are widely present in plant tissue cell walls and endosperms of dicotyledonous plants, such as palm cake, coffee grounds and other agricultural wastes (linear mannans), guar beans and other seed endosperms (galactomannans), and konjac tubers (glucomannans). Mannans have complex structures and require the synergistic hydrolysis of multiple glycoside hydrolases for complete degradation, such as mannanase (EC 3.2.1.78), mannosidase (EC 3.2.1.25), glucosidase (EC 3.2.1.21), α-galactosidase (EC 3.2.1.23) and mannan acetyl esterase (EC 3.1.1.6) (Zhou et al. Bioresource Technology, 2024, 395: 130373).

[0003] As the most important hydrolytic enzyme in the mannanase system, mannanase can randomly degrade the 1,4-glycosidic bonds in the main chain of mannans to produce low molecular weight mannooligosaccharides of different polymerization degrees (Basmak et al. International Journal of Biological Macromolecules, 2024, 261: 129798). According to the similarity of their amino acid sequences, mannanases belong to glycoside hydrolase families 5, 26, 113 and 134. At present, many mannanases have been cloned and expressed, but the existing mannanases have certain defects (poor temperature stability, low expression, etc.), which limit their application in actual production. Therefore, it is of great significance to develop mannanases with better enzymatic properties through molecular modification for their application in the food, feed and other industries.

[0004] Guar gum is a natural thickening agent and potential dietary fiber supplement rich in mannan and galactose. Guar gum is often used as an emulsifying stabilizer and thickening agent in various foods. In the food industry, mannanase can be used to degrade guar gum into partially hydrolyzed guar gum or mannan oligosaccharide with functionality and low viscosity. However, industrial production of guar gum requires high temperature and multiple crushing processes, which is costly. The complicated process and high cost limit the wide application of partially hydrolyzed guar gum and guar oligosaccharide. Therefore, developing guar oligosaccharide from guar gum pieces can reduce production costs. It is of great significance to explore mannanase with high catalytic activity for guar gum pieces for the food industry.

[0005] Chinese invention patent ZL201510174562.2 discloses a method for preparing oligomannogalactose by using mannanase, xylanase and endoglucanase to hydrolyze guar gum. Chinese invention patent ZL201610808818.5 discloses a method for preparing soluble dietary fiber and mannan oligosaccharide by using mannanase and galactosidase to hydrolyze guar gum. Chinese invention patent CN201910933670.1 discloses a method for producing galactose, mannose and mannan oligosaccharide by using mannanase and galactosidase to hydrolyze guar gum. The above methods all use guar gum as raw material and need multiple enzymes for complex production steps. At present, there is no literature and patent report on the preparation of guar gum mannan oligosaccharide from guar gum pieces. SUMMARY

[0006] The technical problem solved by the present application is how to efficiently use guar gum pieces to prepare guar gum mannan oligosaccharide.

[0007] To solve the above technical problem, the first aspect of the present application provides a mannanase mutant, which is any one of the following proteins:

[0008] A1) the amino acid sequence of the protein is the amino acid sequence of the wild-type mannanase shown in SEQ ID NO: 3, wherein the 38th Gln is mutated to Lys, the 224th Asp is mutated to Ala, and the 272nd Ala is mutated to Thr, to obtain;

[0009] A2) a fusion protein obtained by connecting a protein tag to the N-terminus or / and C-terminus of the protein shown in A1).

[0010] In the above mutant, the amino acid sequence of the protein shown in A1) is SEQ ID NO: 1.

[0011] The second aspect of the present application provides a nucleic acid molecule encoding the mannanase mutant of the first aspect.

[0012] The nucleic acid molecule described above is a DNA molecule described in B1) or B2) or B3) below:

[0013] B1) the nucleotide sequence of the DNA molecule is sequence 2;

[0014] B2) a DNA molecule having 85% or more identity to the nucleotide sequence defined in B1) and encoding the mannanase mutant of the first aspect;

[0015] B3) a DNA molecule hybridizing to the nucleotide sequence defined in B1) under stringent conditions and encoding the mannanase mutant of the first aspect.

[0016] In a third aspect, the present application provides a biological material containing the nucleic acid molecule of the second aspect, which is any one of the following:

[0017] C1) an expression cassette containing the nucleic acid molecule of the second aspect;

[0018] C2) a recombinant vector containing the nucleic acid molecule of the second aspect, or a recombinant vector containing the expression cassette of C1);

[0019] C3) a recombinant microorganism containing the nucleic acid molecule of the second aspect, or a recombinant microorganism containing the expression cassette of C1), or a recombinant microorganism containing the recombinant vector of C2).

[0020] In the above, the recombinant microorganism is a recombinant Escherichia coli or a recombinant Aspergillus niger.

[0021] In an embodiment of the present application, the recombinant Escherichia coli is obtained by introducing a recombinant plasmid into Escherichia coli, wherein the recombinant plasmid is a plasmid obtained by replacing the fragment between the BamHI and XhoI enzyme cutting sites of the pET28a vector with the mannanase mutant gene shown in sequence 2, and the plasmid expresses the mannanase mutant shown in sequence 1.

[0022] The recombinant Aspergillus niger is obtained by transforming a recombinant plasmid into Aspergillus niger to obtain a recombinant Aspergillus niger containing the recombinant plasmid; wherein the recombinant plasmid is a plasmid obtained by inserting the mannanase mutant gene shown in sequence 2 into the Aspergillus niger vector pEASY-Blunt.

[0023] In a fourth aspect, the present application provides the use of the mutant of the first aspect or the nucleic acid molecule of the second aspect or the biological material of the third aspect in any one of the following:

[0024] D1) preparing a mannanase;

[0025] D2) preparing a product containing mannanase activity;

[0026] D3) preparing a manno-oligosaccharide;

[0027] D4) hydrolyzing guar splits or guar gum to obtain mannan oligosaccharides.

[0028] The product containing mannanase activity can be a mannanase enzyme preparation.

[0029] The optimal pH of the mannanase is 6.5, the enzyme is stable between pH 5.0-8.0, the optimal temperature is 45℃, and the enzyme retains more than 80% activity below 55℃.

[0030] In the fifth aspect, the present application provides a method for preparing mannanase, comprising the following steps: fermenting the recombinant microorganism of the third aspect, and collecting the fermentation product to obtain mannanase.

[0031] In the embodiment of the present application, the recombinant microorganism is exemplified as a recombinant Aspergillus niger, and in the method, after collecting the fermentation product, the following step is further included: taking the supernatant of the fermentation product to obtain mannanase.

[0032] In the sixth aspect, the present application provides a method for preparing mannan oligosaccharides, comprising the following steps: using guar splits or guar gum as the substrate, and using the mutant of the first aspect or the fermentation product of the fifth aspect as the enzyme to perform hydrolysis to obtain mannan oligosaccharides.

[0033] In the embodiment of the present application, the enzyme is exemplified as the supernatant of the fermentation product of the recombinant Aspergillus niger.

[0034] In the embodiment of the present application, the enzyme is exemplified as the supernatant of the fermentation product of the recombinant Aspergillus niger.

[0035] In the embodiment of the present application, the enzyme is exemplified as the supernatant of the fermentation product of the recombinant Aspergillus niger.

[0036] In the embodiment of the present application, the enzyme is exemplified as the supernatant of the fermentation product of the recombinant Aspergillus niger.

[0037] In the embodiment of the present application, the enzyme is exemplified as the supernatant of the fermentation product of the recombinant Aspergillus niger.

[0038] In the embodiment of the present application, the enzyme is exemplified as the supernatant of the fermentation product of the recombinant Aspergillus niger.

[0039] The application provides a mannanase mutant, which is derived from Aspergillus niger, is molecularly modified, and is highly expressed in Aspergillus niger, and has good enzymatic properties and hydrolysis properties. BRIEF DESCRIPTION OF DRAWINGS

[0040] Figure 1 It is a purification electrophoretogram of the mannanase mutant.

[0041] Figure 2 It is a curve graph for measuring the optimum pH of the mannanase mutant, wherein (■) represents a citric acid buffer (pH 3.0-6.0), (●) represents a MOPS buffer (pH 6.0-8.0), (◆) represents a CHES buffer (pH 8.0-9.5), and (▲) represents a CAPS buffer (pH 9.5-11.0).

[0042] Figure 3 It is a curve graph for measuring the pH stability of the mannanase mutant, wherein (■) represents a citric acid buffer (pH 3.0-6.0), (●) represents a MOPS buffer (pH 6.0-8.0), (◆) represents a CHES buffer (pH 8.0-9.5), and (▲) represents a CAPS buffer (pH 9.5-11.0).

[0043] Figure 4 It is a curve graph for measuring the optimum temperature of the mannanase mutant.

[0044] Figure 5 It is a curve graph for measuring the temperature stability of the mannanase mutant.

[0045] Figure 6 It is a hydrolysis property graph of the mannanase mutant, wherein M1 represents mannose, M2 represents mannobiose, M3 represents mannotriose, M4 represents mannotetraose, M5 represents mannotetraose, and M6 represents mannohexaose.

[0046] Figure 7Process map of high-density fermentation of Aspergillus niger for secreting mannanase mutants.

[0047] Figure 8 High-performance anion exchange chromatogram of mannan oligosaccharides prepared by hydrolysis of guar gum flake with mannanase mutants. DETAILED DESCRIPTION

[0048] The experimental methods used in the following examples are conventional methods unless otherwise specified.

[0049] The materials, reagents, etc. used in the following examples can be obtained commercially unless otherwise specified.

[0050] The method for determining the activity of mannanase in the following examples is as follows:

[0051] Take 0.1 mL of appropriately diluted enzyme solution and add it to 0.9 mL of 0.5% (w / v) locust bean gum substrate solution (prepared with 50 mM MOPS buffer, pH 6.5), and react in a 45°C water bath for 10 min. The amount of reducing sugar released is determined by the 3,5-dinitrosalicylic acid (DNS) method, using mannose as the standard.

[0052] 1 mannanase enzyme activity unit is defined as the amount of enzyme required to decompose 0.5% locust bean gum substrate to release 1 μmol of mannose per minute at pH 6.5 and 45°C. The enzyme activity calculation formula is: H = Cx x n / (T x V), where H represents enzyme activity (U / mL), Cx represents the amount of mannose substance generated (μmol), n represents the dilution factor of the enzyme solution, T represents the reaction time (min), and V represents the volume of the diluted enzyme solution added (mL).

[0053] Specific enzyme activity is defined as the enzyme activity unit possessed by 1 mg of protein, expressed as U / mg.

[0054] The amino acid sequence of the wild-type mannanase in the following examples is SEQ ID NO: 3.

[0055] The mass / volume ratio or w / v in the following examples is g:L unless otherwise specified.

[0056] Example 1, Obtaining of mannanase mutant gene

[0057] 1. Error-prone PCR

[0058] According to the recombinant plasmid of Aspergillus niger mannanase (pPIC9K-AnMan26) (Wang et al. Process Biochemistry, 2021, 100: 90-97), a primer pair for error-prone PCR without signal peptide was designed. The primer sequences are as follows:

[0059] Upstream primer:

[0060] 5'-CGCGGATCCGAATTCGCTTCCAACCAGACTCTGTCC-3';

[0061] Downstream primer:

[0062] 5'-GCTTGTCGACGGAGCTCTTAAGCCCCCTCCCAGTTCA-3'.

[0063] The error-prone PCR system (50 μL) is as follows: 7 mmol / L Mg2+, 0.3 mmol / L Mn2+, 0.2 mmol / L dATP, 0.2 mmol / L dGTP, 1 mmol / L dCTP, 1 mmol / L dTTP, 0.2 μmol AnMan5AR, 2.5 U Tap polymerase, 20 ng recombinant plasmid, and the total volume is 50 μL.

[0064] The error-prone PCR reaction conditions are as follows: pre-denaturation (98°C, 5 min), denaturation (98°C, 30 s), annealing (55°C, 30 s), extension (72°C, 2 min) for a total of 34 cycles, and total extension (72°C, 10 min).

[0065] A random mutant library of Aspergillus niger mannanase is obtained.

[0066] 2. Mutant expression library

[0067] The random mutant library and the pET28a vector are double-digested by restriction endonucleases BamHI and Xhol, and the digested products are recovered. The digested skeletons are ligated, and the ligation product is transformed into E. coli BL21 (DE3) by using the electroporation method. All the transformants are collected, and the mutant expression library is obtained.

[0068] 3. Directional screening of the mutant expression library

[0069] After the mutant expression library is appropriately diluted, it is uniformly coated on LB plates (containing 50 μg / mL kanamycin) and cultured at 37°C for 24 h. Single colonies are collected, numbered, and transferred to screening plates (LB plates containing 0.5% locust bean gum, 1 mM IPTG, and 50 μg / mL kanamycin) and cultured at 37°C for 1 day. After the culture, the screening plates are placed at 70°C for 1 h to lyse the bacterial cells, and then dyed with 0.1% Congo red solution for 15 min and decolorized with 1 M NaCl solution. The corresponding colonies are numbered according to the transparent circles, and the corresponding colonies are picked from the original plates for re-screening.

[0070] 4. Re-screening of the mutant expression library

[0071] The fermentation broth of each strain capable of producing transparent zone was centrifuged at 10000 x g for 5 min, the bacterial cells were resuspended with distilled water, and the bacterial cells were broken by ultrasonic treatment and centrifuged at 10000 x g for 10 min to obtain the supernatant (i.e. crude enzyme solution). The optimal pH of the crude enzyme solution of each mutant strain was determined at different temperatures, and the mutant with changed optimal pH was a positive mutant. After re-screening, one positive mutant was obtained.

[0072] 5. Sequence analysis of the positive mutant

[0073] The plasmid carrying the mutant gene of mannanase was sequenced, and the gene encoded the mutant of mannanase.

[0074] Compared with the wild-type gene of mannanase, the mutant gene of mannanase had 7 nucleotide mutations, i.e. C→A (the 112th nucleotide from the 5' end of SEQ ID NO: 2), C→A (the 664th nucleotide from the 5' end of SEQ ID NO: 2), and G→A (the 814th nucleotide from the 5' end of SEQ ID NO: 2). The above nucleotide mutations caused the mutation of three amino acid residues of the wild-type protein of mannanase, i.e. Gln→Lys (the 38th amino acid residue from the N-terminus of SEQ ID NO: 1), Asp→Ala (the 224th amino acid residue from the N-terminus of SEQ ID NO: 1), and Ala→Thr (the 272th amino acid residue from the N-terminus of SEQ ID NO: 1), thereby obtaining the mutant of mannanase.

[0075] The nucleotide sequence of the mutant gene of mannanase is SEQ ID NO: 2, and the gene can also be obtained by artificial synthesis.

[0076] The amino acid sequence of the mutant of mannanase is SEQ ID NO: 1.

[0077] The recombinant plasmid carrying the mutant gene of mannanase is a plasmid obtained by replacing the fragment between the BamHI and XhoI enzyme sites of the pET28a vector with the mutant gene of mannanase shown in SEQ ID NO: 2, and the plasmid expresses the mutant of mannanase shown in SEQ ID NO: 1.

[0078] The recombinant plasmid carrying the mutant gene of mannanase was transformed into E. coli BL21 (DE3) to obtain a recombinant E. coli, and the recombinant E. coli was detected to contain the recombinant plasmid carrying the mutant gene of mannanase.

[0079] Example 2, preparation of the mutant of mannanase and determination of its enzymatic properties

[0080] I. Preparation of the mutant of mannanase

[0081] 1. Induced expression of the mutant of mannanase

[0082] The recombinant Escherichia coli constructed in Example 1 was inoculated into liquid LB medium (containing 50 μg / mL kanamycin) and cultured with shaking at 37°C until the OD600 reached between 0.6 and 0.8. IPTG (final concentration 1 mM) was added, and the cells were induced and cultured overnight at 30°C. The cells were collected at 10,000 × g, resuspended in buffer A (20 mM disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, pH 8.0, 300 mM NaCl, 20 mM imidazole), and then sonicated. The cells were centrifuged at 10,000 × g for 10 min, and the supernatant was collected as the crude enzyme solution.

[0083] 2. Purification of mannanase mutant

[0084] The mannanase mutant was purified using an agarose Ni-IDA affinity column. The specific steps are as follows:

[0085] Equilibrate the Ni-IDA column to 5-10 column volumes with buffer A. Load the crude enzyme solution at a flow rate of 0.5 mL / min. Elute with buffer A and buffer B (20 mM disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, pH 8.0, 300 mM NaCl, 50 mM imidazole) at a flow rate of 1 mL / min until OD280 < 0.05. Finally, elute with buffer C (20 mM disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, pH 8.0, 300 mM NaCl, 200 mM imidazole) and collect the target protein to obtain the purified product. This purified product is a mannanase mutant solution (solvent: buffer C, enzyme activity: 3000 U / mL).

[0086] The purified product was subjected to SDS-PAGE electrophoresis.

[0087] The results are as follows Figure 1 As shown, lane M is the molecular weight standard, lane 1 is the crude enzyme solution of the recombinant bacteria, and lane 2 is the purified product of the crude enzyme solution of the recombinant bacteria. The results show that the target product with a size of 40 kDa was obtained, which is consistent with the expected size. This purified product is the mannanase mutant solution.

[0088] II. Determination of the enzymatic properties of mannanase mutants

[0089] 1. Determination of the optimal pH for mannanase mutants

[0090] The purified product (mannanase mutant solution) obtained above was used as the test enzyme solution, and its mannanase activity was measured under different buffer conditions according to the aforementioned method. The various buffer solutions are as follows:

[0091] 1) 50mM citrate buffer (citric acid-trisodium citrate) (pH 3.0-6.0);

[0092] 2) 50mM MOPS buffer (Beijing Biodi Biotech Co., Ltd., BN30670) (pH 6.0-8.0);

[0093] 3) 50mM CHES buffer (Beijing Biodi Biotech Co., Ltd., BN30392) (pH 8.0-9.5);

[0094] 4) 50mM CAPS buffer (Beijing Biodi Biotech Co., Ltd., DE4020) (pH 9.5-11.0).

[0095] The reaction temperature during the determination was 45℃, and the corresponding enzyme activity of the mannanase mutant at the pH with the highest enzyme activity was taken as 100%, and the relative enzyme activity at other pHs was calculated.

[0096] The results are shown in Figure 2 As can be seen from the figure, the optimal pH of the mannanase mutant is 6.5, and the buffer used is 50mM MOPS buffer.

[0097] 2. Determination of pH stability of the mannanase mutant

[0098] The purified product (mannotriose solution) obtained above was diluted with the above-mentioned buffers 1)-4), treated in a 30℃ water bath for 30min, quickly cooled in ice water for 30min, and then the enzyme activity was determined according to the foregoing method. The relative enzyme activity of the mannanase mutant after treatment at different pHs was calculated based on the untreated mannanase mutant as 100%. The enzyme activity determination conditions were: 50mmol / L MOPS buffer (pH 6.5), reaction temperature 45℃.

[0099] The results are shown in Figure 3 As can be seen from the figure, the mannanase mutant has good pH stability and remains stable between pH 5.0-8.0.

[0100] 3. Determination of optimal temperature of the mannanase mutant

[0101] The purified product (mannotriose solution) obtained above was diluted with 50mM MOPS buffer with pH 6.5, and the enzyme activity was determined according to the foregoing method at different temperatures (30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80℃). The relative enzyme activity at other pHs was calculated based on the enzyme activity at the optimal temperature as 100%.

[0102] The results are shown in Figure 4 As can be seen from the figure, the optimal temperature of the mannanase mutant is 45℃.

[0103] 4. Temperature stability assay of the mannanase mutant

[0104] The purified product (mannotase mutant solution) obtained above was diluted with 50 mM MOPS buffer at pH 6.5 as appropriate, and incubated at different temperatures (30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80 °C) for 30 min, and then rapidly cooled in ice water for 30 min. The activity was then determined according to the method described above. The relative enzyme activity of the mannanase mutant after treatment at different temperatures was calculated based on the untreated mannanase mutant as 100%. The enzyme activity was determined under the following conditions: 50 mmol / L MOPS buffer (pH 6.5), reaction temperature 45 °C.

[0105] The results are shown in Table 1. Figure 5 As can be seen from Table 1, the mannanase mutant has good temperature stability, retaining more than 80% of the enzyme activity below 55 °C.

[0106] 5. Hydrolysis characteristics of the mannanase mutant

[0107] Guar gum (1%, w / v) was dissolved in 50 mM MOPS acid buffer (pH 6.5), and the purified product (mannotase mutant solution) obtained above was added to give a final concentration of 5 U / mL of the mutant, and then hydrolyzed at 40 °C for 8 h. Samples were taken at intervals, and all samples were inactivated in a boiling water bath for 10 min. All samples were subjected to thin layer chromatography analysis. The results are shown in Table 2. Sample 2 μL, developing agent is n-butanol: acetic acid: water (2:1:1), color developing agent is methanol: sulfuric acid (95:5). Figure 6

[0108] The results of thin layer chromatography are shown in Table 2. Amount is water, percentage mass volume ratio); after fermentation 96 h, feeding amount is changed to 100 mL, As can be seen from Table 2, the mannanase mutant mainly produces mannooligosaccharides with a degree of polymerization of 2-6 and a series of mannooligosaccharides with a degree of polymerization greater than 6 when hydrolyzing guar gum.

[0109] Example 3. Expression of the mannanase mutant by high-density fermentation of Aspergillus niger

[0110] 1. Construction of the recombinant bacteria

[0111] The mannanase mutant gene obtained in Example 1 was used as a template, and the following primer pair was used for PCR amplification to obtain the PCR amplification product. The primer sequences are as follows:

[0112] Upstream primer:

[0113] 5'-GTCTGCACAGGGTTGGCAATGTTCGCCAAATTGTCCCTC-3';

[0114] Downstream primer:

[0115] 5'-TTGATGATTTCAGTAACGTTAAGTTTAAGCCCCCTCCCAGTTCA-3'.

[0116] The gene fragment was connected with Aspergillus niger vector pEASY-Blunt (Wang et al., Foods. 2021, 10, 2949) by seamless cloning to obtain a recombinant plasmid.

[0117] The recombinant plasmid was obtained by inserting the mannanase mutant gene shown in SEQ ID NO: 2 into Aspergillus niger vector pEASY-Blunt.

[0118] The recombinant plasmid was linearized by double enzyme digestion with NotI and XhoI, and the digested product was recovered by pre-cooled ethanol. The product was transformed into prepared Aspergillus niger protoplast cells (Wang et al., Foods. 2021, 10, 2949), and cultured in CD solid medium (34.22% sucrose, 0.3% NaNO3, 0.2% KCl, 0.1% KH2PO4, 0.05% MgSO4·7H2O, 0.001% FeSO4·7H2O, pH 5.5, 2% agar powder, mass percentage) at 34°C for 5-7 days to obtain recombinant Aspergillus niger containing the recombinant plasmid.

[0119] 2. High-density fermentation

[0120] The recombinant Aspergillus niger single colony was inoculated into 10 mL DPY medium (2% glucose, 1% tryptone, 0.5% yeast extract powder, 0.5% KH2PO4, 0.05% MgSO4·7H2O, the rest was water, mass percentage) and cultured for 72 h as a primary seed liquid. The primary seed liquid was inoculated into 300 mL DPY medium at a 5% (v / v) inoculation amount, and cultured under the same conditions for 60 h as a secondary seed liquid.

[0121] The whole high-density fermentation was carried out in a 5L fermenter (fermentation medium 2.7L, formula: 4% defatted soybean powder, 3% corn syrup, 5% corn starch hydrolysate (5% corn starch was hydrolyzed by high-temperature α-amylase (from leaf, S10005) at 25℃ for 12h, and the obtained product), 2% glucose, 1% ammonium sulfate, 0.5% CaCl2, 0.3% KH2PO4, and the rest was water, pH 5.5, mass percentage volume ratio) in a fed-batch fermentation mode. The fermenter and the feed were sterilized at 121℃ for 21min. The fermenter was taken out and cooled at room temperature for 2-3h. The fermenter was kept at 30℃, with aeration amount of 2vvm, pH 5.5, and stirring at 300rpm. The secondary seed liquid was inoculated into the fermenter for 36h, and this time was set as the 0h of the formal fermentation. From the 36th hour of fermentation, samples were taken at 24h intervals to determine the enzyme activity and protein concentration. After 48h of fermentation, 200mL of feed (30% corn starch hydrolysate, 1% corn syrup, 0.1% KH2PO4, and the rest was water) was added at 24h intervals. Fermentation length is 168 h in total. Figure 7 The dissolved oxygen value of the fermenter was monitored during the fermentation, and ammonia water was automatically added to maintain pH 5.5.

[0122] 3. Fermentation results

[0123] The fermentation product obtained in the above 2 was centrifuged at 10000xg for 15min, and the fermentation supernatant was collected.

[0124] The enzyme activity of the mannanase mutant in the fermentation supernatant was detected. The enzyme activity determination conditions were: 50mM MOPS buffer (pH 6.5), reaction temperature 45℃.

[0125] During the fermentation, the growth of the recombinant Aspergillus niger and the secretion of the mannanase mutant were as shown in the table below: Enzyme amount (U / g guar gum slice) The results showed that after 168h of fermentation, the extracellular mannanase activity of the recombinant Aspergillus niger was 1800U / mL, and the protein concentration was 3.44mg / mL.

[0126] Example 4, Application of the mannanase mutant in the preparation of guar manno-oligosaccharides

[0127] 1. Hydrolysis of different amounts of guar gum pieces by the mannanase mutant

[0128] Take 1, 5, 10, 20, 30, 40 g guar gum pieces into 100 mL distilled water (pH value is natural pH, generally about 6.5; distilled water can also be replaced by MOPS buffer with pH 6.5), according to the ratio of 2000 U / g of guar gum pieces to add the fermentation supernatant prepared in Example 3, and place it in 40℃ for 8h hydrolysis, enzyme hydrolysis in boiling water bath inactivation for 10 min, to obtain the enzyme hydrolysate. The obtained enzyme hydrolysate was centrifuged at 10000 rpm for 10 min, and the supernatant was collected, which was the crude sugar solution. The content of reducing sugar in the crude sugar solution was determined by 3, 5-dinitrosalicylic acid method, and the yield of reducing sugar was calculated. The precipitate was washed with pure water for two times and dried, and the hydrolysis rate of guar gum pieces was calculated. The hydrolysis rate and the yield of reducing sugar were calculated according to the following formula:

[0129] Hydrolysis rate = (raw material dry weight - hydrolysis residue dry weight) / raw material dry weight x 100%;

[0130] Reducing sugar yield = (crude sugar solution reducing sugar concentration x volume) / raw material dry weight x 100%.

[0131] The hydrolysis rate and the yield of reducing sugar after the mannase mutant hydrolyzed different amounts of guar gum pieces are shown in Table 1.

[0132] Table 1 is the hydrolysis rate and the yield of reducing sugar after the mannase mutant hydrolyzed different amounts of guar gum pieces

[0133]

[0134] As can be seen from Table 1, with the gradual increase of the amount of guar gum pieces, the hydrolysis rate and the yield of reducing sugar of the guar gum pieces gradually decrease. When the amount of guar gum pieces is 10% (w / v), the hydrolysis rate of the guar gum pieces is 76.1%, and the yield of reducing sugar is 56.2%; when the amount of guar gum pieces is 20% (w / v), the hydrolysis rate of the guar gum pieces is 73.5%, and the yield of reducing sugar is 56.2%.

[0135] 2, Mannase mutant hydrolyzes guar gum pieces with different amounts

[0136] Take 100 mL distilled water (pH value is natural pH; distilled water can also be replaced by MOPS buffer solution with pH 6.5), add 10 g guar gum slices and stir well, add the fermentation supernatant prepared in Example 3 according to the ratio of 250 U / g, 500 U / g, 1000 U / g, 2000 U / g, 3000 U / g of guar gum slices respectively, and hydrolyze at 40°C for 8h. After enzyme hydrolysis, inactivate in boiling water bath for 20 min to obtain the enzyme hydrolysate. After centrifugation at 10000 rpm for 10 min, the supernatant, i.e. the crude sugar solution, is collected. The content of reducing sugar in the crude sugar solution is determined by 3,5-dinitrosalicylic acid method and the yield of reducing sugar is calculated. The precipitate is washed with pure water for two times and then dried to weigh, and the hydrolysis rate of guar gum slices is calculated. The hydrolysis rate and the yield of reducing sugar are calculated according to the following formula:

[0137] Hydrolysis rate = (dry weight of raw material - dry weight of hydrolysis residue) / dry weight of raw material x 100%;

[0138] Yield of reducing sugar = (concentration of reducing sugar in crude sugar solution x volume) / dry weight of raw material x 100%.

[0139] The hydrolysis rate and the yield of reducing sugar of guar gum slices hydrolyzed by different amounts of mannanase mutants are shown in Table 2.

[0140] Table 2 is the hydrolysis rate and the yield of reducing sugar of guar gum slices hydrolyzed by different amounts of mannanase mutants

[0141] Hydrolysis rate (%) Reducing sugar yield (%) Figure 8 250 45.1 35.9 500 66.5 45.5 1000 71.8 52.6 2000 76.1 56.2 3000 77.6 58.3

[0142] As can be seen from Table 2, with the gradual increase of enzyme amount, the hydrolysis rate and the yield of reducing sugar of guar gum slices are gradually increased. When the enzyme amount is 2000 U / g of guar gum slices, after hydrolysis by the mannanase mutant for 8h, the hydrolysis rate and the yield of reducing sugar of guar gum slices are 76.1% and 56.2% respectively. Subsequently, when the enzyme amount is increased to 3000 U / g of guar gum slices, the hydrolysis rate and the yield of reducing sugar of guar gum slices are 77.6% and 58.3% respectively.

[0143] Example 5, HPLC analysis of guar gum manno-oligosaccharides

[0144] Take 10 g of guar gum slices, dissolve in 100 mL of distilled water (pH value is natural pH; distilled water can also be replaced by MOPS buffer solution with pH 6.5), add 2000 U / g of the fermentation supernatant prepared in Example 3, and hydrolyze at 40°C for 8h. After enzyme hydrolysis, inactivate in boiling water bath for 20 min to obtain the enzyme hydrolysate. After centrifugation at 10000 rpm for 10 min, the supernatant, i.e. the crude sugar solution, is collected. After vacuum freeze-drying of the crude sugar solution, a powder product, i.e. guar gum manno-oligosaccharides, is obtained.

[0145] The guar mannan oligosaccharide sample 6 mg was weighed, dissolved in 3 mL of distilled water, filtered with a 0.22 μm filter membrane, and then subjected to high performance anion exchange chromatography analysis. The chromatographic column was CarboaPacTM PA200, and the column temperature was 25°C. The mobile phase A was distilled water. The mobile phase B was 1 mol / L NaOH aqueous solution. The mobile phase C was 1 mol / L sodium acetate aqueous solution. The flow rate was 1.0 min / mL. The elution program was as follows: 0-5 min, 10% B; 5-15 min, 10% B+5% C; 15.1-20 min, 10% B+7.5% C; 20.1-21 min, 50% B+50% C; 21-24 min, 10% B. Mannose (M1, sigma company, 4440-M), mannobiose (M2, Megazyme company, O-MBI), mannotriose (M3, Megazyme company, O-MTR), mannotetraose (M4, Megazyme company, O-MTE), mannopentaose (M5, Megazyme company, O-MPE), mannohexaose (M6, Megazyme company, O-MHE) and maltodecaose (M10, Shanghai Yuanye company, B27566) were used as standard samples.

[0146] The high performance anion exchange chromatogram of the guar mannan oligosaccharide is shown in ​ According to the calculation, after 8 h of enzymatic hydrolysis, the contents of mannobiose to mannodecaose in the obtained guar mannan oligosaccharide were greater than 90% (by mass percentage), and the oligosaccharides with a degree of polymerization of 2-6 accounted for 40.4%, and the oligosaccharides with a degree of polymerization of 6-10 accounted for 53.9%.

[0147] Example 6, Application of wild-type mannanase in preparation of guar mannan oligosaccharide

[0148] A wild-type Aspergillus niger mannanase (i.e., the wild-type mannanase shown in SEQ ID NO: 3, Wang et al., Process Biochemistry. 2021, 100, 90-97) was prepared, and the optimum pH value thereof was 5.

[0149] Guar gum powder 10 g was dissolved in 100 mL of distilled water (pH value was natural pH, generally about 6.5) to obtain a guar gum powder aqueous solution. Then, a part of the guar gum powder aqueous solution was directly added with 2000 U / g substrate of wild-type Aspergillus niger mannanase, and another part of the guar gum powder aqueous solution was adjusted to pH 5 (adjusted by adding 50 mM citric acid-trisodium citrate buffer (pH 5)) and then added with 2000 U / g substrate of wild-type Aspergillus niger mannanase. Both of the above two systems were hydrolyzed at 40°C for 8 h. After enzyme hydrolysis, boiling water bath inactivation was performed for 20 min to obtain an enzyme hydrolysate. After centrifugation at 10000 rpm for 10 min, the supernatant, i.e., a crude sugar solution, was collected. The content of reducing sugar in the crude sugar solution was determined by the 3,5-dinitrosalicylic acid method, and the yield of reducing sugar was calculated. The precipitate was washed with pure water twice, dried, weighed, and the hydrolysis rate of the guar gum powder was calculated. The hydrolysis rate and the yield of reducing sugar were calculated according to the following formula:

[0150] Hydrolysis rate = (dry weight of raw material - dry weight of hydrolysis residue) / dry weight of raw material × 100%;

[0151] Yield of reducing sugar = (concentration of reducing sugar in crude sugar solution × volume) / dry weight of raw material × 100%.

[0152] When the wild-type Aspergillus niger mannanase was used to hydrolyze the guar gum powder, the hydrolysis rate and the yield of reducing sugar of the guar gum powder were 60.5% and 41.3% respectively at pH 5, and the hydrolysis rate and the yield of reducing sugar of the guar gum powder were 13.7% and 8.2% respectively at pH 6.5. It can be seen that the wild-type mannanase has low efficiency in hydrolyzing the guar gum powder at natural pH, and has hydrolysis effect at the optimal pH 5, but the hydrolysis effect is not as good as that of the mannanase mutant.

[0153] The above merely describes preferred specific embodiments of the present application, but the protection scope of the present application is not limited thereto. Any changes or replacements within the technical scope disclosed by the present application can be easily conceived by those skilled in the art, and should be covered within the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.

[0154] The contents not described in detail in the specification belong to the prior art known to those skilled in the art.

Claims

1. A mutant of mannanase, which is any one of the following proteins: A1) the amino acid sequence of the protein is the amino acid sequence of wild-type mannanase shown in SEQ ID NO: 3, wherein the Gln at position 38 is mutated to Lys, the Asp at position 224 is mutated to Ala, and the Ala at position 272 is mutated to Thr, to obtain; A2) a fusion protein obtained by connecting a protein tag to the N-terminus or / and C-terminus of the protein shown in A1).

2. The mutant according to claim 1, wherein: The amino acid sequence of the protein shown in A1) is SEQ ID NO:

1.

3. A nucleic acid molecule encoding the mutant of mannanase according to claim 1 or 2.

4. The nucleic acid molecule of claim 3, wherein: The nucleic acid molecule is a DNA molecule according to any one of the following B1), B2) or B3): B1) the nucleotide sequence of the DNA molecule is SEQ ID NO: 2; B2) a DNA molecule having 85% or more identity to the nucleotide sequence defined in B1), and encoding the mutant of mannanase according to claim 1; B3) a DNA molecule which hybridizes to the nucleotide sequence defined in B1) under stringent conditions, and encoding the mutant of mannanase according to claim 1.

5. Biomaterials containing the nucleic acid molecule according to claim 3 or 4, characterized in that: The biological material is any one of the following: C1) an expression cassette containing the nucleic acid molecule according to claim 3 or 4; C2) a recombinant vector containing the nucleic acid molecule according to claim 3 or 4, or a recombinant vector containing the expression cassette according to C1); C3) a recombinant microorganism containing the nucleic acid molecule according to claim 3 or 4, or a recombinant microorganism containing the expression cassette according to C1), or a recombinant microorganism containing the recombinant vector according to C2).

6. Use of the mutant according to claim 1 or 2, or the nucleic acid molecule according to claim 3 or 4, or the biological material according to claim 5, in any one of the following: D1) preparation of mannanase; D2) preparation of a product containing mannanase activity; D3) preparation of manno-oligosaccharides; D4) hydrolysis of guar splits or guar gum to obtain manno-oligosaccharides.

7. A method for preparing mannanase, comprising the steps of: fermenting the recombinant microorganism according to claim 5, and collecting the fermentation product, to obtain mannanase.

8. A method for preparing manno-oligosaccharides, comprising the steps of: hydrolysis of guar splits or guar gum with the mutant according to claim 1 or 2, or the fermentation product according to claim 7, as enzyme, to obtain manno-oligosaccharides.

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