Novel glycosyl transferase and application thereof in synthesis of slowly digestible starch

Abstract

The invention relates to the technical field of enzyme engineering, and discloses novel glycosyl transferase and application thereof in synthesis of slowly digestible starch, glycosyl transferase protein is named as HsGT, and the amino acid sequence of the HsGT is shown as SEQ ID NO: 1. The invention discloses a new type of 4, 6-alpha-glucosyltransferase, and particularly relates to HsGT glycosyltransferase. Compared with the sequence recorded in the existing database, the similarity of the amino acid sequence of the HsGT glycosyl transferase is 58.6%, so that the innovation of the HsGT glycosyl transferase is manifested in the field of protein sequences, and the HsGT glycosyl transferase shows the highest enzymatic activity in the known same class of enzymes.

Description

Technical Field

[0001] The invention relates to the technical field of enzyme engineering, and in particular to a novel glycosyltransferase and application thereof in synthesizing slowly digestible starch. Background Art

[0002] The incidence of diabetes, cardiovascular disease and obesity continues to rise, which is closely related to the long-term intake of foods rich in rapidly digestible starch. In contrast, foods containing more slowly digestible starch and resistant starch are digested more slowly in the body and are low-glycemic index foods, which help maintain stable blood sugar levels before and after meals. 4,6-α-glucosyltransferase can break the (α1→4) glycosidic bond in the starch molecule and transfer the resulting glucan to another glycosyl group with an (α1→6) glycosidic bond, forming starch containing a (α1→4) and (α1→6) chimeric glycosidic bond structure. Since amylase has a weak ability to hydrolyze this structure, it can significantly slow down the release of glucose.

[0003] At present, the known 4,6-α-glucosyltransferases (4,6-α-GTs) include GtfB, GtfC, GtfD, GtfW, GtfML4, GtfX and GtfY. However, the enzyme activity of 4,6-α-GTs reported in the existing literature has not reached the ideal level, and these enzymes have high molecular weight and complex multi-domain characteristics, which makes their soluble expression in heterologous systems difficult, which limits their large-scale production and application. Summary of the invention

[0004] In view of this, the present invention proposes a novel glycosyltransferase and its application in the synthesis of slowly digestible starch, aiming to evaluate the performance of the enzyme-catalyzed product and characterize its enzymatic properties.

[0005] The present invention provides a glycosyltransferase protein, the glycosyltransferase protein is named HsGT, and the amino acid sequence of the HsGT is shown in SEQ ID NO:1.

[0006] The present invention also provides a glycosyltransferase protein, the glycosyltransferase protein is named HsGT, and the HsGT is expressed by a gene with a Genbank ID of B4102_3700.

[0007] The present invention also provides an application of the above-mentioned glycosyltransferase protein, wherein the application comprises at least one of the following:

[0008] A1) Application in the synthesis of slowly digestible starch;

[0009] A2) Application in synthetic oligosaccharides;

[0010] A3) Application as food additive;

[0011] A4) Application in the preparation of food additives.

[0012] The present invention also provides a composition, which comprises the above-mentioned glycosyltransferase protein.

[0013] The present invention also provides a method for preparing the above-mentioned glycosyltransferase protein, the method comprising the following steps:

[0014] Expressing a gene containing Genbank ID B4102_3700 in a host cell;

[0015] The host cells are collected and disrupted to release the glycosyltransferase protein.

[0016] The present invention also proposes a method for preparing slowly digestible starch by using the above-mentioned glycosyltransferase protein catalyzed reaction, the method comprising the following steps:

[0017] Using amylose as a substrate, the above-mentioned glycosyltransferase protein is mixed with the substrate to carry out a catalytic reaction to obtain the slowly digestible starch.

[0018] The present invention also provides a method for synthesizing oligosaccharides using the above-mentioned glycosyltransferase protein, the method comprising the following steps:

[0019] Maltooligosaccharide is used as a substrate, the above-mentioned glycosyltransferase protein is mixed with the substrate, and a catalytic reaction is carried out to obtain the oligosaccharide.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The present invention is based on the disclosed 4,6-α-glucosyltransferase as a reference, and through database screening, a new 4,6-α-glucosyltransferase sequence is discovered, named HsGT glycosyltransferase. The amino acid sequence of the enzyme has a similarity of 58.6% with the known sequence in the database, showing its innovation at the protein sequence level. Among the known enzymes of the same type, HsGT glycosyltransferase exhibits the highest enzyme activity, and its enzyme activity can reach 3895.63U / μmol. The optimal reaction temperature of the enzyme is 50°C, and the optimal pH value is 6.6. In the process of catalyzing amylose, HsGT glycosyltransferase can produce products containing (α1→4) and (α1→6) glycosidic bonds, so the product has a high resistance to α-amylase and belongs to the category of slowly digestible starch. In summary, the HsGT glycosyltransferase in the present invention is a new enzyme species that can synthesize slowly digestible starch. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Various other advantages and benefits will become apparent to those of ordinary skill in the art by reading the detailed description of the preferred embodiments below. The accompanying drawings are only for the purpose of illustrating the preferred embodiments and are not to be considered as limiting the present invention. Moreover, the same reference symbols are used throughout the accompanying drawings to represent the same components. In the accompanying drawings:

[0023] Figure 1 The analysis results of the reaction products after being treated with different enzymes, where M is the maltooligosaccharide standard (G1-G7), 1 is α-amylase treatment, and 2 is pullulanase treatment;

[0024] Figure 2 The specific results of HsGT glycosyltransferase for different substrates, where M is an oligosaccharide standard (G1-G7), 1 is dextran, 2 is amylose, and 3 is maltoheptaose;

[0025] Figure 3 This is the HPAEC spectrum of maltoheptaose produced by HsGT glycosyltransferase;

[0026] Figure 4 The effect of different temperatures on the activity of HsGT glycosyltransferase;

[0027] Figure 5 The effect of different pH on the activity of HsGT glycosyltransferase. DETAILED DESCRIPTION

[0028] Exemplary embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. Although exemplary embodiments of the present disclosure are shown in the accompanying drawings, it should be understood that the present disclosure can be implemented in various forms and should not be limited by the embodiments described herein. On the contrary, these embodiments are provided in order to enable a more thorough understanding of the present disclosure and to fully convey the scope of the present disclosure to those skilled in the art. It should be noted that, in the absence of conflict, the embodiments of the present invention and the features described in the embodiments can be combined with each other. The present invention will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.

[0029] Example 1

[0030] 1. Screening of 4,6-α-glucosyltransferase

[0031] 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, and six sequences annotated as potential 4,6-α-glycosyltransferases were screened out through conservative domain analysis. Table 1 shows the sources of candidate genes for 4,6-α-glucosyltransferase. These sequences were synthesized by the company and constructed into the pCold-TF plasmid, followed by expression and purification. Using amylose as a substrate, the 4,6-α-glucosyltransferase activity of the recombinant protein was determined. Table 2 shows the results of the determination of 4,6-α-glucosyltransferase activity. The results showed that the protein expressed by the gene with Genbank ID B4102_3700 exhibited 4,6-α-glucosyltransferase activity, and its molar activity reached 3895.63U / μmol, which is the highest catalytic activity reported in existing literature. The amino acid homology of this gene with the existing sequences in the database is 58.6%, thus showing its uniqueness in protein sequence. The enzyme was named HsGT.

[0032] Amino acid sequence of HsGT (SEQ ID NO: 1):

[0033] 。

[0034] Table 1

[0035]

[0036] Table 2

[0037] 4,6-α-GTase Specific activity (U / μmol) GtfC (control) 2661.67 HkDJ 3895.63

[0038] 2. Structural characteristics of HsGT catalytic products

[0039] The product of the enzyme catalyzed reaction was analyzed by thin layer chromatography (TLC) after adding 5 units of α-amylase and pullulanase. The results are shown in Figure 1 . The analysis showed that when amylose was used as a substrate, a small amount of disaccharides were detected in the hydrolysis products of α-amylase. It can be inferred that there are a small amount of (α1→4) glycosidic bonds in the catalytic products of HsGT glycosyltransferase, which shows strong resistance to amylase and belongs to the category of slowly digestible starch. After hydrolysis by pullulanase, the catalytic products of HsGT glycosyltransferase produced a large amount of monosaccharides to tetrasaccharides, indicating that its catalytic products contain a large amount of (α1→6) branched structures. Therefore, the structure of the catalytic product of HsGT enzyme has both (α1→4) and (α1→6) glycosidic bonds, which makes the product show strong resistance to α-amylase.

[0040] 3. Substrate specificity of HsGT glycosyltransferase

[0041] After HsGT glycosyltransferase acts on dextran, amylose and maltoheptaose, the products are detected by thin layer chromatography (TLC). The results are shown in Figure 2 . Analysis of the graphical data shows that HsGT glycosyltransferase does not show significant activity towards dextran containing a large number of branched structures. However, when amylose is used as a substrate, the enzyme is able to synthesize polysaccharides with different degrees of polymerization. In the case of maltoheptaose as a substrate, oligosaccharides with a degree of polymerization exceeding 7 are produced after the enzyme action. High performance anion exchange chromatography (HPAEC) was further used to separate and identify the enzyme-catalyzed products of the maltoheptaose substrate at the high performance liquid chromatography (HPLC) level, and the results are presented in Figure 3 The analysis showed that there were heterogeneous high-polymerization oligosaccharides in the products treated by HsGT glycosyltransferase, which suggested that the enzyme had transglycosidic activity.

[0042] 4. Enzymatic properties of HsGT glycosyltransferase

[0043] 1. Effect of different temperatures on HsGT glycosyltransferase activity

[0044] The enzymatic activity of HsGT glycosyltransferase on amylose was measured under different temperature conditions, and the results are shown in Figure 4 The study found that HsGT glycosyltransferase exhibited high enzyme activity in the temperature range of 35-55°C, and its most suitable reaction temperature was 50°C.

[0045] 2. Effect of different pH on HsGT glycosyltransferase activity

[0046] like Figure 5 As shown in Figure 2, the activity of HsGT glycosyltransferase was significantly affected by pH. The enzyme activity was higher when the pH was between 6.1 and 7.6, and the optimal pH was 6.6.

[0047] V. Overview

[0048] The discovery and research of HsGT glycosyltransferase has brought new breakthroughs in the field of starch metabolism. This enzyme not only has high 4,6-α-glucosyltransferase activity, but also has potential application value in the food industry and biotechnology due to its unique substrate specificity and enzymatic properties. HsGT glycosyltransferase's efficient conversion ability to amylose makes it an ideal candidate enzyme for the synthesis of slowly digestible starch. As a functional food ingredient, slowly digestible starch can slow down the digestion rate and help control blood sugar levels, which is of great significance for diabetic patients.

[0049] In addition, HsGT glycosyltransferase performs well in synthesizing oligosaccharides with specific structures, which provides the possibility for the development of new prebiotics. As a nutrient source for probiotics, prebiotics can promote the growth of beneficial bacteria in the intestine and improve intestinal health. The transglycosidic activity of HsGT glycosyltransferase enables it to synthesize oligosaccharides with complex structures, which may provide new ideas for the development of new prebiotics.

[0050] In terms of industrial applications, the thermal stability and pH adaptability of HsGT glycosyltransferase give it high operational flexibility in the production process. The optimal reaction temperature of the enzyme is 50°C, which facilitates starch conversion under mild conditions. At the same time, the enzyme is most active at pH 6.6, which makes it possible to carry out enzyme reactions under near-neutral conditions, reducing corrosion to equipment and impact on the environment.

[0051] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, ordinary technicians in the relevant field should understand that the specific implementation methods of the present invention can still be modified or replaced by equivalents. Any modification or equivalent replacement that does not depart from the spirit and scope of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A glycosyltransferase protein, characterized in that The glycosyltransferase protein is named HsGT, and the amino acid sequence of HsGT is shown in SEQ ID NO:

1.

2. A glycosyltransferase protein, characterized in that The glycosyltransferase protein is named HsGT, and the HsGT is expressed by the gene with Genbank ID B4102_3700.

3. Use of the glycosyltransferase protein according to claim 1 or 2, characterized in that: The application includes at least one of the following: A1) Application in the synthesis of slowly digestible starch; A2) Application in synthetic oligosaccharides; A3) Application as food additive; A4) Application in the preparation of food additives.

4. A composition, characterized in that The composition comprises the glycosyltransferase protein according to claim 1 or 2.

5. A method for preparing the glycosyltransferase protein according to claim 1 or 2, characterized in that: The method comprises the following steps: Expressing a gene containing Genbank ID B4102_3700 in a host cell; The host cells are collected and disrupted to release the glycosyltransferase protein.

6. A method for preparing slowly digestible starch by using the glycosyltransferase protein catalyzed reaction according to claim 1 or 2, characterized in that: The method comprises the following steps: Using amylose as a substrate, the glycosyltransferase protein according to claim 1 or 2 is mixed with the substrate to carry out a catalytic reaction to obtain the slowly digestible starch.

7. A method for synthesizing oligosaccharides using the glycosyltransferase protein according to claim 1 or 2, characterized in that: The method comprises the following steps: Maltooligosaccharide is used as a substrate, the glycosyltransferase protein according to claim 1 or 2 is mixed with the substrate, and a catalytic reaction is carried out to obtain the oligosaccharide.

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