WO2023155404A1 - 一种具有4,3/6-α-葡萄糖基转移酶活性的GtfB酶 - Google Patents

一种具有4,3/6-α-葡萄糖基转移酶活性的GtfB酶 Download PDF

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WO2023155404A1
WO2023155404A1 PCT/CN2022/114438 CN2022114438W WO2023155404A1 WO 2023155404 A1 WO2023155404 A1 WO 2023155404A1 CN 2022114438 W CN2022114438 W CN 2022114438W WO 2023155404 A1 WO2023155404 A1 WO 2023155404A1
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gtfb
application according
gtfb enzyme
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柏玉香
董晶晶
金征宇
王艳丽
纪杭燕
李晓晓
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Jiangnan University
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)
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    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
    • C12P19/08—Dextran
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    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/18—Preparation of compounds containing saccharide radicals produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins

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  • the invention relates to a GtfB enzyme with 4,3/6- ⁇ -glucosyltransferase activity and belongs to the technical field of starch modification.
  • ⁇ -glucan is an oligo/polysaccharide composed of several glucose linked by ⁇ -1,2, ⁇ -1,3, ⁇ -1,4 or ⁇ -1,6 glycosidic bonds.
  • the differences in the degree of branching, molecular weight and conformation of ⁇ -glucan can lead to different functional properties of ⁇ -glucan, which has a variety of industrial application prospects.
  • ⁇ -glucans synthesized by lactic acid bacteria (LAB) are used as tougheners, thickeners and emulsifiers in the health and food industries.
  • GtfB enzyme (EC 2.4.1.-) is a glycosyltransferase that uses maltodextrin/starch as a substrate to synthesize ⁇ -glucan, and belongs to the subfamily of the glycosyl hydrolase GH70 family.
  • the currently reported GtfB enzymes mainly exhibit 4,6- ⁇ -glucosyltransferase activity, which can cut ( ⁇ 1 ⁇ 4) glycosidic bonds of maltodextrin/starch and synthesize ( ⁇ 1 ⁇ 6) glycosidic bonds.
  • the invention discloses the GtfB enzyme whose sequence is shown in SEQ ID NO.1, which can cut ( ⁇ 1 ⁇ 4) glycosidic bonds of maltodextrin or starch, and synthesize ( ⁇ 1 ⁇ 3) and ( ⁇ 1 ⁇ 6) glycosidic bonds, which can be used for Synthesis of ⁇ -glucans with ( ⁇ 1 ⁇ 4), linear ( ⁇ 1 ⁇ 6) glycosidic linkages, ( ⁇ 1 ⁇ 3,4) branched glycosidic linkages and ( ⁇ 1 ⁇ 4,6) branched glycosidic linkages.
  • N1 GtfB enzyme The GtfB enzyme whose sequence is shown in SEQ ID NO.1 comes from Lactobacillus reuteri N1, we named it N1 GtfB enzyme, the results of substrate and product specificity experimental analysis showed that N1 GtfB enzyme has 4,3/ 6- ⁇ -glucanotransferase activity, able to use starch as a substrate to synthesize ( ⁇ 1 ⁇ 4) and ( ⁇ 1 ⁇ 6) alternately, accompanied by ( ⁇ 1 ⁇ 3,4) and ( ⁇ 1 ⁇ 4,6) Branched alpha-glucans. To date, this activity has not been reported in any GtfB enzymes, and this bond-type combined ⁇ -glucan has not been synthesized in any wild-type glucanases (GSs) or GtfB enzymes. The specificity of this product expands the diversity of ⁇ -glucans, which opens up new prospects for the synthesis of novel glucans from starch.
  • GSs wild-type glucanases
  • the invention discloses the application of GtfB enzyme in the preparation of ⁇ -glucan, comprising the following steps:
  • the amino acid sequence of the GtfB enzyme is shown in SEQ ID NO.1, and the ⁇ -glucan has ( ⁇ 1 ⁇ 4), linear ( ⁇ 1 ⁇ 6) glycosidic bonds, ( ⁇ 1 ⁇ 3,4) branched glycosidic bonds and ( ⁇ 1 ⁇ 4,6) branched glycosidic linkages.
  • the amino acid sequence of the GtfB enzyme is shown in SEQ ID NO.1, which is a novel GtfB from Lactobacillus reuteri N1, which we named as N1 GtfB enzyme.
  • the starch is amylose, and the substrate concentration is 0.6%; the dextrin is maltoheptaose, and the substrate concentration is 1%.
  • the buffer used may be a 10-50 mM, pH 4-6 buffer, such as 25 mM, pH 6 sodium acetate buffer.
  • the reaction temperature for substrate modification catalyzed by the N1 GtfB enzyme is 37-40°C, 24h-48h, for example, 37°C, 24h.
  • the amount of N1 GtfB enzyme added is 12-20U/g dry basis starch or dextrin.
  • N1 GtfB enzyme has 4,3/6- ⁇ -glucanotransferase activity, and can synthesize ( ⁇ 1 ⁇ 4) and ( ⁇ 1 ⁇ 6) with starch as substrate. ) alternately with ( ⁇ 1 ⁇ 3,4) and ( ⁇ 1 ⁇ 4,6) branched ⁇ -glucans.
  • this activity has not been found in any reported GtfB enzymes, and the ⁇ -glucan product of this bond type combination has not been synthesized in any wild-type glucanases (GSs) or GtfB enzymes.
  • the method of the present invention uses starch or dextrin as a substrate, and synthesizes a ⁇ -glucan with a unique structure after being modified by N1 GtfB enzyme.
  • Four glycosidic linkages ⁇ 1 ⁇ 4), ( ⁇ 1 ⁇ 6), ( ⁇ 1 ⁇ 3,4) and ( ⁇ 1 ⁇ 4,6) existed in the product at the same time.
  • the specificity of the product expands the diversity of ⁇ -glucan, opens up new prospects and provides new ideas for the synthesis of new glucans from starch.
  • Fig. 1 is the substrate specificity analysis of N1 GtfB enzyme in embodiment 1.
  • G1 represents glucose
  • G2-G8 represent maltose to maltooctaose, respectively
  • AML and AMP represent amylose and amylopectin, respectively.
  • Fig. 2 is the molecular weight distribution diagram of the products obtained by reacting with maltoheptaose and amylose as substrates for 24 hours in Example 2.
  • G7 stands for maltoheptaose
  • amylose stands for amylose.
  • Fig. 3 is the molecular weight 1 H NMR chart of the product obtained by reacting N1 GtfB enzyme and amylose for 24 hours in Example 3.
  • Figure 4 shows the situation of oligosaccharides synthesized in real time by N1 GtfB enzyme respectively modifying maltoheptaose and amylose in Example 4.
  • G1 is glucose
  • G2-G7 are maltose to maltoheptaose respectively
  • IsoG2 IsoG3 and Panose are isomaltose, isomaltotriose and panose respectively.
  • Fig. 5 is a two-dimensional nuclear magnetic spectrum ( 1 H- 1 H COZY and 13 C- 1 H HSQC) of the product synthesized by N1 GtfB enzyme modified amylose in Example 6.
  • Fig. 6 is the structural model of N1 GtfB enzyme modified amylose product constructed in embodiment 7.
  • the artificially synthesized nucleotide sequence of the gene fragment encoding the N1 GtfB enzyme shown in SEQ ID NO: 2 was used to construct a recombinant plasmid with pET-15b, and the E.coli BL21 (DE3) expression strain was used for expression. It was placed in LB liquid medium containing 100 ⁇ g/mL ampicillin, and cultured at 37° C. and 200 r/min until the OD600 value was 0.4-0.6. After taking it out, put it in an ice bath for 15 minutes, add isopropylthiogalactoside to a final concentration of 1 mM, and culture at 16°C and 160 r/min for 20 hours to induce enzyme production.
  • the crude enzyme solution was purified by nickel affinity chromatography, eluted sequentially with 20mM Tris-HCl (250mM NaCl, pH 8) and 20mM Tris-HCl (250mM NaCl, pH 8) containing different concentrations of imidazole, and each fraction was collected The flow-through solution was verified by polyacrylamide gel electrophoresis.
  • Enzyme activity definition the enzyme required to consume 1mg of amylose per minute is defined as 1U.
  • Example 1 Using N1 GtfB enzyme to catalyze maltooligosaccharides, amylose and amylopectin respectively and analyze the substrate specificity of N1 GtfB enzyme
  • Amylose and amylopectin need to be pretreated before the reaction.
  • Amylose needs to be dissolved in alkali (NaOH, 1M) first, then neutralized by acid, and then add sodium acetate buffer (pH 6, 25mM) to prepare a 0.6% (ie 0.6g/100mL) solution.
  • N1 GtfB enzyme to a final concentration of 20 ⁇ g/mL, react at 37°C for 24 hours, and terminate the reaction in a boiling water bath.
  • Amylopectin is dissolved in sodium acetate buffer (pH 6, 25mM), prepared into a 0.6% (ie 0.6g/100mL) solution, vortexed, gelatinized, and then cooled to 37°C.
  • N1 GtfB enzyme can act on maltodextrin and starch substrates with DP ⁇ 3.
  • the substrate is maltodextrin
  • a large amount of oligo/polysaccharides with DP ⁇ 2 and a small amount of glucose are synthesized in the product.
  • the substrate is When amylose and amylopectin are used, the amount of oligo/polysaccharides synthesized in the product is significantly smaller than that produced when maltodextrin is used as a substrate.
  • N1 GtfB enzyme has both hydrolytic and transgenic properties for dextrin or starch substrates with DP ⁇ 3.
  • Glycosylase (dismutation) activity was generated in the modified products, indicating that the N1 GtfB enzyme has both hydrolytic and transgenic properties for dextrin or starch substrates with DP ⁇ 3.
  • Example 2 Using N1 GtfB enzyme to catalyze maltoheptaose and amylose respectively and analyze the molecular weight of the products
  • N1 GtfB enzyme to a final concentration of 20 ⁇ g/mL, react at 37°C for 24 hours, and terminate the reaction in a boiling water bath.
  • Example 3 N1 GtfB enzyme catalyzes amylose and analyzes the one-dimensional proton nuclear magnetic spectrum of the product
  • N1 GtfB enzyme to a final concentration of 20 ⁇ g/mL, react at 37°C and pH 6.0 for 24 hours, terminate the reaction in a boiling water bath, and freeze-dry.
  • the two split peaks at ⁇ 4.97ppm indicate that there are at least two different structural units in the (-) ⁇ -D-Glcp-(1 ⁇ 6)-structure.
  • R ⁇ ( ⁇ 5.237ppm) and R ⁇ ( ⁇ 4.653ppm) represent the anomeric hydrogen signal of 4-substituted reducing terminal glucose.
  • G ⁇ ( ⁇ 5.231ppm) and G ⁇ ( ⁇ 4.640ppm) are ectopic signals of free glucose.
  • the t4 signal between ⁇ 3.40 ⁇ 3.45ppm proves the existence of ( ⁇ 1 ⁇ 4,6) branch. Overall, the results of 1 HNMR clearly confirmed the presence of ( ⁇ 1 ⁇ 6) bond in the product.
  • Example 4 Oligosaccharide analysis of real-time synthesis of maltoheptaose and amylose modified by N1 GtfB enzyme
  • N1 GtfB enzyme to a final concentration of 20 ⁇ g/mL, react at 37°C for 24 hours, and terminate the reaction in a boiling water bath.
  • HPAEC-PAD high-pressure anion chromatography
  • Example 5 Methylation analysis of real-time products during amylose modification using N1 GtfB enzyme
  • the method for modifying amylose with N1 GtfB enzyme is the same as that of (2) in Example 2.
  • the reaction solution is directly freeze-dried for future use. 20 mg of lyophilized sample was dissolved in 20 mL of DMSO at 60°C, then 20 mL of NaOH solution dissolved in DMSO was added to react for 1 h, and then CH 3 I liquid was added dropwise for methylation reaction at room temperature for 2 h. After the reaction, the methylated product was dialyzed with a 1000Da dialysis bag, extracted with chloroform, dehydrated with sodium sulfate, and dried with nitrogen.
  • Example 6 Analysis of two-dimensional NMR spectra of products synthesized by N1 GtfB enzyme modified amylose
  • N1 GtfB enzyme modified amylose and the sample pretreatment method before NMR spectrum determination is the same as in Example 3.
  • the different glycosyl structural units in the modified product were analyzed by 1 HNMR, 2D 1 H COZY and 2D 13 C- 1 H HSQC spectrum results.
  • glucose units in the product there are 7 differently substituted glucose units in the product, namely A: -(1 ⁇ 4)- ⁇ -D-Glcp-(1 ⁇ 4)-; B: -(1 ⁇ 3 ,4) ⁇ -D-Glcp-(1 ⁇ 4)-; C: ⁇ -D-Glcp-(1 ⁇ 4)-; D: -(1 ⁇ 6) ⁇ -D-Glcp-(1 ⁇ 4) -;E: -(1 ⁇ 4,6) ⁇ -D-Glcp-(1 ⁇ 4)-;F:-(1 ⁇ 4)- ⁇ -D-Glcp-(1 ⁇ 4,6);G: -(1 ⁇ 4)- ⁇ -D-Glcp-(1 ⁇ 6).
  • Embodiment 7 Construct the structure model of N1 GtfB enzyme modified amylose product
  • Example 6 Combined with the one/two-dimensional NMR of Example 3 and Example 6, the results of real-time detection of oligosaccharides by high-pressure ion chromatography and the results of methylation experiments, the bond types and ratios contained in the N1 GtfB enzyme modified products were determined, thereby constructing The corresponding product structure model, the results are shown in Figure 6.
  • the substrates in Example 1 were respectively adjusted to glucose, maltose, sucrose, isomaltose, isomaltotriose, aspergillus niger or panose, and the rest of the reaction conditions or parameters were consistent with those in Example 1. Specifically, these substrates were dissolved in pH 6 and 25mM sodium acetate buffer systems respectively, and prepared into 1% solutions respectively, and N1 GtfB enzyme was added to make the final concentration (20 ⁇ g/mL), and the substrates were prepared at 37°C. Reaction 24h, boiling water bath to terminate the reaction.
  • the reaction was monitored by thin-layer chromatography (TLC), and it was found that the N1 GtfB enzyme had no activity on glucose, maltose, sucrose, isomaltose, isomaltotriose, nigerose, and panose.
  • TLC thin-layer chromatography
  • Example 2 The enzyme treatment in Example 2 is adjusted to no enzyme treatment. All the other reaction conditions or parameters are consistent with Example 2. Specifically, maltoheptaose was dissolved in a pH 6, 25mM sodium acetate buffer system to prepare a 1% solution, and incubated at 37°C for 24h. Amylose was first dissolved in alkali (NaOH, 1M), then neutralized by acid, and then buffered with sodium acetate to prepare a 0.6% solution, which was kept at 37°C for 24 hours.
  • alkali NaOH, 1M
  • Example 4 The enzyme treatment in Example 4 was adjusted to no enzyme treatment. The remaining reaction conditions or parameters are consistent with those in Example 4. Specifically, maltoheptaose is dissolved in a pH 6, 25mM sodium acetate buffer system to prepare a 1% solution and incubated at 37°C for 24h. Amylose was first dissolved in alkali (NaOH, 1M), then neutralized by acid, and then buffered with sodium acetate to prepare a 0.6% solution, which was kept at 37°C for 24 hours.
  • alkali NaOH, 1M
  • the oligosaccharides in the samples were monitored by high-pressure ion chromatography. It can be seen from the results that the standard maltoheptaose contains a certain amount of maltopentaose and maltohexaose, while the content of oligosaccharides in the standard amylose is very small.

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Abstract

一种具有4,3/6-α-葡萄糖基转移酶活性的GtfB酶，属于淀粉改性技术领域。制备了一种(α1→4)和(α1→6)交替并伴有(α1→3,4)和(α1→4,6)分支的α-葡聚糖。同时提供了一种以直链淀粉为底物制备的高支化且低分子量聚合物的方法。

Description

一种具有4,3/6-α-葡萄糖基转移酶活性的GtfB酶 技术领域
本发明涉及一种具有4,3/6-α-葡萄糖基转移酶活性的GtfB酶，属于淀粉改性技术领域。
背景技术
α-葡聚糖是由若干个葡萄糖以α-1,2、α-1,3、α-1,4或α-1,6糖苷键连接而成的寡/多糖。α-葡聚糖在分枝程度、分子质量和构象方面的差异可以导致α-葡聚糖表现出不同的功能性质，从而具有多种多样的工业应用前景。例如，由乳酸菌(LAB)合成的α-葡聚糖在健康和食品行业中可用作增韧剂、增稠剂和乳化剂。
GtfB酶(EC 2.4.1.-)是一种以麦芽糖糊精/淀粉为底物，合成α-葡聚糖的糖基转移酶，属于糖基水解酶GH70家族的亚家族。目前报道的GtfB酶主要展现4,6-α-葡萄糖基转移酶活性，能够切断麦芽糖糊精/淀粉的(α1→4)糖苷键，合成(α1→6)糖苷键。迄今只有来自发酵乳杆菌NCC 2970的GtfB酶展现4,3-α-葡萄糖基转移酶活性，可以切断麦芽糖糊精/淀粉的(α1→4)糖苷键，合成(α1→3)糖苷键。缺乏能够合成多种糖苷键组合的新型GtfB酶限制了以淀粉为底物合成结构不同的α-葡聚糖。因此，缺乏能够合成多种糖苷键组合的新型GtfB酶，限制了以淀粉为底物合成不同结构的α-葡聚糖。
发明内容
本发明披露序列如SEQ ID NO.1所示的GtfB酶，能够切断麦芽糖糊精或淀粉的(α1→4)糖苷键，合成(α1→3)和(α1→6)糖苷键，可以用于合成具有(α1→4)、线性(α1→6)糖苷键、(α1→3,4)分支糖苷键和(α1→4,6)分支糖苷键的α-葡聚糖。
所述序列如SEQ ID NO.1所示的GtfB酶来自罗伊氏乳杆菌N1，我们将其命名为N1 GtfB酶，底物和产物特异性实验分析的结果表明N1 GtfB酶具有4,3/6-α-葡聚糖转移酶活性，能够以淀粉为底物合成具有(α1→4)和(α1→6)交替，并伴有(α1→3,4)和(α1→4,6)分支的α-葡聚糖。迄今为止，该活性未在任何GtfB酶中被报道，同时该键型组合的α-葡聚糖未在任何野生型葡聚蔗糖酶(GSs)或GtfB酶中被合成。该产物的特异性扩大了α-葡聚糖的多样性，这为以淀粉合成新型葡聚糖开辟了新的前景。
本发明披露了GtfB酶在制备α-葡聚糖中的应用，包括如下步骤：
(1)以淀粉或糊精为底物，溶解淀粉或糊精，
(2)添加GtfB酶，催化底物生成α-葡聚糖；
所述GtfB酶的氨基酸序列如SEQ ID NO.1所示，所述α-葡聚糖具有(α1→4)、线性(α1→6) 糖苷键、(α1→3,4)分支糖苷键和(α1→4,6)分支糖苷键。
所述GtfB酶的氨基酸序列如SEQ ID NO.1所示，是来自罗伊氏乳杆菌N1的新型GtfB，我们将其命名为N1 GtfB酶。
在本发明的某些实施方式中，包括如下步骤：
(1)以淀粉为底物时，将直链淀粉先用氢氧化钠溶液溶解，再用盐酸中和后，加缓冲液，
(2)再添加N1 GtfB酶催化底物改性，得到合成的α-葡聚糖。
在本发明的某些实施方式中，包括如下步骤：
(1)以糊精为底物时，加缓冲液，
(2)再添加N1 GtfB酶催化底物改性，得到合成的α-葡聚糖。
在本发明的某些实施方式中，所述淀粉为直链淀粉，底物浓度为0.6％；所述糊精为麦芽七糖，底物浓度为1％。
在本发明的某些实施方式中，所使用的缓冲液可以是10～50mM、pH 4～6的缓冲液，例如25mM、pH为6的醋酸钠缓冲液。
在本发明的某些实施方式中，所述N1 GtfB酶催化底物改性的反应温度为37～40℃，24h～48h，例如37℃、24h。
在本发明的某些实施方式中，N1 GtfB酶的添加量为12-20U/g干基淀粉或糊精。
本发明通过底物和产物特异性实验分析，发现N1 GtfB酶具有4,3/6-α-葡聚糖转移酶活性，能够以淀粉为底物合成具有(α1→4)和(α1→6)交替，并伴有(α1→3,4)和(α1→4,6)分支的α-葡聚糖。迄今为止，该活性未在任何已报道的GtfB酶中发现，且该键型组合的α-葡聚糖产物未在任何野生型葡聚蔗糖酶(GSs)或GtfB酶中被合成。
本发明的方法以淀粉或糊精为底物，经N1 GtfB酶改性后，合成一种结构独特的α-葡聚糖。该产物中的同时存在(α1→4)、(α1→6)、(α1→3,4)和(α1→4,6)四种糖苷键。该产物的特异性扩大了α-葡聚糖的多样性，为以淀粉合成新型葡聚糖开辟了新的前景，提供了新的思路。
附图说明
图1为实施例1中N1 GtfB酶的底物特异性分析。G1代表葡萄糖，G2-G8分别代表麦芽糖至麦芽八糖，AML和AMP分别代表直链淀粉和支链淀粉。
图2为实施例2中以分别以麦芽七糖和直链淀粉为底物反应24h反应所得产物分子量分布图。G7代表麦芽七糖，amylose代表直链淀粉。
图3为实施例3中N1 GtfB酶和直链淀粉反应24h反应所得产物分子量 1HNMR图。
图4为实施例4中N1 GtfB酶分别改性麦芽七糖和直链淀粉实时合成的寡糖情况。G1为 葡萄糖，G2-G7分别麦芽糖至麦芽七糖，IsoG2、IsoG3和Panose分别为异麦芽糖、异麦芽三糖和潘糖。
图5为实施例6中N1 GtfB酶改性直链淀粉合成的产物二维核磁谱图( 1H- 1H COSY和 13C- 1H HSQC)。
图6为实施例7中构建的N1 GtfB酶改性直链淀粉产物的结构模型。
具体实施方式
来自罗伊氏乳杆菌N1的GtfB酶的制备及纯化：
人工合成核苷酸序列如SEQ ID NO:2所示的编码N1 GtfB酶的基因片段，以pET-15b构建重组质粒，利用E.coli BL21(DE3)表达菌株进行表达。将其置于含有100μg/mL氨苄青霉素的LB液体培养基中，于37℃、200r/min的条件下培养，直至OD600值为0.4-0.6。取出后，冰浴15min，加入异丙基硫代半乳糖苷至终浓度为1mM，于16℃、160r/min的条件下培养20h诱导产酶。在4℃、10000r/min的条件下离心10min，收集菌体。按照1g菌体溶于10mL 25mM Tris-HCl(250mM NaCl，pH 8)将菌体重悬，于冰浴中超声破碎20min。破碎后的菌液在4℃、10000r/min的条件下离心30min，收集上清液即为粗酶液。利用镍亲和层析对粗酶液进行纯化，依次用20mM Tris-HCl(250mM NaCl，pH 8)和含有不同浓度咪唑的20mM Tris-HCl(250mM NaCl，pH 8)进行洗脱，收集每部分的流穿液，进行聚丙烯酰胺凝胶电泳条带验证。
N1 GtfB酶的比酶活的检测计算：
准确称量100mg直链淀粉溶于2mL的2mol/L的NaOH溶液中，待直链淀粉完全溶解后加入适量的1M的HCl调节pH至7，再补足去离子水定容至10mL，配置成1％的直链淀粉溶液。250μL的0.25％的直链淀粉溶液，加入纯化的N1 GtfB酶(终浓度为90nM)用pH 6的缓冲液(25mM Tris-HCl)补足至1mL，于40℃保温10min。分别在1、2、4、6、8min取出100μL反应液，分别加入50μL 0.4M的NaOH溶液中进行碱灭活，然后加入50μL 0.4M的HCl溶液进行酸中和。取上述混合液150μL与预先配制好的15μL的碘溶液(将0.2g I 2和2g KI溶解在100ml蒸馏水中以制备20倍浓缩液，使用前稀释20倍)进行显色反应，测量反应液在660nm处吸光值。
酶活力定义：每分钟消耗1mg直链淀粉所需的酶定义为1U。
实施例1：利用N1 GtfB酶分别催化麦芽低聚糖、直链淀粉和支链淀粉并分析N1 GtfB酶的底物特异性
(1)利用N1 GtfB酶催化麦芽低聚糖
①将麦芽低聚糖(G3-G7)溶于pH 6、25mM的醋酸钠缓冲体系中，配制成1％(即1g/100mL)的溶液，
②加入N1 GtfB酶，使其终浓度为20μg/mL，于37℃下反应24h，沸水浴终止反应。
(2)利用N1 GtfB酶催化直链淀粉
直链淀粉和支链淀粉在反应前提前需要进行预处理。
①直链淀粉需先进行碱溶(NaOH,1M)，再进行酸中和，然后加入醋酸钠缓冲液(pH 6、25mM)，配制成0.6％(即0.6g/100mL)的溶液，
②再加N1 GtfB酶，使其终浓度为20μg/mL，于37℃下反应24h，沸水浴终止反应。
(3)利用N1 GtfB酶催化支链淀粉
①支链淀粉则是溶于醋酸钠缓冲液(pH 6、25mM)，配制成0.6％(即0.6g/100mL)溶液，涡旋后进行糊化处理，然后冷却至37℃，
②加入N1 GtfB酶，使其终浓度为20μg/mL，于37℃下反应24h，沸水浴终止反应。
反应结束后，利用薄层色谱(TLC)监测反应中麦芽低聚糖合成情况。测定结果如图1所示。N1 GtfB酶能够作用于DP≥3的麦芽糊精和淀粉类底物，当底物为麦芽糊精时，产物中合成了大量DP≥2的寡/多糖及其微量的葡萄糖，当底物为直链淀粉和支链淀粉时，产物中合成的寡/多糖的量要明显小于以麦芽糊精为底物时产生的量。总的来说，改性产物中均生成了一系列较底物更短和更长的寡糖/多糖，表明N1 GtfB酶对DP≥3的糊精或淀粉类底物同时具有水解酶和转糖基酶(歧化)活性。
实施例2：利用N1 GtfB酶分别催化麦芽七糖、直链淀粉并分析产物的分子量
(1)利用N1 GtfB酶催化麦芽七糖
①将麦芽七糖溶于pH 6、25mM的醋酸钠缓冲体系中，配制成1％的溶液，
②加入N1 GtfB酶，使其终浓度为20μg/mL，于37℃下反应24h，沸水浴终止反应。
(2)利用N1 GtfB酶催化直链淀粉
①将直链淀粉先进行碱溶(NaOH,1M)，再进行酸中和，然后加入醋酸钠缓冲液(pH 6、25mM)，配制成0.6％的溶液，
②再加N1 GtfB酶，使其终浓度为20μg/mL，于37℃下反应24h，沸水浴终止反应。
终止反应后，进行冻干处理，得酶合成的改性产物。将冻干样品用超纯水配制成5mg/mL，利用高效凝胶渗透色谱监测反应中产物分子量的情况。测定结果如图2所示。麦芽七糖和直链淀粉经N1 GtfB修饰后的分子量分别为3.4×10 3Da和5.7×10 3Da，麦芽七糖的分子量较酶处理前的1.2×10 3Da有了明显增大，直链淀粉分子量较酶处理前的5.7×10 3Da略微减少， 但产物分子量数量级都在10 3Da，这说明N1 GtfB酶在反应过程中展现出显著的转糖基活力。
实施例3：N1 GtfB酶催化直链淀粉并分析产物的一维核磁氢谱
①将直链淀粉先进行碱溶(NaOH,1M)，再进行酸中和，然后加入醋酸钠缓冲液(pH 6、25mM)，配制成6mg/mL的溶液。
②加入N1 GtfB酶，使其终浓度20μg/mL，在37℃、pH 6.0条件下反应24h，沸水浴终止反应，冻干。
取20mg冻干样品，加入1mL D 2O，煮沸1h，立即冻干。冻干后再加入1mL D 2O，煮沸1h，立即冻干备用。最后此样品溶于D 2O，在沸水浴中煮1h，然后在AVANCE-600MHz光谱仪(Bruker,Germany)上记录一维核磁共振氢谱结果，测试探针温度为65℃。在δ5.40-5.35ppm处的存在5个分裂峰，表明在(-)α-D-Glcp-(1→4)-结构中至少存在5个不同结构单元。在δ～4.97ppm的两个分裂峰，表明在(-)α-D-Glcp-(1→6)-结构中存在至少存两个不同结构单元。Rα(δ5.237ppm)和Rβ(δ4.653ppm)代表4-取代还原末端葡萄糖的异头氢信号。Gα(δ5.231ppm)和Gβ(δ4.640ppm)是游离葡萄糖的异位信号。δ3.40～3.45ppm之间的t4信号佐证了(α1→4,6)分支的存在。总的来说， 1HNMR的结果清楚地证实了产物中存在(α1→6)键。
实施例4：N1 GtfB酶分别改性麦芽七糖和直链淀粉实时合成的寡糖分析
(1)利用N1 GtfB酶催化麦芽七糖
①将麦芽七糖溶于pH 6、25mM的醋酸钠缓冲体系中，配制成1％的溶液，
②加入N1 GtfB酶，使其终浓度为20μg/mL，于37℃下反应24h，沸水浴终止反应。
(2)利用N1 GtfB酶催化直链淀粉
①将直链淀粉先进行碱溶(NaOH,1M)，再进行酸中和，然后加入醋酸钠缓冲液(pH6、25mM)，配制成0.6％的溶液，
②再加N1 GtfB酶，使其终浓度为20μg/mL，于37℃下反应24h，沸水浴终止反应。
用高压阴离子色谱(HPAEC-PAD)分别记录反应0min、10min、1h、24h时合成的寡糖的情况。用商业标准低聚糖鉴定了合成产物谱峰的性质。如图4所示，当底物在24h后几乎耗尽时，异麦芽糖和潘糖的合成明显表明(α1→6)键的形成。同时图中可见，产物中还存在了一些系列出峰时间不符合麦芽低聚糖的寡/多糖的峰，这说明这些产物中可能含有(α1→3)或(α1→6)糖苷键。N1 GtfB酶改性直链淀粉的情况与改性G7大致相同，仅仅只是产量相对较小。
实施例5：利用N1 GtfB酶改性直链淀粉过程中实时产物的甲基化分析
N1 GtfB酶改性直链淀粉的方法与实施例2的(2)相同，改性反应结束后，将反应液直接进行冻干处理，备用。取20mg冻干样品在60℃下溶于20mL DMSO中，然后加入20mL 溶于DMSO的NaOH溶液反应1h，再逐滴滴加CH 3I液体在室温下甲基化反应2h。反应结束后，甲基化产物用1000Da透析袋进行透析，然后用氯仿萃取，硫酸钠除水，再进行氮吹干。接着依次用三氟乙酸(2M)水解，用NaBH 4还原，然后用乙酸酐和吡啶(1:1)乙酰化。最后用GC-MS分析甲基化糖产物。将得到的离子片段与CCRC光谱数据库-PMAA进行比较，以确定糖苷键的类型和比例。如表1所示，甲基化糖产物的分子碎片峰片段验证了(α1→6)、(α1→3,4)和(α1→4,6)糖苷键的存在。4-取代、6-取代、3,4-二取代和4,6-二取代葡萄糖基的摩尔百分比为53：8：6：12。
表1 GC-MS分析甲基化糖产物
Figure PCTCN2022114438-appb-000001
*分子碎片是不同甲基化糖产物的GC-MS特征片段。
实施例6：N1 GtfB酶改性直链淀粉合成的产物二维核磁谱图分析
N1 GtfB酶改性直链淀粉以及核磁谱图测定前样品前处理方法的方法与实施例3中相同。 通过 1HNMR、2D  1H COSY和2D  13C- 1H HSQC谱结果分析改性产物中的不同糖基结构单元。如图5和表2所示，产物中存在7个不同取代的葡萄糖单元，即A：-(1→4)-α-D-Glcp-(1→4)-；B：-(1→3,4)α-D-Glcp-(1→4)-；C：α-D-Glcp-(1→4)-；D：-(1→6)α-D-Glcp-(1→4)-；E：-(1→4,6)α-D-Glcp-(1→4)-；F：-(1→4)-α-D-Glcp-(1→4,6)；G：-(1→4)-α-D-Glcp-(1→6)。
表2 N1 GtfB改性直链淀粉产物中结构单元的 1H和 13C化学位移
Figure PCTCN2022114438-appb-000002
实施例7：构建N1 GtfB酶改性直链淀粉产物的结构模型
结合实施例3、实施例6的一/二维核磁，高压离子色谱实时检测寡糖结果以及甲基化实验结果，确定了N1 GtfB酶改性产物所包含的键型类型和比例，从而构建了相应的产物结构模型，结果如图6所示。
对比例1：
将实施例1中的底物分别调整为葡萄糖、麦芽糖、蔗糖、异麦芽糖、异麦芽三糖、黑曲霉糖或潘糖，其余反应条件或者参数与实施例1一致。具体的，是分别将这些底物溶于pH 6、25mM的醋酸钠缓冲体系中，分别配制成1％的溶液，加入N1 GtfB酶，使其终浓度为(20μg/mL)，于37℃下反应24h，沸水浴终止反应。反应结束后，利用薄层色谱(TLC)监测反应情况，发现N1 GtfB酶对葡萄糖、麦芽糖、蔗糖、异麦芽糖、异麦芽三糖、黑曲霉糖、潘糖无活性。
对比例2：
将实施例2中的加酶处理调整为无酶处理。其余反应条件或者参数与实施例2一致。具体地，是将麦芽七糖溶于pH 6、25mM的醋酸钠缓冲体系中，配制成1％的溶液，于37℃下保温24h。直链淀粉则是先进行碱溶(NaOH，1M)，再进行酸中和，然后加入醋酸钠缓冲，配制成0.6％的溶液，于37℃下保温24h。
分别利用高效凝胶渗透色谱监测反应中产物分子量的情况。测定结果如图2所示。麦芽七糖和直链淀粉的分子量分别为1.2×10 3Da和5.7×10 3Da，
对比例3：
将实施例4中的加酶处理调整为无酶处理。其余反应条件或者参数与实施例4一致，具体地，是将麦芽七糖溶于pH 6、25mM的醋酸钠缓冲体系中，配制成1％的溶液，于37℃下保温24h。直链淀粉则是先进行碱溶(NaOH，1M)，再进行酸中和，然后加入醋酸钠缓冲，配制成0.6％的溶液，于37℃下保温24h。
分别利用高压离子色谱监测样品中寡糖的情况。结果中可见麦芽七糖标品中含一定量的麦芽五糖和麦芽六糖，而直链淀粉标品中寡糖含量很少。
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。

Claims (16)

  1. GtfB酶在制备α-葡聚糖中的应用，其特征在于，包括如下步骤：
    (1)以淀粉或糊精为底物，溶解淀粉或糊精，
    (2)添加GtfB酶，催化底物生成α-葡聚糖；
    所述GtfB酶的氨基酸序列如SEQ ID NO.1所示，所述α-葡聚糖具有(α1→4)、线性(α1→6)糖苷键、(α1→3,4)分支糖苷键和(α1→4,6)分支糖苷键。
  2. 根据权利要求1所述的应用，其特征在于，所述淀粉包括支链淀粉、直链淀粉，所述糊精包括麦芽糊精、麦芽低聚糖(聚合度≥3)。
  3. 根据权利要求2所述的应用，其特征在于，包括如下步骤：
    (1)以直链淀粉为底物，将淀粉先用碱溶解，再用酸中和，加缓冲液，
    (2)再添加氨基酸序列如SEQ ID NO.1所示的GtfB酶进行改性，得到合成的α-葡聚糖。
  4. 根据权利要求2所述的应用，其特征在于，包括如下步骤：
    (1)以支链淀粉为底物，向淀粉中加缓冲液，
    (2)再添加氨基酸序列如SEQ ID NO.1所示的GtfB酶进行改性，得到合成的α-葡聚糖。
  5. 根据权利要求2所述的应用，其特征在于，包括如下步骤：
    (1)以糊精为底物，加缓冲液，
    (2)再添加氨基酸序列如SEQ ID NO.1所示的GtfB酶进行改性，得到合成的α-葡聚糖。
  6. 根据权利要求3所述的应用，其特征在于，加入缓冲液后，底物浓度为0.6％～1.0％。
  7. 根据权利要求4所述的应用，其特征在于，加入缓冲液后，底物浓度为0.6％～1.0％。
  8. 根据权利要求5所述的应用，其特征在于，加入缓冲液后，底物浓度为0.6％～1.0％。
  9. 根据权利要求3所述的应用，其特征在于，所使用的缓冲体系浓度是10～50mM、pH4～6的缓冲液，包括25mM、pH为6的醋酸钠缓冲液。
  10. 根据权利要求4所述的应用，其特征在于，所使用的缓冲体系浓度是10～50mM、pH 4～6的缓冲液，包括25mM、pH为6的醋酸钠缓冲液。
  11. 根据权利要求5所述的应用，其特征在于，所使用的缓冲体系浓度是10～50mM、pH 4～6的缓冲液，包括25mM、pH为6的醋酸钠缓冲液。
  12. 根据权利要求3所述的应用，其特征在于，反应的温度为37～40℃，24h～48h。
  13. 根据权利要求4所述的应用，其特征在于，反应的温度为37～40℃，24h～48h。
  14. 根据权利要求5所述的应用，其特征在于，反应的温度为37～40℃，24h～48h。
  15. GtfB酶的应用，其特征在于，所述GtfB酶的氨基酸序列如SEQ ID NO.1所示，能够切断麦芽糖糊精或淀粉的(α1→4)糖苷键，合成(α1→3)和(α1→6)糖苷键。
  16. 氨基酸序列如SEQ ID NO.1所示的GtfB酶或其合成的α-葡聚糖在食品、保健品、饮料、医药和饲料添加剂方面的应用。
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