High-salt-tolerant endo-beta-1,4-glucanase mutants and methods for making same

Abstract

The application discloses a high-concentration salt-resistant endo-beta-1,4-glucanase mutant and a preparation method thereof, wherein at least one amino acid in three positions of 39th, 137th and 183rd in the amino acid sequence shown in SEQ ID No. 1 is replaced; the aspartic acid Asp in the 39th position is mutated into threonine Thr, the glutamic acid Glu in the 137th position is mutated into asparagine Asn, and the aspartic acid Asp in the 183rd position is mutated into serine Ser. The mutation sites are determined through three-dimensional modeling of the wild-type endo-beta-1,4-glucanase, the obtained mutant can still maintain good catalytic efficiency and stability in the environment containing a large amount of salt, and the application of the endo-beta-1,4-glucanase in the high-concentration salt environment is optimized.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of enzyme engineering and genetic engineering, and particularly relates to a high-salt-tolerant endo-beta-1,4-glucanase mutant and a preparation method thereof. BACKGROUND

[0002] Cellulose is the main component of lignocellulosic biomass, and is the most widely distributed and most abundant polysaccharide on earth. As a linear polymer connected by beta-1,4-glucosidic bonds, cellulose is usually used as a raw material for producing soluble sugars, bioethanol and other important industrial chemicals. In the bioconversion technology using cellulose as a raw material, cellulase plays a core role in hydrolyzing cellulose into fermentable monosaccharides, which can be utilized by other microorganisms to convert into fuel ethanol and a variety of other high-value chemicals. In the paper 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 on cotton and flax fibers, improving fiber quality and softness; 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 for degrading cellulose is an efficient and environmentally friendly approach. Currently, various degradation processes have been developed, and various types of cellulases have been used to achieve effective utilization of biomass resources. Endo-beta-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 beta-1,4-glucosidic bonds of cellulose, reducing the crystallinity and degree of polymerization of cellulose, and producing cellulose oligosaccharides. These properties make endo-beta-1,4-glucanase have wide application value in the fields of energy, food, papermaking, washing, etc., leading to a significant increase in demand for endo-beta-1,4-glucanase, and thus the development of endo-beta-1,4-glucanase with more optimal properties, especially greater breakthroughs in pH and temperature. However, there are relatively few studies on salt-tolerant endo-beta-1,4-glucanase, which to some extent limits the development and utilization of cellulose resources. Salt-tolerant cellulase can exhibit good hydrolysis in harsh industrial environments and can be applied in the fields of agricultural waste treatment, sewage treatment, and treatment of various types of waste containing a large amount of salt. Therefore, it is of great significance and application value to construct beta-1,4-glucanase industrial strains with salt-tolerant properties. SUMMARY

[0004] The first object of the present application is to provide a salt-tolerant endo-beta-1,4-glucanase mutant with improved salt tolerance, and the second object of the present application is to provide a preparation method of the endo-beta-1,4-glucanase mutant.

[0005] Technical solution: The endo-beta-1,4-glucanase mutant resistant to high concentration of salt provided by the application is obtained by substituting at least one amino acid in the three positions of 39th, 137th and 183rd in the amino acid sequence shown in SEQ ID No. 1; the 39th aspartic acid Asp is mutated into threonine Thr, the 137th glutamic acid Glu is mutated into asparagine Asn, and the 183rd aspartic acid Asp is mutated into serine Ser.

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

[0007] The mutant is resistant to salt of 100-150 g / L.

[0008] The wild-type endo-beta-1,4-glucanase is from Clostridium cellulovorans , the amino acid sequence is SEQ ID NO. 1, and the gene sequence is SEQ ID NO. 2.

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

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

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

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

[0013] The mutant E137N / D183S is that the 137th glutamic acid Glu is mutated into asparagine Asn and the 183rd aspartic acid Asp is mutated into serine Ser.

[0014] The mutant D39T / E137N / D183S is that the 39th aspartic acid Asp is mutated into threonine Thr, the 137th glutamic acid Glu is mutated into asparagine Asn, and the 183rd aspartic acid Asp is mutated into serine Ser.

[0015] The nucleotide sequence provided by the application is a gene sequence encoding the endo-beta-1,4-glucanase mutant protein.

[0016] The vector provided by the application comprises the above-mentioned nucleotide sequence.

[0017] The vector preferably comprises a cloning vector or an expression vector. The vector is a plasmid or a virus.

[0018] The recombinant bacteria of the present application comprise the nucleotide sequence or the vector described above.

[0019] The host bacteria preferably comprise Escherichia coli.

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

[0021] (1) Designing point mutation primers, using the plasmid with the original endo-β-1, 4-glucanase gene as a template, and performing PCR reaction using the point mutation primers to obtain the point mutation gene sequence, using DpnI endonuclease to digest the template plasmid, and then performing recombination reaction on the DpnI digestion product;

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

[0023] (3) Collecting the host bacteria expressing the endo-β-1, 4-glucanase mutant, resuspending the bacterial bodies, breaking the cells, centrifuging to obtain the supernatant, and obtaining the crude enzyme liquid containing the endo-β-1, 4-glucanase mutant.

[0024] The host bacteria preferably comprise Escherichia coli. E. coli BL21 (DE3).

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

[0026]

[0027] Note: The underlined part in the primer is the mutation site, and "F" represents the upstream primer and "R" represents the downstream primer.

[0028] The system of the PCR reaction is:

[0029]

[0030] The conditions of the PCR reaction are:

[0031]

[0032] The system of the DpnI endonuclease digestion is:

[0033]

[0034] The conditions of the DpnI endonuclease digestion are: 37 °C for 2 h.

[0035] The recombination reaction system is:

[0036]

[0037] The conditions of the recombination reaction are: 37℃ reaction for 1 h.

[0038] Beneficial effects: compared with the prior art, the present application has the following remarkable advantages: (1) by determining the mutation sites of the endo-β-1, 4-glucanase from the source through three-dimensional modeling, the amino acid mutations of D39T, E137N and D183S are realized by using point mutation technology, and the mutant genes are introduced into the host bacteria to express the mutant enzymes, and the obtained mutants can still maintain good catalytic efficiency and stability in the environment containing a large amount of salt, thereby optimizing the application of the endo-β-1, 4-glucanase in the high-concentration salt environment; (2) the activity of the mutant D39T / E137N enzyme in the high-concentration salt solution (150 g / L MgCl2, 200 g / L NaCl or 100 g / L CaCl2) is increased by 7.72 times compared with the original enzyme. Clostridium BRIEF DESCRIPTION OF DRAWINGS cellulovorans

[0039] Figure 1 For Clostridium cellulovorans The protein structure diagram of the endo-β-1, 4-glucanase from the source and the schematic diagram of the 39th, 137th and 183rd amino acid sites;

[0040] Figure 2 The standard curve diagram for determining the enzyme activity by colorimetry;

[0041] Figure 3 The relative enzyme activity diagram of the original enzyme and the mutant enzyme in different salt solutions. DETAILED DESCRIPTION

[0042] The technical solutions of the present application will be further described below in combination with examples.

[0043] Example 1

[0044] The endo-β-1, 4-glucanase mutant D39T is obtained by mutating the 39th aspartic acid of the wild-type endo-β-1, 4-glucanase original enzyme sequence to threonine; the preparation method is as follows:

[0045] 1. Construction of recombinant plasmid

[0046] The plasmid pET-22b (purchased from Jinweizhi (Suzhou) Company), BL21 (DE3) (purchased from Shenzhen Kangti Life Science and Technology Co., Ltd.) and the endo-β-1, 4-glucanase from the source are used. E. coli Clostridium cellulovorans ​​The wild-type endo-β-1,4-glucanase gene was synthesized by Jinweizhi (Suzhou) Company. The primers for introducing mutation sites are shown in Table 1, the PCR reaction system is shown in Table 2, the template for point mutation PCR is pET-22b plasmid with the sequence of the wild-type endo-β-1,4-glucanase gene, and other components used for point mutation PCR are from MutUFO Fast Mutagenesis Kit (Nanjing Julong Biological). The PCR reaction conditions are shown in Table 3.

[0047] Table 1 D39T mutant point mutation primer

[0048]

[0049] Note: The underlined part in the primer is the mutation site, and “F” represents the upstream primer and “R” represents the downstream primer.

[0050] Table 2 PCR reaction system

[0051]

[0052] Table 3 PCR reaction conditions

[0053]

[0054] After the PCR, since the product contains the original template plasmid, in order to prevent the formation of false positive transformants in the subsequent transformation, DpnI is used for digestion. The digestion reaction system is shown in Table 4.

[0055] Table 4 DpnI digestion system

[0056]

[0057] The above reaction system is placed at 37°C for constant temperature reaction for 2 h.

[0058] After the DpnI digestion reaction is completed, the DpnI digestion product is subjected to recombination reaction, and the reaction system is shown in Table 5.

[0059] Table 5 Recombination reaction system

[0060]

[0061] The above reaction system is placed at 37°C for constant temperature reaction for 1 h, and the obtained product is the recombinant plasmid containing the designed point mutation, which is identified and confirmed by sequencing by Jinweizhi (Suzhou) Company.

[0062] 2. Expression of mutant enzyme and preparation of crude enzyme solution

[0063] The recombinant plasmid is transformed into E. coliBL21(DE3) host bacteria, and coated on the surface of LB solid medium containing 100 μg / mL ampicillin, 37°C overnight culture. E. coli single colony was picked and inoculated in 3 mL of LB liquid medium containing 100 μg / mL ampicillin, 37°C culture for 10 h. The seed liquid was inoculated into a 250 mL conical flask containing 30 mL of TB medium at a 5% inoculation amount, and cultured at 37°C, 200 rpm. When the culture reached 6 h, IPTG was added to a final concentration of 0.4 mM, and the culture temperature was set to 22°C, and the culture was continued for 18 h. The fermentation broth was centrifuged at 4000 rpm for 10 min, and the bacteria were resuspended with a pre-set high-concentration salt solution (150 g / L MgCl2, 200 g / L NaCl, 100 g / L CaCl2), and the cells were broken by ultrasonic for 5-8 min until the liquid was slightly clear. The supernatant was centrifuged at 4000 rpm for 10 min, and the supernatant was used as the crude enzyme solution of mutant D39T. The above strain culture and crude enzyme solution preparation experiment was carried out in triplicate.

[0064] Example 2

[0065] Endo-β-1, 4-glucanase mutant E137N, which is a mutant in which glutamic acid at position 137 of the original enzyme sequence of wild-type endo-β-1, 4-glucanase is mutated to asparagine; the preparation method is as follows:

[0066] The rest are the same as Example 1, except that the point mutation primer is as follows:

[0067] Table 6 E137N mutant point mutation primer

[0068]

[0069] Example 3

[0070] Endo-β-1, 4-glucanase mutant D183S, which is a mutant in which aspartic acid at position 183 of the original enzyme sequence of wild-type endo-β-1, 4-glucanase is mutated to serine; the preparation method of mutant D183S is as follows:

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

[0072] Table 7 D183S mutant point mutation primer

[0073]

[0074] Example 4

[0075] Endo β-1,4-glucanase mutant D39T / E137N, which is an endo β-1,4-glucanase mutant D39T sequence mutated at position 137 glutamic acid to asparagine; the preparation method is as follows:

[0076] The rest are the same as example 2, except that the template for point mutation PCR is pET-22b plasmid with endo β-1,4-glucanase mutant D39T.

[0077] Example 5

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

[0079] The rest are the same as example 2, except that the template for point mutation PCR is pET-22b plasmid with endo β-1,4-glucanase mutant E137N.

[0080] Example 6

[0081] Endo β-1,4-glucanase mutant D39T / E137N / D183S, which is an endo β-1,4-glucanase mutant D39T / E137N sequence mutated at position 183 aspartic acid to serine; the preparation method is as follows:

[0082] The rest are the same as example 3, except that the template for point mutation PCR is pET-22b plasmid with endo β-1,4-glucanase mutant D39T / E137N.

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

[0084] The bacteria extracted in examples 1-6 were resuspended with 150 g / L MgCl2, 200 g / L NaCl, and 150 g / L CaCl2, respectively, and ultrasonically broken. After centrifugation to obtain the supernatant, the enzyme activity was determined by DNS method.

[0085] The DNS enzyme activity determination method is to prepare 1.00, 0.80, 0.60, 0.40, and 0.20 mg / mL glucose solutions, respectively, take 150 μL into a clean EP tube, add 150 μL of DNS solution, boil in water bath for 10 min, measure the absorbance at 540 nm, and draw the standard curve, and the results are as follows: Figure 2The reaction was terminated by adding 150 μL DNS solution and boiling in a water bath for 10 min. After rapidly cooling to room temperature, the absorbance was measured at 540 nm after appropriate dilution with deionized water. In addition, 30 μL of the crude enzyme solution was added to 120 μL of deionized water, and the same DNS solution and treatment were used as a negative control. The enzyme activity was calculated according to the above method, 1 enzyme activity was defined as the amount of enzyme that catalyzed the decomposition of 1 μmol of reducing sugar in 1 min under the set reaction conditions, and the original enzyme activity was defined as 100%, and the relative enzyme activity of the mutant enzyme was further calculated, and the results are shown in Table 8 and Figure 3 as shown in Table 8.

[0086] Table 8 Endo-β-1, 4-glucanase mutant salt-tolerant activity

[0087]

[0088] As shown in Table 8 and Figure 3 Table 8, the enzyme activity of the six mutants of the enzyme was improved compared with the original enzyme in high-concentration salt solutions. In a 150 g / L MgCl2solution, the enzyme activity of mutant D39T / E137N was improved by 234% compared with the original enzyme; in a 100 g / L CaCl2solution, the enzyme activity of mutant D39T / E137N / D183S was improved by 293% compared with the original enzyme; and in a 200 g / L NaCl solution, the enzyme activity of mutant D39T / E137N was improved by 268%.

[0089] The above results show that the six mutations constructed in the present application improve 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 mutants are D39T, E137N, D183S, D39T / E137N, E137N / D183S, or D39T / E137N / D183S. Mutant D39T is formed by mutating aspartic acid (Asp) at position 39 of the amino acid sequence shown in SEQ ID No. 1 to threonine (Thr); mutant E137N is formed by mutating glutamic acid (Glu) at position 137 of the amino acid sequence shown in SEQ ID No. 1 to asparagine (Asn); mutant D183S is formed by mutating aspartic acid (Asp) at position 183 of the amino acid sequence shown in SEQ ID No. 1 to serine (Ser); mutant D39T / E137N is formed by mutating aspartic acid (Asp) at position 39 of the amino acid sequence shown in SEQ ID No. 1 to threonine (Thr) and glutamic acid (Glu) at position 137 to asparagine (Asn); mutant E137N / D183S ... of the amino acid sequence shown in SEQ ID No. 1 to asparagine (Asn); mutant E137N / D183S is formed by mutating aspartic acid (Asp) at position 39 of the amino acid sequence shown in SEQ ID No. 1 to threonine ( The amino acid sequence shown in SEQ ID No. 1 is modified by mutating glutamic acid Glu at position 137 to asparagine Asn and aspartic acid Asp at position 183 to serine Ser; the mutant D39T / E137N / D183S is modified by mutating aspartic acid Asp at position 39 to threonine Thr, glutamic acid Glu at position 137 to asparagine Asn, and aspartic acid Asp at position 183 to serine Ser in the amino acid sequence shown in SEQ ID No.

1.

2. The endo-β-1,4-glucanase mutant according to claim 1, characterized in that, The mutants are tolerant to salt concentrations of 100-200 g / L.

3. A polynucleotide, characterized in that, The polynucleotide is the gene sequence encoding the endo-β-1,4-glucanase mutant protein of claim 1.

4. A carrier, characterized in that, The vector comprises the polynucleotide of claim 3.

5. The carrier according to claim 4, characterized in that, The vector includes a cloning vector or an expression vector.

6. A recombinant bacterium, characterized in that, The recombinant bacteria comprises the polynucleotide of claim 3 or the vector of claim 4.

7. The microorganism according to claim 6, characterized in that, The host bacterium is Escherichia coli.

8. A method for preparing the endo-β-1,4-glucanase mutant according to claim 1, characterized in that, Includes the following steps: (1) Design point mutation primers, use the plasmid with the original endonuclease β-1,4-glucanase gene as a template, use the point mutation primers to perform PCR reaction to obtain the point mutation gene sequence, use DpnI endonuclease to digest the template plasmid, and then use the DpnI digest to perform recombination reaction. (2) The expression vector carrying the mutant gene is transferred into the host bacteria for fermentation expression; (3) Collect host bacteria expressing the endo-β-1,4-glucanase mutant, resuspend the bacterial cells, break the cells, centrifuge and collect the supernatant to obtain the crude enzyme solution containing the endo-β-1,4-glucanase mutant.

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