WO2021243821A1 - Maltotriose amylase mutant for producing maltotriose at high specificity - Google Patents

Abstract

A maltotriose amylase mutant for producing maltotriose at high specificity, relating to the field of genetic engineering and enzyme engineering. TfAmyA from Thermobifida fusca NTU22 is modified, and is truncated, and on this basis, site-directed mutagenesis is performed on leucine at position 47, glycine at position 101, glycine at position 103, and glycine at position 153. The main product maltotriose of the obtained mutant enzyme has the specificity increased and the yield increased, which is more suitable for the production of maltotriose.

Description

Maltotriose amylase mutant with high specificity for producing maltotriose Technical field

The invention relates to a maltotriose amylase mutant for producing maltotriose with high specificity, and belongs to the fields of genetic engineering and enzyme engineering.

Background technique

Maltotriose (α-D-Glc-(1→4)-α-D-Glc-(1→4)-D-Glc, G3 for short) belongs to a kind of functional oligosaccharide, composed of three glucoses The units are connected by α-(1→4) glycosidic bonds. Its molecular formula is C ₁₈ H ₃₂ O ₁₆ , relative molecular mass is 504.438, density is 1.8±0.1g·cm ^-3 , melting point is 132-135°C, and it is a white solid powder at room temperature. G3 has a variety of excellent physicochemical and physiological properties. It is a digestible nutritious sweetener. It has the characteristics of non-cariogenic, low sweetness, low viscosity, low calorie, anti-crystallization and low osmotic pressure. It can be used to change freezing The freezing temperature of food and the control of the browning variable caused by Maillard reaction in hot processed food. G3 provides high moisture retention capacity, prevents excessive drying and low water activity, facilitates the control of microbial contamination, has strong stability, and is a strong inhibitor of starch retrogradation. G3 is widely used in food, beverage, chemical, pharmaceutical and other industries. Most of the G3 products on the market come from Japan. "Fuji Oligomer G3" from Japan Food & Chemical Corporation, "New Malto-oligosaccharide" from Japan Corn Starch Corporation, "Linear Oligosaccharide origo" from Mitsubishi Chemical Food Co., Ltd., and "puretose" from Kunei Chemical Industry Co., Ltd., The main ingredients are all G3.

Maltotriose amylase (EC 3.2.1.116, maltotriose forming α-amylase, AmyA for short) is encoded by the gene tfa, which is an α-amylase with the ability to form maltotriose. AmyA uses starch or dextrin as the substrate to hydrolyze the α-(1→4) glycosidic bond in the sugar chain through endo or exocytosis to produce maltotriose and a small amount of by-products. The discovery of this enzyme provides a new idea for the preparation of G3.

At present, the preparation process of G3 is divided into chemical method and enzymatic method. In the chemical method, pullulan is used as the starting material, and G3 can be prepared through two steps of acetylation and deacetylation. In the two-step reaction, high-risk reagents such as concentrated sulfuric acid, petroleum ether, methanol, and sodium methoxide are used. Although the steps are short and the operation is simple, the cost of the substrate is high and the environmental hazard is high. As a green, energy-saving, efficient and safe alternative method, enzymatic method can significantly reduce production costs, improve product quality, and meet consumer demand. It is the driving force behind the sustainable development of the maltotriose industry. Because of the different enzymes used, enzymatic preparation is divided into two types. Enzymatic preparation is divided into two types because of the different enzymes used. The first enzymatic method is to hydrolyze pullulan by pullulanase, which can continuously and automatically prepare pharmaceutical grade G3 (purity above 99%), but this method has the problem of high substrate cost. Compared with pullulan, starch is a rich natural renewable resource and the main storage carbohydrate in the seeds, roots or tubers of many important food crops (such as rice, corn, wheat, rice, potatoes and cassava) , More economical. Maltodextrin is a liquefied product obtained by adding α-amylase after starch gelatinization. Due to the action of α-amylase, maltodextrin has a low degree of polymerization and a large solubility, which is conducive to the action of other enzymes. The second method is to use AmyA as the main enzyme and use cheap raw materials such as starch or maltodextrin as the substrate to produce G3. Generally speaking, pullulanase needs to be added to the reaction system for debranching. However, AmyA currently has the problem of more by-products of glucose and maltose, which increases the difficulty and cost of separation and purification. Therefore, obtaining AmyA with high G3 specificity through molecular modification and other means can increase the yield of G3, simplify the production process, and reduce the production cost.

Summary of the invention

In order to solve the existing problems, the present invention provides a maltotriose amylase mutant. The maltotriose amylase TfAmyA derived from Thermobifida fusca NTU22 used in the present invention produces G1-G3 products at the early stage of the enzyme conversion reaction, and belongs to a kind of endonuclease, which reduces the yield of the substrate when TfAmyA produces G3. At the same time, it also increases the difficulty of separation and purification. Therefore, further improving the G3 production capacity and specificity of the enzyme is beneficial to the reduction of G3 production costs and large-scale production.

The present invention provides a maltotriose amylase mutant with high specificity to produce maltotriose, which is truncated at the carboxyl terminal relative to the parent TfAmyA, or truncated amino acids at positions 47, 101, The amino acid at either position 103 or 153 is mutated.

In one embodiment of the present invention, the amino acid sequence of the parent TfAmyA is shown in SEQ ID NO.1.

In one embodiment of the present invention, the mutant is any one of the following (a) to (e):

(a) Truncating amino acids 451-572 of the parent, the amino acid sequence is shown in SEQ ID NO.4;

(b) Mutate the leucine at position 47 of the amino acid shown in SEQ ID NO. 4 to lysine or arginine, and name the mutants L47K and L47R, respectively;

(c) Mutation of the glycine at position 101 of the amino acid shown in SEQ ID NO. 4 to serine, threonine, aspartic acid, asparagine, glutamate, or glutamine, respectively, and the mutants Named as G101S, G101T, G101D, G101N, G101E, G101Q;

(d) Mutation of glycine at position 103 of the amino acid shown in SEQ ID NO. 4 to histidine, and name the mutant G103H;

(e) Mutate the glycine at position 153 of the amino acid shown in SEQ ID NO. 4 to serine, aspartic acid, asparagine, glutamic acid, glutamine or histidine, and name the mutants respectively It is G153S, G153D, G153N, G153E, G153Q, G153H.

The present invention provides genes encoding the mutants.

The present invention provides a recombinant expression vector carrying the gene, and the expression vector is any one of the pET series, the Duet series, the pGEX series, the pHY300, the pHY300PLK, the pPIC3K or the pPIC9K series.

The present invention provides microbial cells expressing the mutant or carrying the gene.

In one embodiment of the present invention, the microbial cell is a prokaryotic cell or a eukaryotic cell.

The present invention provides a method for improving the specificity of maltotriose production. The method uses dextrin as a substrate and the mutant as a catalyst to produce maltotriose.

In one embodiment of the present invention, the dextrin uses rice, corn, wheat, rice, potato, sweet potato, kudzu root and/or cassava as a raw material.

The present invention also protects the application of the mutant, or the gene, or the recombinant expression vector, or the microbial cell, or the method for improving the specificity of maltotriose production in the preparation of maltotriose.

The beneficial effects of the present invention: the present invention obtains a variety of mutants through truncated mutation and site-directed mutation of TfAmyA, the activity of maltotriose amylase is enhanced; the ratio of maltotriose in the product is increased, which is beneficial to simplify the maltotriose The subsequent purification process and the realization of large-scale production, and can significantly reduce the production cost of maltotriose, has a wide range of industrial application prospects.

Description of the drawings

Figure 1 shows the SDS-PAGE images before and after truncation; a is the wild type, and b is the mutant after truncation.

detailed description

The technical solution of the present invention will be further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited to this.

The medium formula described in the examples is as follows:

LB medium (g/L): peptone 10, yeast extract 5, NaCl 10.

TB medium (g/L): peptone 10, yeast powder 24, glycerol 5, K ₂ HPO ₄ ·3H ₂ O 16.43, KH ₂ PO ₄ 2.31.

The pullulanase described in the examples is any debranching enzyme with starch debranching effect.

The detection method of G3 content in the examples:

The enzyme-converted sample was boiled in a water bath for 10 minutes, ^{centrifuged at 12000 r·min -1} for 10 min, and then co-precipitated with acetonitrile with a final concentration of 50% (volume ratio). After standing for 2 hours, ^{centrifuged at 12000 r·min -1} for 10 min, and the supernatant was taken , Pass the 0.22μM filter membrane with a syringe. The mobile phase was prepared with the ratio of acetonitrile: water (74:26), and the sample was detected with an Agilent 1200 HPLC chromatograph and a HYPERSIL APS2 amino column. The column temperature was set to 40°C and the flow rate was 0.8 mL·min ^-1 . The amount of maltotriose produced was calculated based on the absorption peak area and the peak area of the standard maltotriose product.

Example 1: Preparation of truncated enzyme

1) Construction of truncated body and wild-type vector

①According to the amino acid sequence and nucleotide sequence of maltotriose amylase derived from Thermobifida fusca NTU22 (the nucleotide sequence is shown in SEQ ID NO. 2), the truncated gene fragment Tftfa-ΔML (nucleotide The sequence is shown in SEQ ID NO.3), and the vector pET20b(+) (the expression vector has a signal peptide to initiate gene expression) is connected with the one-step cloning enzyme Exnase Ⅱ (Nanjing Novezan Biotechnology Co., Ltd.) to construct the recombination Plasmid pET20b(+)-Tftfa-ΔML.

② Connect the gene fragment with the nucleotide sequence shown in SEQ ID NO. 2 to the vector pET20b(+) in the same way as above to construct the recombinant plasmid pET20b(+)-Tftfa.

2) Expression and purification of truncated body and wild enzyme

① Transform the recombinant plasmid pET20b(+)-Tftfa-ΔML into host E. coli BL21(DE3) competent cells, culture at 37°C for 12-14h to grow a single clone, pick the single clone, and perform sequencing verification; The correct positive transformants were connected to LB liquid medium (containing 100μg/mL ampicillin), cultured at 37°C and 200rpm for 8-10h, and the seed fermentation broth was connected to TB liquid medium (containing 100μg/mL) at 5% inoculum. mL ampicillin and 7.5g/L glycine); Escherichia coli was cultured in a shaker at 37°C to OD ₆₀₀ =0.6～0.8, IPTG was added at a final concentration of 0.134mM to induce extracellular expression, and the culture was continued on a shaker at 25°C. 48 After hours, the fermentation broth was centrifuged at 4°C and 10,000 g for 15 min to remove the bacteria, and the supernatant was collected and purified to obtain the purified truncated enzyme.

②The recombinant plasmid pET20b(+)-Tftfa was transformed into host E. coli BL21(DE3) competent cells by the same method as step ①, and then cultured and induced to obtain purified wild-type enzyme.

Example 2: Preparation of mutant enzyme

1) Site-directed mutation

Using plasmid pET20b(+)-Tftfa-ΔML as a template, using synthetic mutant primers, PCR was performed to construct mutants.

Table 1 Primers

PCR system: 2×Phanta Max Master Mix 25μL, template 1μL, upstream primer 2μL, downstream primer 2μL, ddH2O 20μL. PCR parameters: 94°C pre-denaturation 4min; into PCR cycle: 98°C denaturation 10s, 55°C annealing 30s, 72°C extension (duration is calculated according to the length of the amplified fragment, 1000bp·min ^-1 ), cycle 25-30 times; finally 72 ℃ 10min, 4 ℃ heat preservation. The PCR product was digested with Dpn I (Fermentas) and transformed into E. coli JM109 competent cells. After the transformed competent cells were cultured overnight in LB solid medium (containing 100μg/mL ampicillin), a single clone was picked up in LB liquid medium ( Containing 100μg/mL ampicillin) cultured, and then extract the plasmid, and sequenced.

2) Expression and purification of mutant enzymes

Transform the plasmid with the correct mutation sequence into the competent cells of the expression host E. coli BL21 (DE3), pick the single clone transferred into the expression host E. coli BL21 (DE3) and place it in LB liquid medium (containing 100 μg/mL ampicillin) at 37°C, Incubate at 200rpm for 8-10h, and transfer the seed fermentation broth to TB liquid medium (containing 100μg/mL ampicillin, 7.5g·L ^-1 glycine) according to the 5% inoculum; E. coli was cultured to OD _{600 on a shaker at 37°C} ＝0.6～0.8, add IPTG at a final concentration of 0.134mM to induce extracellular expression, and continue to culture in a shaker at 25°C. After fermentation for 48 hours, the fermentation broth is centrifuged at 4°C and 10000g for 15min to remove the bacteria, and the supernatant is collected and purified , And get mutant enzyme samples respectively.

Example 3: Determination of enzyme activity of maltotriose amylase

Enzyme activity determination adopts DNS method, and the system is as follows.

Substrate preparation: 1% mass concentration of soluble starch is suspended in the corresponding Buffer, heated and stirred to gelatinize.

Reaction system: Blank group: 1mL substrate (2g/100mL soluble starch) + 1mL Buffer.

Control group: 1mL substrate + 0.9mL Buffer + 0.1mL enzyme solution diluted by an appropriate multiple.

Add the substrate and Buffer components to the test tube with stopper in advance, and place it in a constant temperature water bath for 10 minutes. Dilute the enzyme to an appropriate multiple, add it to the control group reaction, and time it accurately for 10 minutes. After 10 minutes, 3mL DNS was added, and all the test tubes with stoppers were placed in a boiling water pot at the same time, accurately timed for 7 minutes, and quickly taken out and placed in an ice bath to cool down. After cooling down to low temperature, add 10mL H ₂ O and mix well. Use a 540nm spectrophotometer to measure the absorbance value Abs ₅₄₀ corresponding to the blank, and substitute it into the standard curve to calculate the enzyme activity.

_{Standard curve: The relationship between Abs 540} and the concentration of reducing sugar can be measured with different concentrations of glucose according to the above system. The linear regression equation is the standard curve.

Enzyme activity definition: The amount of enzyme required to release 1 μmol reducing sugar per minute is defined as an enzyme activity unit (U).

After culturing and fermentation on a shaker at 25°C for 48 hours, the supernatant was collected and the activity of wild enzyme and mutant enzyme shake flask was measured. The results are shown in Table 1. The enzyme activity of the truncated TfAmyA-ΔML has little change. Except for the increased enzyme activity of L47 and G103 mutants, most of the enzyme activities of G101 and G153 mutations decreased or slightly decreased.

Table 2 Comparison table of shake flask enzyme activity between mutant and wild type

Example 4: HPLC method to analyze the amount of maltotriose produced

Prepare a 5% mass concentration maltodextrin solution (pH 5.5) with a DE value of 5-7, add a certain amount of maltotriose amylase wild enzyme and mutant enzyme, and the amount of enzyme added is 60U·g ^-1 substrate. Lulanase was added to the reaction system with an enzyme amount of 32 U·g ^{-1 as a} substrate, placed in a water bath shaker with a rotation speed of 150 rpm and a temperature of 55° C., and reacted for 11 hours. Take a sample, boil it in a boiling water bath for 10 minutes to inactivate the enzyme, centrifuge and take the supernatant to obtain the product solution. Take 500μL of the product solution and mix 1:1 with acetonitrile, leave it at room temperature for 2h to precipitate large molecular weight dextrin or limit dextrin, then centrifuge at 12000rpm for 20min, and pass the supernatant through a 0.22μM filter membrane with a syringe to obtain HPLC analysis sample. The mobile phase was prepared with the ratio of acetonitrile: water (74:26), and the sample was detected with an Agilent 1200 HPLC chromatograph and a HYPERSIL APS2 amino column. The column temperature was set to 40°C and the flow rate was 0.8 mL·min ^-1 . According to the absorption peak area and the G3 standard peak area, the amount of G3 produced is calculated.

Table 2 shows the results of enzyme conversion analysis of wild enzymes and mutant enzymes. The truncated body TfAmyA-ΔML increased the yield of G3, and the ratio of G3 also increased slightly. Except for the G103 mutations G103V and G103P, the other mutations of L47, G101, G103 and G153 significantly increased the G3 ratio of the enzyme conversion product. The single mutant improves the specificity of G3, reduces other sugars in the product, and increases the main product G3. The ratio refers to the ratio of the mass concentration of G3 in the product to the mass concentration of the total product. The yield refers to the ratio of the G3 mass concentration of the product to the mass concentration of the substrate.

Table 3 G3 yield and ratio comparison table between mutant and wild type

Although the present invention has been disclosed as above in preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. The protection scope of the present invention should be defined by the claims.

Claims (20)

1. The mutant of the maltotriose amylase is characterized in that the mutant has the amino acid sequence of the leucine at position 47 of the maltotriose amylase shown in SEQ ID NO. 4 mutated to arginine.
2. The mutant of the maltotriose amylase is characterized in that the mutant is based on the maltotriose amylase whose amino acid sequence is as shown in SEQ ID NO. 1, and the ends are truncated.
3. The mutant according to claim 2, characterized in that, the mutant is truncated amino acids 451-272 of the parent, and its amino acid sequence is shown in SEQ ID NO.4.
4. The mutant of maltotriose amylase is characterized in that the mutant uses the maltotriose amylase whose amino acid sequence is as shown in SEQ ID NO. 4 as a parent, and the amino acid at position 47 of the parent is mutated.
5. The mutant according to claim 4, wherein the mutant is the mutation of leucine at position 47 of the parent to lysine.
6. A mutant of maltotriose amylase, characterized in that the mutant is based on the maltotriose amylase whose amino acid sequence is as shown in SEQ ID NO. The amino acid at any position at position 153 is mutated.
7. The mutant of claim 6, wherein the mutant is any one of the following (a) to (c):

   (a) Mutation of the glycine at position 101 of the amino acid shown in SEQ ID NO. 4 to serine, threonine, aspartic acid, asparagine, glutamic acid, or glutamine;

   (b) Mutation of glycine at position 103 of the amino acid shown in SEQ ID NO.4 to histidine;

   (c) Mutation of the glycine at position 153 of the amino acid shown in SEQ ID NO. 4 to serine, aspartic acid, asparagine, glutamic acid, glutamine or histidine.
8. A gene encoding the mutant of any one of claims 1-7.
9. A recombinant expression vector carrying the gene of claim 8.
10. The recombinant expression vector according to claim 9, wherein the expression vector is any one of pET series, Duet series, pGEX series, pHY300, pHY300PLK, pPIC3K or pPIC9K series.
11. A microbial cell expressing the mutant of any one of claims 1-7.
12. The microbial cell of claim 11, wherein the microbial cell is a prokaryotic cell or a eukaryotic cell.
13. A microbial cell carrying the gene of claim 8.
14. The microbial cell according to claim 13, wherein the microbial cell is a prokaryotic cell or a eukaryotic cell.
15. A method for improving the specificity of maltotriose production, which is characterized in that dextrin is used as a substrate, and the mutant described in any one of claims 1 to 7 is used as a catalyst to produce maltotriose.
16. The method according to claim 15, wherein the dextrin uses rice, corn, wheat, rice, potato, sweet potato, kudzu root and/or cassava as a raw material.
17. Use of the mutant of any one of claims 1-7 or the recombinant expression vector of claim 9 or 10 in the preparation of maltotriose.
18. Use of the gene of claim 7 or the microbial cell of claim 11 in the preparation of maltotriose.
19. The use of the microbial cells of any one of claims 12-14 in the preparation of maltotriose.
20. Use of the method of claim 15 or 16 in the preparation of maltotriose.

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