Endo-beta-1, 4-glucanase mutant resistant to high-concentration salt and preparation method of endo-beta-1, 4-glucanase mutant

Abstract

The invention discloses an internally tangent beta-1, 4-glucanase mutant resistant to high-concentration salt and a preparation method of the internally tangent beta-1, 4-glucanase mutant. According to the mutant, at least one amino acid in the 39 site, the 137 site and the 183 site of an amino acid sequence shown in SEQ ID No.1 is replaced; aspartic acid Asp at the 39th site is mutated into threonine Thr, glutamic acid Glu at the 137th site is mutated into asparagine Asn, and aspartic acid Asp at the 183rd site is mutated into serine Ser. A mutation site is determined by performing three-dimensional modeling on the wild type internally-cut beta-1, 4-glucanase, the obtained mutant can still keep relatively good catalytic efficiency and stability in an environment containing a large amount of salt, and the application of the internally-cut beta-1, 4-glucanase in a high-concentration salt environment is optimized.

Description

Technical Field

[0001] The invention relates to the technical fields of enzyme engineering and genetic engineering, and in particular to a high-concentration salt-resistant endo-beta-1,4-glucanase mutant and a preparation method thereof. Background Art

[0002] Cellulose is the main component of lignocellulosic biomass and is the most widely distributed and abundant polysaccharide on earth. As a linear polymer connected by β-1,4-glucosidic bonds, cellulose is often used as a raw material for the production of soluble sugars, bioethanol and other important industrial chemicals. In the bioconversion technology using cellulose as raw material, cellulase plays a core role in hydrolyzing cellulose into fermentable monosaccharides, which can be used by other microorganisms to convert into fuel ethanol and a variety of other high value-added chemicals. In the papermaking industry, the use of cellulase to treat pulp can remove small particles and hemicellulose that affect quality, and improve paper strength, freedom and whiteness. In the textile industry, cellulase can help remove impurities from cotton and linen fibers and improve the quality and softness of fibers; in the food industry, it can be used to prepare low-calorie foods or improve the taste and shelf life of products such as bread and dairy products.

[0003] Using microorganisms to produce enzymes to degrade cellulose is an efficient and environmentally friendly approach. Currently, a variety of degradation processes have been developed, and various types of cellulases have been used to achieve effective utilization of biomass resources. Among them, endo-β-1,4-glucanase, also known as endo-cellulase, is an important component of the cellulase system. It acts on the non-crystalline region of cellulose and can randomly hydrolyze the β-1,4-glucosidic bonds of cellulose, reduce the crystallinity and polymerization degree of cellulose, and produce cellooligosaccharides. These characteristics make endo-β-1,4-glucanase have a wide range of application value in the fields of energy, food, papermaking, washing, etc., resulting in a significant increase in the demand for endo-β-1,4-glucanase, and further research on endo-β-1,4-glucanase with better properties, especially with greater breakthroughs in pH and temperature. However, there are relatively few studies involving salt-tolerant endo-β-1,4-glucanase, which to a certain extent limits the development and utilization of cellulose resources. Salt-tolerant cellulase can show good hydrolysis in harsh industrial environments and can be used in agricultural waste treatment, sewage treatment, and various wastes containing a large amount of salt. Therefore, it is of great significance and application value to construct industrial strains of β-1,4-glucanase with salt tolerance. Summary of the invention

[0004] Purpose of the invention: The first purpose of the present invention is to provide an endo-β-1,4-glucanase mutant with improved salt tolerance; the second purpose of the present invention is to provide a method for preparing the endo-β-1,4-glucanase mutant.

[0005] Technical solution: The high-salt-resistant endo-β-1,4-glucanase mutant of the present invention is a mutant in which at least one amino acid is replaced at positions 39, 137 and 183 of the amino acid sequence shown in SEQ ID No.1; the aspartic acid Asp at position 39 is mutated to threonine Thr, the glutamic acid Glu at position 137 is mutated to asparagine Asn, and the aspartic acid Asp at position 183 is mutated to serine Ser.

[0006] Preferably, the mutant comprises D39T, E137N, D183S, D39T / E137N, E137N / D183S or D39T / E137N / D183S.

[0007] The mutant has a salt tolerance of 100 to 150 g / L.

[0008] The wild-type endo-β-1,4-glucanase is from Clostridium cellulovorans, and has an amino acid sequence of SEQ ID NO.1 and a gene sequence of SEQ ID NO.2.

[0009] The mutant D39T, that is, the 39th aspartic acid Asp is mutated to threonine Thr.

[0010] The mutant E137N, that is, the 137th glutamic acid Glu is mutated to asparagine Asn.

[0011] The mutant D183S, that is, the 183rd aspartic acid Asp is mutated to serine Ser.

[0012] The mutant D39T / E137N, that is, the aspartic acid Asp at position 39 is mutated to the threonine Thr, and the glutamic acid Glu at position 137 is mutated to the asparagine Asn.

[0013] In the mutant E137N / D183S, the glutamic acid Glu at position 137 is mutated to asparagine Asn and the aspartic acid Asp at position 183 is mutated to serine Ser.

[0014] The mutant D39T / E137N / D183S, namely, the aspartic acid Asp at position 39 is mutated to threonine Thr, the glutamic acid Glu at position 137 is mutated to asparagine Asn and the aspartic acid Asp at position 183 is mutated to serine Ser.

[0015] The nucleotide sequence of the present invention is a gene sequence encoding the above-mentioned endo-β-1,4-glucanase mutant protein.

[0016] The vector of the present invention comprises the above-mentioned nucleotide sequence.

[0017] Preferably, the vector includes a cloning vector or an expression vector. The vector is a plasmid or a virus.

[0018] The recombinant bacteria of the present invention comprises the above-mentioned nucleotide sequence or the above-mentioned vector.

[0019] Preferably, the host bacteria is Escherichia coli.

[0020] The method for preparing the β-1,4-glucanase mutant of the present invention comprises the following steps:

[0021] (1) Designing point mutation primers, using the plasmid carrying the original endoglucanase gene as a template, using the point mutation primers to perform PCR reaction to obtain the gene sequence of the point mutation, using the DpnI endonuclease to digest the template plasmid, and then performing a recombination reaction on the DpnI digest;

[0022] (2) transferring the expression vector carrying the mutant gene into the host bacteria for fermentation expression;

[0023] (3) collecting host bacteria expressing the endo-β-1,4-glucanase mutant, resuspending the bacteria and breaking the cells, and centrifuging and collecting the supernatant to obtain a crude enzyme solution containing the endo-β-1,4-glucanase mutant.

[0024] Preferably, the host bacteria is E. coli BL21 (DE3).

[0025] In step (1), the sequence of the point mutation primer includes:

[0026]

[0027]

[0028] Note: The underlined part in the primers indicates the mutation site, “F” represents the upstream primer and “R” represents the downstream primer.

[0029] The system of the PCR reaction is:

[0030]

[0031] The conditions for the PCR reaction were:

[0032]

[0033] The system for DpnI endonuclease digestion is:

[0034]

[0035] The conditions for DpnI endonuclease digestion were: 37°C for 2h.

[0036] The recombination reaction system is:

[0037]

[0038] The conditions for the recombination reaction were: 37°C for 1 h.

[0039] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: (1) The mutation site is determined by three-dimensional modeling of the endo-β-1,4-glucanase from Clostridium cellulovorans, and the D39T, E137N, and D183S amino acid mutations are achieved by using point mutation technology, and the mutant genes are introduced into the host bacteria to express the mutant enzyme body. The obtained mutant can still maintain good catalytic efficiency and stability in an environment containing a large amount of salt, thereby optimizing the application of endo-β-1,4-glucanase in a high-concentration salt environment; (2) The activity of the mutant D39T / E137N enzyme in a high-concentration salt solution (150 g / LMgCl2, 200 g / L NaCl or 100 g / L CaCl2) is 7.72 times higher than that of the original enzyme. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 The protein structure diagram of the endo-β-1,4-glucanase from Clostridium cellulovorans and the schematic diagram of the amino acid sites 39, 137, and 183;

[0041] Figure 2 This is the standard curve for the colorimetric determination of enzyme activity;

[0042] Figure 3 The relative enzyme activities of the original enzyme and the mutant enzyme in different salt solutions. DETAILED DESCRIPTION

[0043] The technical solution of the present invention is further described below in conjunction with embodiments.

[0044] Example 1

[0045] Endo-β-1,4-glucanase mutant D39T, wherein the 39th aspartic acid in the original enzyme sequence of the wild-type endo-β-1,4-glucanase is mutated to threonine; the preparation method is as follows:

[0046] 1. Construction of recombinant plasmid

[0047] Plasmid pET-22b (purchased from Jinweizhi (Suzhou) Company), E. coli BL21 (DE3) (purchased from Shenzhen Kangti Life Science Co., Ltd.). The wild-type endo-β-1,4-glucanase gene from Clostridium cellulovorans was synthesized by Jinweizhi (Suzhou) Company. The primers used to introduce mutation sites are shown in Table 1, the PCR reaction system is shown in Table 2, the template for point mutation PCR is the pET-22b plasmid with the wild-type endo-β-1,4-glucanase gene sequence, and the other components used in point mutation PCR are all from MutUFO Fast Mutagenesis Kit (Nanjing Jujiang Biological). The PCR reaction conditions are shown in Table 3.

[0048] Table 1 Primers for point mutation of D39T mutant

[0049]

[0050] Note: The underlined part in the primers indicates the mutation site, “F” represents the upstream primer and “R” represents the downstream primer.

[0051] Table 2 PCR reaction system

[0052]

[0053] Table 3 PCR reaction conditions

[0054]

[0055] After the PCR was completed, since the product contained the original template plasmid, it was digested with DpnI to prevent it from forming false positive transformants in subsequent transformation. The reaction system for digestion is described in Table 4.

[0056] Table 4 DpnI digestion system

[0057]

[0058] The above reaction system was placed in a constant temperature reaction at 37°C for 2 hours.

[0059] After the DpnI digestion reaction was completed, the DpnI digestion product was subjected to a recombination reaction, and the reaction system was as described in Table 5.

[0060] Table 5 Recombination reaction system

[0061]

[0062] The reaction system was placed in a constant temperature reaction at 37°C for 1 hour. The resulting product was a recombinant plasmid containing the designed point mutation, which was sequenced and confirmed by GENEWIZ (Suzhou) Co., Ltd.

[0063] 2. Expression of mutant enzymes and preparation of crude enzyme solution

[0064] Transform the recombinant plasmid into the host bacteria E.coli BL21 (DE3), and spread it on the surface of LB solid culture medium containing 100μg / mL ampicillin, and culture it at 37℃ overnight. Pick a single colony of E. coli and inoculate it into 3mL LB liquid culture medium containing 100μg / mL ampicillin, and culture it at 37℃ for 10h. Inoculate the seed liquid with a 5% inoculum into a 250mL conical flask containing 30mL TB culture medium, and culture it at 37℃ and 200rpm. When the culture reaches 6h, add IPTG to a final concentration of 0.4mM, set the culture temperature to 22℃, and continue to culture for 18h. The fermentation broth was centrifuged at 4000 rpm for 10 min, the bacteria were collected, and resuspended with a preset high concentration salt solution (150 g / L MgCl2, 200 g / L NaCl, 100 g / L CaCl2), and the cells were ultrasonically disrupted for 5 to 8 minutes until the liquid became slightly clear. The supernatant was taken as the crude enzyme solution of mutant D39T after centrifugation at 4000 rpm for 10 min. The above strain culture and crude enzyme solution preparation experiments were carried out in triplicate.

[0065] Example 2

[0066] The endo-β-1,4-glucanase mutant E137N is obtained by mutating the 137th glutamic acid in the original enzyme sequence of the wild-type endo-β-1,4-glucanase to asparagine; the preparation method is as follows:

[0067] The rest are the same as Example 1, except that the point mutation primers are as follows:

[0068] Table 6 Primers for point mutation of E137N mutant

[0069]

[0070] The endo-β-1,4-glucanase mutant D183S is obtained by mutating the 183rd aspartic acid in the original enzyme sequence of the wild-type endo-β-1,4-glucanase to serine; the preparation method of the mutant D183S is as follows:

[0071] The rest is the same as Example 1, except that the point mutation primers are as follows:

[0072] Table 7D183S mutant point mutation primers

[0073]

[0074] Example 4

[0075] Endo-β-1,4-glucanase mutant D39T / E137N, wherein the 137th glutamic acid in the sequence of the endo-β-1,4-glucanase mutant D39T is mutated to asparagine; the preparation method is as follows:

[0076] The rest is the same as Example 2, except that the template of the point mutation PCR is the pET-22b plasmid carrying the endonuclease mutant D39T.

[0077] Example 5

[0078] Endo-β-1,4-glucanase mutant E137N / D183S, wherein the 183rd aspartic acid Asp of the sequence of the endo-β-1,4-glucanase mutant E137N is mutated to serine Ser; the preparation method is as follows:

[0079] The rest is the same as Example 2, except that the template of the point mutation PCR is the pET-22b plasmid carrying the endonuclease mutant E137N.

[0080] Example 6

[0081] Endo-β-1,4-glucanase mutant D39T / E137N / D183S, wherein the 183rd aspartic acid of the endo-β-1,4-glucanase mutant D39T / E137N is mutated to serine; the preparation method is as follows:

[0082] The rest is the same as Example 3, except that the template of the point mutation PCR is the pET-22b plasmid carrying the endoglucanase mutant D39T / E137N.

[0083] Performance test: Determination of enzyme activity of original enzyme and mutant enzyme in high concentration salt solution

[0084] The cells extracted in Examples 1 to 5 were resuspended in 150 g / L MgCl2, 200 g / L NaCl, and 150 g / L CaCl2, respectively, and ultrasonically disrupted. The supernatant was obtained by centrifugation and the enzyme activity was determined using the DNS method.

[0085] The method for determining DNS enzyme activity is to prepare 1.00, 0.80, 0.60, 0.40, and 0.20 mg / mL glucose solutions, take 150 μL into a clean EP tube, add 150 μL DNS solution, boil in water for 10 min, measure the absorbance at 540 nm and draw a standard curve. The results are as follows: Figure 2As shown. Add 30μL crude enzyme solution and 120μL 1% cellulose solution to the EP tube, react at 60℃ for 30min, quickly cool to room temperature, add 150μL DNS solution to terminate the reaction, and boil in a boiling water bath for 10min. After quickly cooling to room temperature, dilute appropriately with deionized water, and measure the absorbance at 540nm. In addition, take 30μL crude enzyme solution and add 120μL deionized water, using the same DNS solution and treatment method as a negative control. The enzyme activity was calculated according to the above method. 1 enzyme activity is defined as the amount of enzyme that catalyzes the decomposition of 1μmol of reducing sugar in 1 minute under the set reaction conditions. The original enzyme activity is defined as 100%, and the relative enzyme activity of the mutant enzyme is further calculated. The results are shown in Tables 8 and Figure 3 shown.

[0086] Table 8 Salt tolerance activity of endo-β-1,4-glucanase mutants

[0087]

[0088] From Table 8 and Figure 3 The results showed that the enzyme activity of the five mutants of the enzyme was improved compared with the original enzyme in high-concentration salt solutions. In 150 g / L MgCl2 solution, the enzyme activity of mutant D39T / E137N was 234% higher than that of the original enzyme; in 100 g / L CaCl2 solution, the enzyme activity of mutant D39T / E137N / D183S was 293% higher than that of the original enzyme; in 200 g / L NaCl solution, the enzyme activity of mutant D39T / E137N was 268% higher.

[0089] The above results show that the five mutations constructed in the present invention enhance the enzyme activity of the endo-β-1,4-glucanase in high concentrations of sodium ions, magnesium ions, and calcium ions.

Claims

1. A high-salt-tolerant endo-β-1,4-glucanase mutant, characterized in that: The mutant is a mutant in which at least one amino acid is replaced in the three positions of position 39, position 137 and position 183 of the amino acid sequence shown in SEQID No.1; the aspartic acid Asp at position 39 is mutated to threonine Thr, the glutamic acid Glu at position 137 is mutated to asparagine Asn, and the aspartic acid Asp at position 183 is mutated to serine Ser.

2. The endo-β-1,4-glucanase mutant according to claim 1, characterized in that The mutants include D39T, E137N, D183S, D39T / E137N, E137N / D183S or D39T / E137N / D183S.

3. The endo-β-1,4-glucanase mutant according to claim 1, characterized in that The mutant has a salt tolerance of 100 to 200 g / L.

4. A nucleotide sequence, characterized in that The nucleotide sequence is a gene sequence encoding the endo-β-1,4-glucanase mutant protein according to claim 1 or 2.

5. A carrier, characterized in that The vector comprises the nucleotide sequence of claim 4.

6. The carrier according to claim 5, characterized in that The vector includes a cloning vector or an expression vector.

7. A recombinant bacterium, characterized in that: The recombinant bacterium comprises the nucleotide sequence of claim 4 or the vector of claim 5.

8. The heavy bacteria according to claim 7, characterized in that: The host bacteria is Escherichia coli.

9. A method for preparing the endo-β-1,4-glucanase mutant according to claim 1, characterized in that: The steps include: (1) Designing point mutation primers, using the plasmid carrying the original endoglucanase gene as a template, using the point mutation primers to perform PCR reaction to obtain the gene sequence of the point mutation, using the DpnI endonuclease to digest the template plasmid, and then performing a recombination reaction on the DpnI digest; (2) transferring the expression vector carrying the mutant gene into the host bacteria for fermentation expression; (3) collecting host bacteria expressing the endo-β-1,4-glucanase mutant, resuspending the bacteria and breaking the cells, and centrifuging and collecting the supernatant to obtain a crude enzyme solution containing the endo-β-1,4-glucanase mutant.

10. The preparation method according to claim 9, characterized in that: In step (1), the sequence of the point mutation primer includes:

Images