A kind of glucanase mutant and its application

Abstract

A dextranase mutant and its application, obtained by mutating the Asp57 and Asn114 sites of the dextranase PfGlu16C as the parent. The optimal temperature of the dextranase mutant is 5-10°C higher than that of the wild type, and the half-life at 55°C is extended by 77.8% and 37.0% respectively compared with the wild type. T 50 The values ​​were 10.7℃ and 2.2℃ higher than those of the wild type, respectively; in terms of catalytic efficiency, the two mutants were on par with the wild type, with no significant decrease. Compared with blind screening or artificial natural mutagenesis, enzyme molecule improvement shortens the time for enzyme property modification. Such a glucanase variant that remains stable in an acidic pH environment and in a medium to low temperature range and has high enzyme activity has been subjected to bran degradation experiments, showing that it has a significant effect on the hydrolysis efficiency of bran, which meets the requirements of feed addition.

Description

Technical Field

[0001] The present invention relates to the field of gene engineering and genetic engineering, and in particular to a 16th family glucanase mutant with improved thermal stability and application thereof. Background Art

[0002] Cellulose, hemicellulose and lignin are the main components of plant cell walls. Cellulose accounts for about 40%-45% of the cell dry weight and is a linear molecule formed by glucose connected by β-1,4 and β-1,3 glycosidic bonds. Hemicellulose (mainly including xylan, glucan and mannan) accounts for about 30%-35% of the cell stem and is the most abundant renewable biomass resource after cellulose. It is composed of heteropolysaccharides and is named according to the main chain composition type as glucan, mannan and glucomannan (Schulze E 1891. Ber Dtsch Chem Ges 24, 2277-2287). β-glucan is a structural non-starch polysaccharide in the cell walls of monocotyledonous grasses, mainly present in the endosperm and endosperm cells. For example, the endosperm cell walls of barley and oats contain about 70%-75% β-glucanase (Philippe S et al., 2016. Planta 224(2), 449-461).

[0003] β-glucanase is a general term for a class of enzymes that can decompose glucan polymers composed of β-glucosidic bonds. According to different modes of action, it can be divided into two types: endo-type and exo-type. Among them, β-1,3-1,4-glucanase (EC 3.2.1.73) can specifically act on β-1,4 glycosidic bonds connected to β-1,3 bonds, degrading them into low molecular weight fragments, losing hydrophilicity and viscosity, reducing the viscosity of intestinal contents of monogastric animals, increasing endogenous digestive enzyme activity, improving intestinal microbial environment, and improving growth performance and feed conversion rate (Mathlouthi N et al. 2002. Amin Res 51, 395-406.). It is widely used in food brewing, especially in the beer brewing process. The glucan in malt makes beer filtration difficult and blocks the filter membrane, increasing the production cost and quality of beer. The synergistic effect of acid glucanase and xylanase can solve the above problems. Therefore, the production, purification, thermal stability, catalytic efficiency and structural basis of acidic glucanase and its application in feed processing, brewing industry, juice processing and energy are being studied in depth. Since different industries have different requirements for the properties of glucanase, it is still of great significance to improve the research of glucanase with application potential. Summary of the invention

[0004] Technical problem to be solved: The present invention provides a 16th family thermostable glucanase mutant and its application. This type of mutant is an acidic glucanase mutant obtained by site-directed mutation of the amino acids of the wild-type glucanase. This type of mutant can withstand high-temperature treatment above 70°C and has very high hemicellulase activity under acidic pH conditions, especially in feed, beer brewing and food industries. It has great application potential.

[0005] Technical solution: A glucanase mutant is obtained by taking the glucanase PfGlu16C as the parent and mutating the Asp57 or Asn114 sites respectively.

[0006] The gene sequence of the parent PfGlu16C is shown in SEQ ID NO: 1. The amino acid sequence is shown in SEQ ID NO: 4.

[0007] The mutant D57N was obtained after the Asp57 site mutation, and the gene sequence is shown in SEQ ID NO: 2. The amino acid sequence is shown in SEQ ID NO: 5.

[0008] The mutant N144L was obtained after the Asn114 site mutation, and the gene sequence is shown in SEQ ID NO: 3. The amino acid sequence is shown in SEQ ID NO: 6.

[0009] A recombinant vector contains the above gene sequence.

[0010] A recombinant bacterial strain contains the above recombinant vector.

[0011] Beneficial effects: Compared with the prior art, the present invention provides a glucanase mutant with excellent thermal stability suitable for use in hemicellulose degradation. The optimal temperature of the glucanase mutant is 5-10°C higher than that of the wild type, and the half-life at 55°C is extended by 77.8% and 37.0% respectively compared with the wild type. 50 The values ​​were 10.7℃ and 2.2℃ higher than those of the wild type, respectively; in terms of catalytic efficiency, the two mutants were on par with the wild type, with no significant decrease. Compared with blind screening or artificial natural mutagenesis, enzyme molecule improvement shortens the time for enzyme property modification. Such a glucanase variant that remains stable in an acidic pH environment and in a medium to low temperature range and has high enzyme activity has been subjected to bran degradation experiments, showing that it has a significant effect on the hydrolysis efficiency of bran, which meets the requirements of feed addition. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 is the optimal pH for the wild type and the two mutants;

[0013] Figure 2 pH stability of the wild type and two mutants;

[0014] Figure 3 is the optimum temperature for the wild type and the two mutants;

[0015] Figure 4 is the half-life of the wild type and two mutants at 55°C 1 / 2 ;

[0016] Figure 5 T of the wild type and two mutants 50 value. DETAILED DESCRIPTION

[0017] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments.

[0018] 1. Strains and vectors: The expression host Pichia pastoris GS115 was purchased from Invitrogen.

[0019] 2. Enzymes and other biochemical reagents: High-fidelity polymerase was purchased from Fermentas, and barley glucan was purchased from Sigma. Other reagents were domestic analytical grade reagents (all available from ordinary biochemical reagent companies).

[0020] 3. Culture medium:

[0021] 1) YPD medium: 2% glucose, 2% peptone, 1% yeast extract;

[0022] 2) LB medium: 1% peptone, 0.5% yeast extract, 1% NaCl, 1% agar powder (solid);

[0023] 3) MD medium: 1.5% agarose, 2% glucose, 0.00004% Biotin, 1.34% YNB;

[0024] 5) BMGY medium: 2% peptone, 1% yeast extract, 1% glycerol (V / V), 0.00004%, Biotin1.34% YNB;

[0025] 6) BMMY medium: 2% peptone, 1% yeast extract, 1.34% YNB, 0.5% methanol (V / V), 0.00004% Biotin.

[0026] Example 1 Obtaining genes encoding thermostable glucanase mutants

[0027] Using the recombinant expression vector pic9r_PfGlu16c of the glucanase gene PfGlu16c from Pseudocercospora fijiensis as a template, site-directed mutagenesis was performed on the D57 and N144 sites respectively. The primers designed are shown in Table 1. The mutation method and cloning method refer to the literature (You, et al., 2016).

[0028] Table 1 Primers for site-directed mutagenesis of xylanase HwXyl10A

[0029]

[0030] Example 2 Preparation of thermostable glucanase mutants

[0031] The linear recombinant expression vector obtained by PCR was directly transformed into DMT competent cells, and colony PCR was used for verification to obtain the nucleic acid sequence of the mutant. The recombinant plasmid was linearized and transformed into Pichia pastoris Gs115 to obtain the recombinant yeast strains Gs115 / D57N and Gs115 / N144L.

[0032] The Gs115 strain containing the recombinant plasmid was inoculated into a 10 mL test tube containing 2 mL of BMGY medium, and cultured in a shaking incubator at 30°C and 220 rpm for 48 h. The culture solution was centrifuged at 3000 g for 5 min, the supernatant was discarded, and the precipitate was resuspended in 2 mL of BMMY medium containing 0.5% methanol, and then induced again at 30°C and 220 rpm for 48 h. The supernatant was used for enzyme activity detection, and mutants D57N and N144L were screened, which had improved thermal stability and catalytic activity compared with the wild enzyme.

[0033] The wild-type strain GS115 / PfGlu16C and two mutant strains Gs115 / D57N and Gs115 / N144L were enlarged into a fermentation system. First, the seed culture was inoculated in YPD medium, and then inoculated into a 1L Erlenmeyer flask with 300mL BMGY medium at a 1% inoculum, and placed at 30°C, 220rpm shaking for 48h; then the culture was centrifuged at 3000g for 5min, the supernatant was discarded, and the precipitate was resuspended in 100mL BMMY medium containing 0.5% methanol, and then placed at 30°C, 220rpm for induction culture again. 0.5mL of methanol was added every 12h to keep the methanol concentration in the bacterial solution at 0.5%, and the supernatant was taken for enzyme activity detection. Finally, the supernatant was concentrated to 20mL, and the protein was purified by anion exchange method for enzymatic property determination and comparison.

[0034] Example 3 Comparative analysis of enzymatic properties of recombinant thermostable glucanase mutants and wild type

[0035] 1. DNS method: The specific method is as follows: Under the optimal pH and temperature conditions, 1 mL of the reaction system includes 100 μL of diluted enzyme solution and 900 μL of substrate. The reaction lasts for 10 minutes. 1.5 mL of DNS is added to terminate the reaction and the reaction is boiled in water for 5 minutes. After cooling, the OD value is measured at 540 nm. One unit of enzyme activity (U) is defined as the amount of enzyme required to decompose glucan to produce 1 μmol of reducing sugar per minute under given conditions.

[0036] 2. Determination of properties of recombinant thermostable glucanase mutants and wild type

[0037] 1. The optimum pH and pH stability of the recombinant thermostable glucanase mutant and wild type are determined as follows:

[0038] The purified xylanase mutants and wild type were subjected to enzymatic reactions at different pH values ​​(1.0-6.5) to determine their optimal pH. The substrate xylan was used to determine the xylanase activity at 45°C in 0.1 mol / L citric acid-sodium hydrogen phosphate buffer at different pH values. Figure 1 As shown in Figure 2, the optimal reaction pH of the wild type and mutants is similar between 4.0-4.5; the residual enzyme activity of the wild type glucanase and mutants was measured after being treated at 37°C for 1 hour in an environment of pH 1.0-12.0. The results are shown in Figure 2. Figure 2 As shown, there is no significant difference in pH stability between the wild type and the two mutants, both of which are stable between pH 3.0 and pH 10.0.

[0039] 2. The optimum temperature determination method for wild-type glucanase and mutants is as follows:

[0040] The optimum temperature of the recombinant thermostable glucanase mutant and wild type was determined by performing an enzymatic reaction in a 0.1 mol / L citric acid-sodium hydrogen phosphate buffer (pH 4.5) buffer system at different temperatures (30-70°C). Figure 3 The results showed that the optimum temperatures of the thermostable glucanase mutants D57N and N144L were 10℃ and 5℃ higher than that of the wild type (45℃), respectively, and the relative enzyme activities of the two mutants at high temperatures were significantly higher than those of the wild enzyme.

[0041] 3. The thermal stability of wild-type glucanase and mutants was determined as follows:

[0042] Half-life at 55℃ (t 1 / 2 ): The mutant and wild type were treated at 55°C for different time periods, up to 120 min, and their respective residual enzyme activities were detected. The half-life determination results at 55°C are as follows: Figure 4 The results showed that the t 1 / 2The thermal stability of mutant D57N was the best, which was 96 minutes and 74 minutes respectively, which were 77.8% and 37.0% longer than that of the wild type (54 minutes).

[0043] T 50 The mutant and wild-type enzymes were treated at 40-80°C for half an hour. The temperature corresponding to when the enzyme activity remained half of the original was the T of the enzyme. 50 The result is as follows Figure 5 The results showed that the T 50 The values ​​were 69.5℃ and 61.0℃, respectively, which were 10.7℃ and 2.2℃ higher than the wild type (58.8℃), and the results were consistent with the trend of the half-life determination results. That is, the order of thermal stability is: D57N>N144L>PfGlu16C.

[0044] 4. The method for determining the kinetic parameters of the recombinant high specific activity thermostable xylanase mutant and wild type is as follows:

[0045] The detection method was referred to the literature (You, et al., 2018) to determine the first-order reaction time of the reaction. m and V max The reaction time was 5 min. Different concentrations of xylan (1.25, 1.0, 0.8, 0.4, 0.2, 0.15 and 0.1 mg / mL) were used as substrates to measure the enzyme activity under the optimal conditions (temperature, pH), and the corresponding reaction rate was calculated. K was calculated using GraFit7 software. m Value and V max .

[0046] The catalytic efficiency (k cat / K m ) were 600 mL / s·mg and 380 mL / s·mg, respectively. The catalytic efficiency of mutant D57N was increased by 58% compared with the wild type (380 mL / s·mg); the specific activities of D57N and N144L were 2700 U / mg and 2100 U / mg, respectively, which had no significant difference with the wild type (Table 2).

[0047] Table 2 Specific activities and kinetic parameters of xylanase mutants and wild type

[0048]

Claims

1. A glucanase mutant, characterized in that The mutants were obtained by mutating the Asp57 or Asn114 sites of the glucanase PfGlu16C as the parent, wherein the Asp57 site was mutated to obtain the mutant D57N, and the amino acid sequence was shown in SEQ ID NO:5; the Asn114 site was mutated to obtain the mutant N144L, and the amino acid sequence was shown in SEQ ID NO:

6.

2. The glucanase mutant according to claim 1, characterized in that When the Asp57 site mutates, the mutant D57N is obtained, and the gene sequence is shown in SEQ ID NO:2; when the Asn114 site mutates, the mutant N144L is obtained, and the gene sequence is shown in SEQ ID NO:

3.

3. A recombinant vector, characterized in that: Contains the gene sequence according to claim 2.

4. A recombinant strain, characterized in that: Contains the recombinant vector according to claim 3.

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