Pullulanase PulW310B and application thereof in synthesis of maltooligosaccharides with high polymerization degree

Abstract

The invention discloses pullulanase PulW310B, the amino acid sequence of the pullulanase PulW310B is shown as SEQ ID NO: 1, the pullulanase PulW310B is type II pullulanase, has 4-alpha transglycosylation activity, and can be used for efficiently synthesizing high-content maltohexaose and maltononaose by taking maltotriose as a donor and a receptor, a new way is provided for synthesizing high-polymerization-degree maltooligosaccharides, and the pullulanase PulY103B is simple to prepare, low in cost and high in yield. And the synthetic product has few byproducts and is suitable for industrial production and market popularization and application.

Description

Technical Field

[0001] The present invention belongs to the technical field of enzyme engineering and synthetic biology, and specifically relates to a type II pullulanase PulW310B with 4-α transglycosidic activity derived from Bacillus aggrad W310, and the ability to convert pullulan into high-content maltotriose, maltohexaose and maltononaose through its hydrolysis and synthesis properties. Background Art

[0002] Pullulan is a linear polysaccharide formed by maltotriose connected by α-1,6 glycosidic bonds. Pullulanase is a type of starch debranching enzyme, named for its ability to hydrolyze pullulan. According to substrate specificity and hydrolysis characteristics, pullulanase can be divided into five types: type I pullulanase, type II pullulanase, type I pullulan hydrolase, type II pullulan hydrolase and type III pullulan hydrolase. According to the characteristics of pullulanase, it can be used in the food industry, and it also has important applications in the environmental protection, medical and washing industries.

[0003] Usually, pullulanase can produce oligosaccharides such as maltotriose, panose and isopanose through its hydrolysis characteristics. In previous reports, the maltooligosaccharides obtained by pullulanase hydrolysis reaction are mainly concentrated in maltooligosaccharides with a degree of polymerization lower than 6, and the degree of polymerization of maltooligosaccharides reaching a certain yield is generally less than or equal to 4. At the same time, glucose is inevitably present in the hydrolyzate, which has a negative impact on the quality of maltooligosaccharides. However, more and more researchers have found that some pullulanases can produce oligosaccharides with new structures through their special synthetic characteristics. At the same time, it was found that the oligosaccharides produced by synthesis have fewer by-products. At the same time, researchers pay more attention to the development of mild pullulanases, and their successful development is more in line with today's economic and environmental protection concepts. Our team previously discovered that pullulanases PulY103A and PulY103B from Bacillus megaterium can synthesize maltohexaose, and it was also found that PulY103B can synthesize trace amounts of maltononaose. This method provides a new idea for the preparation of high-polymerization maltooligosaccharides. According to previous reports, high-polymerization maltooligosaccharides have better moisturizing and prebiotic effects, and have a far-reaching impact on the development of functional foods and health foods. At present, the production of high-polymerization maltooligosaccharides is mainly prepared by maltooligosaccharide amylase and cyclodextrin enzyme hydrolysis characteristics, but the output of maltononaose and maltodecose is relatively small, resulting in its functionality is not clear at present, so maltooligosaccharides rich in maltononaose will have important theoretical and social value in the development of functional foods and health foods. Summary of the invention

[0004] The present invention provides a pullulanase PulW310B, which is derived from Bacillus aggradiensis ( Bacillus aryabhattai)W310, the amino acid sequence of which is shown in SEQ ID NO: 1, the nucleotide sequence of which is shown in SEQ ID NO: 2, and the pullulanase PulW310B is a type II pullulanase.

[0005] The pullulanase PulW310B of the present invention can convert pullulan into high-content maltotriose, maltohexaose and maltononaose through hydrolysis and synthesis characteristics.

[0006] In order to achieve the above-mentioned object of the present invention, the present invention provides the following technical solutions: 1. Extract genomic DNA from Bacillus aggregatis W310, use DNA as template, and use primers pulW310BF: 5'-GGAATTCCATATGACTGGTGATAATAAGTTTCAG-3' and pulW310BR: 5'-CCGCTCGAGTCGTTTGAAATAAAATAAGAA-3' to clone the pullulanase gene pulW310B , construct the recombinant plasmid PulW310-PET23b, construct the recombinant strain PulW310- pET23b- E. coli BL21 (DE3), heterologous expression of the recombinant protein PulW310B; protein purification by Ni-IDA affinity chromatography to obtain pullulanase PulW310B; 2. Activity analysis of pullulanase PulW310B The pullulanase PulW310B of the present invention has an optimum reaction pH of 7.0 and an optimum reaction temperature of 40°C. It can exert nearly 50% enzyme activity at 25°C. The pullulanase can hydrolyze pullulan and has 4-α transglycosidic activity, and can convert pullulan into high-content maltotriose, maltohexaose and maltononaose.

[0007] Advantages and technical effects of the present invention: The present invention provides a novel type II pullulanase, which has 4-α transglycosidic activity and can synthesize high-polymerization maltooligosaccharide (maltononaose) with maltotriose as donor and acceptor. The present invention provides a new way for the efficient synthesis of maltohexaose and maltononaose, and also provides a novel raw material for the development of functional foods and health foods. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 The results of SDS-PAGE detection of purified pullulanase PulW310B, wherein lane M: high molecular weight protein standard; lane 1: lysate; lane 2: electrophoresis pure PulW310B; Figure 2The optimum pH measurement results of pullulanase PulW310B, in which ◇ is 2-morpholineethanesulfonic acid buffer; ■ is 3-(N-morpholinyl)propanesulfonic acid buffer; △ is tris(hydroxymethyl)aminomethane hydrochloride buffer; ● is borax buffer; Figure 3 The optimum temperature determination result of pullulanase PulW310B; Figure 4 This is a thin layer chromatography result diagram of pullulanase PulW310B hydrolyzing maltotriose; Figure 5 The figure is the result of thin layer chromatography of pullulan hydrolysis by pullulanase PulW310B; Figure 6 The figures are the HPLC chromatograms of the hydrolysis and synthesis of pullulan by pullulanase PulW310B using pullulan as substrate, wherein figure a is the HPLC chromatogram of the maltooligosaccharide standard; figure b is the HPLC chromatogram of the pullulan product. DETAILED DESCRIPTION

[0009] The present invention is further described in detail below by way of examples, but the content of the present invention is not limited thereto. In the present examples, the methods are operated according to conventional methods unless otherwise specified, and the reagents used are conventional reagents or reagents prepared according to conventional methods unless otherwise specified; Example 1: Cloning, expression and preparation of pullulanase PulW310B The genomic DNA of Bacillus megaterium Y103 was extracted using the bacterial genome extraction kit (Cat﹟: DP1502) of Biotech Company; the upstream and downstream primers pulW310BF and pulW310BR were designed and synthesized according to the conservative sequence of the pullulanase gene: pulW310BF: 5'-GGAATTCCATATGACTGGTGATAATAAGTTTCAG-3'; pulW310BR: 5'-CCGCTCGAGTCGTTTGAAATAAAATAAGAA-3'; The extracted DNA was used as a template for PCR amplification. The reaction conditions were as follows: pre-denaturation at 95°C for 5 min; 30 cycles of amplification at 94°C for 30 s, 5°C for 30 s, and 72°C for 2 min; extension at 72°C for 10 min, and recovery of the PCR amplification product. The target fragment was connected to the pET-23b vector and transformed into Escherichia coli by heat shock method. E. coli BL21 (DE3), selected positive clones for sequencing, and obtained the full-length of the target gene of 2016 bp. Bacillus megaterium The similarity of pullulanase BmP from P6 was 93.1%.

[0010] Take the recombinant plasmid pulW310B -PET23b was inoculated into 10 mL of LB liquid medium containing 50 µg / mL ampicillin sodium salt and cultured at 37°C with a shaker at 250 rpm until the bacterial solution concentration OD 600 When the p-value was about 0.8 (logarithmic growth phase), the inducer IPTG was added to make the final concentration reach 1 mmol / L, and the cells were induced at 180 rpm at 20°C constant temperature shaker for 12 h.

[0011] The cells in the culture medium were collected, and the cells were broken by ultrasound. The supernatant was collected as the crude enzyme solution of pullulanase PulW310B. The crude enzyme solution was concentrated by ultrafiltration and Ni affinity chromatography to obtain electrophoresis grade pure pullulanase PulW310B. The results of SDS-PAGE detection are shown in Figure 1 The results showed that pullulanase PulW310B was expressed in Escherichia coli.

[0012] Example 2: Enzymatic properties of pullulanase PulW310B 1. Optimal pH test of pullulanase PulW310B Determination of the optimal pH of pullulanase: Pullulanase PulW310B was reacted at 40°C and 50 mmol / L in different buffers with a pH of 5.5-10; the buffers were 2-morpholineethanesulfonic acid buffer (pH 5.5-7.5), 3-(N-morpholino)propanesulfonic acid buffer (pH 6.5-8.0), tris(hydroxymethylaminomethane)hydrochloric acid buffer (pH 7.5-9.0), and borax buffer (pH 8.5-10.0); Results Figure 2 , it can be seen from the figure that the optimum pH of pullulanase PulW310B is 7.0.

[0013] 2. Optimal reaction temperature test of pullulanase PulW310B Determination of the optimal temperature: The reaction was carried out at 25-50°C in 3-(N-morpholino)propanesulfonic acid buffer at pH 7.0; Results Figure 3 From the figure, we can see that the optimum reaction temperature of pullulanase PulW310B is 40℃, and it can exert nearly 50% enzyme activity at 25℃, indicating that the reaction conditions of the enzyme are mild.

[0014] Example 3: Ability of PulW310B to synthesize maltohexaose and maltononaose 1. Experiment on the hydrolysis performance of pullulanase PulW310B 0.2U / mL PulW310B was mixed with 5 mmol / L maltotriose solution, and reacted in a water bath at 40℃ and pH 7.0. Samples were taken at 0 min, 15 min, 30 min, 1 h, 2 h, and 4 h, respectively. The enzyme solution was inactivated in a boiling water bath for 5 min. The results were analyzed by thin layer chromatography. Figure 4 ,It can be seen from the figure that PulW310B has the ability to hydrolyze α-1,4 glycosidic bonds and 4-α transglycosidic activity.

[0015] 2. Mix 0.2 U / mL PulW310B with 1% pullulan, react in a water bath at 40°C and pH 7.0, take samples at 0 min, 15 min, 30 min, 1 h, 2 h, and 4 h, inactivate the enzyme solution in a boiling water bath for 5 min, and analyze the results by thin layer chromatography. Figure 5 , it can be seen from the figure that PulW310B has the ability to hydrolyze α-1,6 glycosidic bonds and transglycosidic activity. The hydrolysis characteristics of PulW310B for maltotriose and pullulan indicate that PulW310B is a type II pullulanase.

[0016] The product of pullulan reaction for 2 hours was analyzed by HPLC. Figure 6 The results showed that the main products of pullulan PulW310B were maltotriose, maltohexaose and maltononaose, with the proportions of 73.6%, 16.7% and 6.7%; the yields of the three were 62.2%, 13.5% and 5.4%, respectively.

Claims

1. A pullulanase PulW310B, whose amino acid sequence is shown in SEQ ID NO:

1.

2. Use of the pullulanase PulW310B according to claim 1 in the synthesis of high-polymerization maltooligosaccharides.

3. The use according to claim 2, characterized in that: Maltohexaose and maltononaose are synthesized through 4-α transglycosidic activity.

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