CN115873832A - Pullulanase mutant with improved stability and construction method thereof - Google Patents

Abstract

The invention discloses a pullulanase mutant with improved stability and a construction method thereof, belonging to the field of genetic engineering and enzyme engineering. On the basis of pullulanase from high-enzyme-activity bacillus longissimus, two mutant strains D147F and G690M with improved single-point thermal stability and a combined mutant D147F/G690M are obtained by screening. And the heat stability of the mutant is obviously improved, and simultaneously, the activity of the enzyme is not influenced. Compared with natural pullulanase, the mutants in the invention are more suitable for industrial production, and have huge application prospect and industrial value.

Description

A pullulanase mutant with improved stability and its construction method

technical field

The invention relates to a pullulanase mutant with improved stability and a construction method thereof, belonging to the technical fields of genetic engineering and enzyme engineering.

Background technique

Isomaltose (isomaltooligosaccharides, IMOs) is a class of functional oligosaccharides, composed of 2 to 10 α-1, 6-glucosidic bond-linked glucose groups, including isomaltose, panose, isomaltotriose and other main functional ingredients. IMOs can promote the proliferation of probiotics such as Bifidobacterium and Lactobacillus in the human intestinal tract, and regulate intestinal flora; they have low calorie value, promote gastrointestinal motility, improve constipation and lipid metabolism, and prevent dental caries. Therefore, IMOs It is widely used as a prebiotic, food additive and feed ingredient.

Pullulanase (EC 3.2.1.41) is one of the starch debranching enzymes, which can specifically hydrolyze the α-1,6 glycosidic bond on the branch point of starch to release amylose, which is beneficial to the starch saccharification and Glucose production, thereby significantly improving the efficiency of starch saccharification and the conversion rate of starch to glucose, is an important and indispensable auxiliary enzyme in the starch sugar industry. Since the 1980s, foreign scholars have successively discovered and analyzed a variety of pullulanase from different sources. But so far, in the starch sugar industry, only type I pullulanase has shown a better compounding or compounding effect with glucoamylase. Among them, Bacillus naganoensis pullulanase (PulA) has high catalytic activity and has practical industrial application value.

At present, IMOs are generally produced by enzymatic conversion, using α-amylase, β-amylase, pullulanase and glucosidase to catalyze the production from starch. The typical production process usually includes 3 steps: First, Liquefaction, in which starch is liquefied by heat-resistant α-amylase to produce oligosaccharides and dextrins; the second is saccharification, oligosaccharides and dextrins are saccharified by glucoamylases such as β-amylase and pullulanase; the third is transglycosylation Furthermore, α-glucosidase (EC 3.2.1.20) can catalyze the release of glucose from the non-reducing end of the substrate and transfer the glucosyl residue to the 6-OH group of the non-reducing glucose unit, generating IMOs.

Most studies have only improved the thermostability of enzymes by immobilizing enzymes or displaying them on the cell surface, but their thermostability is still unsatisfactory for industrial applications. Therefore, the research on the molecular modification of pullulanase to improve its thermal stability will be the research focus of the present invention.

Contents of the invention

In order to solve the current problem of low thermal stability of pullulanase, the present invention provides a pullulanase mutant, which is the 147th and 147th positions of pullulanase shown in SEQ ID NO.1. /or obtained by mutating the 690th amino acid.

In one embodiment, the mutant is obtained by mutating the aspartic acid at the 147th position of the pullulanase shown in SEQ ID NO.1 to phenylalanine, named: D147F , the amino acid sequence of which is shown in SEQ ID NO.2.

In one embodiment, the mutant is obtained by mutating the glycine at position 690 of the pullulanase shown in SEQ ID NO.1 to methionine, named: G690M, and its amino acid The sequence is shown in SEQ ID NO.3.

In one embodiment, the mutant is to mutate the aspartic acid at position 147 of the pullulanase shown in SEQ ID NO.1 to phenylalanine, and change the glycine at position 690 to obtained by mutation to methionine, named:

The amino acid sequence of D147F/G690M is shown in SEQ ID NO.4.

The present invention also provides a method for preparing any of the mutants described above, comprising the following steps:

(1) Using the nucleotide sequence shown in SEQ ID NO.5 as a template, designing primers for site-directed mutagenesis, performing PCR amplification to obtain a gene containing the mutation site, and connecting the gene with the vector to construct a recombinant expression vector;

(2) Transforming the recombinant expression vector constructed in step (1) into host cells.

The present invention also provides the gene encoding the mutant.

In one embodiment, the gene contains the nucleotide sequences shown in SEQ ID NO.6-8.

The invention also provides a recombinant vector carrying the gene.

In one embodiment of the present invention, the recombinant vector uses pET-28a(+) vector as the expression vector.

The present invention also provides recombinant microbial cells carrying the gene or containing the recombinant vector.

In one embodiment, the recombinant microbial cell uses Escherichia coli as an expression host.

The present invention also provides a genetically engineered bacterium, the genetically engineered bacterium uses Escherichia coli as a host, and expresses the pullulanase mutant.

In one embodiment, the genetically engineered bacteria use Escherichia coli BL21(DE3) as the expression host.

In one embodiment, the genetically engineered bacteria use the pET-28a(+) vector as the expression host.

The invention provides a method for improving the thermostability of pullulanase, the method is, the amino acid sequence of pullulanase as shown in SEQ ID NO.1 D147 and/or amino acid 690 Make mutations.

The present invention also provides the above-mentioned pullulanase mutant, or the gene encoding the above-mentioned mutant, or the above-mentioned recombinant vector, or the application of the above-mentioned recombinant cell in transforming starch to produce isomalto-oligosaccharide.

Beneficial effect:

(1) On the basis of natural pullulanase, the present invention transforms the molecular structure of pullulanase through rational design, combined with site-directed mutagenesis biotechnology, analyzes the influence of residues after mutation on the thermal stability of the enzyme, and finally obtains Two mutant strains D147F and G690M with improved single point mutation stability.

(2) The pullulanase mutant D147F provided by the invention has a half-life of 3.7h at 55°C, compared with the natural pullulanase (half-life of 2.5h), the half-life is 1.48 times that of the natural pullulanase; Mutant G690M had a half-life of 5.1 hours at 55°C, which was 2.04 times that of natural pullulanase; D147F/G690M had a half-life of 6 hours at 55°C, which was 2.4 times that of natural pullulanase.

(3) The heat stability of the pullulanase mutant provided by the present invention is significantly improved, and at the same time, the activity of the enzyme does not decrease significantly. Among them, mutant D147F retained 80.5%, 83.2% and 91.2% of the relative enzyme activity of mutant D147F and G690M and combined mutant D147F/G690M after heat treatment at 55°C for 2 h while the catalytic activity of the enzyme was basically unchanged. The group retained 55.7% of the relative enzyme activity.

(4) The pullulanase mutant obtained in the present invention is more suitable for the application of catalyzing starch hydrolysis than the wild type, and is more conducive to the flexibility of the production process.

Description of drawings

Figure 1 is the SDS-PAGE analysis of the pure enzyme solution of wild-type pullulanase and pullulanase mutant; wherein: M, protein maker; 1, pure enzyme solution of wild-type pulA; 2-8 respectively: containing S17E, D147F, P207W, G670R, G690M, T696F, D147F/G690M pure enzyme solution.

Figure 2 shows the residual activity of wild-type pullulanase and its mutants after incubation at 55°C and pH 5.0 for 2 hours.

Figure 3 shows the half-life of wild-type pullulanase and pullulanase mutants D147F, G690M, D147F/G690M at 55°C.

Figure 4 shows the effect of pH on the enzyme activity of wild-type pullulanase and pullulanase mutants D147F, G690M, and D147F/G690M.

Fig. 5 is the effect of temperature on the activity of wild-type pullulanase and pullulanase mutants D147F, G690M, and D147F/G690M.

Detailed ways

The pET-28a(+) vector involved in the following examples was purchased from Invitrogen.

The medium involved in the following examples is as follows:

(1) LB liquid medium: peptone 10g/L, yeast extract 5g/L, NaCl 10g/L.

(2) LB solid medium: peptone 10g/L, yeast extract 5g/L, NaCl 10g/L, agar 15g/L.

The detection methods involved in the following examples are as follows:

The enzyme activity of pullulanase was determined by DNS method. In the experimental group, 200 μL of 2% (w/v) pullulan solution was mixed with 200 μL of enzyme solution, reacted at 55°C for 20 minutes, and immediately placed in an ice-water bath for 5 minutes to terminate the reaction. The control group was incubated at 55° C. for 20 minutes, and then 200 uL of 2% (w/v) pullulan solution was added. After cooling, 600 μL of DNS reagent was added to both the experimental group and the control group, and boiled in a boiling water bath for 5 minutes. After cooling in an ice-water bath, the corresponding absorbance was measured at 540 nm.

Enzyme activity definition: Under the reaction conditions of 55°C and pH 5.0, 1 mL of enzyme solution produces 1 μmol of glucose per 1 minute of reaction, which is 1 enzyme activity unit.

Specific enzyme activity: defined as the enzyme activity of a unit of protein U/ml.

Embodiment 1: Construction of the recombinant plasmid containing pullulanase mutant

(1) Construction of recombinant plasmid containing wild-type pullulanase

Chemically synthesized wild-type pullulanase gene pulA whose nucleotide sequence is shown in SEQ ID NO.1, and pET-28a(+) vector were digested with NdeI and MluI enzymes and ligated to prepare the recombinant vector pET- 28a(+)-pulA.

(2) Obtaining recombinant vectors containing mutants:

Using the whole plasmid PCR technology, the recombinant vector pET-28a(+)-pulA prepared in step (1) was used as a template to carry out site-directed mutagenesis to obtain recombinant plasmids pET-28a(+)-S17E and pET-28a containing mutant genes (+)-D147F, pET-28a(+)-P207W, pET-28a(+)-G670R, pET-28a(+)-G690M, pET-28a(+)-T696F, pET-28a(+)-D147F /G690M.

The designed primer sequences are as follows:

S17E-F: GTTCTGGCCCCGAAGGAACTGGGCTTTGAC;

S17E-R: GTCAAAGCCCAGTTCCTTCGGGGCCAGAAC;

D147F-F: GTGCATCCGCACTTATTTGAGATCCGCTGTG;

D147F-R: ACACAGCGGATCTCAAATAAGTGCGGATGC;

P207W-F: AAGCTGGCACCTTGGAGTCTGCCGCTGCCG;

P207W-R: CGGCAGCGGCAGACTCCAAGGTGCCAGCTT;

G670R-F: GACGGTCAGCGCCGTGGTACAACACCGTTTG;

G670R-R: ACGGTGTTGTACCACGGCGCTGACCGTCAC;

G690M-F: CGGTCAGCGCTGCATGACAACACCGTTTGGC;

G690M-R: GCCAAACGGTGTTGTCATGCAGCGCTGACCG;

T696F-F: GCGCTGCGGTACATTTCCGTTTGGCCAAG;

T696F-R: CTTGGCCAAACGGAAATGTACCGCAGCGC.

Among them, the PCR amplification program was set as follows: first, pre-denaturation at 95°C for 5 minutes; then 30 cycles; denaturation at 95°C for 30 seconds, annealing at 72°C for 30 seconds, extension at 58°C for 3.5 minutes, and incubation at 4°C. PCR products were detected by 0.8% agarose gel electrophoresis.

The final amplified fragment was treated with Dpn I enzyme in a 37°C water bath for 1 h to remove the template, and then the PCR mixture was chemically transformed into E.coli JM109 competent cells, and the transformation solution was coated with kanapenicillin (50 μg/mL ) on LB solid medium, the plasmid was extracted and sequenced, and the sequencing work was completed by Suzhou Jinweizhi. Verify that the correct plasmids were named pET-28a(+)-pulA, pET-28a(+)-S17E, pET-28a(+)-D147F, pET-28a(+)-P207W, pET-28a(+)- G670R, pET-28a(+)-G690M, pET-28a(+)-T696F, pET-28a(+)-D147F/G690M.

Embodiment 2: the construction of the recombinant escherichia coli producing pullulanase mutant

Respectively the recombinant plasmids pET-28a(+)-pulA, pET-28a(+)-S17E, pET-28a(+)-D147F, pET-28a(+)-P207W, pET-28a(+) obtained in Example 1 )-G670R, pET-28a(+)-G690M, pET-28a(+)-T696F, pET-28a(+)-D147F/G690M were transformed into E.coli BL21 competent cells, and genetically engineered bacteria were prepared respectively : E.coli BL21/pET-28a(+)-pulA, E.coli BL21/pET-28a(+)-S17E, E.coli BL21/pET-28a(+)-D147F, E.coli BL21/pET- 28a(+)-P207W, E.coli BL21/pET-28a(+)-G670R, E.coliBL21/pET-28a(+)-G690M, E.coli BL21/pET-28a(+)-T696F, E. coli BL21/pET-28a(+)-D147F/G690M.

Example 3: Expression of pullulanase mutants

The genetically engineered bacteria constructed in Example 2 were respectively inoculated into 10 mL of LB liquid medium containing 50 μg/mL kanamycin sulfate, and cultured overnight at 37° C. and 200 rpm to prepare seed liquid;

The prepared seed solution was transferred to 100 mL of LB liquid medium containing 50 μg/mL kanamycin sulfate according to the inoculum size of 2% (v/v), and cultured at 30° C. for 20 h to obtain a fermentation liquid. The prepared fermentation broth was centrifuged at 8000×g at 4°C for 5 minutes to obtain cell thalli, and the cells were washed 3 times, and then re-dissolved with 10 mL of disodium hydrogen phosphate-sodium dihydrogen phosphate buffer (pH 7.0) suspended.

Treat the resuspended cells with a sonicator in an ice bath for 30 minutes, centrifuge for 30 minutes (8000×g, 4°C), and remove the supernatant to obtain the crude enzyme solution;

The supernatant fraction was filtered through a 0.22-μm filter, and then further loaded onto a 1 mL Ni affinity column, which was pre-equilibrated with 50 mM wash buffer (20 mM Tris and 500 mM NaCl, pH 7.4), followed by elution buffer (20mM Tris, 500mM NaCl and 500mM imidazole, pH 7.4) were used to elute unbound protein and pullulanase with a linear gradient; the pure enzyme solution of wild-type pullulanase, the pure enzyme solution of mutant S17E, and the mutant Pure enzyme solution of D147F, pure enzyme solution of mutant P207W, pure enzyme solution of mutant G670R, pure enzyme solution of mutant G690M, pure enzyme solution of mutant T696F, pure enzyme solution of mutant D147F/G690M;

The above pure enzyme solutions were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The results are shown in Figure 1, and the results showed that there was an obvious band at 63kDa, which proved that the pullulanase was expressed.

In order to test the influence of single point mutations on thermal stability, thermal stability experiments were carried out on the prepared pure enzymes. After incubating the pure enzymes prepared in step (2) in a water bath at 55°C for 2 hours, 1 mL was taken, according to Pullulan enzyme activity assay method is used to measure the residual enzyme activity of the remaining enzyme, and the enzyme activity of the pure enzyme solution that has not been subjected to high temperature treatment is used as a blank control to obtain the percentage of residual enzyme activity, and the enzyme activity changes of related wild-type proteins and mutants The results are shown in Figure 2.

The results showed that the mutants D147F and G690M and the combined mutant D147F/G690M retained 80.5%, 83.2% and 91.2% of the relative enzyme activities, respectively, while the control group only retained 15.7% of the relative enzyme activities; the relative enzyme activities of the other mutants were the same. Below 15%.

The specific enzyme activities of wild-type pullulanase, mutant D147F, mutant G690M and combined mutant D147F/G690M were detected respectively, and the results are shown in Table 1:

Table 1 Specific activity of different pullulanases

Embodiment 4: Pullulanase mutant enzymatic properties

1. Thermal stability

Take the pure enzyme solution of wild-type pullulanase prepared in Example 3, the pure enzyme solution of mutant D147F, the pure enzyme solution of mutant G690M, and the pure enzyme solution of mutant D147F/G690M and place them in a constant temperature water bath at 55°C In the process, samples were taken at intervals, and the residual enzyme activity was measured according to the pullulanase enzyme activity assay method, and its thermal stability was compared to obtain the half-life results of wild-type pullulanase and its mutants as shown in Table 2 and Figure 3 shown. The half-life of the mutant D147F/G690M at 55°C can reach 6.0h, which is 2.4 times that of the natural pullulanase.

Table 2 Half-life of different pullulanases

2. Optimal pH

The pure enzyme solution of wild-type pullulanase prepared in Example 3, the pure enzyme solution of mutant D147F, the pure enzyme solution of mutant G690M, and the pure enzyme solution of mutant D147F/G690M were respectively placed in In the 50mM buffer solution of acetic acid/sodium acetate (pH 3.5～5.0), citric acid/sodium phosphate (pH 5.5～7.0), take the unincubated initial enzyme activity as 100%, measure the enzyme activity, the results are shown in Figure 4, The optimum pH of the mutant was 5.0, which was similar to that of the wild type.

3. Optimum temperature

The pure enzyme solution of wild-type pullulanase prepared in Example 3, the pure enzyme solution of mutant D147F, the pure enzyme solution of mutant G690M, and the pure enzyme solution of mutant D147F/G690M were respectively placed in acetic acid/acetic acid In the 50mM buffer solution of sodium (pH 5.0), setting reaction temperature is 40～70 ℃, with unincubated initial enzyme activity as 100%, measure enzyme activity, the result is as shown in Figure 5, the optimal temperature of mutant is 55 ℃, similar to wild type.

4. Kinetic parameters of pullulanase

Using pullulan as a substrate, the pure enzyme solution of wild-type pullulanase, the pure enzyme solution of mutant D147F, the pure enzyme solution of mutant G690M, and the pure enzyme solution of mutant D147F/ Kinetic parameters of pure enzyme solution of G690M. Among them, the concentration of pullulan substrate was 0.25, 0.5, 1, 2, 4, 6, 8, 10, 12, 14 and 16mg/mL; After the reaction was completed for 10 minutes, GraphPad Prism 8.0 was used to perform regression analysis on the experimental data to determine the Vmax and Km values. The results are shown in Table 3. Compared with the wild type, the catalytic performance of the mutant has little change.

Table 3 Kinetic parameters of different pullulanases

Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore The scope of protection of the present invention should be defined by the claims.

Claims (10)

1. A pullulanase mutant, characterized in that the amino acid at position 147 and/or 690 of the pullulanase whose amino acid sequence is shown in SEQ ID NO.1 is mutated. 2. the pullulanase mutant according to claim 1, is characterized in that, the mutant is (a), (b) or (c): (a) mutating the aspartic acid at position 147 of the pullulanase whose amino acid sequence is shown in SEQ ID NO.1 to phenylalanine; (b) mutating the glycine at position 690 of the pullulanase whose amino acid sequence is shown in SEQ ID NO.1 to methionine; (c) Mutating aspartic acid at position 147 of the pullulanase whose amino acid sequence is shown in SEQ ID NO.1 to phenylalanine, and mutating glycine at position 690 to methionine. 3. The gene encoding the pullulanase mutant described in claim 1 or 2. 4. The recombinant vector carrying the gene of claim 3. 5. The recombinant vector according to claim 4, wherein pET-28a(+) is used as the expression vector. 6. Carrying the gene according to claim 3, or the recombinant microbial cell containing the recombinant vector according to claim 4 or 5. 7. A recombinant Escherichia coli, characterized in that, using pET-28a(+) as an expression vector to express the pullulanase mutant described in claim 1 or 2. 8. The recombinant Escherichia coli according to claim 7, wherein Escherichia coli BL21(DE3) is used as the expression host. 9. A method for improving the thermostability of pullulanase, characterized in that the 147th aspartic acid of the pullulanase whose amino acid sequence is shown in SEQ ID NO.1 is mutated into phenylalanine, And/or mutate glycine at position 690 to methionine. 10. The mutant according to claim 1 or 2, or the gene according to claim 3, or the recombinant vector according to claim 4 or 5, or the recombinant microbial cell according to claim 6, or the recombinant microbial cell according to claim 7 or 8 The application of the recombinant Escherichia coli described above in the production of isomaltooligosaccharide by hydrolyzing starch.

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