4,6-alpha-glucosyltransferase, method for producing the same, and use thereof

Abstract

The application provides a 4,6-alpha-glucosyltransferase and a preparation method and application thereof, and belongs to the technical field of 4,6-alpha-glucosyltransferase synthesis. The preparation method of the 4,6-alpha-glucosyltransferase comprises the following steps: taking the amino acid sequence of the 4,6-alpha-glucosyltransferase as a template, searching, and obtaining a first amino acid sequence; constructing the first amino acid sequence to a plasmid for transformation and expression, and obtaining an expression product; purifying the expression product, obtaining the 4,6-alpha-glucosyltransferase, and naming the 4,6-alpha-glucosyltransferase as PiGT glycosyltransferase. The 4,6-alpha-glucosyltransferase obtained in the application, namely the PiGT glycosyltransferase, can catalyze amylose to produce isomaltulose with more superior anti-alpha-amylase hydrolysis performance, and the application has more remarkable effects compared with the prior art.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of 4,6-alpha-glucosyltransferase synthesis, in particular to a 4,6-alpha-glucosyltransferase and a preparation method and application thereof. BACKGROUND

[0002] The continuous increase in the incidence of diabetes, cardiovascular disease and obesity is related to long-term consumption of food containing high rapidly digestible starch. Food with high content of slowly digestible starch and resistant starch has a lower digestion rate in the body than ordinary starch, and belongs to low glycemic index food, which is beneficial to control the homeostasis of blood glucose concentration before and after meals. Isomaltulose is a soluble dietary fiber with prebiotic characteristics, which can be connected to the non-reducing end of starch fragments by linear (alpha1→6)-glucan chains under the catalysis of 4,6-alpha-glucosyltransferase, thereby forming starch containing (alpha1→6) glycosidic bond structure, especially the proportion of (alpha1→6) bond in the isomaltulose product obtained by taking long-chain and linear (alpha1→4) glucan chain as substrate is the highest, and the hydrolysis ability of amylase to this structure of isomaltulose is poor, so it is a dietary fiber with broad application prospect.

[0003] 4,6-alpha-glucosyltransferase (4,6-alpha-GTs) belongs to GH70 family, and the (beta / alpha)8 barrel of the catalytic center is composed of three domains A, B and C and four conserved domains. According to the bacterial source, product specificity and conserved domain structure, 4,6-alpha-GT can be divided into three subfamilies of GtfB-like, GtfC-like and GtfD-like. GtfC can cleave alpha-1,4 bonds and synthesize oligomeric isomaltulose (IMMO) with continuous alpha-1,6 bonds, and it is also found that the product of GtfC enzyme has alternating alpha-1,4 and alpha-1,6 bonds. Therefore, it is very valuable to further improve the activity of 4,6-alpha-glucosyltransferase and reduce the digestion rate of denatured starch.

[0004] Therefore, it is very valuable to further improve the activity of 4,6-alpha-glucosyltransferase and reduce the digestion rate of denatured starch. SUMMARY

[0005] The present application provides a 4,6-alpha-glucosyltransferase and a preparation method and application thereof, and aims to solve the technical problems of low activity of 4,6-alpha-glucosyltransferase and low efficiency of synthesizing isomaltulose in the prior art.

[0006] In order to achieve the above-mentioned application purposes, the present application provides the following technical solutions:

[0007] The present application provides a preparation method of 4,6-alpha-glucosyltransferase, comprising the following steps:

[0008] Taking the amino acid sequence of the 4,6-alpha-glucosyltransferase as a template, a first amino acid sequence is obtained by searching;

[0009] The first amino acid sequence is constructed into a plasmid for transformation and expression, and an expression product is obtained;

[0010] The expression product is purified to obtain the 4,6-alpha-glucosyltransferase, which is named as PiGT glycosyltransferase.

[0011] Further, the amino acid sequence of the 4,6-alpha-glucosyltransferase preferably adopts the amino acid sequence of NCBI accession number AKM18207.

[0012] Further, the searching is preferably BLASTp searching in Uniport protein database.

[0013] Further, the first amino acid sequence is preferably one sequence from Propioniferax innocua annotated as potential 4,6-alpha-glucosyltransferase, Genbank ID FB460_0880.

[0014] Further, the plasmid is pCold-TF plasmid.

[0015] Further, the expression conditions are as follows: culturing at 15 DEG C and 180 rpm, and inducing for 20 hours under 0.2mmol / L IPTG concentration.

[0016] The application also provides the 4,6-alpha-glucosyltransferase obtained by the preparation method of the 4,6-alpha-glucosyltransferase.

[0017] The application also provides the 4,6-alpha-glucosyltransferase obtained by the preparation method of the 4,6-alpha-glucosyltransferase or the application of the 4,6-alpha-glucosyltransferase in synthesis of isomaltulose and prevention of diabetes, cardiovascular disease and obesity.

[0018] Compared with the prior art, the technical scheme of the application has the following beneficial effects:

[0019] When taking straight-chain starch as a substrate, the activity of the PiGT glycosyltransferase is 2239.42 U / μmol, which has high activity; secondly, the PiGT glycosyltransferase obtained by the application can catalyze straight-chain starch to produce isomaltulose which has more superior anti-alpha-amylase hydrolysis performance, and has more significant effect in application than other transferases, and can further treat or prevent diabetes, cardiovascular disease and obesity and other diseases of the same type. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 A schematic comparison chart of the effects of 4,6-alpha-glucosyltransferase on different groups of substrates in the test example 2 of the present application is shown,

[0021] Wherein: 1 is a negative control group, 2 is a dextran group, 3 is a maltotriose group, and 4 is a amylose group;

[0022] Figure 2 A schematic diagram of the separation and identification results in the test example 3 of the present application is shown;

[0023] Figure 3 A schematic diagram of the H NMR technical analysis results shown in the test example 4 of the present application is shown; 1 A schematic diagram of the H NMR technical analysis results shown in the test example 4 of the present application is shown;

[0024] Figure 4 A schematic diagram of the TLC analysis results shown in the test example 5 of the present application is shown,

[0025] Wherein: M is an oligomaltose standard (G1-G7); 1 is an alpha-amylase group; and 2 is a pullulanase group. DETAILED DESCRIPTION

[0026] The present application provides a preparation method of 4,6-alpha-glucosyltransferase, comprising the following steps:

[0027] Taking the amino acid sequence of 4,6-alpha-glucosyltransferase as a template, a first amino acid sequence is obtained by searching;

[0028] The first amino acid sequence is constructed into a plasmid for transformation and expression, and an expression product is obtained.

[0029] The expression product is purified to obtain the 4,6-alpha-glucosyltransferase, which is named as PiGT glycosyltransferase.

[0030] In the present application, the amino acid sequence of 4,6-alpha-glucosyltransferase preferably adopts the amino acid sequence of NCBI accession number AKM18207.

[0031] In the present application, the searching preferably is BLASTp searching in Uniport protein database.

[0032] In the present application, the first amino acid sequence preferably is one sequence from Propioniferax innocua annotated as potential 4,6-alpha-glucosyltransferase, Genbank ID FB460_0880.

[0033] In the present application, the plasmid preferably is pCold-TF plasmid.

[0034] In this invention, the preferred expression conditions are: culture at 15°C and 180 rpm, and induction at a concentration of 0.2 mmol / L IPTG for 20 h.

[0035] The present invention also provides 4,6-α-glucosyltransferase obtained by the preparation method of the 4,6-α-glucosyltransferase described in the above technical solution.

[0036] This invention also provides the 4,6-α-glucosyltransferase prepared by the above-described method, or the application of the 4,6-α-glucosyltransferase in the synthesis of isomaltoglycan and in the prevention of diabetes, cardiovascular disease and obesity.

[0037] In this invention, unless otherwise specified, all raw materials required for preparation are commercially available products well known to those skilled in the art.

[0038] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0039] Example 1

[0040] Using the amino acid sequence of 4,6-α-glucosyltransferase (NCBI accession number AKM18207) as a template, a BLASTp search was performed in the Uniport protein database. After conserved domain analysis, a sequence from Propioniferax innocua annotated as a potential 4,6-α-glucosyltransferase was screened. The Genbank ID of this sequence was FB460_0880. After synthesizing this sequence, it was constructed into the pCold-TF plasmid, transformed, and then cultured at 15℃ and 180 rpm. Expression was induced for 20 h at a concentration of 0.2 mmol / L IPTG. Finally, the 4,6-α-glucosyltransferase, namely PiGT glycosyltransferase, was obtained.

[0041] Performance and Testing

[0042] Test Example 1

[0043] The activity of PiGT glycosyltransferase obtained in Example 1 was determined as follows: 200 μL of 0.25% amylose V solution (w / v, 0.1 mol / L citrate buffer) was incubated in a 45°C water bath for 2 min. PiGT glycosyltransferase solution with a final concentration of 50 μg / mL was added, and the reaction was continued at 45°C for 15 min. The reaction was then terminated by inactivating the enzyme at 95°C for 1 min. 200 μL of the reaction solution was mixed thoroughly with 3800 μL of iodine reagent and incubated in the dark for 2 min. The OD660 absorbance of 200 μL of the mixture was measured at 660 nm using a microplate reader. One unit of enzyme activity is defined as the amount of enzyme required to consume 1 mg of Amylose V per minute. The activity of PiGT glycosyltransferase was determined to be 2239.42 U / μmol.

[0044] Test Example 2

[0045] Using dextran, maltoheptaose, and amylose as substrates, dextran, maltoheptaose, and amylose groups were respectively prepared, and 25 mmol / L solutions were prepared for each group. The PiGT glycosyltransferase obtained in Example 1 was then applied to each of the three groups. The reaction process was as follows: excess PiGT glycosyltransferase was incubated with the three substrates at 37°C for 12 h, followed by incubation in a boiling water bath for 5 min to terminate the enzymatic reaction. The product type was then detected by TLC. The results are as follows: Figure 1 ;based on Figure 1 It is known that PiGT glycosyltransferase has no significant activity for dextran with a large number of branched structures. When maltoheptaose is used as a substrate, oligosaccharides with a degree of polymerization greater than 7 can be generated. When amylose is used as a substrate, PiGT glycosyltransferase can synthesize polysaccharides with different degrees of polymerization.

[0046] Test Example 3

[0047] The products of the maltoheptaose group and the amylose group obtained in Test Example 2 were separated and identified by high performance anion exchange chromatography. The results are as follows: Figure 2 As shown; based on Figure 2 It is known that PiGT glycosyltransferase catalyzes the formation of oligosaccharides with a higher degree of polymerization in the products of amylose and maltheptaose, suggesting that the enzyme has transglycosylation activity.

[0048] Test Example 4

[0049] The product of the amylose group obtained in test 2 was used... 1 Further identification was performed using 1H NMR technology, and the results were as follows: Figure 3 As shown; based on Figure 3It can be seen that characteristic peaks corresponding to α-1,4 glycosidic bonds and newly formed α-1,6 glycosidic bonds in the product appear at δ 5.12 and δ 4.78, respectively, and no characteristic peaks of other glycosidic bond types are identified. According to the peak area integral, the content of α-1,6 bonds is about 70.7%, which is significantly higher than the basic content of 2% in the amylose substrate. This proves that PiGT glycosyltransferase can catalyze the production of isomaltan from amylose with superior resistance to α-amylase hydrolysis.

[0050] Test Example 5

[0051] The amylose group product obtained from Test Example 2 was mixed with 5 U of α-amylase and pullulanase, respectively, and designated as the α-amylase group and pullulanase group. TLC analysis was performed, and the results are as follows: Figure 4 As shown; based on Figure 4 It can be seen that when amylose is used as a substrate, a small amount of glucose was detected in the hydrolysis products of α-amylase, suggesting that the catalytic products of glycosyltransferase contain a small amount of (α1→4) glycosidic bonds; pullulanase hydrolyzes polysaccharides to produce a large number of monosaccharides to tetrasaccharides, and the polysaccharides treated with glycosyltransferase have a large number of (α1→6) branched structures. Therefore, the structure of the enzyme catalytic products has both (α1→4) and (α1→6) glycosidic bonds.

[0052] In summary, the PiGT glycosyltransferase described in this invention has higher activity, and isomaltan with superior performance can be obtained based on it, thus exhibiting better application effects in the prevention of similar diseases such as diabetes, cardiovascular disease, and obesity.

[0053] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for producing a 4,6-α-glucosyltransferase, characterized by, The method comprises the following steps: Taking the amino acid sequence of the 4,6-alpha-glucosyltransferase as a template, a first amino acid sequence is obtained by searching; The nucleotide sequence of the first amino acid sequence is constructed into a plasmid for transformation and expression, to obtain an expression product; The expression product is purified to obtain the 4,6-alpha-glucosyltransferase, which is named as PiGT glycosyltransferase; The amino acid sequence of the 4,6-alpha-glucosyltransferase is the amino acid sequence with the NCBI accession number of AKM18207; The searching is BLASTp searching in the Uniport protein database; The first amino acid sequence is one sequence from Propioniferax innocua, which is annotated as potential 4,6-alpha-glucosyltransferase, and the Genbank ID is FB460_0880; The plasmid is a pCold-TF plasmid; The expression condition is culturing at 15 DEG C and 180 rpm, and inducing for 20 hours under the concentration of 0.2 mmol / L IPTG.

2. Application of the 4,6-alpha-glucosyltransferase obtained by the preparation method of the 4,6-alpha-glucosyltransferase in claim 1 in synthesis of isomaltulose.

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