Pullulanase mutant with acid-base tolerance and application thereof

Abstract

The invention belongs to the field of gene engineering, and particularly relates to a pullulanase mutant with acid-base tolerance and application of the pullulanase mutant. On the basis of parent pullulanase with an amino acid sequence as shown in SEQ ID NO.1, lysine at the 205th site, lysine at the 288th site and lysine at the 387th site are mutated into phenylalanine, and the pullulanase mutant is obtained; the pullulanase mutant has better enzyme activity than parent pullulanase when the pH value is 2.0-10.0 and the temperature is 30-90 DEG C, has better thermal stability and acid-base tolerance, can be widely applied to industrial production, and has a wide application prospect.

Description

Technical Field

[0001] The invention belongs to the field of enzyme engineering, and particularly relates to a pullulanase mutant with acid-base tolerance and application thereof. Background Art

[0002] Pullulanase is a type of starch debranching enzyme, named for its ability to specifically hydrolyze pullulan (a polymer of maltotriose connected by α-1,6 glycosidic bonds). It belongs to the amylase class and can specifically cut the α-1,6 glycosidic bonds in the branch points of amylopectin, cutting off the entire branch structure to form straight-chain starch.

[0003] However, the existing pullulanases still have many defects. First, the more significant defect is low enzyme activity. Different researchers expressed pullulanases from different sources (Bacillus acidopullulyticus, Bacillus deramificans, Bacillus naganoensis) in Escherichia coli, but their enzyme activities were far from meeting the requirements of industrial production. Second, the thermal stability is poor. For example, the pullulanases from Shewanella arctica have a half-life of only 20 minutes at 40°C. The pullulanases from Bacillus megaterium can only have 40% of residual enzyme activity after being treated at 40°C, while the process of starch hydrolysis often requires a certain temperature (about 40°C). Third, the enzyme has poor acid resistance. The saccharification process of starch is carried out in the range of pH 4.5-5.0, and most types of pullulanases have very low activity within this range.

[0004] At present, the improvement of pullulanase by genetic engineering technology mainly focuses on the improvement of heat resistance and acid-base tolerance. However, the pullulanase obtained so far still has the disadvantages of poor heat stability, poor acid-base tolerance, and low enzyme activity, and cannot be widely used in the hydrolysis of pullulan polysaccharides under various pH values ​​and different temperature conditions.

[0005] Therefore, how to obtain a pullulanase with good thermal stability and acid-base tolerance is of great importance for industrial production. The present invention unexpectedly found that a pullulanase mutant was obtained by simultaneously mutating the lysine at positions 205, 288 and 387 in the sequence of the parent pullulanase (amino acid sequence as shown in SEQ ID NO.1) to phenylalanine, and the pullulanase mutant had better enzyme activity than the parent pullulanase at pH 2.0-10.0 and temperature between 30°C and 90°C, and had better thermal stability and acid-base tolerance. Summary of the invention

[0006] In view of the above technical problems, the object of the present invention is to provide a pullulanase and its application, which specifically include the following contents:

[0007] In a first aspect, the present invention provides a pullulanase mutant, wherein the pullulanase mutant is obtained by simultaneously mutating the lysine at positions 205, 288 and 387 of a parent pullulanase with an amino acid sequence as shown in SEQ ID NO.1 to phenylalanine.

[0008] Preferably, the amino acid sequence of the pullulanase mutant is shown as SEQ ID NO.3.

[0009] In a second aspect, the present invention provides a use of the pullulanase mutant described in the first aspect in a product for enzymatically hydrolyzing a polysaccharide containing α-1,6 glycosidic bonds.

[0010] Preferably, the polysaccharide is pullulan.

[0011] In a third aspect, the present invention provides a gene encoding a pullulanase mutant, wherein the gene sequence is shown as SEQ ID NO.4.

[0012] In a fourth aspect, the present invention provides a recombinant expression plasmid / vector containing the pullulanase mutant gene described in the second aspect.

[0013] In a fifth aspect, the present invention provides a host cell containing the pullulanase mutant gene described in the second aspect above.

[0014] Preferably, the host cell is a bacterium or a fungus.

[0015] Preferably, the bacteria is Escherichia coli.

[0016] In a sixth aspect, the present invention provides a method for preparing the pullulanase mutant according to the first aspect, the method comprising:

[0017] The host cell described in the fifth aspect is inoculated into a fermentation medium for fermentation, and the fermentation broth is collected and centrifuged to separate and obtain the pullulanase mutant described in the first aspect.

[0018] The beneficial effects of the present invention are as follows: the present invention provides a pullulanase mutant, wherein the pullulanase mutant is obtained by simultaneously mutating the lysine at positions 205, 288 and 387 in the sequence of a parent pullulanase (amino acid sequence as shown in SEQ ID NO.1) to phenylalanine; the pullulanase mutant has better enzyme activity than the parent pullulanase at pH 2.0-10.0 and temperature 30°C-90°C, has better thermal stability and acid-base tolerance, and can be widely used in industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Specific enzyme activities of pullulanase mutants under different pH conditions;

[0020] Figure 2 pH stability of pullulanase mutants;

[0021] Figure 3 Specific enzyme activities of pullulanase mutants under different temperature conditions;

[0022] Figure 4 Thermostability of pullulanase mutants. DETAILED DESCRIPTION

[0023] The present invention is described in detail below through specific embodiments, but the protection scope of the present invention is not limited to the following embodiments, and any technical solutions that can be thought of by any technician in the field based on the present invention and combined with the common knowledge in the field belong to the protection scope of the present invention.

[0024] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0025] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0026] Example 1 Expression and enzyme activity of pullulanase mutants

[0027] 1. Expression of the Parent Pullulanase

[0028] The target gene encoding the parent pullulanase (GenBank: KX576675.1) was synthesized by chemical synthesis, wherein the amino acid sequence of the parent pullulanase is shown in SEQ ID NO.1, and the CDS sequence is shown in SEQ ID NO.2; the target gene was purified by BamH After double digestion with I and Xho, the cells were connected with the expression vector pET-24a(+), and the connected products were transformed into Escherichia coli K12, cultured on LB solid medium at 37°C for 12 hours, and then the transformants were picked and inoculated into LB liquid medium for culture, and cultured at 37°C for 12 hours. The plasmid was extracted and sequenced to obtain the recombinant plasmid Pull-pET24a(+) with correct sequencing; the recombinant plasmid Pull-pET24a((+) with correct sequencing was heat-shock transformed into Escherichia coli K12 strain, and cultured on LB solid medium at 37°C for 12 hours, and then the transformants were picked and inoculated into LB liquid medium for culture, and cultured at 37°C for 12 hours to obtain seed solution; the seed solution was inoculated into TB medium at a 5% inoculum, and cultured at 37°C until OD 600 The pH value was 0.6, and then 0.05 mM isopropylthio-D-galactoside (IPTG) was used, and the shaking flask was continued to induce culture at 37°C for 48 hours to obtain the fermentation broth 1 containing the parent pullulanase.

[0029] 2. Expression of pullulanase single-site mutants

[0030] According to the parent pullulanase with an amino acid sequence as shown in SEQ ID NO.1, the lysine at positions 205, 288 and 387 in the sequence were mutated to phenylalanine to obtain pullulanase single-site mutants, which were named: Pull-K205F, Pull-K288F, Pull-K387F, respectively; according to the mutated amino acid sequence, a target gene encoding the pullulanase single-site mutant was synthesized by chemical synthesis, and the target gene was connected to the expression vector pET-24a(+) with reference to the method described in the above "1", and Escherichia coli K12 was transformed, and fermentation broths 2-4 containing the pullulanase single-site mutants Pull-K205F, Pull-K288F, and Pull-K387F were obtained by culture.

[0031] 3. Expression of pullulanase mutants

[0032] According to the parent pullulanase with an amino acid sequence as shown in SEQ ID NO.1, the lysine at positions 205, 288 and 387 in the sequence were mutated to phenylalanine to obtain a pullulanase mutant, which was named: Pull-K205 / 288 / 387F; according to the mutated amino acid sequence (shown in SEQ ID NO.3), a target gene encoding a pullulanase single-site mutant (shown in SEQ ID NO.4) was synthesized by chemical synthesis, and codon optimization was performed. The optimized gene sequence is shown in SEQ ID NO.5. Referring to the method described in the above "1", the optimized gene sequence was connected to the expression vector pET-24a(+), transformed into Escherichia coli K12, and cultured to obtain a fermentation broth 5 containing the pullulanase mutant Pull-K205 / 288 / 387F.

[0033] 4. Isolation and purification of different pullulanases and mutants

[0034] The fermentation broths 1-5 obtained above were centrifuged at 4°C and 12000g for 8 min, and the fermentation supernatants were collected; the pH of the fermentation supernatants was adjusted to 5.0, and the fermentation supernatants were placed in a 50°C water bath for heat treatment for 1 h, and the fermentation supernatants after water bath treatment were centrifuged at 4°C and 12000g for 8 min, and the supernatants were collected; 70% (NH4)2SO4 was slowly added to the supernatant, and the solution was placed at 4°C for salting out for 12 h to obtain a salting-out solution; the salting-out solution was centrifuged at 4°C and 12000g for 8 min, and the precipitate was collected; the precipitate was treated with 20 mmol / L phosphate buffer was redissolved, and dialyzed in 20mmol / L phosphate buffer for 12h to obtain a dialysate; the dialysate was filtered through a 0.22μm membrane to prepare a loading sample; the loading sample was purified using an AKTA protein purifier to obtain an eluate; the eluate was dialyzed in 50mM pH 5.0 acetate buffer for 12h to obtain purified pullulanase Pull and mutants Pull-K205F, Pull-K288F, Pull-K387F, and Pull-K205 / 288 / 387F.

[0035] 5. Pullulanase mutant enzyme activity detection

[0036] The enzyme activities of the purified pullulanase Pull and its mutants Pull-K205F, Pull-K288F, Pull-K387F, and Pull-K205 / 288 / 387F were determined.

[0037] Enzyme activity detection: 3,5-dinitrosalicylic acid (DNS) method was used. The specific method was as follows: 100 μL of reaction system (10 μL of potassium phosphate buffer with a final concentration of 100 mM pH 5.0, 50 μL of pullulan with a final concentration of 3% and 40 μL of enzyme solution) was reacted at 50°C for 15 min, 100 μL of DNS reaction solution was added, and the mixture was placed in a boiling water bath for 6 min, cooled to room temperature with running water, diluted with distilled water, mixed, and the OD was measured. 476 .

[0038] Enzyme activity definition: One unit of enzyme activity (U) is defined as the amount of enzyme required to release reducing sugars equivalent to the reducing power of 1 μmol of glucose per minute under the assay conditions.

[0039] Determination of pullulanase specific enzymatic activity: The enzyme activity (U / mL) of the purified pullulanase was determined, and the protein content (mg / mL) of the purified pullulanase was determined by Bradford method to calculate the specific enzymatic activity of the pullulanase; wherein, the calculation formula of the pullulanase specific enzymatic activity is as follows:

[0040] Pullulanase specific enzyme activity (U / mg) = enzyme activity of the purified pullulanase (U / mL) / protein content of the purified pullulanase (mg / mL).

[0041] The results of the enzyme activity test of the above-purified pullulanase and mutants are shown in Table 1.

[0042] Table 1 Results of pullulanase and mutant activity tests (U / mg)

[0043] Group Sample 1 Sample 2 Sample 3 average Pull 381.51 362.37 375.69 373.19±8.01 Pull-K205F 371.19 359.83 381.74 370.92±8.95 Pull-K288F 419.87 456.57 432.13 436.19±15.26 Pull-K387F 408.31 399.09 423.55 410.32±10.09 Pull-K205 / 288 / 387F 522.63 555.47 560.85 546.32±16.89

[0044] The above results show that the specific enzyme activity of the parent pullulanase is 373.19U / mg. Compared with the parent pullulanase, the specific enzyme activity of the pullulanase single-site mutants Pull-K205F, Pull-K288F, and Pull-K387F did not change significantly, while the pullulanase mutant Pull-K205 / 288 / 387F described in the present application has a significantly increased specific enzyme activity compared with the parent pullulanase.

[0045] Example 2 pH and thermal stability of pullulanase mutants

[0046] 1. pH stability of pullulanase mutants

[0047] Phosphate buffer solutions with pH values ​​of 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 and 10.0 were prepared respectively to replace the phosphate buffer solution in the pullulanase activity assay method, and the reaction was carried out at 50° C. for 15 min to determine the specific enzyme activities of the purified pullulanase Pull obtained in Example 1 and the mutants Pull-K205F, Pull-K288F, Pull-K87F and Pull-K205 / 288 / 387F.

[0048] The results are as follows Figure 1 As shown, compared with the parent pullulanase Pull and the single-site mutant pullulanase mutants Pull-K205F, Pull-K288F, and Pull-K87F, the pullulanase mutant Pull-K205 / 288 / 387F described in the present application has significant specific enzyme activity under any pH conditions, has good specific enzyme activity under different pH conditions, and has good acid-base tolerance.

[0049] Phosphate buffers with pH values ​​of 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 and 10.0 were prepared respectively to replace the phosphate buffer in the pullulanase activity determination method, and the reaction was carried out at 50° C. for 15 min, 30 min, 60 min, 120 min, 240 min and 480 min, respectively, to determine the specific enzyme activity of the purified pullulanase mutant Pull-K205 / 288 / 387F obtained in Example 1.

[0050] The results are as follows Figure 2As shown, the specific enzyme activity of the pullulanase mutant Pull-K205 / 288 / 387F described in the present application is basically stable when treated at different pH values ​​for different time periods, indicating that the pullulanase mutant Pull-K205 / 288 / 387F described in the present application has good pH stability.

[0051] 2. Thermal stability of pullulanase mutants

[0052] 100 μL of the reaction system (10 μL of potassium phosphate buffer with a final concentration of 100 mM pH 5.0, 50 μL of pullulan with a final concentration of 3% and 40 μL of enzyme solution) was reacted at 20°C, 30°C, 40°C, 50°C, 60°C, 70°C, 80°C and 90°C for 15 min, 100 μL of DNS reaction solution was added, the mixture was placed in a boiling water bath for 6 min, cooled to room temperature with running water, diluted with distilled water, mixed and the OD was measured. 476 The specific enzyme activities of the purified pullulanase Pull obtained in Example 1 and its mutants Pull-K205F, Pull-K288F, Pull-K87F, and Pull-K205 / 288 / 387F were determined.

[0053] The results are as follows Figure 3 As shown, compared with the parent pullulanase Pull and the single-site mutant pullulanase mutants Pull-K205F, Pull-K288F, and Pull-K87F, the pullulanase mutant Pull-K205 / 288 / 387F described in the present application has significant specific enzyme activity under the temperature conditions of 30°C-90°C, and significant specific enzyme activity between the temperatures of 30°C-80°C, all of which are higher than 400U / mg, indicating that it has good specific enzyme activity under different temperature conditions and good heat resistance.

[0054] Determination of the thermal stability of the enzyme: The enzyme solution with the same amount of enzyme was placed at 50°C, 60°C, 70°C, 80°C, and 90°C for 15 min, 30 min, 60 min, 120 min, 240 min, and 480 min, respectively. Then, the solution was reacted at 50°C for 15 min in a phosphate buffer solution with a pH of 5.0, and the specific enzyme activity of the purified pullulanase mutant Pull-K205 / 288 / 387F obtained in Example 1 was determined.

[0055] The results are as follows Figure 4 As shown, the specific enzyme activity of the pullulanase mutant Pull-K205 / 288 / 387F described in the present application is basically stable after being treated at different temperatures for different times, indicating that the pullulanase mutant Pull-K205 / 288 / 387F described in the present application has good thermal stability, and the enzyme activity loss after being treated at 100°C for 6 hours is less than 60%.

[0056] The above are only some preferred embodiments of the present invention, and the present invention is not limited to the contents of the embodiments. For those skilled in the art, various changes and modifications can be made within the scope of the technical solution of the present invention, and any changes and modifications made are within the protection scope of the present invention.

Claims

1. A pullulanase mutant, characterized in that: The pullulanase mutant is obtained by simultaneously mutating the lysine at positions 205, 288 and 387 of the parent pullulanase with an amino acid sequence as shown in SEQ ID NO.1 to phenylalanine.

2. The pullulanase mutant according to claim 1, characterized in that The amino acid sequence of the pullulanase mutant is shown in SEQ ID NO.

3.

3. Use of the pullulanase mutant according to claim 1 or 2 in enzymatic hydrolysis of polysaccharides containing α-1,6 glycosidic bonds.

4. The use according to claim 3, characterized in that The polysaccharide is pullulan.

5. A gene encoding the pullulanase mutant according to claim 1 or 2, characterized in that: The gene sequence is shown in SEQ ID NO.

4.

6. A recombinant expression plasmid or vector containing the gene according to claim 5.

7. A host cell containing the gene according to claim 5.

8. The host cell according to claim 7, characterized in that The host cell is a bacterium or a fungus.

9. The host cell according to claim 8, characterized in that The bacteria is Escherichia coli.

10. The method for preparing a pullulanase mutant according to claim 1 or 2, characterized in that: The host cell according to any one of claims 7 to 9 is inoculated into a fermentation medium for fermentation, and the fermentation broth is collected and centrifuged to separate and obtain the pullulanase mutant according to claim 1 or 2.

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