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 thereof and application of the pullulanase mutant enzyme. The pullulanase mutant enzyme 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 specific enzyme activity of the mutant enzyme is increased by 1.42 times, and the enzyme stability under application conditions is increased by 23.5% compared with the starting enzyme. The mutant enzyme has potential application value in hydrolysis of raw starch together with raw starch hydrolysis enzymes.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a pullulanase mutant enzyme, a coding gene thereof, an expression strain and application thereof. BACKGROUND

[0002] Pullulanase (EC 3.2.1.41) is a starch-debranching enzyme that can specifically cleave the alpha-1, 6-glucosidic bonds in pullulan, amylopectin and dextran. Most plant-derived starches contain about 80% amylopectin, and most amylases can only specifically hydrolyze alpha-1, 4-glucosidic bonds and have no hydrolysis activity on alpha-1, 6-glucosidic bonds at branch points. Pullulanase can hydrolyze small molecular weight amylopectin and part of limit dextrin, and usually plays a key role in complete starch hydrolysis. With the development and continuous optimization of production technology, pullulanase has been widely used in starch sugar industry, production of resistant starch, alcohol fermentation and detergent industry, and has great industrial application potential.

[0003] It is reported that most pullulanases are suitable for starch processing at high temperature. Developing cold starch debranching under low and medium temperature conditions can save cost and reduce energy consumption. Therefore, obtaining a pullulanase suitable for cold starch hydrolysis with good catalytic performance will help to develop a starch cold hydrolysis process. SUMMARY

[0004] The present application provides a pullulanase mutant enzyme, a coding gene thereof, an expression strain and application thereof to overcome the deficiencies of the prior art. The present application is based on pullulanase PulBs from marine uncultured microorganisms, and a mutation site is designed by Funclib after whole gene synthesis to obtain a mutant gene. After induction expression of the engineering bacteria containing the mutant plasmid, a pullulanase mutant enzyme with improved specific enzyme activity and stability is obtained. When pullulan sugar is used as a substrate, the specific enzyme activity of the mutant enzyme is improved by 1.42 times. While the specific activity is improved, the stability of the mutant is also improved. Under the condition of 30℃ and pH 7.5, the stability of the mutant enzyme is improved by 23.5% compared with the starting enzyme PulBs, and the hydrolysis rate of raw starch when the mutant enzyme and raw starch hydrolysis enzyme are used together is improved by 6.9% compared with the starting enzyme, which has application prospect in raw starch hydrolysis.

[0005] The amino acid sequence of the pullulanase mutant enzyme of the present application is shown in SEQ ID NO: 1, which is obtained by mutating valine at position 376, glutamine at position 466 and cysteine at position 528 in the amino acid sequence of the starting enzyme to cysteine, histidine and serine, respectively, and mutating glycine at position 584 to serine. The amino acid sequence of the pullulanase mutant enzyme of the present application can also include a combination of nonsense mutations or synonymous mutations in the sequence.

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

[0007] The mutant plasmid of the present application contains the coding gene of the pullulanase mutant enzyme as shown in SEQ ID NO: 2.

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

[0009] The engineered strain expressing the pullulanase mutant enzyme of the present application is named Escherichia coli BL21(DE3) / pET-28a(+)-PulBs-20, which has been deposited with the China Center for Type Culture Collection (CCTCC) on November 1, 2023, and the deposit number is CCTCC NO: M 20232095, and the deposit address is Wuhan, China, Wuhan University.

[0010] The construction method of the engineered strain expressing the pullulanase mutant enzyme of the present application comprises the following steps:

[0011] Firstly, the crystal structure of pullulanase from anaerobic Bacillus is used as a template to perform homology modeling of the structure of pullulanase PulBs using Swiss-Model. A semi-rational design strategy is adopted, and non-fully conserved amino acid residues near the catalytic active center are selected as candidate sites. Through Funclib site-specific analysis and free energy calculation, the top 50 mutations with the lowest free energy are obtained, and each of them is subjected to molecular docking using AutoDockVina. The mutation with higher affinity to the pentasaccharide containing an alpha-1, 6 glycosidic bond is selected to determine the target amino acid of the mutation. The full gene is synthesized and connected to the expression vector pET-28a(+), and the expression host is E. coli BL21(DE3), thereby obtaining the engineered strain containing the mutant gene of the present application.

[0012] The expression plasmid vector in the above construction method includes the listed 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 application can be obtained by fermentation of the engineered strain.

[0015] The application of the pullulanase mutant enzyme is to apply the pullulanase mutant enzyme to assist the hydrolysis of raw starch hydrolysis enzyme to raw starch. The optimal addition amount of the pullulanase mutant enzyme is 6 mg when the mass concentration of the starch milk is 30%, the raw starch hydrolysis enzyme is 0.17 mg, and the addition ratio between the pullulanase mutant enzyme and the raw starch hydrolysis enzyme is about 35:1. The pH of the hydrolysis system is controlled to be 5.5-7.0, and the temperature is controlled to be 25-50℃.

[0016] When the pullulan sugar is used as the substrate, the specific enzyme activity of the mutant enzyme is increased by 1.42 times under the condition of 45℃ and pH 6.5. The stability of the mutant enzyme is also improved. The stability of the mutant enzyme is increased by 23.5% than the original enzyme under the condition of 30℃ and pH 7.5. The hydrolysis rate of the raw starch is increased by 6.9% than that of the raw starch hydrolysis enzyme alone when the mutant enzyme is used to hydrolyze the raw starch together with the raw starch hydrolysis enzyme, and the mutant enzyme has a good application prospect in the cold hydrolysis of starch.

[0017] The specific enzyme activity, the optimal pH, the optimal temperature and the stability of the mutant enzyme protein and the original wild-type protein are determined and compared. The determination results show that the specific enzyme activity of the mutant enzyme obtained by the application is increased by 1.42 times than the original enzyme when the pullulan sugar is used as the substrate. The stability of the mutant enzyme is also improved, and the half-life of the mutant enzyme is prolonged by 23.5% than the original enzyme. Compared with the original enzyme, the optimal temperature and the optimal pH of the mutant enzyme are similar, but the specific enzyme activity and the stability are obviously improved. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 The SDS-PAGE patterns of the purified mutant protein and the original enzyme are as follows: 1 is the supernatant of PulBs broken, 2 is the purified penetration liquid, 3 is the pure enzyme of PulBs, 6 is the supernatant of PulBs-20 broken, 5 is the purified penetration liquid, 4 is the pure enzyme of PulBs-20, and M is the protein marker.

[0019] Figure 2 In the table, a is the optimal temperature, b is the optimal pH, c is the temperature stability under the condition of 30℃, and d is the determination of the optimal addition amount of the pullulanase hydrolysis reaction.

[0020] Figure 3 The amount of reducing sugar produced by the pullulanase and the mutant enzyme and the amylase to hydrolyze raw starch for 6h. DETAILED DESCRIPTION

[0021] In the following examples, the implementation methods are conventional methods unless otherwise specified.

[0022] (I) Construction of the expression strain containing the pullulanase mutant gene of the application

[0023] 1. Selection of mutation site of pullulanase gene

[0024] 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 the pullulanase PulBs is modeled using Swiss-Model (http: / / swissmodel.expasy.org / ) based on the structure of Anoxybacillus sp. LM18-11 as a template.

[0025] According to the simulated structure and multiple sequence alignment information, the amino acid residues near the catalytic active center that are not completely conserved are selected as candidate sites. Through site-specific analysis and free energy calculation by Funclib (https: / / funclib.weizmann.ac.il / bin / steps), the free energy ranking is obtained. Using AutoDockVina, the candidate enzymes with higher free energy ranking are subjected to molecular docking energy simulation with the pentasaccharide containing alpha-1, 6-glycosidic bond. The mutation is designed to select the amino acid with higher affinity to the pentasaccharide small molecule. The valine at position 376 is mutated to cysteine, the glutamine at position 466 is mutated to histidine, the cysteine at position 528 is mutated to serine, and the glycine at position 584 is mutated to serine.

[0026] 2. Construction of pullulanase mutant gene engineering strain

[0027] The mutant gene of pullulanase PulBs in step 1 is subjected to whole gene synthesis and linked to the expression vector pET-28a(+). The expression host is Escherichia coli BL21(DE3), and the engineering strain containing the mutant gene of the application, Escherichia coli BL21(DE3) / pET-28a(+)-PulBs-20, is obtained.

[0028] The engineering strain expressing the mutant pullulanase of the application is classified and named as Escherichia coli BL21(DE3) / pET-28a(+)-PulBs-20, which has been preserved in the China Center for Type Culture Collection (CCTCC) with the preservation number CCTCC NO: M 20232095, the preservation time being November 01, 2023, and the preservation address being Wuhan, China, Wuhan University.

[0029] (II) Expression of the engineering strain containing the mutant pullulanase gene of the application and protein purification

[0030] The engineering strain E. coli BL21(DE3) / pET-28a(+)-PulBs-20 obtained in (one) was inoculated into 400 mL LB liquid medium containing kanamycin, and was placed in a 37°C, 200 rpm incubator to culture until OD 600 was 0.6, 0.2 mM IPTG was added for induction, and the culture was continued at 16°C, 120 rpm for 15 hours. The bacterial cells were collected by centrifugation at 4°C, 8000 g, 3 times the volume of the bacterial liquid of Citrate-Na2HPO4 (pH 7.5, 50 mM) buffer was added, and the cells were broken by ultrasonic treatment at 350 W under ice bath conditions for 30 min. The supernatant was collected by centrifugation at 12000 g, and the crude enzyme solution was obtained. 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 purified by Ni 2+ -NTA column chromatography, and the protein was detected to reach SDS-PAGE purity.

[0031] (three) Detection of the optimum pH and the optimum temperature of the pullulanase mutant enzyme containing the application

[0032] The principle of the pullulanase detection reaction is as follows:

[0033] The activity of the pullulanase was determined in Citrate-Na2HPO4 buffer (50 mM, pH 6.5), and the enzyme activity analysis was performed by the 3,5-dinitrosalicylic acid (DNS) method.

[0034] The reaction system included 300 μL 1% pullulan, 270 μL Citrate-Na2HPO4 buffer (50 mM, pH 6.5), 30 μL enzyme solution, and the reaction was performed for 10 min. 300 μL DNS was added to terminate the reaction, and the reaction was boiled in a water bath for 15 min. After cooling with cold water, the OD 540 absorbance value was measured. Three parallel experiments were set up, and the boiled enzyme solution was used as the zero point. The enzyme activity unit (U) was defined as the amount of enzyme that hydrolyzed 1 μmol of maltose per minute.

[0035] The detection results showed that when pullulan was used as the substrate, the optimum pH of the mutant enzyme obtained in the application was 6.5, and the enzyme could show more than 60% enzyme activity in the pH range of 5.5-7.0. The optimum temperature of the mutant was 45°C, and the enzyme could show more than 50% enzyme activity in the temperature range of 25°C-50°C.

[0036] (four) Detection of the stability of the pullulanase mutant enzyme containing the application at 30°C

[0037] The mutant enzyme was incubated at 30°C and pH 7.5, and the enzyme activity was detected at intervals. The residual enzyme activity after a certain time was calculated based on the initial enzyme activity of 100%. The determination results showed that the half-life of the mutant obtained in the application was 340 h under the condition, which was increased by 23.5% compared with the starting enzyme.

[0038] (V) Application of the mutant pullulanase in the application in the hydrolysis of raw starch by the auxiliary raw starch hydrolyzing enzyme

[0039] The processing industry usually uses a starch slurry with a concentration of 20-30% as a starting substrate. Therefore, the hydrolysis rate of raw starch hydrolyzing enzyme and pullulanase in the hydrolysis of raw starch was determined at a corn raw starch concentration of 30%. The reaction system included: 30% concentration of raw starch milk, 0.17 mg of raw starch hydrolyzing enzyme, 6 mg of pullulanase, and the reaction condition was 35°C, pH 6.5, and 1 mM of Ca 2+ .

[0040] The degree of raw starch hydrolysis was calculated using the following formula:

[0041]

[0042] Figure 3 The detection results showed that when the same amount of enzyme was added, the hydrolysis rate of the mutant proteinase obtained in the application was increased by 6.9% compared with the starting enzyme.

Claims

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

1.

2. A gene encoding the mutant pullulanase enzyme of claim 1, characterized in that The nucleotide sequence is shown as SEQ ID NO:

2.

3. The engineering strain expressing the pullulanase mutant enzyme of claim 1, characterized in that: The taxonomic name of the engineering strain is Escherichia coli BL21(DE3) / pET-28a(+)-PulBs-20, which has been preserved in the China Center for Type Culture Collection (CCTCC) with the preservation number CCTCC NO: M 20232095 on November 1, 2023, and the preservation address is Wuhan, China, Wuhan University.

4. The application of the pullulanase mutant enzyme of claim 1, characterized in that: The pullulanase mutant enzyme is applied to assist the hydrolysis of raw starch by a raw starch hydrolysis enzyme.

5. The application of claim 4, characterized in that: The pH of the hydrolysis system is controlled at 5.5-7.0, and the temperature is controlled at 25-50℃.

6. The application of claim 4, characterized in that: With 30% corn raw starch as the substrate, the addition amount of the pullulan mutant enzyme and the raw starch hydrolysis enzyme is 6 mg and 0.17 mg respectively, and the hydrolysis rate of raw starch by the combined action is increased by 6.9% compared with the hydrolysis rate by the raw starch hydrolysis enzyme alone.

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