阅读说明：本技术 一种多酶耦合生产低聚异麦芽糖的方法 (Method for producing isomaltooligosaccharide by multi-enzyme coupling ) 是由 吴敬 王蕾 陈晟 易子玲 魏贝贝 于 2021-02-06 设计创作，主要内容包括：本发明公开了一种多酶耦合生产低聚异麦芽糖的方法,属于生物技术领域。本发明所将淀粉加入水或缓冲液中进行糊化和液化,得到液化淀粉溶液；将合成酶和淀粉脱支酶添加至液化液中进行合成反应得到中间反应液；将α-淀粉酶及α-葡萄糖苷酶和/或糖化酶依次添加至中间反应液去除未反应的底物及产物中的直链寡糖部分得到处理后反应液；再将右旋糖酐水解酶添加至处理后反应液进行水解反应得到低聚异麦芽糖。本发明主要解决了原料使用成本高,操作工艺流程复杂,产品纯度低等问题,优势体现在包含非功能性成分含量低,无其他副产物。(The invention discloses a method for producing isomaltooligosaccharide by multi-enzyme coupling, belonging to the technical field of biology. Adding starch into water or buffer solution for gelatinization and liquefaction to obtain liquefied starch solution; adding synthetase and starch debranching enzyme into the liquefied solution to perform synthetic reaction to obtain intermediate reaction solution; sequentially adding alpha-amylase and alpha-glucosidase and/or glucoamylase to the intermediate reaction solution to remove unreacted substrates and straight-chain oligosaccharide parts in the products to obtain a treated reaction solution; and adding dextran hydrolase into the treated reaction solution for hydrolysis reaction to obtain isomaltooligosaccharide. The invention mainly solves the problems of high use cost of raw materials, complex operation process flow, low product purity and the like, and has the advantages of low content of non-functional components and no other by-products.)

1. A method for producing isomaltooligosaccharide is characterized in that starchy raw materials are used as substrates, and the dextran is synthesized by synthetic reaction of synthetic enzyme under the assistance of starch debranching enzyme; after the unreacted substrate and the straight chain oligosaccharide in the product are treated by alpha-amylase, alpha-glucosidase and/or glucoamylase, the product obtained in the step of synthesizing is hydrolyzed by dextran hydrolase to obtain the isomaltooligosaccharide.

2. The method of producing isomaltooligosaccharides according to claim 1, wherein,

adding starch into water or buffer solution for gelatinization and liquefaction to obtain liquefied starch solution; adding synthetase and starch debranching enzyme into the liquefied solution to perform synthetic reaction to obtain intermediate reaction solution; sequentially adding alpha-amylase and alpha-glucosidase and/or glucoamylase into the intermediate reaction liquid to remove unreacted substrates and linear chain oligosaccharides in products to obtain a treated reaction liquid; and adding dextran hydrolase into the reaction solution after the intermediate treatment for hydrolysis reaction to obtain a hydrolysis reaction solution, thus obtaining the isomaltooligosaccharide.

3. The method of producing isomaltooligosaccharides according to claim 1, wherein,

the method comprises the steps of adding malt dextrin into dextrin solution in water or buffer solution; adding synthetase and starch debranching enzyme into dextrin solution for synthetic reaction to obtain intermediate reaction liquid; sequentially adding alpha-amylase and alpha-glucosidase and/or glucoamylase into the intermediate reaction liquid to remove unreacted substrates and linear chain oligosaccharides in products to obtain a treated reaction liquid; and adding dextran hydrolase into the reaction solution after the intermediate treatment for hydrolysis reaction to obtain isomaltooligosaccharide.

4. The method for producing isomaltooligosaccharides according to any one of claims 1 to 3, wherein,

the synthetase is one or two of 4, 6-alpha-glucosyltransferase and dextran dextrinase; the starch debranching enzyme is one or two of pullulanase and isoamylase; the dextran hydrolase is one or two of dextranase and isomaltotriose dextranase.

5. The method for producing isomaltooligosaccharides according to any one of claims 1 to 4, wherein the synthesis reaction is carried out at a temperature of 25 to 55 ℃, at a pH of 4 to 7.5, and for a period of 12 to 24 hours;

when the synthetase is 4, 6-alpha-glucosyltransferase, the addition amount of the 4, 6-alpha-glucosyltransferase in the liquefied starch solution is 100-5000U/g starch;

when the synthetase is dextran dextrinase, the addition amount of the dextran dextrinase in the liquefied starch solution is 5-100U/g maltodextrin or starch;

when the starch debranching enzyme is pullulanase, the addition amount of the pullulanase in the liquefied starch solution is 10-100U/g of maltodextrin or starch.

6. The process for producing isomaltooligosaccharides according to any one of claims 1 to 5, wherein when the linear oligosaccharides contained in the unreacted substrate and the unreacted product are treated with the α -amylase and the saccharifying enzyme, the reaction temperature and time of the α -amylase are 90 to 100 ℃ and 30 to 60 minutes, respectively, and the reaction temperature and time of the saccharifying enzyme are 50 to 70 ℃ and 30 to 60 minutes, respectively;

the addition amount of the alpha-amylase is not less than 1000U/g of maltodextrin or starch, and when the saccharifying enzyme is added, the addition amount of the saccharifying enzyme is not less than 660U/g of maltodextrin or starch.

7. The method for producing isomaltooligosaccharides according to any one of claims 1-6, wherein the temperature of the hydrolysis reaction of dextran hydrolase is 30-70 ℃, the pH is 3.5-7, and the time is 2-8 h;

when the dextran hydrolase is dextranase, the addition amount of the dextranase in the reaction solution after treatment is 5-500U/g maltodextrin or starch.

When the dextran hydrolase is isomaltotriose dextranase, the addition amount of the isomaltotriose dextranase in the treated reaction solution is 10-200U/g maltodextrin or starch.

8. The method for producing isomaltooligosaccharides according to any one of claims 1 to 7, wherein the isomaltooligosaccharides have an average degree of polymerization of 2 to 3 and an IMO content of 60 to 81%.

9. The method for producing isomaltooligosaccharides according to claim 1, wherein the starch is selected from the group consisting of mung bean starch, potato starch, wheat starch, sweet potato starch, water chestnut starch, lotus root starch, water chestnut starch, corn starch, sweet potato starch, arrowroot starch, tapioca starch.

10. Isomaltooligosaccharides obtainable by the process according to any one of claims 1 to 9.

Technical Field

The invention relates to a method for producing isomaltooligosaccharide by multi-enzyme coupling, belonging to the technical field of biology.

Background

Isomaltooligosaccharides (IMO), also known as isomaltooligosaccharides, branched oligosaccharides, and the like, are one type of starch sugar, and have a main component of polyglucose containing one or more α -1,6 glycosidic bonds. IMO is not easily utilized by digestive enzymes of human body, has low glycemic index characteristic, and can be used as health sugar substitute for diabetic patients. In addition, IMO can be decomposed and utilized by intestinal probiotics, particularly bifidobacteria, to generate short-chain fatty acid and inhibit intestinal harmful bacteria, so that IMO is used as an important functional oligosaccharide and is widely applied to industries such as food and the like. The isomaltooligosaccharide belongs to a non-digestible oligosaccharide, and has multiple physiological functions of hypoglycemia, preventing decayed teeth, promoting the growth of probiotics, improving the immunity of a human body and the like, so that the isomaltooligosaccharide can play a good probiotic role while promoting the growth of bifidobacteria in intestinal tracts after being eaten by the human body.

The polymerization degree of isomaltooligosaccharide is usually 2-10, and the main functional components are isomaltose, panose, isomaltose and isomaltotriose. The method is characterized in that Japanese researchers firstly discover and develop commercial production technology and apply the technology to food development, the technology is widely applied to crops such as potatoes, barley, wheat and the like, and also applied to fermentation products such as sake, soy sauce and honey, the fermentation products are pushed to the market for sale in 1985, China also starts to invest in research and development work on isomaltooligosaccharide in 1994, and invests the isomaltooligosaccharide in industrial production to enter the market of China, and meanwhile, many countries in the United states, Europe and the like also vigorously develop the application potential of the isomaltooligosaccharide.

At present, there are three major categories of methods related to isomaltooligosaccharides, including the conversion of biological enzymes, wherein the first category is the processing and production of starchy raw materials, and mainly involves the steps of raw material liquefaction, saccharification and transglycosylation reaction using, for example, α -glucosidase, but the yield of isomaltooligosaccharides produced by this method is low, and the subsequent product separation cost is high (see, for example, patent CN1721545A production process of glycosylated isomaltooligosaccharide, CN104131051A preparation method of isomaltooligosaccharides, etc.); the second type is to prepare isomaltooligosaccharides by retropolymerization using a high-concentration glucose solution as a raw material, but this method has a high cost of raw materials and a low efficiency of conventional polymerases, and thus the yield of isomaltooligosaccharides produced by this method is low (see, specifically, patent CN108546724A for high-purity isomaltooligosaccharides and methods for preparing them); THE third category is THE method OF using sucrose as raw material, firstly synthesizing dextran by dextran sucrase and then preparing isomaltooligosaccharide by hydrolysis, but this method requires sucrose raw material, which is relatively expensive compared with starch raw material, and moreover, this method can only use glucose part in sucrose, so THE theoretical yield OF isomaltooligosaccharide to sucrose does not exceed 50% (see patent WO2016029198A1PROCESS FOR THE PRODUCTION OF ALOMOOGOSACCHARIDES, etc.).

The above disadvantages greatly limit the further development of the isomaltooligosaccharide market. Therefore, there is an urgent need to find a method for producing isomaltooligosaccharides with high yield and low cost.

Disclosure of Invention

[ problem ] to

The invention aims to solve the technical problem of providing isomaltose hypgather production with high yield and low cost.

[ solution ]

In order to solve the problems, the invention provides a method for producing isomaltose hypgather, which comprises the steps of adding starch into water or buffer solution for gelatinization and liquefaction to obtain liquefied starch solution; adding synthetase and starch debranching enzyme into the liquefied solution to perform synthetic reaction to obtain intermediate reaction solution; sequentially adding alpha-amylase and alpha-glucosidase and/or glucoamylase to the intermediate reaction solution to remove unreacted substrates and straight-chain oligosaccharides in the product to obtain a treated reaction solution; adding dextran hydrolase into the treated reaction solution to perform hydrolysis reaction to obtain isomaltooligosaccharide; the starch is corn starch, wheat starch, rice starch, mung bean starch, pea starch, cassava starch, potato starch and/or sweet potato starch;

or, the method is that the malt dextrin is added into the dextrin solution in water or buffer solution; adding synthetase and starch debranching enzyme into dextrin solution for synthetic reaction to obtain intermediate reaction liquid; sequentially adding alpha-amylase and alpha-glucosidase and/or glucoamylase to the intermediate reaction solution to remove unreacted substrates and straight-chain oligosaccharides in the product to obtain a treated reaction solution; and adding dextran hydrolase into the treated reaction solution for hydrolysis reaction to obtain isomaltooligosaccharide.

The synthetase is one or two of 4, 6-alpha-glucosyltransferase (EC 2.4.1.-), dextran dextrinase (EC 2.4.1.2). The amino acid sequence of the 4, 6-alpha-glucosyltransferase is shown in SEQ ID NO 1. The starch debranching enzyme can catalyze an enzyme for hydrolyzing 1, 6-beta-D-glycosidic bonds at the branching points of starch chains, and can be selected from the following enzymes: pullulanase and isoamylase.

The temperature of the synthesis reaction is 25-55 ℃, the pH value is 4-7.5, and the time is 12-24 h.

In one embodiment of the present invention, when the synthase is 4,6- α -glucosyltransferase, the amount of the 4,6- α -glucosyltransferase added to the liquefied starch solution is 100 to 5000U/g starch.

In one embodiment of the invention, when the synthetase is dextran dextrinase, the addition amount of the dextran dextrinase in the liquefied starch solution is 5-100U/g maltodextrin or starch.

In one embodiment of the present invention, when the starch debranching enzyme is pullulanase, the pullulanase is added to the liquefied starch solution in an amount of 10 to 100U/g of maltodextrin or starch.

The alpha-amylase is 1, 4-D-glucan hydrolase, and high-temperature amylase can be selected. The saccharifying enzyme is alpha-1, 4-glucohydrolase.

In the reaction process of removing unreacted substrates and linear chain oligosaccharides in the products, when starch or maltodextrin subjected to the first-step synthesis reaction is taken as a substrate, high-temperature amylase and saccharifying enzyme are used for treatment; the reaction temperature and time of the high-temperature amylase are respectively 90-100 ℃ and 30-60min, and the reaction temperature and time of the saccharifying enzyme are respectively 50-70 ℃ and 30-60 min.

In one embodiment of the present invention, the amount of the α -amylase added to the intermediate reaction solution is not less than 1000U/g maltodextrin or starch.

In one embodiment of the present invention, when the intermediate treatment reaction is performed by adding α -glucosidase to the intermediate reaction solution, the amount of α -glucosidase added to the intermediate reaction solution is 1 to 20U/g maltodextrin or starch.

In one embodiment of the present invention, when the saccharifying enzyme is added to the intermediate reaction solution to carry out the reaction, the amount of the alpha-glucosidase added to the intermediate reaction solution is not less than 660U/g maltodextrin or starch.

The dextran hydrolase comprises a dextranase (EC 3.2.1.11) and/or an isomaltotriose dextranase (EC3.2.1.95). The amino acid sequence of the isomaltotriose dextranase is shown as SEQ ID NO. 2.

The temperature of the hydrolysis reaction is 30-70 ℃, the pH value is 3.5-7, and the time is 2-8 h.

In one embodiment of the invention, when the dextran hydrolase is dextranase, the addition amount of the dextranase in the reaction solution after treatment is 5-500U/g maltodextrin or starch.

In one embodiment of the invention, when the dextran hydrolase is isomaltotriose dextranase, the addition amount of the isomaltotriose dextranase in the treated reaction solution is 10-200U/g maltodextrin or starch.

The invention also provides the isomaltooligosaccharide prepared by the method.

The method for preparing isomaltooligosaccharide can be further used for preparing products containing isomaltooligosaccharide, such as healthy sugar substitutes for diabetics, for example products for controlling blood sugar, preventing decayed teeth, promoting growth of probiotics and improving human immunity.

[ advantageous effects ]

(1) The invention provides a method for producing isomaltooligosaccharide with high yield and low cost, which takes cheap starch or maltodextrin as raw material, the starch is firstly treated by 4, 6-alpha-glucosyltransferase and/or dextranase, and then treated by dextranase and/or isomaltotriose dextranase to produce the isomaltooligosaccharide, wherein, the alpha-4, 6-glucosyltransferase can synthesize dextran with continuous or discontinuous continuous alpha-1, 6-bonds by taking starch or starch degradation products as substrates, the dextranase can synthesize dextran with continuous alpha-1, 6-bonds by taking starch or starch degradation products as substrates, the dextranase can hydrolyze the dextran with continuous alpha-1, 6-bonds and generate isomaltooligosaccharide mixtures with different polymerization degrees mainly comprising isomaltose, the isomaltotriose dextranase can identify the non-reducing end of glucan with continuous alpha-1, 6-bonds and hydrolyze to generate isomaltotriose; the yield of isomaltose hypgather produced by the method can reach 73 percent.

(2) The invention provides a method for producing isomaltooligosaccharide, which is simple in separation and purification steps because the average polymerization degree of the isomaltooligosaccharide prepared by the method is 2-3 and the IMO content is 60-81%.

(3) The content of trisaccharides such as isomaltotriose or panose in IMO is a main component for reflecting the functionality of isomaltooligosaccharide, and the content reflects the quality of the product, and also influences the price and application prospect of the product. The invention provides a method for producing isomaltooligosaccharide, which can adjust the ratio of trisaccharide to disaccharide in IMO by changing the ratio of dextranase and isomaltotriose dextranase, and the ratio of isomaltotriose can reach 90 percent at most, so the quality of the isomaltooligosaccharide prepared by the method is good and the application prospect is wide.

Drawings

FIG. 1 shows the HPLC detection spectrum of dextran product in the synthesis step. Wherein, the peaks at 7.385min and 5.377min are trisaccharide and above effective dextran, the peak at 8.550min is isomaltose, the peak at 10.588min is glucose, and other small peaks and almost non-peak positions are impurities in the system.

FIG. 2 shows the HPLC detection pattern of the degradation step with an amino column, wherein 3.511min is a solvent peak (acetonitrile), 9.854min is an isomaltotriose peak, and other peaks are impurities or trace oligosaccharides.

In the synthesis step of fig. 3, when 4,6- α -glucanotransferase and pullulanase were added, bond type detection of the produced dextran was performed by 1H NMR nuclear magnetic hydrogen spectroscopy, about 5.4ppm was α -1,4, near 4.99ppm was α -1,6, the ratio of the two was 1: 8.31.

Detailed Description

Coli JM109 and BL21(DE3) competent cells referred to in the following examples were obtained by the laboratory; referred to in the following examples; pET-20b (+) plasmid and pET-15b (+) plasmid referred to in the following examples were synthesized from Shanghai Jie Co., Ltd; the phosphate citrate buffers referred to in the following examples are configured according to the table; the maltodextrins referred to in the examples below were obtained from the company of Zizetian Bangbang; the tapioca starch referred to in the following examples was purchased from heze tengbang; pullulanase referred to in the following examples was purchased from Shandong-Longkont enzyme preparations, Inc.; the high temperature alpha-amylase referred to in the examples below was purchased from Shandong-Longkont enzyme preparations, Inc.; the α -glucosidase referred to in the examples below was purchased from Shandong Kete enzyme preparation, Inc.; the saccharifying enzymes referred to in the examples below were purchased from Shandong-Longkont enzyme preparations, Inc.

The media involved in the following examples are as follows:

LB liquid medium: 10g/L of peptone and 5g/L, NaCl 10g/L of yeast extract.

LB solid medium: 10g/L of peptone, 5g/L, NaCl 10g/L of yeast extract and 20g/L of agar.

TB liquid medium: 10g/L of peptone, 24g/L of yeast powder and 5g/L, K of glycerol₂HPO₄·3H₂O 16.43g/L、KH₂PO₄ 2.31g/L。

MD solid medium: 20g/L glucose, 20g/L agarose and 13.4g/L YNB.

YPD medium: peptone 20g/L, glucose 20g/L and yeast powder 10 g/L.

BMMY medium: peptone 20g/L, yeast powder 10g/L, YNB 13.4g/L, ammonium sulfate 10g/L, K₂HPO₄·3H₂O3g/L，KH₂PO₄ 11.8g/L。

BMGY medium: glycerol is added by 10g/L on the basis of BMMY.

The preparation methods referred to in the following examples are as follows:

the preparation method of the alpha-4, 6-glucanotransferase comprises the following steps:

synthesizing the gene of alpha-4, 6-glucosyltransferase with the amino acid sequence shown in SEQ ID NO. 1.

The resulting gene was digested with pET-15b (+) plasmid using restriction enzymes Nde I and Hind III, and then T was used₄Connecting the two obtained enzyme digestion products by using a ligase to obtain a recombinant plasmid pET-15b (+) -gtfb; transforming the recombinant plasmid pET-15b (+) -gtfb into competent cells of Escherichia coli E.coli JM109 to obtain recombinant Escherichia coli JM109/pET-15b (+) -gtfb; the recombinant Escherichia coli JM109/pET-15b (+) -gtfb is streaked on an LB solid culture medium, and is cultured in a constant temperature incubator at 37 ℃ for 8-10 hours to obtain a single colony; inoculating the single colony into an LB liquid culture medium, culturing for 8-10 h in a constant-temperature incubator at 37 ℃, and performing plasmid extraction on the obtained seed liquid; then transforming into E.coli BL21(DE3) competent cells according to the same operation, coating the recombinant E.coli BL21(DE3)/pET-15b (+) -gtfb on an LB solid culture medium, and culturing for 8-10 h in a constant temperature incubator at 37 ℃ to obtain a single colony, namely a stable transformant suitable for fermentation; inoculating a single colony into an LB liquid culture medium, culturing for 8-10 h in a constant-temperature incubator at 37 ℃ to obtain a seed solution, transferring the seed solution into a TB liquid culture medium (containing 0.4 mu mol/L ampicillin) according to the inoculation amount of 5% (v/v), culturing for 2h in the constant-temperature incubator at 37 ℃, and continuing to perform induced fermentation for 12h in the constant-temperature incubator at 25 ℃ to obtain a fermentation liquid; centrifuging the fermentation liquid at 4 deg.C for 20min to obtain supernatant as crude enzyme liquid.

The preparation method of the isomaltotriose dextranase comprises the following steps:

synthesizing a gene encoding isomaltotriose dextranase with an amino acid sequence shown as SEQ ID NO. 2; the resulting gene was digested with pET-20b (+) plasmid using restriction enzymes Nco I and Hind III, and then the resulting DNA fragment was digested with T₄Connecting the two obtained enzyme digestion products by using ligase to obtain a recombinant plasmid pET-20b (+) -imtd; transforming the recombinant plasmid pET-20b (+) -imtd into an Escherichia coli E.coli JM109 competent cell to obtain recombinant Escherichia coli JM109/pET-20b (+) -imtd; coating the recombinant Escherichia coli JM109/pET-20b (+) -imtd on an LB solid culture medium, and culturing for 8-10 h in a constant-temperature incubator at 37 ℃ to obtain a single colony; inoculating the single colony into an LB liquid culture medium, culturing for 8-10 h in a constant-temperature incubator at 37 ℃, and performing plasmid extraction on the obtained seed liquid; then transforming into an escherichia coli E.coli BL21(DE3) competent cell by the same operation, coating the recombinant escherichia coli BL21(DE3)/pET-20b (+) -imtd on an LB solid culture medium, and culturing for 8-10 h in a constant temperature incubator at 37 ℃ to obtain a single colony, namely a stable transformant suitable for fermentation; inoculating a single colony into an LB liquid culture medium, culturing for 8-10 h in a constant-temperature incubator at 37 ℃ to obtain a seed solution, transferring the seed solution into a TB liquid culture medium (containing 0.4 mu mol/L ampicillin) according to the inoculation amount of 5% (v/v), culturing for 2h in the constant-temperature incubator at 37 ℃, and continuing to perform induced fermentation for 48h in the constant-temperature incubator at 25 ℃ to obtain a fermentation liquid; centrifuging the fermentation liquid at 4 deg.C for 20min to obtain supernatant as crude enzyme liquid.

The detection methods referred to in the following examples are as follows:

the method for detecting the content and yield of the dextran in the reaction solution comprises the following steps:

detecting the content of dextran in the reaction solution by using a High Performance Liquid Chromatography (HPLC) method; performing sample injection detection on a sample with the flow rate of 0.5mL/min and the sample injection amount of 10 mu L by using an Agilent 1260HPLC chromatograph, a differential refraction detector and a Waters Ca chromatographic column, wherein the detection column temperature is 80 ℃, and the mobile phase is pure water;

wherein, the calculation formula of the yield of the dextran in the reaction solution is as follows: the yield is the sum of peak areas of products with trisaccharide and above polymerization degree/total peak area of the products multiplied by 100 percent.

The method for detecting the content, yield and bond type of isomaltose, glucose and isomaltotriose in the reaction solution comprises the following steps:

detecting the contents of isomaltose, glucose and isomaltose hypgather in the reaction solution by a High Performance Liquid Chromatography (HPLC) method; detecting by an Agilent 1260HPLC chromatograph, a differential refraction detector and an Agilent ZORBAX NH2(4.6 multiplied by 250mm) chromatographic column, wherein the detection column temperature is 35 ℃, the mobile phase is 70-78% acetonitrile, and the sample introduction detection is carried out at the flow rate of 0.8mL/min and the sample introduction amount of 10 mu L;

wherein the calculation formula of the yield of isomaltose, glucose and isomaltotriose in the reaction solution is as follows: yield is the peak area of each product/peak area of standard × standard concentration/substrate concentration × 100%.

The bond type ratio of the resistant dextrins was determined by means of an AVANCE III 400MHz digital NMR nuclear magnetic resonance spectrometer (Bruker Biospin International AG). Accurately weighing 30-40 mg of sample, dissolving the sample in 500 mu L D2O (D, 99.9%), shaking in a vortex till the sample is completely dissolved, and then pumping the sample into a nuclear magnetic tube by using a pipette for measurement at the temperature of 60 ℃. Trimethylsilylpropionic acid (TMSP 0.03%) was dissolved in the sample as an internal standard.

The detection method of the enzyme activity of the alpha-4, 6-glucanotransferase comprises the following steps: detecting by an iodine solution color development method: preparing a substrate: 1g/L amylose mother liquor: adding 2mL of distilled water into 40mg of amylose for fully wetting, adding 2mL of 2M NaOH solution, and carrying out vortex oscillation for fully dissolving; the substrate was prepared by adding 250uL of 2M HCl solution to 500uL of amylose mother liquor, and then adding 3250 uL of phosphate-citrate buffer (pH 7.0) to the solution to prepare 0.125%.

Lugol iodine solution: 0.26g iodine and 2.60g potassium iodide were dissolved in a 10mL volumetric flask (prepared 3 days in advance to ensure complete dissolution of iodine); when in use, 100 mu L of Lugol iodine solution is taken, 50 mu L of 2M HCl solution is added, and then water is supplemented to 26mL to prepare iodine color developing solution.

During the reaction, 200uL of the substrate was placed in a 1.5mL centrifuge tube and incubated at 35 ℃ for 10 min. 200. mu.L of GtfB enzyme solution was added thereto and reacted at 35 ℃ for 10min, 200. mu.L of the reaction solution was added to 3800. mu.L of iodine color developing solution after the reaction was completed and displayed for 5min, and the absorbance at 660nm was measured with a spectrophotometer. The control buffer was used instead of the enzyme solution, and 200. mu.L of the buffer was added to 3800. mu.L of the iodine developing solution for 5 min.

Definition of the enzyme activity of α -4, 6-glucanotransferase: the unit time absorbance value is reduced by one percent at the temperature of 37 ℃ and is defined as an enzyme activity unit.

Enzyme activity (U/mL) ═ 100 xd (dilution factor) x (a control-a experiment) ]/[10min x 0.1mL x (a control-a blank) ].

The detection method of the enzyme activity of the isomaltotriose dextranase comprises the following steps: detecting enzyme activity by using a DNS method, firstly preparing 2 ten thousand substrates of 1 percent glucan, putting 1ml in a test tube with a plug, then adding 0.9ml of buffer solution, putting the test tube in a constant-temperature water bath kettle at 40 ℃ for warm bath for 10min, then adding 0.1ml of enzyme solution, reacting for 10min, and then adding 3ml of DNS to terminate the reaction. Placing the test tube with the plug into boiling water, boiling for 7min, immediately placing into ice water mixture, cooling, adding 10ml water into the test tube until the system is 15ml, mixing uniformly, detecting light absorption value at 540nm, and calculating according to the light absorption value to obtain enzyme activity.

Definition of the enzyme activity of isomaltotriose dextranase: the amount of reducing sugar produced by enzymatic hydrolysis of the glucan substrate within 1min was one enzyme activity unit (1U) at 40 ℃.

Method for detecting DE value (reduction value) (DNS colorimetry):

specific references are found in: the content of reducing sugar in bagasse is measured by the method of pressing, etc. the content of reducing sugar in bagasse is measured by the method of DNS, food research and development, 36(02): 126-128.

Definition of DE value (reduction value): the amount of reducing sugar in the system is the ratio of the total solid content.

Example 1: preparation of isomaltooligosaccharide

The method comprises the following specific steps:

adding maltodextrin into a phosphate buffer solution, and heating and dissolving to obtain a dextrin solution with the concentration of 15%; adding 4, 6-alpha-glucanotransferase (SEQ ID NO:1), pullulanase and chloramphenicol into a dextrin solution according to the addition amounts of 400U/g maltodextrin, 30U/g maltodextrin and 0.04g/L, respectively, and reacting in a water bath shaker at 37 ℃ and 150rpm for 24 hours to obtain an intermediate reaction solution; adding high-temperature alpha-amylase into the intermediate reaction liquid according to the addition amount of 6000U/g maltodextrin or starch, reacting for 30min at 95 ℃, then adjusting the pH to 4.2, adding saccharifying enzyme into the reaction liquid according to the addition amount of 750U/g maltodextrin or starch, and reacting for 30min at 60 ℃ to obtain treated reaction liquid; adding crude enzyme solution of isomaltotriose dextranase (SEQ ID NO:2) and chloramphenicol (for preventing infectious microbes during reaction) into the treated reaction solution according to the addition amounts of 150U/g dextran and 0.04g/L, respectively, and reacting in a water bath shaker at 37 deg.C for 4h to obtain hydrolysis reaction solution.

Detecting the content and yield of dextran in the intermediate reaction solution, boiling and centrifuging the intermediate reaction solution through intermediate treatment, diluting the intermediate reaction solution by 4 times, filtering the intermediate reaction solution by using a filter membrane, and detecting the dextran in the intermediate reaction solution through high performance liquid chromatography; boiling the hydrolysis reaction liquid, centrifuging to obtain a supernatant, mixing the supernatant with pure acetonitrile at a ratio of 1:1(v/v), precipitating for 2h, centrifuging again after precipitation is finished, filtering the supernatant obtained by centrifuging again with a filter membrane, and placing the filtered supernatant in a detection bottle to detect the content and yield of isomaltose, glucose and isomaltose hypgather in the hydrolysis reaction liquid by high performance liquid chromatography.

According to the detection result, the ratio of dextran in the intermediate reaction solution is 79%, and the detection spectrum is shown in figure 1; the alpha-1, 6 bond type ratio is about 88.9%, the NMR spectrum is shown in figure 3, the yield of oligomeric isomaltose in the hydrolysis reaction solution is 79%, and more than 90% of the products in the hydrolysis reaction solution are isomaltotriose.

Example 2: preparation of isomaltooligosaccharide

The method comprises the following specific steps:

adding cassava starch into a proper amount of phosphate buffer solution (50mM pH6.0) to prepare 15% suspension for gelatinization and liquefaction, stirring until the suspension becomes suspension, adding 10U/g starch CGTase, placing in a water bath kettle at 85 ℃, starting to time 7min when the suspension is completely viscous to carry out liquefaction, and then boiling to inactivate enzyme for 10 min. Adding 4, 6-alpha-glucanotransferase (SEQ ID NO:1), pullulanase and chloramphenicol into the starch liquefaction liquid according to the addition amounts of 400U/g starch, 20U/g starch and 0.04g/L, respectively, and reacting in a water bath shaker at 37 ℃ and 150rpm for 24h to obtain an intermediate reaction liquid; adding high-temperature alpha-amylase into the intermediate reaction liquid according to the addition amount of 6000U/g starch, reacting for 30min at 95 ℃, then adjusting the pH to 4.2, adding saccharifying enzyme into the reaction liquid according to the addition amount of 750U/g starch, and reacting for 30min at 60 ℃ to obtain treated reaction liquid; adding crude enzyme solution of isomaltotriose dextranase (SEQ ID NO:2) and chloramphenicol into the treated reaction solution according to the addition amounts of 150U/g dextran and 0.04g/L, respectively, and reacting in a water bath shaker at 37 ℃ for 4h to obtain a hydrolysis reaction solution.

Detecting the content and yield of dextran in the intermediate reaction solution, boiling and centrifuging the intermediate reaction solution, diluting the intermediate reaction solution by 4 times, filtering the intermediate reaction solution by using a filter membrane, and detecting the dextran in the intermediate reaction solution by using a high performance liquid chromatography (calcium column, the same below); boiling the hydrolysis reaction liquid, centrifuging to obtain a supernatant, mixing the supernatant with pure acetonitrile at a ratio of 1:1(v/v), precipitating for 2h, centrifuging again after precipitation is finished, filtering the supernatant obtained by centrifuging again with a filter membrane, placing the filtered supernatant in a detection bottle, and detecting the content and yield of isomaltose, glucose and isomaltose hypgather in the reaction liquid 4 by high performance liquid chromatography (calcium column, the same below).

As is clear from the results of the examination, the yield of dextran in the intermediate reaction solution was 72.4%, and the yield of isomaltose hypgather in the hydrolysis reaction solution was 77.3%, and the isomaltotriose content was 90% or more.

In FIG. 3, about 5.4nnm is α -1,4, and about 5.0nnm is α -1, 6.

Example 3: preparation of isomaltooligosaccharide

The method comprises the following specific steps: adding maltodextrin into a phosphate buffer solution, and heating and dissolving to obtain a dextrin solution with the concentration of 15%; adding 4, 6-alpha-glucanotransferase (SEQ ID NO:1), pullulanase and chloramphenicol into a maltodextrin solution according to the addition amounts of 400U/g maltodextrin, 30U/g maltodextrin and 0.04g/L, respectively, and reacting in a water bath shaker at 37 ℃ and 150rpm for 24 hours to obtain an intermediate reaction solution; adding high-temperature alpha-amylase into the intermediate reaction liquid according to the addition amount of 6000U/g maltodextrin, reacting for 30min at 95 ℃, then adjusting the pH to 4.2, adding saccharifying enzyme into the reaction liquid according to the addition amount of 750U/g maltodextrin, and reacting for 30min at 60 ℃ to obtain treated reaction liquid; adding crude enzyme solution of isomaltotriose dextranase (SEQ ID NO:2) and chloramphenicol into the treated reaction solution according to the addition amounts of 150U/g dextran and 0.04g/L, respectively, and reacting in a water bath shaker at 37 ℃ for 4h to obtain a hydrolysis reaction solution.

Detecting the content and yield of dextran in the intermediate reaction solution, boiling and centrifuging the intermediate reaction solution for dilution, filtering the intermediate reaction solution by using a filter membrane, and detecting the dextran in the intermediate reaction solution by using a high performance liquid chromatography; boiling the hydrolysis reaction liquid, centrifuging to obtain a supernatant, mixing the supernatant with pure acetonitrile at a ratio of 1:1(v/v), precipitating for 2h, centrifuging again after precipitation is finished, filtering the supernatant obtained by centrifuging again with a filter membrane, and placing the filtered supernatant in a detection bottle to detect the content and yield of isomaltose, glucose and isomaltose hypgather in the reaction liquid 4 by high performance liquid chromatography.

According to the detection results, the yield of dextran in the intermediate reaction solution is 75.33%, and the yield of isomaltotriose in the hydrolysis reaction solution is 78%.

Comparative example 1

The method comprises the following specific steps:

on the basis of example 1, a crude enzyme solution of isomaltotriose dextranase (SEQ ID NO:2) was replaced with a commercial dextranase (purchased from Hirsheng), and the amount of addition of the commercial dextranase was replaced with 5000U/g dextran, to obtain reaction solution 4.

And (3) boiling the reaction solution 4, centrifuging to obtain a supernatant, mixing the supernatant with pure acetonitrile at a ratio of 1:1(v/v), precipitating for 2 hours, centrifuging again after precipitation is finished, filtering the supernatant obtained by centrifuging again with a filter membrane, and placing the filtered supernatant in a detection bottle to detect the content and yield of isomaltose, glucose and isomaltose hypgather in the reaction solution 4 by high performance liquid chromatography.

As a result of the examination, the yield of isomaltose in the hydrolysis reaction mixture was 50%, and isomaltotriose was hardly detected.

Comparative example 2

The method comprises the following specific steps:

on the basis of example 1, the crude enzyme solution of isomaltotriose dextranase (SEQ ID NO:2) was replaced by commercial dextranase (purchased from Hirsheng), and the amount of commercial dextranase was replaced by 500U/g dextran, and the reaction was carried out at 50 ℃ for 2h to obtain a hydrolysis reaction solution.

And (3) boiling the reaction solution 4, centrifuging to obtain a supernatant, mixing the supernatant with pure acetonitrile at a ratio of 1:1(v/v), precipitating for 2 hours, centrifuging again after precipitation is finished, filtering the supernatant obtained by centrifuging again with a filter membrane, and placing the filtered supernatant in a detection bottle to detect the content and yield of isomaltose, glucose and isomaltose hypgather in the reaction solution 4 by high performance liquid chromatography.

From the results of the detection, it was found that the yield of isomaltose in the reaction mixture 4 was 21.5%, the yield of isomaltotriose was 28.1%, the yield of glucose was 1.6%, and the yield of isomaltooligosaccharide was low.

The sequence is as follows:

SEQ ID NO:1

QANDGHWYLFTADGTAASRVAKWAGTYYYFDPQTHLRVDDNYVQSQWGDWYMFGKDGRIATGLYKWDKNNQWYYFDPVTYLKVTNKWVDGNYYDEDGAQAISKLVTINNRLYYFDDQGKEISNQFRTIHGDKYYFGNDSAAVTGQQTIDGKVYKFSNYGYLLGNRYGKIENGKLNIYSLADNSLIKTVEAGPWENMAYSMDSNSINNIDGYISYTGWYRPYGTSQDGKTWYPTTVADWRPILMYVWPSKDVQVKFIQYFVNHGYENSNYGLTAGSVKDLSENTASIKLNEVAQNLRYVIEQHVVAAKSTSQLANDINNFITTIPELSKASELSVVNSYGYKPDNSGSVDDDQVIFVNNDSKNQKIGNTSYADSNYRLMNRTINNQNGDNNSDDSPELLVGNDIDNSNPVVQAENLNWEYFLLNYGKFMNYNPNGNFDGFRIDAADNIDADVLDQAAQLINSIYNTKGNQANANDHLIYNEGYHLGAANMLDRKSNPELYMDSGYFYTLENVLGRASDRDDINNLITNSIVNRQNDVSENVATPNWSFVTNHDQRKNLINQIVIDDHPGVADIMSDGYKAEYVNQAWKEFYADQARTDKKYTQYNLPAQYALLLTNKDTVPQVYYGDLYDETDQYMQNKSVYYDAITTLMKARKSYVSGGQSMIKINDHLLTSVRYGKGIIDGNVSMTDILGRNSGIAVVVGNDAQMANQTISINMGKAHANQAYKQLLGTIDSGLTSSDTTIYHTDSNGVLNVTVKGYSNPYVSGYLGVWVPLNGGANITTKASEVTNQSDKTYSSNAALDSHVIYEDFSLFQPEPTSKAEHAYNIIADNASLFNELGITDFWMAPAYTPFNTSRYNEGYSMTDRYNLGTEANLTKYGSGEELSNAIAALHDAGLKVQEDLVMNQMIGFSGQEAVTVTRTDGHAKQLTVDGKTFANQIYFAYTRGGGEGQKNYGGKYLDELQKKYPELFTTKAVSTGVAPDPSVHITEWSAKYQNGTSLQNIGIGLAVKLANGDYAYLNDSNNKAFNTTLPETMSSADYYANIEDD

SEQ ID NO:2

NPTKPVEDAPVTADVGNLHTWWHDNAVYNTDSPTENGEVRRSSFYDVQVAQAHQPDKFFDSFAYMSIPRSGKGKVGYTKEDGAEFSSEANLSMSWSSFEYAKDVWVDVSLKTGQTISSADEVQIRPSSYDFEKKLVDEDTIRIKVPYSDAGYRFSVEFDPQLYTSYNDMSGNSGKLTTVAEGNRPIHTEPMNSMMIFAEPKLQGEEEKRLIPNPSSGSIHYPEEGEVKDLNTVTEEIIYFKPGTYHMGSDYHAVLPPNVKWVYLAPGAYVKGAFRFFHDNQAQYKVTGYGVLSGEQYVYEADTANNYNALSGASNCHVTCVKMLQFESSNIGQQLDLQGVTINEPPYHSFVVYAHEGEKEIGVENFRMNVENYKQVGSWYWQTDGIELYQGGTMKNTFFNANDDVLKMYHSDVTIDNTVIWKNENGPVIQWGWTPRNIDNVNVTDTTVIHNRMYWKDPKYNTCILNSSSHWEDMGSTAKADPNTTVKNMRFENITVEGMTNCAMRIYALSNTENIHVKNLSIDSWNGLDWTSQVSHLKRYTNSAGEKVTIGNEIPDGNGLALENYSVGGEIIEKSGDNWNDYKLGRLGFDGENWDSWNAWKSTP

although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

SEQUENCE LISTING

<110> university of south of the Yangtze river

<120> method for producing isomaltooligosaccharide by multi-enzyme coupling

<130> BAA200816A

<160> 2

<170> PatentIn version 3.3

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Gln Ala Asn Asp Gly His Trp Tyr Leu Phe Thr Ala Asp Gly Thr Ala

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Ala Ser Arg Val Ala Lys Trp Ala Gly Thr Tyr Tyr Tyr Phe Asp Pro

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Gln Thr His Leu Arg Val Asp Asp Asn Tyr Val Gln Ser Gln Trp Gly

35 40 45

Asp Trp Tyr Met Phe Gly Lys Asp Gly Arg Ile Ala Thr Gly Leu Tyr

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Lys Trp Asp Lys Asn Asn Gln Trp Tyr Tyr Phe Asp Pro Val Thr Tyr

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Leu Lys Val Thr Asn Lys Trp Val Asp Gly Asn Tyr Tyr Asp Glu Asp

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Gly Ala Gln Ala Ile Ser Lys Leu Val Thr Ile Asn Asn Arg Leu Tyr

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Tyr Phe Asp Asp Gln Gly Lys Glu Ile Ser Asn Gln Phe Arg Thr Ile

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His Gly Asp Lys Tyr Tyr Phe Gly Asn Asp Ser Ala Ala Val Thr Gly

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Gln Gln Thr Ile Asp Gly Lys Val Tyr Lys Phe Ser Asn Tyr Gly Tyr

145 150 155 160

Leu Leu Gly Asn Arg Tyr Gly Lys Ile Glu Asn Gly Lys Leu Asn Ile

165 170 175

Tyr Ser Leu Ala Asp Asn Ser Leu Ile Lys Thr Val Glu Ala Gly Pro

180 185 190

Trp Glu Asn Met Ala Tyr Ser Met Asp Ser Asn Ser Ile Asn Asn Ile

195 200 205

Asp Gly Tyr Ile Ser Tyr Thr Gly Trp Tyr Arg Pro Tyr Gly Thr Ser

210 215 220

Gln Asp Gly Lys Thr Trp Tyr Pro Thr Thr Val Ala Asp Trp Arg Pro

225 230 235 240

Ile Leu Met Tyr Val Trp Pro Ser Lys Asp Val Gln Val Lys Phe Ile

245 250 255

Gln Tyr Phe Val Asn His Gly Tyr Glu Asn Ser Asn Tyr Gly Leu Thr

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Ala Gly Ser Val Lys Asp Leu Ser Glu Asn Thr Ala Ser Ile Lys Leu

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Asn Glu Val Ala Gln Asn Leu Arg Tyr Val Ile Glu Gln His Val Val

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Ala Ala Lys Ser Thr Ser Gln Leu Ala Asn Asp Ile Asn Asn Phe Ile

305 310 315 320

Thr Thr Ile Pro Glu Leu Ser Lys Ala Ser Glu Leu Ser Val Val Asn

325 330 335

Ser Tyr Gly Tyr Lys Pro Asp Asn Ser Gly Ser Val Asp Asp Asp Gln

340 345 350

Val Ile Phe Val Asn Asn Asp Ser Lys Asn Gln Lys Ile Gly Asn Thr

355 360 365

Ser Tyr Ala Asp Ser Asn Tyr Arg Leu Met Asn Arg Thr Ile Asn Asn

370 375 380

Gln Asn Gly Asp Asn Asn Ser Asp Asp Ser Pro Glu Leu Leu Val Gly

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Asn Asp Ile Asp Asn Ser Asn Pro Val Val Gln Ala Glu Asn Leu Asn

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Trp Glu Tyr Phe Leu Leu Asn Tyr Gly Lys Phe Met Asn Tyr Asn Pro

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Asn Gly Asn Phe Asp Gly Phe Arg Ile Asp Ala Ala Asp Asn Ile Asp

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Ala Asp Val Leu Asp Gln Ala Ala Gln Leu Ile Asn Ser Ile Tyr Asn

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Thr Lys Gly Asn Gln Ala Asn Ala Asn Asp His Leu Ile Tyr Asn Glu

465 470 475 480

Gly Tyr His Leu Gly Ala Ala Asn Met Leu Asp Arg Lys Ser Asn Pro

485 490 495

Glu Leu Tyr Met Asp Ser Gly Tyr Phe Tyr Thr Leu Glu Asn Val Leu

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Gly Arg Ala Ser Asp Arg Asp Asp Ile Asn Asn Leu Ile Thr Asn Ser

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Ile Val Asn Arg Gln Asn Asp Val Ser Glu Asn Val Ala Thr Pro Asn

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Trp Ser Phe Val Thr Asn His Asp Gln Arg Lys Asn Leu Ile Asn Gln

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Ile Val Ile Asp Asp His Pro Gly Val Ala Asp Ile Met Ser Asp Gly

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Tyr Lys Ala Glu Tyr Val Asn Gln Ala Trp Lys Glu Phe Tyr Ala Asp

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Gln Ala Arg Thr Asp Lys Lys Tyr Thr Gln Tyr Asn Leu Pro Ala Gln

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Tyr Ala Leu Leu Leu Thr Asn Lys Asp Thr Val Pro Gln Val Tyr Tyr

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Gly Asp Leu Tyr Asp Glu Thr Asp Gln Tyr Met Gln Asn Lys Ser Val

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Tyr Tyr Asp Ala Ile Thr Thr Leu Met Lys Ala Arg Lys Ser Tyr Val

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Ser Gly Gly Gln Ser Met Ile Lys Ile Asn Asp His Leu Leu Thr Ser

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Val Arg Tyr Gly Lys Gly Ile Ile Asp Gly Asn Val Ser Met Thr Asp

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Ile Leu Gly Arg Asn Ser Gly Ile Ala Val Val Val Gly Asn Asp Ala

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Gln Met Ala Asn Gln Thr Ile Ser Ile Asn Met Gly Lys Ala His Ala

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Asn Gln Ala Tyr Lys Gln Leu Leu Gly Thr Ile Asp Ser Gly Leu Thr

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Ser Ser Asp Thr Thr Ile Tyr His Thr Asp Ser Asn Gly Val Leu Asn

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Val Thr Val Lys Gly Tyr Ser Asn Pro Tyr Val Ser Gly Tyr Leu Gly

755 760 765

Val Trp Val Pro Leu Asn Gly Gly Ala Asn Ile Thr Thr Lys Ala Ser

770 775 780

Glu Val Thr Asn Gln Ser Asp Lys Thr Tyr Ser Ser Asn Ala Ala Leu

785 790 795 800

Asp Ser His Val Ile Tyr Glu Asp Phe Ser Leu Phe Gln Pro Glu Pro

805 810 815

Thr Ser Lys Ala Glu His Ala Tyr Asn Ile Ile Ala Asp Asn Ala Ser

820 825 830

Leu Phe Asn Glu Leu Gly Ile Thr Asp Phe Trp Met Ala Pro Ala Tyr

835 840 845

Thr Pro Phe Asn Thr Ser Arg Tyr Asn Glu Gly Tyr Ser Met Thr Asp

850 855 860

Arg Tyr Asn Leu Gly Thr Glu Ala Asn Leu Thr Lys Tyr Gly Ser Gly

865 870 875 880

Glu Glu Leu Ser Asn Ala Ile Ala Ala Leu His Asp Ala Gly Leu Lys

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Val Gln Glu Asp Leu Val Met Asn Gln Met Ile Gly Phe Ser Gly Gln

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Glu Ala Val Thr Val Thr Arg Thr Asp Gly His Ala Lys Gln Leu Thr

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Val Asp Gly Lys Thr Phe Ala Asn Gln Ile Tyr Phe Ala Tyr Thr Arg

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Gly Gly Gly Glu Gly Gln Lys Asn Tyr Gly Gly Lys Tyr Leu Asp Glu

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Leu Gln Lys Lys Tyr Pro Glu Leu Phe Thr Thr Lys Ala Val Ser Thr

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Gly Val Ala Pro Asp Pro Ser Val His Ile Thr Glu Trp Ser Ala Lys

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Tyr Gln Asn Gly Thr Ser Leu Gln Asn Ile Gly Ile Gly Leu Ala Val

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Lys Leu Ala Asn Gly Asp Tyr Ala Tyr Leu Asn Asp Ser Asn Asn

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Lys Ala Phe Asn Thr Thr Leu Pro Glu Thr Met Ser Ser Ala Asp

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Tyr Tyr Ala Asn Ile Glu Asp Asp

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Asn Pro Thr Lys Pro Val Glu Asp Ala Pro Val Thr Ala Asp Val Gly

1 5 10 15

Asn Leu His Thr Trp Trp His Asp Asn Ala Val Tyr Asn Thr Asp Ser

20 25 30

Pro Thr Glu Asn Gly Glu Val Arg Arg Ser Ser Phe Tyr Asp Val Gln

35 40 45

Val Ala Gln Ala His Gln Pro Asp Lys Phe Phe Asp Ser Phe Ala Tyr

50 55 60

Met Ser Ile Pro Arg Ser Gly Lys Gly Lys Val Gly Tyr Thr Lys Glu

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Asp Gly Ala Glu Phe Ser Ser Glu Ala Asn Leu Ser Met Ser Trp Ser

85 90 95

Ser Phe Glu Tyr Ala Lys Asp Val Trp Val Asp Val Ser Leu Lys Thr

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Gly Gln Thr Ile Ser Ser Ala Asp Glu Val Gln Ile Arg Pro Ser Ser

115 120 125

Tyr Asp Phe Glu Lys Lys Leu Val Asp Glu Asp Thr Ile Arg Ile Lys

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Val Pro Tyr Ser Asp Ala Gly Tyr Arg Phe Ser Val Glu Phe Asp Pro

145 150 155 160

Gln Leu Tyr Thr Ser Tyr Asn Asp Met Ser Gly Asn Ser Gly Lys Leu

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Thr Thr Val Ala Glu Gly Asn Arg Pro Ile His Thr Glu Pro Met Asn

180 185 190

Ser Met Met Ile Phe Ala Glu Pro Lys Leu Gln Gly Glu Glu Glu Lys

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Arg Leu Ile Pro Asn Pro Ser Ser Gly Ser Ile His Tyr Pro Glu Glu

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Gly Glu Val Lys Asp Leu Asn Thr Val Thr Glu Glu Ile Ile Tyr Phe

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Lys Pro Gly Thr Tyr His Met Gly Ser Asp Tyr His Ala Val Leu Pro

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Pro Asn Val Lys Trp Val Tyr Leu Ala Pro Gly Ala Tyr Val Lys Gly

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Ala Phe Arg Phe Phe His Asp Asn Gln Ala Gln Tyr Lys Val Thr Gly

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Tyr Gly Val Leu Ser Gly Glu Gln Tyr Val Tyr Glu Ala Asp Thr Ala

290 295 300

Asn Asn Tyr Asn Ala Leu Ser Gly Ala Ser Asn Cys His Val Thr Cys

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Val Lys Met Leu Gln Phe Glu Ser Ser Asn Ile Gly Gln Gln Leu Asp

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Leu Gln Gly Val Thr Ile Asn Glu Pro Pro Tyr His Ser Phe Val Val

340 345 350

Tyr Ala His Glu Gly Glu Lys Glu Ile Gly Val Glu Asn Phe Arg Met

355 360 365

Asn Val Glu Asn Tyr Lys Gln Val Gly Ser Trp Tyr Trp Gln Thr Asp

370 375 380

Gly Ile Glu Leu Tyr Gln Gly Gly Thr Met Lys Asn Thr Phe Phe Asn

385 390 395 400

Ala Asn Asp Asp Val Leu Lys Met Tyr His Ser Asp Val Thr Ile Asp

405 410 415

Asn Thr Val Ile Trp Lys Asn Glu Asn Gly Pro Val Ile Gln Trp Gly

420 425 430

Trp Thr Pro Arg Asn Ile Asp Asn Val Asn Val Thr Asp Thr Thr Val

435 440 445

Ile His Asn Arg Met Tyr Trp Lys Asp Pro Lys Tyr Asn Thr Cys Ile

450 455 460

Leu Asn Ser Ser Ser His Trp Glu Asp Met Gly Ser Thr Ala Lys Ala

465 470 475 480

Asp Pro Asn Thr Thr Val Lys Asn Met Arg Phe Glu Asn Ile Thr Val

485 490 495

Glu Gly Met Thr Asn Cys Ala Met Arg Ile Tyr Ala Leu Ser Asn Thr

500 505 510

Glu Asn Ile His Val Lys Asn Leu Ser Ile Asp Ser Trp Asn Gly Leu

515 520 525

Asp Trp Thr Ser Gln Val Ser His Leu Lys Arg Tyr Thr Asn Ser Ala

530 535 540

Gly Glu Lys Val Thr Ile Gly Asn Glu Ile Pro Asp Gly Asn Gly Leu

545 550 555 560

Ala Leu Glu Asn Tyr Ser Val Gly Gly Glu Ile Ile Glu Lys Ser Gly

565 570 575

Asp Asn Trp Asn Asp Tyr Lys Leu Gly Arg Leu Gly Phe Asp Gly Glu

580 585 590

Asn Trp Asp Ser Trp Asn Ala Trp Lys Ser Thr Pro

595 600