JP7238158B2 - Maltotriose-producing amylase mutants that produce maltotriose with high specificity - Google Patents

Description

The present invention relates to mutants of maltotriose-producing amylases that produce maltotriose with high specificity, and belongs to the fields of genetic engineering and enzyme engineering.

Maltotriose (abbreviated as Maltotriose, α-D-Glc-(1→4)-α-D-Glc-(1→4)-D-Glc, G3) is an α-(1→4) glycoside It belongs to functional oligosaccharides consisting of three glucose units linked via bonds. Its molecular formula is C18H32O16, its relative molecular weight is 504.438, its density is 1.8±0.1 g·cm ⁻³ , its melting point is 132-135° C., and it is a white solid powder at normal temperature. G3 has various excellent physico-chemical and physiological properties, and also has caries-preventing characteristics, low sweetness, low viscosity, low calorie, crystal resistance, and low osmotic pressure. It is a possible nutritive sweetener and can be used to modify the freezing temperature of frozen foods as well as control the amount of browning caused by the Maillard reaction in thermally processed foods. G3 provides high moisturizing capacity to prevent excessive dryness and low water activity, easy to control microbial contamination, has strong stability and strong inhibition against starch retrogradation. is an agent. G3 is widely used in industries such as food, beverage, chemical industry, and medicine. Most of the G3 products on the market come from Japan. Nippon Shokuhin Kako Co., Ltd.'s "Fujioligo#360", Nippon Corn Starch Co., Ltd.'s "New Maltooligosaccharide", Nippon Mitsubishi Chemical Foods Co., Ltd.'s "Linear Oligosaccharide Origo", and Gunei Chemical Industry Co., Ltd.'s "Pure Tose" are , the main component of which is G3.

Maltotriose-forming amylase (EC 3.2.1.116, maltotriose forming α-amylase, abbreviated as AmyA) is an α-amylase encoded by the gene tfa and capable of forming maltotriose. Using starch or dextrin as a substrate, AmyA hydrolyzes α-(1→4) glycosidic bonds in sugar chains by end-cutting or exo-cutting to produce maltotriose and a small amount of by-products. Finding this enzyme provides new knowledge for the preparation of G3.

Currently, the manufacturing process of G3 is divided into two, chemical method and enzymatic method. The chemical method uses pullulan as a starting material, and G3 can be prepared by two steps of acetylation and deacetylation. This two-step reaction requires high-risk reagents such as concentrated sulfuric acid, petroleum ether, methanol and sodium methoxide, is short in steps and simple to operate, but has high substrate costs and is harmful to the environment. is large. As an environmentally friendly, energy-saving, efficient and safe alternative, the enzymatic method can significantly reduce production costs, improve product quality, and meet consumer demand for the maltotriose industry. is the driving force behind the sustainable development of The enzymatic preparation is divided into two types according to the difference in the enzymes used. The first enzymatic method hydrolyzes pullulan with pullulanase and can continuously and automatically prepare pharmaceutical grade G3 (purity of 99% or more), but this method has the problem of high substrate cost. Starch is an abundant, natural and renewable resource compared to pullulan and is the primary storage of seeds, roots or tubers of many important food crops such as rice, maize, wheat, rice, potato and cassava. Carbohydrate and more economical. Maltodextrin is the product of gelatinizing starch and then liquefying it with the addition of α-amylase. Due to the action of α-amylase, maltodextrin has a low degree of polymerization and high solubility, which is advantageous for the action of other enzymes. The second method is a method of producing G3 using AmyA as the main enzyme and inexpensive raw materials such as starch and maltodextrin as substrates. Generally, it is necessary to add pullulanase to the reaction system for debranching. At present, however, AmyA contains many by-products such as glucose and maltose, and there are problems such as difficulty in separation and purification and an increase in cost. Therefore, by obtaining AmyA with high G3 specificity by means of molecular modification or the like, it is possible to improve the production amount of G3, simplify the production process, and reduce the production cost.

The present invention provides mutants of maltotriose-producing amylase to solve the above conventional problems. The maltotriose-producing amylase TfAmyA from Thermobifida fusca NTU22 used in the present invention belongs to a class of endonucleases producing G1-G3 products at the initial stage of the enzymatic conversion reaction. Therefore, when TfAmyA produces G3, the yield relative to the substrate decreases and the difficulty of separation and purification increases. Therefore, further enhancing the G3-producing ability and specificity of the enzyme is advantageous for reducing the production cost of G3 and increasing the scale of production.

The present invention provides mutants of maltotriose-producing amylases that produce maltotriose with high specificity, which are carboxyl-terminally truncated or truncated mutants relative to the parent TfAmyA amino acids 47, 101, 103, or 153 of any one amino acid is mutated.

In one embodiment of the invention, the amino acid sequence of said parental TfAmyA is that shown in SEQ ID NO:1.

One embodiment of the present invention is a mutant according to any of (a)-(e) below:
(a) a mutant in which amino acids 451-572 of the parent are truncated and whose amino acid sequence is shown in SEQ ID NO:4;
(b) mutants obtained by mutating leucine at position 47 to lysine or arginine in the amino acid sequence shown by SEQ ID NO: 4 and named L47K and L47R, respectively;
(c) G101S, G101T, G101D, G101N, G101E, obtained by mutating glycine at position 101 to serine, threonine, aspartic acid, asparagine, glutamic acid, or glutamine in the amino acid sequence shown in SEQ ID NO: 4, respectively; a mutant named G101Q;
(d) A mutant named G103H obtained by mutating glycine at position 103 to histidine in the amino acid sequence shown in SEQ ID NO: 4;
(e) mutants obtained by mutating glycine at position 153 to serine, aspartic acid, asparagine, glutamic acid, glutamine, or histidine in the amino acid sequence shown in SEQ ID NO: 4, respectively, G153S, G153D, G153N, Mutants named G153E, G153Q, G153H.

The present invention provides genes encoding said mutants.

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

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

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

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

In one embodiment of the present invention, the dextrin is sourced from rice, corn, wheat, paddy rice, potato, sweet potato, arrowroot and/or cassava.

The present invention further provides a use for preparing maltotriose using said mutant, said gene, said recombinant expression vector, said microbial cell, or a method for increasing the specificity of maltotriose production. .

In the present invention, truncation and site-directed mutagenesis were performed on TfAmyA to obtain various mutants, which increased the activity of maltotriose-producing amylase and increased the activity of maltotriose-producing amylase. The ose ratio is improved, which is advantageous for simplifying the subsequent purification process of maltotriose and realizing large-scale production, and can significantly reduce the production cost of maltotriose, and has a wide industrial application. is expected.

FIG. 1 is an illustration of SDS-PAGE before and after truncation, panel a is the wild type and panel b is the mutant after truncation.

Hereinafter, the invention according to the present disclosure will be described based on specific embodiments. The invention according to the present disclosure is not limited to the following embodiments.

The media formulations described in the examples are as follows.
LB medium (g/L): peptone 10, yeast extract 5, NaCl 10.
TB medium (g/L): peptone 10, yeast powder 24, glycerol 5, K2HPO4.3H2O 16.43, KH2PO4 2.31.

The pullulanase described in the examples is a type of debranching enzyme that has a debranching effect on starch.

Method for measuring G3 content in Examples:
The enzymatically converted sample was placed in a boiling water bath for 10 minutes, centrifuged at 12000 rmin ⁻¹ for 10 minutes, co-precipitated with 50% final concentration (by volume) of acetonitrile, and left for 2 hours. After that, it was centrifuged at 12000 r·min ⁻¹ for 10 minutes, and the supernatant was taken and passed through a 0.22 μM filtration membrane with a syringe. The mobile phase was prepared in a ratio of acetonitrile:water (74:26) using an Agilent 1200 HPLC chromatography and a HYPERSIL APS2 amino column, setting the column temperature to 40°C and the flow rate to 0.8 mL min ^-1 . and measured the sample. The amount of maltotriose produced was calculated from the absorption peak area and the peak area of the maltotriose standard product.

Example 1: Preparation of truncated enzyme 1) Construction of truncated vector and wild type vector Based on this, the truncated gene fragment Tftfa-ΔML (nucleotide sequence is shown in SEQ ID NO: 3) and the vector pET20b(+) (having a signal peptide that initiates gene expression on the expression vector) were seamlessly combined. Ligation was performed with the cloning enzyme Exnase II (Vazyme Biotech Co., Ltd.) to construct the recombinant plasmid pET20b(+)-Tftfa-ΔML.
2. The gene fragment whose nucleotide sequence is represented by SEQ ID NO: 2 was ligated with the vector pET20b(+) in the same manner as above to construct the recombinant plasmid pET20b(+)-Tftfa.

2) Expression and purification of truncated and wild-type enzymes 1. Transform recombinant plasmid pET20b(+)-Tftfa-ΔML into competent cells of host E. coli BL21(DE3) and culture at 37°C for 12-14 hours. A single colony was then extracted and sequence verified; positive transformants verified as correct were inoculated into LB liquid medium (containing 100 µg/mL ampicillin) and incubated at 37°C. , at 200 rpm for 8-10 hours, seed fermentation broth was inoculated at 5% inoculum into TB liquid medium (containing 100 μg/mL ampicillin and 7.5 g/L glycine); After culturing to 0.6-0.8 and inducing extracellular expression by adding IPTG to a final concentration of 0.134 mM and continuing the fermentation culture on a shaker at 25°C for a further 48 hours, the fermentation broth was incubated at 4°C. , 10,000 g for 15 minutes, the cells were removed, and the supernatant was collected and purified to obtain a purified truncated enzyme.
2. The recombinant plasmid pET20b(+)-Tftfa was transformed into competent cells of the host E. coli BL21(DE3) in the same manner as in step 1, and cultured and induced to obtain the purified wild-type enzyme. rice field.

Example 2: Preparation of Mutant Enzymes 1) Site-directed mutagenesis Using the plasmid pET20b(+)-Tftfa-ΔML as a template, PCR is performed using synthesized mutagenesis primers to construct mutants.

PCR system: 2x Phanta Max Master Mix 25 μL, template 1 μL, upstream primer 2 μL, downstream primer 2 μL, ddH2O 20 μL. PCR parameters are as follows. A PCR cycle was entered: denaturation at 98°C for 10 seconds, annealing at 55°C for 30 seconds, extension at 72°C (length of time depends on the length of the amplified fragment). calculated, 1000 bp·min ⁻¹ ), cycled 25-30 times; final incubation at 72° C. for 10 minutes, incubation at 4° C.; The PCR product was digested with Dpn I (Fermentas), transformed into Escherichia coli JM109 competent cells, and the transformed competent cells were cultured overnight in LB solid medium (containing 100 μg/mL ampicillin) to form single colonies. was extracted and cultured in LB liquid medium (containing 100 μg/mL ampicillin), after which plasmids were extracted and sequenced.

2) Expression and Purification of Mutant Enzymes Correctly mutated and sequenced plasmids were transformed into competent cells of host E. coli BL21(DE3) and single colonies of transformed host E. coli BL21(DE3) were extracted. The seed fermentation broth was cultured at 5% inoculum in LB liquid medium (containing 100 μg/mL ampicillin and 7.5 g/L glycine) in LB liquid medium (containing 100 μg/mL ampicillin) for 8-10 hours at 37°C. E. coli was cultured on a 37°C shaker to OD600 = 0.6-0.8, IPTG was added to a final concentration of 0.134 mM to induce extracellular expression, and then placed on a 25°C shaker to induce extracellular expression. After continuing the fermentation culture for 48 hours, the fermentation broth is centrifuged at 10000 g for 15 minutes at 4° C. to remove the cells, the supernatant is collected and purified to yield the purified mutant enzyme. Obtained.

Example 3: Enzyme activity measurement of maltotriose-forming amylase Enzyme activity was measured using the DNS method, and the system is as follows.
Substrate preparation: Soluble starch at a concentration of 1% by weight was suspended in a corresponding buffer, heated and stirred to gelatinize.
Reaction system: Blank set: 1 mL substrate (2 g/100 mL soluble starch) + 1 mL buffer.
Control group: 1 mL of substrate + 0.9 mL of buffer + 0.1 mL of appropriately diluted enzyme solution.

Substrate and buffer components were preloaded into stoppered test tubes and placed in a constant temperature water bath to incubate for 10 minutes. Enzymes were diluted appropriately and added to control pairs to perform reactions. 10 minutes were accurately counted. After 10 minutes, 3 mL of DNS was added, all stoppered tubes were placed simultaneously in a pan with boiling water to accurately count 7 minutes, quickly removed and placed in an ice bath to cool. After cooling down, 10 mL of H2O was added and mixed well. The enzymatic activity was calculated by measuring the absorbance value Abs540 with the corresponding blank removed using a 540 nm spectrophotometer and substituting it into the standard curve.

Calibration curve: Using the above system, measure the relationship between Abs540 and reducing sugar concentration at different glucose concentrations, and use the linear regression equation as the calibration curve.

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

After 48 hours of fermentation culture in a shaker at 25° C., the supernatant was collected and the enzymatic activity in the shaker of the wild-type and mutant enzymes was determined. Table 1 shows the results. The enzymatic activity of the truncated TfAmyA-ΔML did not change significantly, and in addition to the improved enzymatic activity of the L47 and G103 mutations, the G101 and G153 mutations reduced or slightly reduced the enzymatic activity. has decreased to

Example 4: Analysis of maltotriose production amount by HPLC method A maltodextrin solution (pH 5.5) with a concentration of 5% by weight with a DE value of 5-7 was prepared, and a fixed amount of the wild enzyme of maltotriose-producing amylase and Mutant enzymes were added with an enzyme amount of 60 U·g ⁻¹ substrate, and pullulanase with an enzyme amount of 32 U·g ⁻¹ substrate was added to the reaction system and placed in a water bath shaker at a rotation speed of 150 rpm and a temperature of 55°C. and reacted for 11 hours. A sample was taken, boiled in a boiling water bath for 10 minutes to deactivate the enzyme, and the supernatant was removed by centrifugation to obtain the product solution. 500 μL of the product solution was mixed 1:1 with acetonitrile and allowed to stand at room temperature for 2 hours to precipitate high molecular weight or limit dextrins, then centrifuged at 12000 rpm for 20 minutes and the supernatant was 0. HPLC-analysable samples were obtained through a 22 μM filtration membrane. The mobile phase was prepared in a ratio of acetonitrile:water (74:26) using an Agilent 1200 HPLC chromatography and a HYPERSIL APS2 amino column, setting the column temperature to 40°C and the flow rate to 0.8 mL min ^-1 , and the sample was measured. The amount of G3 produced was calculated from the absorption peak area and the peak area of the G3 standard product.

Table 2 shows the enzymatic conversion assay results for the wild-type and mutant enzymes. Truncated TfAmyA-ΔML improved the yield of G3 and slightly improved the proportion of G3. Besides the G103 mutations G103V and G103P, the other L47, G101, G103 and G153 mutations significantly enhanced the G3 ratio of enzymatic conversion products. A single mutant increased the specificity of G3, reduced other sugars in the product, and increased the major product G3. Here, the ratio refers to the ratio of the mass concentration of G3 in the product to the mass concentration of the entire product. Yield refers to the ratio of the mass concentration of G3 in the product to the mass concentration of the substrate.

While preferred embodiments of the present invention have been disclosed above, they are not so limited. Various design changes and modifications made by those skilled in the art fall within the scope of the present invention without departing from the spirit and scope of the present invention. Accordingly, the scope of the present invention shall be based on the subject matter defined in the appended claims.

Claims (10)

A mutant of a maltotriose-generating amylase having an amino acid sequence represented by SEQ ID NO: 4, wherein the parent is a maltotriose-generating amylase, and leucine at position 47 of the parent is mutated to lysine . In the amino acid sequence shown by SEQ ID NO: 4, the parent is a maltotriose-generating amylase, and an amino acid at any one of the 101st, 103rd, and 153rd positions of the parent is mutated,
A mutant of maltotriose-producing amylase, characterized by being any one of the following (a) to (c):
(a) in the amino acid sequence shown in SEQ ID NO: 4, glycine at position 101 is mutated to serine, threonine, aspartic acid, asparagine, glutamic acid, or glutamine;
(b) in the amino acid sequence shown in SEQ ID NO: 4, glycine at position 103 is mutated to histidine;
(c) in the amino acid sequence shown in SEQ ID NO: 4, glycine at position 153 is mutated to serine, aspartic acid, asparagine, glutamic acid, glutamine or histidine; A gene encoding a mutant according to claim 1 or 2 . A recombinant expression vector comprising the gene of claim 3 . The recombinant expression vector according to claim 4 , wherein the expression vector is any one series of pET series, Duet series, pGEX series, pHY300 series, pHY300PLK series, pPIC3K series, or pPIC9K series. . A microbial cell comprising the gene of claim 3 . A method for improving production specificity of maltotriose, which comprises producing maltotriose using dextrin as a substrate and the mutant according to claim 1 or 2 as a catalyst. The method according to claim 7 , characterized in that said dextrin is sourced from rice, corn, wheat, paddy rice, potato, sweet potato, arrowroot and/or cassava. Use of the mutant according to claim 1 or 2 or the recombinant expression vector according to claim 4 or 5 for preparing maltotriose. Use of the method according to claim 7 or 8 for preparing maltotriose.

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