Eurythermal, broad-ph and salt-tolerant dual-enzyme-coupled recombinant fused β-mannanase and use thereof

Abstract

The present invention belongs to the technical field of the preparation of fused enzymes. Disclosed are a eurythermal, broad-pH and salt-tolerant dual-enzyme-coupled recombinant fused β-mannanase and the use thereof. For the first time, a computer-aided molecular design is used to couple a cold-active enzyme from Bacillus clausii to a protein sequence-modified thermophilic enzyme from Thermotoga maritima via linker L, and to add a C-terminal polypeptide sequence for improving the structural stability of the enzyme at the C-terminus, and a fused enzyme is obtained by means of the induced expression of constructed recombinant Escherichia coli. The obtained fused enzyme can tolerate a pH value of 4-11, a temperature of 20-100°C, and salinity of up to 20%, has the characteristic of initiating an enzymatic reaction at both low and high temperatures, respectively, and can be widely used in pulp bleaching, detergents, foods, feeds, coffee processing, petroleum exploitation, drug development, biofuel production, and the enzymatic production of mannooligosaccharides. The enzyme can simplify the problem of the simultaneous requirement for different types of β-mannanases in the production process, can be used for multiple purposes, and has broad application prospects.

Description

A pan-temperature and pan-pH salt-tolerant double-enzyme coupled recombinant beta-mannan fusion enzyme and application thereof TECHNICAL FIELD

[0001] The present application belongs to the technical field of fusion enzyme preparation, and particularly relates to a pan-temperature and pan-pH salt-tolerant double-enzyme coupled recombinant beta-mannan fusion enzyme and application thereof. BACKGROUND

[0002] Beta-mannanase is an endohydrolase that can degrade mannopolysaccharides containing beta-1, 4-D-mannoside bonds, including mannan, galactomannan, glucan and mannooligosaccharide, and belongs to hemicellulase, and widely exists in animals, plants and microorganisms. At present, the most studied is the beta-mannanase from microorganisms, and the reported ones are, for example, Bacillus in bacteria, sphingomonad, enterobacter, Trichoderma in fungi, Rhizopus, Aspergillus and Streptomyces in actinomycetes, etc.

[0003] β-mannanase is one of the important industrial enzymes, which has a wide range of applications in feed, textile printing and dyeing, food, papermaking and medicine. At room temperature, the β-mannanase in the range of weak acid to weak base has been mass-produced and popularized in China. The challenge of medium temperature enzyme is mainly to screen or design enzymes that can tolerate high concentration of surfactants to meet the application of detergents. This is mainly monopolized by several international companies, including Novozymes (merged with Genencor, renamed Novonesis), DSM, Dupont, which are still in the leading position. Since Dalian Baotaite Technology Co., Ltd. introduced Pyrolase160 from Diversa Company in the United States in 2005 and first promoted the application of thermophilic mannanase in domestic oil fields, universities, research institutes and enterprises have also responded, and the industrialization scale and popularization of medium and high temperature β-mannanase have been basically realized. In recent years, there has been great progress in the research and development of this type of enzyme, involving 3268 patent applications, 418 granted invention patents, 9 utility model patents, and 153 still valid granted invention patents. In the past five years, a large number of researchers have been continuously optimizing the activity, function, adaptive conditions, expression system and high-yield biological processing technology of this type of enzyme.For example, Chinese invention patent CN 117025572A discloses a heat-resistant mannanase mutant No. 4 and its mutant gene and application, Chinese invention patent CN 117106754A discloses a heat-resistant mannanase mutant No. 74 and its mutant gene and application; Chinese invention patent CN 116445456A discloses a beta-mannanase mutant N186D with improved acid resistance and its preparation and application; Chinese invention patent CN 116334042A discloses a method for improving the stability of neutral mannanase PLM5A and a mutant; Chinese invention patent CN 116042575A discloses a high-temperature-resistant beta-mannanase ReTMan26 and its gene and application; Chinese invention patent CN 114350638 A discloses a method for producing high-temperature-resistant acid beta-mannanase; Chinese invention patent CN 115838706 A discloses a high-temperature-resistant mannanase mutant; Chinese invention patent CN 115838707 A discloses a mannanase mutant; Chinese invention patent CN 113584003 A discloses a beta-mannanase mutant with improved heat resistance and its encoding gene and application; Chinese invention patent CN 113293154A discloses a beta-mannanase heat-resistant mutant M18, a recombinant bacterium and its application; Chinese invention patent CN 113073107A discloses a mannanase gene AbMan5, a recombinant expression plasmid, a recombinant expression strain, a mannanase and its application; Chinese invention patent CN 113151025 A discloses a Pichia pastoris for fusion expression of beta-mannanase and alpha-galactosidase, a preparation method and its application. However, in industrial production, harsh engineering conditions, the demand for higher performance, high stability, and wide adaptability of industrial enzymes is still difficult to meet, especially in the trend of green and low-carbon development, the development of these efficient and economical enzyme catalysts is the mainstream of industrial enzymes, and beta-mannanase is no exception. Overall trend, breaking the temperature limit of such enzymes, such as developing extreme thermophilic enzymes, or high as 100℃, or psychrophilic enzymes 10℃ to minus, still have high activity, acid and alkali resistance, high salinity resistance and other extreme engineering environment of new beta-mannanase still exist great challenge.

[0004] SUMMARY

[0005] In view of this, the purpose of the present application is to provide a pan-temperature and pan-pH salt-tolerant double-enzyme coupled recombinant beta-mannanase fusion enzyme and its application. The beta-mannanase fusion enzyme of the present application has high activity under the conditions of 20-100℃, pH 4-11, 20% NaCl or KCl high salinity, which provides a new way for developing a new generation of pan-type fusion mannanase.

[0006] To achieve the above-mentioned purpose, the present application provides the following technical solutions:

[0007] The application provides a salt-tolerant dual-enzyme coupled recombinant beta-mannan fusion enzyme with pan-temperature and pan-pH resistance, which is dual-enzyme coupled according to a general formula (1):

[0008] E1-L-E2-ψ (1)

[0009] wherein the amino acid sequence of E1 is shown in SEQ ID NO: 1; L is a linker for protein fusion, E2 is obtained by amino acid mutation based on the amino acid sequence shown in SEQ ID NO: 2; and ψ is a C-terminal polypeptide sequence for improving the structural stability of the enzyme.

[0010] Based on the above technical scheme, further, the amino acid mutation includes any one of the following:

[0011] (1) the lysine at the 85th position in the amino acid sequence shown in SEQ ID NO: 2 is mutated into tyrosine, glycine, alanine, glutamic acid, aspartic acid, threonine, asparagine, histidine, tyrosine, preferably aspartic acid or tyrosine, i.e. K85D or K85Y;

[0012] (2) the leucine at the 112th position in the amino acid sequence shown in SEQ ID NO: 2 is mutated into glycine, threonine, valine, isoleucine, preferably isoleucine, i.e. L112I;

[0013] (3) the alanine at the 113th position in the amino acid sequence shown in SEQ ID NO: 2 is mutated into valine, isoleucine, preferably valine, i.e. A113V;

[0014] (4) the leucine at the 114th position in the amino acid sequence shown in SEQ ID NO: 2 is mutated into alanine, threonine, valine, preferably valine, i.e. L114V;

[0015] (5) the lysine at the 116th position in the amino acid sequence shown in SEQ ID NO: 2 is mutated into histidine, arginine, preferably arginine, i.e. K116R;

[0016] (6) the lysine at the 123th position in the amino acid sequence shown in SEQ ID NO: 2 is mutated into histidine, arginine, preferably arginine, i.e. K123R;

[0017] (7) the glutamic acid at the 148th position in the amino acid sequence shown in SEQ ID NO: 2 is mutated into tyrosine, aspartic acid, preferably aspartic acid, i.e. E148D;

[0018] (8) the serine at the 159th position in the amino acid sequence shown in SEQ ID NO: 2 is mutated into glycine, alanine, threonine, preferably threonine, i.e. S159T;

[0019] (9) the isoleucine at position 160 of the amino acid sequence set forth in SEQ ID NO: 2 is mutated to a glycine, leucine, serine, threonine, preferably serine, i.e. I160S;

[0020] (10) the glutamic acid at position 180 of the amino acid sequence set forth in SEQ ID NO: 2 is mutated to a glycine, alanine, valine, isoleucine, tyrosine, preferably tyrosine, i.e. E180Y;

[0021] (11) the serine at position 183 of the amino acid sequence set forth in SEQ ID NO: 2 is mutated to a glycine, alanine, histidine, aspartic acid, glutamic acid, threonine, lysine, preferably lysine, i.e. S183K;

[0022] (12) the leucine at position 218 of the amino acid sequence set forth in SEQ ID NO: 2 is mutated to an aspartic acid, glutamic acid, methionine, tyrosine, preferably methionine, i.e. L218M;

[0023] (13) the lysine at position 221 of the amino acid sequence set forth in SEQ ID NO: 2 is mutated to an alanine, serine, threonine, tyrosine, preferably tyrosine, i.e. K221Y;

[0024] (14) the isoleucine at position 232 of the amino acid sequence set forth in SEQ ID NO: 2 is mutated to a valine, leucine, threonine, preferably leucine, i.e. I232L;

[0025] (15) the glutamic acid at position 233 of the amino acid sequence set forth in SEQ ID NO: 2 is mutated to a valine, histidine, lysine, tyrosine, preferably tyrosine, i.e. E233Y;

[0026] (16) the phenylalanine at position 237 of the amino acid sequence set forth in SEQ ID NO: 2 is mutated to a tyrosine, i.e. F237Y;

[0027] (17) the asparagine at position 245 of the amino acid sequence set forth in SEQ ID NO: 2 is mutated to a valine, leucine, isoleucine, preferably isoleucine, i.e. N245I;

[0028] (18) the lysine at position 246 of the amino acid sequence set forth in SEQ ID NO: 2 is mutated to an aspartic acid, glutamic acid, tyrosine, preferably tyrosine, i.e. K246Y;

[0029] (19) the arginine at position 299 of the amino acid sequence set forth in SEQ ID NO: 2 is mutated to a glycine, alanine, serine, threonine, isoleucine, histidine, aspartic acid, asparagine, glutamic acid, glutamine, tyrosine, preferably tyrosine, i.e. R299Y;

[0030] (20) the lysine at position 304 of the amino acid sequence set forth in SEQ ID NO: 2 is mutated to glycine, alanine, serine, tyrosine, preferably serine, i.e., K304S;

[0031] (21) the glutamic acid at position 308 of the amino acid sequence set forth in SEQ ID NO: 2 is mutated to leucine, isoleucine, tyrosine, preferably tyrosine, i.e., E308Y.

[0032] Based on the above technical scheme, further, the amino acid mutation is K85Y, L112I, A113V, L114V, K116R, K123R, E148D, S159T, I160S, E180Y, S183K, L218M, K221Y, I232L, E233Y, F237Y, N245I, K246Y, R299Y, K304S, E308Y in SEQ ID NO: 2.

[0033] Based on the above technical scheme, further, the amino acid sequence of the linker L is X-(EAAAK) m -Y, X-(GGGGS) m -Y, X-(GS) m -Y, wherein X and Y are flexible sequences ALA, and m = 4-8; preferably m = 4.

[0034] Based on the above technical scheme, further, the amino acid sequence of the linker L is ALAEAAAKEAAAKEAAAKEAAAKALAEAAAKEAAAKEAAAKEAAAKALA.

[0035] Based on the above technical scheme, further, ψ is a short peptide (GT)n consisting of glycine G and threonine T, n = 2-8, preferably n = 4, to improve the stability of the enzyme.

[0036] The present application provides a gene encoding the above-mentioned pan-temperature and pan-pH salt-tolerant double-enzyme-coupled recombinant β-mannan fusion enzyme.

[0037] The present application provides a recombinant vector carrying the above-mentioned gene.

[0038] Based on the above technical scheme, further, the backbone vector of the recombinant vector is a pET series vector.

[0039] Based on the above technical scheme, further, the pET series vector includes pET-3a or pET-28a.

[0040] Based on the above technical scheme, further, the recombinant vector contains a T7 promoter.

[0041] The present application provides a recombinant strain carrying the above-mentioned recombinant vector.

[0042] Based on the above technical solution, further, the host cell of the recombinant strain is bacteria, fungi or yeast.

[0043] Based on the above technical solution, further, the host cell of the recombinant strain is one of Escherichia coli BL21 (DE3), Origami (DE3), Origami B (DE3) or Rosetta-gami (DE3).

[0044] The present application provides a method for preparing a pan-temperature and pan-pH salt-tolerant double-enzyme coupled recombinant β-mannanase fusion enzyme, inoculating the recombinant strain into a fermentation medium for culture, inducing the expression of the pan-temperature and pan-pH salt-tolerant double-enzyme coupled recombinant β-mannanase fusion enzyme protein, and separating and purifying to obtain the pan-temperature and pan-pH salt-tolerant double-enzyme coupled recombinant β-mannanase fusion enzyme.

[0045] Based on the above technical solution, further, the inoculation amount is 1-10% of the volume of the fermentation medium; the culture conditions are 30-38℃ and 150-220rpm for 3-24h, and the induction culture is 2-20h, and the inducer is IPTG with a final concentration of 0.05-5mM.

[0046] The present application provides an enzyme preparation containing the above-mentioned pan-temperature and pan-pH salt-tolerant double-enzyme coupled recombinant β-mannanase fusion enzyme, and the content is 0.05-1wt%.

[0047] The present application also provides the application of the above-mentioned pan-temperature and pan-pH salt-tolerant double-enzyme coupled recombinant β-mannanase fusion enzyme and enzyme preparation in food, feed, coffee processing, drug development, pulp bleaching, oil exploitation, detergent, biofuel production and enzymatic preparation of mannose oligosaccharide.

[0048] Based on the above technical solution, further, the raw materials for the enzymatic preparation of mannose oligosaccharide include konjac gum, tara gum, locust bean gum and guar gum.

[0049] Based on the above technical solution, further, the pH is controlled at 6-10, the temperature is controlled at 40-80℃, and the addition amount of the double-enzyme coupled recombinant β-mannanase is 30-500ppm.

[0050] The present application has the following beneficial effects compared with the prior art:

[0051] 1) The double-enzyme coupled recombinant β-mannanase of the present application has more persistent catalytic efficiency by the fusion of low-temperature mannanase and thermophilic mannanase, realizes higher catalytic reaction efficiency by combining the advantages of the two enzymes, can maintain activity in the extreme conditions of both poles, improves the enzyme's resistance to denaturation, adapts to more harsh process conditions, and can efficiently act on the hydrolysis of various plant gums of the mannan type with β-glycosidic bonds.

[0052] 2) The double-enzyme coupled recombinant β-mannanase of the present application does not start the high-temperature catalytic domain at low temperature 10-40℃, and does not start the low-temperature catalytic domain at 40-100℃, and the low-temperature catalytic domain still maintains catalytic activity when returning to normal temperature. Such a fusion enzyme not only inherits the characteristics of low-temperature enzymes and thermophilic enzymes, but also can function in a wider temperature range, while improving the enzyme's resistance to salt and surfactants, making the β-mannan fusion enzyme have a wider application range and adapt to various biological reaction conditions.

[0053] 3) The C-terminal stabilizing sequence of the double-enzyme coupled recombinant β-mannanase of the present application strengthens the enzyme's resistance to protease degradation, prolongs its service life in industrial applications; the stabilizing sequence improves the solubility of individual enzymes at their respective isoelectric points, reduces the risk of aggregation and precipitation in solution through hydrophilic surface structure modification, improves solubility and anti-precipitation ability, and at the same time widens the pH adaptation range.

[0054] 4) The C-terminal stabilizing sequence of the double-enzyme coupled recombinant β-mannanase of the present application slows down the conformational change and inactivation rate of the enzyme, the linker-helix structure is conducive to the Coil-to-Coil interaction on the back of the catalytic center, plays a role in stabilizing the double enzyme, and prolongs its effective period during storage and use.

[0055] 5) The double-enzyme coupled recombinant β-mannanase of the present application only needs to operate one fusion enzyme instead of two independent enzymes, is suitable for a wider range of biotechnology and industrial application fields, simplifies the use of multiple enzymes, improves its flexibility in production and scientific research, and saves time and resources. BRIEF DESCRIPTION OF DRAWINGS

[0056] In order to more clearly illustrate the embodiments of the present application, the drawings involved in the embodiments will be briefly introduced below.

[0057] Figure 1 is a β-mannan protein three-dimensional structure diagram of SEQ ID NO: 2.

[0058] Figure 2 is a β-mannan protein three-dimensional structure diagram of SEQ ID NO: 3.

[0059] Figure 3 is a double-enzyme coupled recombinant β-mannan fusion enzyme protein three-dimensional structure diagram of SEQ ID NO: 4.

[0060] Figure 4 is a protein surface map of the fusion enzyme of SEQ ID NO: 4 (dark parts are engineered amino acids).

[0061] Figure 5 is a comparison chart of the amino acid sequences of SEQ ID NO: 2 and SEQ ID NO: 3.

[0062] Figure 6 is a chart of the SDS-PAGE results of BIT280 WT crude enzyme solution, wherein M: standard protein band,

[0063] 1: BIT280 WT crude enzyme solution.

[0064] Figure 7 is a schematic diagram of the constructed plasmid for inserting the fusion enzyme of SEQ ID NO: 4.

[0065] Figure 8 is a chart of the determination results of the optimum temperature of BIT280 WT crude enzyme solution, wherein "■" is the fusion enzyme BIT280 WT,

[0066] "●" is the wild type Man6, all measurements are at least in duplicate, and the data points are the average values of individual measurements.

[0067] Figure 9 is a chart of the determination results of the optimum pH of BIT280 WT crude enzyme solution, wherein "■" is the fusion enzyme BIT280 WT,

[0068] "●" is the wild type Man6, all measurements are at least in duplicate, and the data points are the average values of individual measurements.

[0069] Figure 10 is a chart of the determination results of the molecular weight of guar gum solution hydrolyzed by BIT280 WT crude enzyme solution, A: BIT280 WT

[0070] molecular weight distribution chart of guar gum solution sample hydrolyzed by BIT280 WT for 4h; B: molecular weight distribution chart of guar gum solution sample hydrolyzed by BIT280 WT for 24h; C: molecular weight distribution chart of guar gum solution sample hydrolyzed by BIT280 WT for 48h. DETAILED DESCRIPTION

[0071] The application will be described in detail below with reference to the examples, but the embodiments of the application are not limited thereto. Obviously, the examples described below are only part of the embodiments of the application, and other similar embodiments obtained by those skilled in the art without creative labor are also within the protection scope of the application.

[0072] Example 1: Construction of a salt-tolerant dual-enzyme coupled recombinant β-mannan fusion enzyme with pan-temperature and pan-pH resistance

[0073] The present application provides a kind of pan temperature pan pH salt-tolerant double enzyme coupling recombination beta-mannan fusion enzyme, the present application utilizes AI modeling, and modeling analysis is carried out by Google alphafold2 Colab, and the structure is optimized and adjusted, the stability parameter of enzyme is improved, and the design of fusion enzyme is carried out with general formula (1) Double enzyme coupling:

[0074] E1-L-E2-psi (1)

[0075] Wherein E1 is the protein sequence SEQ ID NO:1 of psychrophilic Clostridium bifermentans enzyme Man6;L is the linking sub of protein fusion, and the sequence of linking sub L is ALAEAAAKEAAAKEAAAKEAAAKALAEAAAKEAAAKEAAAKEAAAKALA;E2 is SEQ ID NO:3 obtained by structural modification design based on wild type Thermus aquaticus enzyme Man5 of SEQ ID NO:2;The specific mutation of SEQ ID NO:3 obtained by site-directed amino acid mutation to SEQ ID NO:2 is: K85Y, L112I, A113V, L114V, K116R, K123R, E148D, S159T, I160S, E180Y, S183K, L218M, K221Y, I232L, E233Y, F237Y, N245I, K246Y, R299Y, K304S, E308Y;Psi is the C-terminal polypeptide sequence for improving the stability of enzyme structure, which is a short peptide (GT) 4 composed of glycine G and threonine T;The amino acid sequence of the obtained double enzyme coupling recombination beta-mannanase is SEQ ID NO:4.

[0076] Table 1 comparison of protein stability

[0077] Table 2 comparison of theoretical parameters of wild type Man5 and mutant

[0078] The three-dimensional structure diagram of wild type mannanase Man5 and mutant is as shown in Figures 1-2, the mutant of wild type mannanase Man5 is composed of eight beta chains and eleven alpha helixes, there are ten alpha helixes between eight beta-folds, and the mannanase mutant shows a typical TIM barrel shape. The overall structure of wild type mannanase and mutant mannanase is highly conserved, and through sequence comparison, it is found that the amino acids of the active site are also highly conserved, which conforms to the characteristics of high activity of mannanase mutant, and the catalytic center is kept from changing configuration.

[0079] The protein three-dimensional structure of the double-enzyme coupling recombinant β-mannan fusion enzyme is shown in Figure 3. The hydrophilic surface isolated amino acid side chains K85Y, E180Y, K221Y, E233Y, F237Y, N245I, K246Y, R299Y, etc. are respectively replaced with tyrosine aromatic side chain or isoleucine side chain; K116R, K123R, E148D are replaced with arginine and aspartic acid, which is more conducive to the formation of hydrophilic surface salt bridge, thereby improving the thermal stability and chemical stability of the free amino acid side chain of the hydrophilic surface under alkaline or acidic conditions and the acid and alkali resistance. S159T, A113V, S183K, L218M, I232L, F237Y are conducive to improving the van der Waals attraction between molecules, the formation of internal salt bridge or hydrogen bond, thereby improving the intrinsic stability of the mutant of SEQ ID NO: 3.

[0080] The double-enzyme coupling recombinant β-mannanase described above is optimized according to the codon bias of Escherichia coli, and the nucleotide sequence is shown as SEQ ID NO: 5. It is inserted into the prokaryotic expression vector pET-3a to obtain a recombinant plasmid. The vector is an IPTG-induced expression vector, which is transformed into Escherichia coli BL21 (DE3) to obtain a recombinant strain BIT280 WT. The recombinant Escherichia coli is fermented in a liquid medium, and the fermentation broth is separated and purified to obtain the double-enzyme coupling recombinant β-mannanase BIT280 WT. The fusion enzyme can tolerate pH 4-11, the temperature is suitable for 20-100℃, and the mineralization degree can be as high as 20%.

[0081] Example 2: Shake flask culture to determine the expression of double-enzyme coupling recombinant β-mannanase BIT280 WT

[0082] Prepare 100 ml of seed liquid medium: according to the following formula, prepare the seed culture medium (g / L): tryptone 20.0 g, yeast extract 20.0 g, sodium chloride 0.5 g, ammonium chloride 1 g, disodium hydrogen phosphate 6 g, potassium dihydrogen phosphate 3 g, pH 7.0, sterilize; the inoculation amount is 0.1%, and the culture is carried out at 37℃, 200 r / min, and pH 7.0 for 16 h (i.e. seed liquid); add inducer IPTG (final concentration 1 mM), continue to culture for 4 h, centrifuge to collect the bacterial cells, and then crush to obtain the supernatant, which is the BIT280 WT β-mannanase crude enzyme liquid. The BIT280 WT β-mannanase crude enzyme liquid is analyzed by SDS-PAGE and subsequent Coomassie staining. The results are shown in Figure 6, which shows that it has a clear band at about 75 kDa, which is the double-enzyme coupling recombinant β-mannanase.

[0083] The seed culture solution is inoculated into the fermentation medium at an inoculation amount of 5-8%, and the fermentation medium is as follows: tryptone 20.0 g, yeast extract 20.0 g, sodium chloride 0.5 g, ammonium chloride 1 g, disodium hydrogen phosphate 6 g, potassium dihydrogen phosphate 3 g, glycerol 20 ml, pH 7.0. The high-density fermentation process is used, the rotation speed is 180 r / min, the pH is 7.0, and the culture time is 48 h at 37°C to obtain the fermentation liquor of the high-efficiency expressed β-mannanase. The fermentation liquor is centrifuged, the bacterial precipitate is collected, the bacterial precipitate is dissolved with the phosphate buffer at a weight ratio of 1:10, the bacterial precipitate is broken by using a high-pressure homogenizer, centrifuged at 6000 r / min for 30 min, the precipitate is discarded, and the obtained supernatant is the β-mannanase liquor. The obtained β-mannanase liquor is purified and concentrated to obtain the concentrated enzyme liquor, and the concentrated enzyme liquor is freeze-dried to obtain the powdery β-mannanase preparation.

[0084] Example 3: Enzyme activity determination of double-enzyme coupling recombinant β-mannanase BIT280WT

[0085] In this example, the pNPG method is used to determine the enzyme activity of the double-enzyme coupling recombinant β-mannanase BIT280WT, and the specific process is as follows:

[0086] The standard curve is drawn as follows:

[0087] 1) Prepare different concentrations of p-nitrophenol solution: accurately weigh 10 mg of p-nitrophenol (PNP) powder into 10 mL of distilled water to prepare a 1 mg / mL standard stock solution. Dilute the p-nitrophenol stock solution to 1 mmol / mL with pH 7.0 phosphate buffer, and dilute it to a series of different concentrations of p-nitrophenol solution with pH 7.0 phosphate buffer. The specific concentrations are shown in Table 3.

[0088] Table 3 Preparation of different concentrations of p-nitrophenol solution

[0089] 2) Place 5 cuvettes in a temperature controller, and add different concentrations of p-nitrophenol solution and phosphate buffer into each cuvette according to Table 3, and add 0.2 mL of termination solution (1 mol / L Na2CO3 solution). Use an electronic thermometer to detect temperature equilibrium to 50℃±0.1. After the temperature reaches 50℃, reset the temperature to 51℃ to maintain the temperature of the cuvettes at 50℃.

[0090] 3) Measure the absorbance value: use a spectrophotometer to measure the absorbance value of each concentration of PNP standard solution at 405 nm wavelength, and record A1, A2, A3, A4 and A5. Record and take the average value of each concentration, draw the p-nitrophenol standard curve and fit, y=0.0069x+0.042 (x is the concentration of PNP, μmol / mL). If the fitting constant R2 Less than 0.97, retest and fit.

[0091] The method for determining the enzyme activity comprises the following steps:

[0092] 1) Preheat the water bath: set the water bath to the desired assay reaction temperature (50℃±0.1).

[0093] 2) Prepare the reaction system: take 3 cuvettes, respectively numbered E1, E2 and B. Add the solutions shown in Table 4 to each cuvette. Note: E1 and E2 are reaction tubes, respectively used to measure the absorbance values of the system before and after the reaction; B is a blank control tube, used to subtract the background interference caused by non-enzymatic reactions.

[0094] Table 4 Reaction system

[0095] 3) Start the reaction: place the E1 and E2 cuvettes in the preheated constant temperature water bath for incubation, and start timing.

[0096] 4) Terminate the reaction: after exactly 3 minutes (or the optimal reaction time determined by pre-experiment), quickly add 0.2 mL of termination solution to the E1 and E2 cuvettes, mix well to terminate the reaction.

[0097] 5) Measure the absorbance value: after the reaction system cools to room temperature, adjust the B cuvette to zero, and then measure E1 and E2 in turn at a wavelength of 405 nm using a spectrophotometer, and record A1 and A2. The difference between the two is substituted into the standard curve of p-nitrophenol to obtain the PNP concentration.

[0098] The sample enzyme activity is calculated according to the following formula:

[0099] U=(C×V) / (t×V0)

[0100] In the formula: U is the enzyme activity (U / mL); C is the PNP concentration μmol / mL; V is the total volume of the reaction system; t is the reaction time; V0 is the volume of the mannanase sample (mL); Enzyme activity definition: the amount of enzyme required to degrade 1 μmol of p-nitrophenol-β-D-galactoside per minute at 50℃, pH 7.0, is defined as one enzyme unit, expressed in U. The enzyme activity of the wild-type β-mannanase with the amino acid sequence shown in SEQ ID NO: 2 is: 2.517 U / mg; the enzyme activity of the modified β-mannanase with the amino acid sequence shown in SEQ ID NO: 3 is: 1.06 U / mg; and the enzyme activity of the double-enzyme coupled recombinant β-mannanase BIT280WT is: 5.217 U / mg.

[0101] Example 4: Determination of the enzyme properties of the double-enzyme coupled recombinant β-mannanase BIT280WT

[0102] 1) Determination of the optimum temperature of β-mannanase

[0103] After the β-mannanase crude enzyme solution was diluted with phosphate buffer (pH 7) as appropriate, the enzyme activity was detected at different temperatures (20, 30, 40, 50, 60, 70, 80, 90, 100 °C) using the above enzyme activity detection method. The relative activity (%) of β-mannanase was calculated by dividing the mannanase activity of the sample by the mannanase activity of the reference sample. In the case of the temperature curve, the reference sample was the sample at the optimum temperature. The detection results are shown in Figure 8. The optimum temperature of the double enzyme coupling recombinant β-mannanase is 50 °C. The enzyme activity can be maintained at more than 80% in the range of 40-80 °C, and at more than 50% in the range of 20-100 °C.

[0104] 2) Determination of the optimum pH of β-mannanase

[0105] The β-mannanase crude enzyme solution was used as the enzyme solution to be detected, and the enzyme activity was detected at 50 °C in different buffer systems. The relative activity (%) of β-mannanase was calculated by dividing the mannanase activity of the sample by the mannanase activity of the reference sample. In the case of the pH curve, the reference sample was the sample at the optimum pH. The various buffers were as follows: citric acid-phosphate buffer (pH 3.0-6.0); phosphate buffer (pH 7.0-8.0); glycine-sodium hydroxide buffer (pH 9.0-11).

[0106] The detection results are shown in Figure 9. The optimum pH of the double enzyme coupling recombinant β-mannanase is 7. The enzyme activity can be maintained at more than 80% in the pH range of 6.0-10.0.

[0107] Example 5: Determination of the mineralization tolerance of the double enzyme coupling recombinant β-mannanase BIT280WT

[0108] In order to understand the mineralization tolerance of the double enzyme coupling recombinant β-mannanase, seven salinity gradients were set, i.e. 2%, 4%, 6%, 10%, 15%, 20% and 25% KCl aqueous solution (w / v), and the effect of different salinity on the enzyme activity of β-mannanase was determined under the optimum conditions of the enzyme. The results are shown in Table 5, which shows that the β-mannanase has strong salt tolerance, and the salt solution has little effect on the enzyme activity.

[0109] Table 5: Results of the mineralization tolerance of β-mannanase

[0110] Example 6: Determination of the compatibility contraindication of the double enzyme coupling recombinant β-mannanase BIT280WT

[0111] In order to understand the compatibility contraindication of the double enzyme coupling recombinant β-mannanase, the experiment sets the metal ions and emulsifier (DOWFAX) to the β-mannanase enzyme activity determination; different metal ions are added to the system according to 5 mmol / L, the emulsifier (DOWFAX) is added to the system according to the weight ratio of 5%, and the enzyme activity is determined under the optimum conditions of the enzyme without adding any ion as the control. The results are shown in Table 6, and the results show that Mg 2+ and Na 2+ have little effect on enzyme activity, while Ca 2+ has a certain inhibitory effect on enzyme activity, and the emulsifier (DOWFAX) has relatively small effect on the enzyme activity of the double enzyme coupling recombinant β-mannanase.

[0112] Table 6 Compatibility contraindication of β-mannanase

[0113] Example 7: Determination of the molecular weight of guar gum solution hydrolyzed by the double enzyme coupling recombinant β-mannanase BIT280WT

[0114] Prepare the guar gum (molecular weight 1000000-2000000 Da) base solution (4.5-5.5 g / L), take 100 ml for each experimental sample, and add β-mannanase solution (5426.08 U / mL) to the sample base solution at a concentration of 30-50 ppm, stir uniformly, then add inorganic boron crosslinking agent (according to the weight ratio of 1%), crosslinking ratio 100:5, stir the base solution to form a gel to the hanging state, and put it into a 70°C constant temperature water bath for hydrolysis for 4, 24 and 48 h. Remove the residue, place the supernatant in boiling water for 10 min, remove the suspended matter again, pass through a 0.22 μm filter membrane, and then detect. Use MALDI-TOF-MS mass spectrometer (Bruker Daltonics Inc. BIFLEX III), nitrogen laser wavelength 337 nm, adopt the working mode of delayed extraction and reflection, acceleration voltage 19.5 kV, reflection voltage 20 kV, delayed extraction voltage 14.5-16.5 kV, and delayed time 50-200 ns, and detect positive ions. The detection results are shown in Figure 10, and the molecular weight of the guar gum solution hydrolyzed by the β-mannanase is reduced to 2000-6000. With the extension of the hydrolysis time, the molecular weight of the guar gum solution hydrolyzed by the β-mannanase is smaller.

[0115] Example 8: Tolerance of the double enzyme coupling recombinant β-mannanase BIT280WT to surfactants

[0116] In order to understand the effect of surfactants on the enzyme activity of β-mannanase, different surfactants (Tween 80, fatty alcohol polyoxyethylene ether sodium sulfate, alkyl alcohol amide, fatty alcohol polyoxyethylene ether and dodecyl dimethyl betaine) were added to the reaction system at 5% (weight ratio) to determine their effects on the enzyme activity of β-mannanase under the optimum conditions of the enzyme. The results are shown in Table 7. The surfactants containing 5% Tween 80, fatty alcohol polyoxyethylene ether sodium sulfate, alkyl alcohol amide and fatty alcohol polyoxyethylene ether had little effect on the activity of β-mannanase, and the surfactant dodecyl dimethyl betaine slightly inhibited the activity of β-mannanase.

[0117] Table 7 Determination of β-mannanase tolerance to surfactants

[0118] Example 9: Determination of hydrolysis of konjac gum at different pH by double-enzyme coupled recombinant β-mannanase BIT280WT

[0119] According to 4.5-5.5 g / L, konjac gum was weighed and dissolved in 50 mmol / L pH 8.5 and pH 9.0 glycine-sodium hydroxide buffer, stirred thoroughly, and double-enzyme coupled recombinant β-mannanase (5426.08 U / mL) was added at 30-50 ppm. After uniform stirring, it was placed in a 50°C constant temperature water bath for 2 h, and the supernatant was the enzyme hydrolysate. The viscosity of the enzyme hydrolysate was measured at room temperature using a DV-1 rotary viscometer. The results are shown in Table 8, which show that under the conditions of pH 8.5 and pH 9.0, β-mannanase can still hydrolyze konjac gum, and the viscosity is below 5 mPa.S.

[0120] Table 8 Determination of β-mannanase hydrolysis of konjac gum at different pH

[0121] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application, and are not limiting; those skilled in the art should understand that they can still modify the technical solutions described in the above examples, or make equivalent substitutions for some or all of the technical features; and these modifications or substitutions do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A thermophilic, pH-tolerant, salt-tolerant dual-enzyme coupled recombinant β-mannan fusion enzyme, characterized in that, The fusion enzyme is coupled with two enzymes according to general formula (1): E1-L-E2-ψ (1) Wherein, the amino acid sequence of E1 is shown in SEQ ID NO:1; L is a protein fusion linker; E2 is obtained by amino acid mutation based on the amino acid sequence shown in SEQ ID NO:2; ψ is a C-terminal polypeptide sequence to improve enzyme structural stability; the amino acid mutation includes any of the following: (1) The lysine at position 85 of the amino acid sequence shown in SEQ ID NO:2 is mutated to tyrosine, glycine, alanine, glutamic acid, aspartic acid, threonine, asparagine, histidine, or tyrosine, preferably aspartic acid or tyrosine, i.e. K85D or K85Y. (2) The leucine at position 112 of the amino acid sequence shown in SEQ ID NO:2 is mutated into glycine, threonine, valine, or isoleucine, with isoleucine being preferred, i.e., L112I; (3) The amino acid sequence shown in SEQ ID NO:2 has alanine at position 113 mutated into valine or isoleucine, preferably valine, i.e. A113V; (4) The leucine at position 114 of the amino acid sequence shown in SEQ ID NO:2 is mutated to alanine, threonine, or valine, with valine being preferred, i.e., L114V. (5) The lysine at position 116 of the amino acid sequence shown in SEQ ID NO:2 is mutated to histidine and arginine, preferably arginine, i.e. K116R. (6) The lysine at position 123 of the amino acid sequence shown in SEQ ID NO:2 is mutated to histidine and arginine, preferably arginine, i.e. K123R; (7) The amino acid sequence shown in SEQ ID NO:2 is mutated at position 148, with glutamic acid being replaced by tyrosine or aspartic acid, preferably aspartic acid, i.e., E148D. (8) The serine at position 159 of the amino acid sequence shown in SEQ ID NO:2 is mutated to glycine, alanine, or threonine, with threonine being preferred, i.e., S159T. (9) The isoleucine at position 160 of the amino acid sequence shown in SEQ ID NO:2 is mutated into glycine, leucine, serine, or threonine, with serine being preferred, i.e., I160S. (10) The glutamic acid at position 180 of the amino acid sequence shown in SEQ ID NO:2 is mutated into glycine, alanine, valine, isoleucine, or tyrosine, preferably tyrosine, i.e. E180Y. (11) The serine at position 183 of the amino acid sequence shown in SEQ ID NO:2 is mutated to glycine, alanine, histidine, aspartic acid, glutamic acid, threonine, lysine, preferably lysine, i.e. S183K. (12) The leucine at position 218 of the amino acid sequence shown in SEQ ID NO:2 is mutated to aspartic acid, glutamic acid, and methionine. Acid, tyrosine, preferably methionine, i.e. L218M; (13) The lysine at position 221 of the amino acid sequence shown in SEQ ID NO:2 is mutated to alanine, serine, threonine, or tyrosine, preferably tyrosine, i.e. K221Y. (14) The isoleucine at position 232 of the amino acid sequence shown in SEQ ID NO:2 is mutated into valine, leucine, or threonine, with leucine being preferred, i.e., I232L; (15) The glutamic acid at position 233 of the amino acid sequence shown in SEQ ID NO:2 is mutated into valine, histidine, lysine, or tyrosine, preferably tyrosine, i.e. E233Y. (16) The amino acid sequence shown in SEQ ID NO:2 has a phenylalanine at position 237 that is mutated to tyrosine, i.e., F237Y; (17) The amino acid sequence shown in SEQ ID NO:2 is mutated at position 245 to valine, leucine, and isoleucine, with isoleucine being preferred, i.e. N245I; (18) The amino acid sequence shown in SEQ ID NO:2 has a lysine at position 246 mutated to aspartic acid, glutamic acid, or tyrosine, preferably tyrosine, i.e. K246Y. (19) The arginine at position 299 of the amino acid sequence shown in SEQ ID NO:2 is mutated to glycine, alanine, serine, threonine, isoleucine, histidine, aspartic acid, asparagine, glutamic acid, glutamine, tyrosine, preferably tyrosine, i.e., R299Y. (20) The lysine at position 304 of the amino acid sequence shown in SEQ ID NO:2 is mutated to glycine, alanine, serine, or tyrosine, preferably serine, i.e. K304S. (21) The glutamic acid at position 308 of the amino acid sequence shown in SEQ ID NO:2 is mutated into leucine, isoleucine, or tyrosine, preferably tyrosine, i.e. E308Y.

2. The thermophilic, pH-tolerant, and salt-tolerant dual-enzyme coupled recombinant β-mannan fusion enzyme according to claim 1, characterized in that, The specific amino acid mutations mentioned are K85Y, L112I, A113V, L114V, K116R, K123R, E148D, S159T, I160S, E180Y, S183K, L218M, K221Y, I232L, E233Y, F237Y, N245I, K246Y, R299Y, K304S, and E308Y in SEQ ID NO:

2.

3. The thermophilic, pH-tolerant, and salt-tolerant dual-enzyme coupled recombinant β-mannan fusion enzyme according to claim 1, characterized in that, The amino acid sequence of the linker L is X-(EAAAK). m -Y、X-(GGGGS) m -Y, X-(GS) m -Y, where X and Y are flexible sequences ALA, m = 4-8; preferably m = 4; ψ is a short peptide (GT)n composed of glycine G and threonine T, n = 2-8, preferably n = 4.

4. The gene encoding the panthermic, panpH-tolerant, salt-tolerant dual-enzyme coupled recombinant β-mannan fusion enzyme as described in any one of claims 1-3.

5. A recombinant vector carrying the gene of claim 4.

6. The recombinant vector as described in claim 5, characterized in that, The backbone vector of the recombinant vector is a pET series vector.

7. A recombinant bacterial strain carrying the recombinant vector according to claim 5 or 6, characterized in that, The host cell of the recombinant strain is bacteria, fungi, or yeast.

8. A method for preparing a thermo- and pH-tolerant salt-tolerant dual-enzyme-coupled recombinant β-mannan fusion enzyme, characterized in that, The recombinant strain described in claim 7 was inoculated into a fermentation medium and cultured to induce the expression of a thermophilic, pH-tolerant, and salt-tolerant dual-enzyme-coupled recombinant β-mannan fusion enzyme protein. The thermophilic, pH-tolerant, and salt-tolerant dual-enzyme-coupled recombinant β-mannan fusion enzyme was then isolated and purified.

9. An enzyme preparation, characterized in that, The enzyme preparation contains the panthermic, panpH-tolerant, and salt-tolerant dual-enzyme coupled recombinant β-mannan fusion enzyme as described in any one of claims 1-3, in a content of 0.05-1 wt%.

10. The application of the panthermic, panpH, salt-tolerant dual-enzyme coupled recombinant β-mannan fusion enzyme according to any one of claims 1-3, and the enzyme preparation according to claim 9, in food, feed, coffee processing, drug development, pulp bleaching, oil extraction, detergents, biofuel production, and enzymatic preparation of mannan oligosaccharides.

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