Pullulanase PulW310B and its use in the synthesis of highly polymerized maltodextrins

Abstract

This invention discloses a pullulanase PulW310B, whose amino acid sequence is shown in SEQ ID NO:1. It is a type II pullulanase with 4-α transglycosylation activity, capable of efficiently synthesizing high-content maltohexasaccharide and maltononose using maltotriose as donor and acceptor. This invention provides a new route for synthesizing highly polymerized maltooligosaccharides. Furthermore, the pullulanase PulY103B of this invention is simple to prepare, produces few byproducts in the synthesized product, and is suitable for industrial production and market application.

Description

Technical Field

[0001] This invention belongs to the fields of enzyme engineering and synthetic biology, specifically relating to a type II pullulanase PulW310B derived from Bacillus aureus W310 with 4-α transglycoside activity, and its ability to convert pullulanose into high-content maltotriose, maltohexaose and maltononose through hydrolysis and synthesis. Background Technology

[0002] Pullulan is a linear polysaccharide composed of maltotriose linked by α-1,6 glycosidic bonds. Pullulanases are a class of starch debranching enzymes, named for their ability to hydrolyze pullulan. Based on substrate specificity and hydrolytic characteristics, pullulanases can be classified into five types: type I pullulanase, type II pullulanase, type I pullulanase hydrolase, type II pullulanase hydrolase, and type III pullulanase hydrolase. Due to their properties, pullulanases have applications in the food industry, as well as in environmental protection, medical, and detergent industries.

[0003] Pullulanases typically produce oligosaccharides, such as maltotriose, panose, and isopanose, through their hydrolytic properties. Previous reports on maltooligosaccharides obtained via pullulanase hydrolysis have primarily focused on those with a degree of polymerization (DOP) below 6, and the DOP of maltooligosaccharides reaching a certain yield is generally less than or equal to 4. Furthermore, the presence of glucose in the hydrolysis products negatively impacts the quality of the maltooligosaccharides. However, increasing research indicates that certain pullulanases can produce oligosaccharides with novel structures through their unique synthetic properties. These synthetically produced oligosaccharides also exhibit fewer byproducts. Simultaneously, researchers are focusing on the development of mild pullulanases, whose successful development aligns better with current economic and environmental principles. Our team previously discovered that pullulanases PulY103A and PulY103B, derived from Bacillus megaterium, can synthesize maltohexasaccharide, and that PulY103B can synthesize trace amounts of maltononose. This method provides a new approach for preparing high-degree-of-polymerization maltooligosaccharides. Previous reports have indicated that highly polymerized maltodextrins possess superior moisturizing and prebiotic effects, significantly impacting the development of functional and health foods. Currently, the production of highly polymerized maltodextrins primarily relies on the hydrolytic properties of maltodextrinase and cyclodextrinase. However, the yields of maltodextrin and maltodecanose are relatively low, leading to a lack of clarity regarding their functionalities. Therefore, maltodextrins rich in maltodextrin hold significant theoretical and social value in the development of functional and health foods. Summary of the Invention

[0004] This invention provides a pullulanase PulW310B, which is derived from Bacillus aureus (…). Bacillus aryabhattaiPullulanase PulW310B, whose amino acid sequence is shown in SEQ ID NO:1 and whose nucleotide sequence is shown in SEQ ID NO:2, is a type II pullulanase.

[0005] The pullulanase PulW310B of this invention can convert pullulanose into high-content maltotriose, maltohexaose and maltononose through hydrolysis and synthesis.

[0006] To achieve the above-mentioned objectives of the present invention, the present invention provides the following technical solution:

[0007] 1. Genomic DNA was extracted from Bacillus argentea W310. Using the DNA as a template, the pullulanase gene was cloned using primers pulW310BF: 5'-GGAATTCCATATGACTGGTGATAATAAGTTTCAG-3′ and pulW310BR: 5'-CCGCTCGAGTCGTTTGAAATAAAATAAGAA-3′. pulW310B Recombinant plasmid PulW310-PET23b was constructed, and recombinant strain PulW310-PET23b- was constructed. E. coli BL21(DE3) was used for heterologous expression of recombinant protein PulW310B; the protein was purified by Ni-IDA affinity chromatography to obtain pullulanase PulW310B;

[0008] 2. Activity analysis of pullulanase PulW310B

[0009] The pullulanase PulW310B of this invention has an optimal reaction pH of 7.0 and an optimal reaction temperature of 40°C. It can exhibit nearly 50% enzyme activity at 25°C. This pullulanase can hydrolyze pullulanose and also has 4-α transglycoside activity, which can convert pullulanose into high-content maltotriose, maltohexaose, and maltononose.

[0010] Advantages and technical effects of the present invention:

[0011] This invention provides a novel type II pullulanase with 4-α transglycosylation activity, which can synthesize highly polymerized maltodextrin (maltononose) using maltotriose as a donor and acceptor. This invention provides a new route for the efficient synthesis of maltohexasaccharide and maltononose, and also provides a novel raw material for the development of functional foods and health foods. Attached Figure Description

[0012] Figure 1 SDS-PAGE results of purified pullulanase PulW310B are shown. Lane M: high molecular weight protein standard; Lane 1: lysis buffer; Lane 2: electrophoretically pure PulW310B.

[0013] Figure 2 The results show the optimal pH determination of pullulanase PulW310B. In the figure, ◇ represents 2-morpholinoethanesulfonic acid buffer; ■ represents 3-(N-morpholino)propanesulfonic acid buffer; △ represents tris(hydroxymethyl)aminomethane hydrochloride buffer; and ● represents borax buffer.

[0014] Figure 3 The optimal temperature for pullulanase PulW310B was determined.

[0015] Figure 4 Thin-layer chromatography results of pullulanase PulW310B hydrolyzing maltotriose;

[0016] Figure 5 Thin-layer chromatography results of pullulanase PulW310B hydrolyzing pullulanose;

[0017] Figure 6 Figure 1 shows the high-performance liquid chromatograms of pullulanase PulW310B hydrolyzing and synthesizing products using pullulanose as a substrate. Figure 2a shows the high-performance liquid chromatogram of maltodextrin standard; Figure 3b shows the high-performance liquid chromatogram of pullulanose product. Detailed Implementation

[0018] The present invention will be further described in detail below through embodiments, but the content of the present invention is not limited thereto. Unless otherwise specified, the methods in this embodiment are operated according to conventional methods, and the reagents used are conventional reagents or reagents prepared according to conventional methods unless otherwise specified.

[0019] Example 1: Cloning, expression and preparation of pullulanase PulW310B

[0020] Genomic DNA was extracted from Bacillus megaterium Y103 using the Biotech Bacterial Genome Extraction Kit (Cat®: DP1502). Upstream and downstream primers pulW310BF and pulW310BR were designed and synthesized based on the conserved sequence of the pullulanase gene.

[0021] pulW310BF: 5'-GGAATTCCATATGACTGGTGATAATAAGTTTCAG-3';

[0022] pulW310BR: 5'-CCGCTCGAGTCGTTTGAAATAAAATAAGAA-3';

[0023] The extracted DNA was used as a template for PCR amplification. The reaction conditions were as follows: 95℃ pre-denaturation for 5 min; 94℃ for 30 s, 5℃ for 30 s, 72℃ for 2 min, for 30 cycles of amplification; extension at 72℃ for 10 min; and recovery of PCR amplification products.

[0024] The target fragment was ligated into the pET-23b vector and transformed into E. coli using the heat shock method. E. coli BL21(DE3), positive clones were selected and sequenced, yielding the full-length target gene of 2016 bp, and... Bacillus megaterium The similarity of pullulanase BmP from P6 was 93.1%.

[0025] Take the recombinant plasmid pulW310B A single colony of BL21 from PET23b was inoculated into 10 mL of LB liquid medium containing 50 µg / mL ampicillin sodium salt and cultured at 37°C on a shaker at 250 rpm until the bacterial concentration reached OD200. 600 When the growth coefficient is around 0.8 (logarithmic growth phase), IPTG is added to achieve a final concentration of 1 mmol / L, and the mixture is induced for 12 h at 180 rpm in a constant temperature shaker at 20℃.

[0026] Cells were collected from the culture medium and disrupted by sonication. The supernatant was collected as the crude pullulanase PulW310B enzyme solution. The crude enzyme solution was concentrated by ultrafiltration and Ni affinity chromatography to obtain electrophoretic grade pure pullulanase PulW310B. SDS-PAGE results are shown below. Figure 1 The results showed that pullulanase PulW310B was expressed in Escherichia coli.

[0027] Example 2: Enzymatic properties of pullulanase PulW310B

[0028] 1. Optimal pH detection experiment for pullulanase PulW310B

[0029] Determination of optimal pH for pullulanase: Pullulanase PulW310B was reacted at 40℃ in different buffer solutions with a pH of 5.5–10 at 50 mmol / L. The buffer solutions were 2-morpholinoethanesulfonic acid buffer (pH 5.5–7.5), 3-(N-morpholino)propanesulfonic acid buffer (pH 6.5–8.0), tris(hydroxymethyl)aminomethane hydrochloride buffer (pH 7.5–9.0), and borax buffer (pH 8.5–10.0).

[0030] See results Figure 2 As can be seen from the figure, the optimal pH for pullulanase PulW310B is 7.0.

[0031] 2. Detection of the optimal reaction temperature of pullulanase PulW310B

[0032] Determination of optimal temperature: The reaction was carried out at 25-50℃ in 3-(N-morpholino)propanesulfonic acid buffer at pH 7.0;

[0033] See results Figure 3As can be seen from the figure, the optimal reaction temperature of pullulanase PulW310B is 40℃, and it can achieve nearly 50% of its enzyme activity at 25℃, indicating that the reaction conditions of this enzyme are mild.

[0034] Example 3: Ability of PulW310B to synthesize maltohexasaccharide and maltononose

[0035] 1. Hydrolytic performance experiment of pullulanase PulW310B

[0036] 0.2 U / 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. The enzyme solution was inactivated by boiling in a water bath for 5 min, and the results were analyzed by thin-layer chromatography. The results are shown in the figure. Figure 4 As can be seen from the figure, PulW310B has the ability to hydrolyze α-1,4 glycosidic bonds and 4-α transglycosylation activity.

[0037] 2. Mix 0.2 U / mL PulW310B with 1% pullulanose and react 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. The enzyme solution was inactivated by boiling in a water bath for 5 min. The results were analyzed by thin-layer chromatography. Figure 5 As can be seen from the figure, PulW310B has the ability to hydrolyze α-1,6 glycosidic bonds and transglycosylation activity. The hydrolytic characteristics of PulW310B on maltotriose and pullulanose indicate that PulW310B is a type II pullulanase.

[0038] The product of pullulanose reaction after 2 hours was analyzed by high performance liquid chromatography. (See attached image.) Figure 6 The results showed that the main products of pullulan PulW310B were maltotriose, maltohexaose and maltononose, with proportions of 73.6%, 16.7% and 6.7%, respectively; and yields of 62.2%, 13.5% and 5.4%, respectively.

Claims

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

1.

2. The application of pullulanase PulW310B as described in claim 1 in the synthesis of high-polymerization-degree maltodextrins, characterized in that: Maltohexaose and maltononose were synthesized via 4-α transglycoside activity.

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