A pullulanase mutant enzyme, its coding gene, expression strain and application thereof

Abstract

The application discloses a pullulanase mutant enzyme, a coding gene thereof, an expression strain and application of the pullulanase mutant enzyme. The pullulanase mutant enzyme with improved enzyme activity is obtained by Funclib design based on a pullulanase from a marine uncultured microorganism as a starting enzyme. When pullulan is used as a substrate, the optimal temperature of the mutant enzyme y is reduced to 35 DEG C, which is the temperature under the application condition, the hydrolysis rate of raw starch when the mutant enzyme y is used in combination with raw starch hydrolysis enzyme is 3.6% higher than that of the starting enzyme, and a higher hydrolysis rate can be achieved in a shorter time. The mutant enzyme has potential application value in hydrolysis of raw starch in combination with raw starch hydrolysis enzyme.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a pullulanase mutant enzyme, a coding gene, an expression strain and an application thereof. Background Art

[0002] Pullulanase (EC3.2.1.41) is a starch debranching enzyme that specifically cleaves α-1,6-glycosidic bonds in pullulan, amylopectin, and glucan. Most plant-derived starches contain approximately 80% amylopectin, while most amylases can only specifically hydrolyze α-1,4-glycosidic bonds and have no hydrolytic activity against α-1,6-glycosidic bonds at branch points. Pullulanase can hydrolyze smaller amylopectin and some limit dextrins, and generally plays a key role in the complete hydrolysis of starch. With the development and continuous optimization of production technology, pullulanase has been widely used in the starch sugar industry, the production of resistant starch, alcohol fermentation, and the detergent industry, and has great potential for industrial application.

[0003] Most pullulanases are reportedly suitable for starch processing at higher temperatures. Developing cold starch debranching methods, which can debranch starch at medium to low temperatures, can save costs and reduce energy consumption. Therefore, obtaining pullulanases with good catalytic performance suitable for cold starch hydrolysis will facilitate the development of cold starch hydrolysis processes. Summary of the Invention

[0004] The present invention addresses the deficiencies of the prior art and provides a pullulanase mutant enzyme, its encoding gene, expression strain, and its application. Based on the pullulanase PulBs from an uncultured marine microorganism, the present invention designs mutation sites using Funclib and then synthesizes the entire gene to obtain the mutant gene. After induction expression in engineered bacteria containing the mutant plasmid, a pullulanase mutant enzyme with enhanced enzyme activity and stability is obtained. When using pullulose as a substrate, the optimal temperature of the mutant enzyme is reduced to 35°C, the temperature under application conditions. When hydrolyzed with raw starch hydrolase, the hydrolysis rate of raw starch is increased by 3.6% compared to the original enzyme, and a higher hydrolysis rate can be achieved in a shorter period of time, showing promising application in the hydrolysis of raw starch.

[0005] The pullulanase mutant enzyme of the present invention has an amino acid sequence as shown in SEQ ID NO: 1, wherein the valine at position 376 in the amino acid sequence of the original enzyme is mutated to threonine, the glutamine at position 466 is mutated to histidine, the threonine at position 479 is mutated to valine, the glycine at position 584 is mutated to glutamine, and the asparagine at position 585 is mutated to aspartic acid. The amino acid sequence of the pullulanase mutant enzyme of the present invention may also include a combination of nonsense mutations or synonymous mutations in the sequence.

[0006] The nucleotide sequence of the gene encoding the pullulanase mutant enzyme is shown in SEQ ID NO: 2.

[0007] The mutant plasmid of the present invention contains the gene encoding the pullulanase mutant enzyme as described in SEQ ID NO: 2.

[0008] The strain expressing the pullulanase mutant enzyme of the present invention contains the mutant plasmid.

[0009] The engineered strain expressing the pullulanase mutant enzyme of the present invention is classified and named Escherichia coli BL21 (DE3) / pET28a (+) -PulBs-21, has been sent to the China Center for Type Culture Collection (CCTCC) for preservation, with a preservation number of CCTCC NO: M20232206, a preservation date of November 13, 2023, and a preservation address of Wuhan University, Wuhan, China.

[0010] The method for constructing an engineered strain expressing a pullulanase mutant enzyme of the present invention comprises the following steps:

[0011] First, using the crystal structure of pullulanase from the anaerobic Bacillus genus as a template, Swiss-Model was used to perform homology modeling of the pullulanase PulBs structure. A semi-rational design strategy was employed, selecting non-completely conserved amino acid residues near the catalytic active center as candidate sites. Funclib site-specific analysis and free energy calculations were used to obtain the top 50 free energy-ranked mutation designs. These designs were then molecularly docked using AutoDockVina. Mutations with high affinity for substrate pentasaccharides containing α-1,6 glycosidic bonds were selected to determine the target amino acids for mutation. The entire gene was synthesized and ligated into the expression vector pET-28a(+). The expression host was E. coli BL21(DE3), resulting in an engineered strain containing the mutant gene of the present invention.

[0012] The expression plasmid vectors described in the above construction method include a series of expression vectors such as pCold, pET15, and pET22.

[0013] The host bacteria in the above construction method include E. coli BL21 (DE3), E. coli DH5α, E. coli JM109 or E. coli Rosetta, etc.

[0014] The pullulanase mutant enzyme of the present invention can be obtained by fermentation of the engineered strain.

[0015] The pullulanase mutant enzyme of the present invention is used to assist raw starch hydrolyzing enzymes in hydrolyzing raw starch. The optimal addition amount of the pullulanase mutant is 6 mg. The optimal addition amount of the raw starch hydrolyzing enzyme is 1 U / mg of raw starch, as reported in a previous laboratory paper. The specific enzymatic activity of the raw starch hydrolyzing enzyme is 9055 U / mg. This means that 1500 U of raw starch amylase is added to 5 mL of 30% starch, which is approximately 0.17 mg. The addition ratio of the pullulanase mutant to the raw starch hydrolyzing enzyme is approximately 36:1. The pH of the hydrolysis system is controlled at 5.5-7.0, and the temperature is controlled at 25°C-50°C.

[0016] The present invention measured and compared the specific enzyme activity, optimal pH, optimal temperature, stability, etc. of the mutant enzyme protein and the original wild-type protein. The measurement results showed that when using prussic acid as a substrate, the optimal temperature of the mutant enzyme obtained by the present invention was reduced to 35°C, which is the temperature under the application conditions. The hydrolysis rate of raw starch by co-action with raw starch hydrolase was increased by 3.6% compared with the starting enzyme, and a higher hydrolysis rate could be achieved in a shorter time. It has a good application prospect in the cold hydrolysis of starch. Compared with the starting enzyme, the optimal pH of the mutant enzyme is consistent, but the optimal temperature is reduced to 35°C, which is consistent with the temperature under the application conditions, and the specific enzyme activity is similar to that of the starting enzyme. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Figure 3 is the SDS-PAGE profile of the purified mutant protein and the starting enzyme: 1 is the starting enzyme PulBs supernatant, 2 is the purified permeate, 3 is the pure PulBs enzyme, 6 is the PulBs-21 supernatant, 5 is the purified permeate, 4 is the pure PulBs-21 enzyme, and M is a protein marker.

[0018] Figure 2 a is the optimum temperature, b is the optimum pH, c is the temperature stability at 30°C, and d is the determination of the optimal addition amount of pullulanase hydrolysis reaction

[0019] Figure 3 It is the amount of reducing sugar produced by the hydrolysis of raw starch by pullulanase and mutant enzyme with amylase for 6 hours. DETAILED DESCRIPTION

[0020] The implementation methods in the following examples are all conventional methods unless otherwise specified.

[0021] (1) Construction of an expression strain containing the pullulanase mutant gene of the present invention

[0022] 1. Selection of pullulanase gene mutation sites

[0023] Based on sequence alignment, the starting enzyme, PulBs, is most similar to the pullulanase from Anoxybacillus sp. LM18-11 (PDB code: 3WDH), with an amino acid sequence identity of 47.84%. The structure of pullulanase PulBs was modeled using the Swiss-Model (http: / / swissmodel.expasy.org / ) using the structure of Anoxybacillus sp. LM18-11 as a template.

[0024] Based on the simulated structure and multiple sequence alignment information, non-completely conserved amino acid residues near the catalytic active center were selected as candidate sites. Free energy rankings were obtained through site-specific analysis and free energy calculation using Funclib (https: / / funclib.weizmann.ac.il / bin / steps). AutoDockVina was used to perform molecular docking energy simulation between the candidate enzymes with higher free energy rankings and the substrate pentasaccharide containing an α-1,6-glycosidic bond. Mutation designs with high affinity for the pentasaccharide small molecule were selected to determine the target amino acids for mutation. The valine at position 376 was mutated to threonine, the glutamine at position 466 was mutated to histidine, the threonine at position 479 was mutated to valine, the glycine at position 584 was mutated to glutamine, and the asparagine at position 585 was mutated to aspartic acid.

[0025] 2. Construction of pullulanase mutant genetically engineered strains

[0026] The mutant gene of pullulanase PulBs in step 1 was fully synthesized and ligated into the expression vector pET-28a(+). The expression host was Escherichia coli BL21(DE3), thereby obtaining the engineered strain Escherichia coli BL21(DE3) / pET-28a(+)-PulBs-21 containing the mutant gene of the present invention.

[0027] The engineered strain expressing the pullulanase mutant enzyme of the present invention is classified and named Escherichia coli BL21 (DE3) / pET28a (+) -PulBs-21, has been sent to the China Center for Type Culture Collection (CCTCC) for preservation, with a preservation number of CCTCC NO: M20232206, a preservation date of November 13, 2023, and a preservation address of Wuhan University, Wuhan, China.

[0028] (2) Expression and protein purification of genetically engineered bacteria containing the pullulanase mutation of the present invention

[0029] The engineered strain E. coli BL21(DE3) / pET-28a(+)-PulBs-21 obtained in (1) was inoculated into 400 mL LB liquid medium containing kanamycin and cultured at 37°C and 200 rpm until OD 600 The cell culture medium was 0.6, IPTG was added to a final concentration of 0.2 mM for induction, and the culture was continued at 16 ° C and 120 rpm for 15 hours. The bacteria were collected by centrifugation at 4 ° C and 8000 g, and Citrate-Na2HPO4 (pH7.5, 50 mM) buffer with a volume of 3 times the bacterial solution was added. The cells were broken by ultrasonication for 30 minutes under 350W ice bath conditions, and the supernatant was collected by centrifugation at 12000 g to obtain a crude enzyme solution. The whole bacterial protein SDS-PAGE showed that the protein expression amount accounted for more than 90% of the whole bacterial protein. The crude enzyme solution was Ni 2+ -NTA column chromatography was used for purification, and the protein was tested to reach SDS-PAGE purity.

[0030] (III) Detection of the optimal pH and temperature of the pullulanase mutant enzyme of the present invention

[0031] The pullulanase detection reaction principle is as follows:

[0032] The pullulanase activity was determined in Citrate-Na2HPO4 buffer (50 mM, pH 6.5) using the 3,5-dinitrosalicylic acid (DNS) method.

[0033] The reaction system included 300 μL 1% pullulan, 270 μL Citrate-Na2HPO4 buffer (50 mM, pH 6.5), 30 μL enzyme solution for 10 min, 300 μL DNS was added to terminate the reaction, and the mixture was boiled in a water bath for 15 min. After cooling with cold water, the OD was measured. 540 The absorbance value of the experiment was set in triplicate and zeroed with boiled enzyme solution. The enzyme activity unit (U) was defined as the amount of enzyme that hydrolyzed pullulanase to produce 1 μmol maltose per minute.

[0034] Test results showed that when pullulan was used as a substrate, the optimal pH for the mutant enzyme obtained in the present invention was 6.5, and the enzyme exhibited over 60% enzyme activity within the pH range of 6.0-7.0. The optimal temperature for the mutant was 35°C, and the enzyme exhibited over 50% enzyme activity within the 20°C-40°C range.

[0035] (IV) Stability testing of the pullulanase mutant enzyme of the present invention at 30°C

[0036] The mutant enzyme was incubated at 30°C and pH 6.5, and samples were taken at intervals to test the enzyme activity. The initial enzyme activity was taken as 100%, and the residual enzyme activity after a certain period of time was calculated. The results showed that the half-life of the mutant obtained in the present invention under these conditions was 120 hours.

[0037] (V) Application of the pullulanase mutant enzyme of the present invention in assisting raw starch hydrolysis by raw starch hydrolyzing enzyme

[0038] The processing industry usually uses 20-30% starch slurry as the starting substrate. Therefore, the hydrolysis rate of raw starch by raw starch hydrolyzing enzyme and pullulanase was determined at 30% corn starch concentration. The reaction system includes: 30% raw starch slurry, 0.17mg raw starch hydrolyzing enzyme, 6mg pullulanase, reaction conditions are 35℃, pH 6.5, and Ca2+ is added to a final concentration of 1mM. 2+ .

[0039] The degree of raw starch hydrolysis was calculated using the following formula:

[0040]

[0041] Figure 3 The test results showed that when the same amount of enzyme was added, the hydrolysis rate of the mutant protease obtained by the present invention was increased by 3.6% compared with the original enzyme, and a good hydrolysis effect was achieved within 0.5 h.

Claims

1. A pullulanase mutant enzyme, characterized in that Its amino acid sequence is shown in SEQ ID NO:

1.

2. The gene encoding the pullulanase mutant enzyme according to claim 1, characterized in that Its nucleotide sequence is shown in SEQ ID NO:

2.

3. An engineered strain expressing the pullulanase mutant enzyme according to claim 1, characterized in that The strain was classified as Escherichia coli BL21(DE3) / pET28a(+)-PulBs-21 and has been deposited in China Center for Type Culture Collection (CCTCC) with the deposit number of CCTCC NO: M20232206.

4. The use of the pullulanase mutant enzyme according to claim 1, characterized in that: The pullulanase mutant enzyme is used to assist raw starch hydrolyzing enzyme in hydrolyzing raw starch.

5. The use according to claim 4, characterized in that: The addition ratio of the pullulan mutase to the raw starch hydrolyzing enzyme is 36:

1.

6. The use according to claim 4, characterized in that: The pH of the hydrolysis system is controlled at 5.5-7.0, and the temperature is controlled at 25°C-50°C.

7. The use according to claim 4, characterized in that: The pH of the hydrolysis system was controlled at 6.5, and the temperature was controlled at 35°C.

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