Phosphatase mutants and methods of making monosaccharides

Abstract

The application belongs to the technical field of biotechnology, and discloses a phosphatase mutant and a preparation method of monosaccharide. The phosphatase mutant provided by the application comprises an amino acid fragment with a sequence shown as SEQ ID NO: 3, and the specificity of amino acid residues obtained by mutation of one or more of the 18th, 46th, 111th, 116th, 134th, 166th, 170th and 188th sites is specifically selected, so that the catalytic activity of the phosphatase mutant is improved, and the phosphatase mutant is endowed with excellent specific binding capacity for specific monosaccharide-6-phosphate, so that a phosphatase with high catalytic activity and specificity is obtained, and the phosphatase is of great significance for realizing large-scale and low-cost biological preparation of monosaccharide with a specific structure.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and in particular relates to a method for preparing a phosphatase mutant and a monosaccharide. Background Technology

[0002] Monosaccharides are carbohydrates that cannot be further broken down into smaller sugar molecules. They are the basic building blocks of oligosaccharides and polysaccharides, and are also important substances for energy metabolism in living organisms. Based on their scarcity, monosaccharides can be divided into common monosaccharides and rare monosaccharides. Common monosaccharides include fructose, glucose, and mannose, which are abundant in nature and easily utilized. Rare monosaccharides include sugars such as tagatose, allulose, and sorbitol, which are scarce in nature. Rare monosaccharides, with their advantages of low calories, biological value, and high sweetness, have become the mainstay of the new generation of sweeteners. Allulose, in particular, exhibits excellent performance in lowering glycemic response, inhibiting hepatic lipase activity, and scavenging free radicals, making it not only a sweetener but also promising for applications in the biomedical field. Tagatose, with the same sweetness as sucrose, has only half the calories, giving it an advantage in controlling energy intake and regulating blood sugar levels. Therefore, efficient and specific monosaccharide biosynthesis is of great significance in the development and utilization of monosaccharides.

[0003] Currently, methods for biosynthesizing monosaccharides include one-step enzymatic catalysis and multi-step enzymatic catalysis. One-step enzymatic catalysis utilizes specific isomerases to catalyze the isomerization of substrates to obtain the target monosaccharide. It has advantages such as simple reaction flow and easy control, but suffers from expensive substrates and high production costs, limiting its application in large-scale industrial production. Multi-step enzymatic catalysis uses inexpensive polysaccharides such as starch as raw materials, utilizing enzyme-catalyzed processes such as hydrolysis, isomerization, phosphorylation, and dephosphorylation to obtain the target monosaccharide. The core of multi-step enzymatic catalysis lies in the phosphatase that catalyzes the synthesis of monosaccharides from monosaccharide-6-phosphate. The high catalytic activity and specificity of the phosphatase directly determine the monosaccharide yield.

[0004] Therefore, obtaining a phosphatase with high catalytic activity and specificity is of great significance for achieving large-scale, low-cost biosynthesis of monosaccharides. Summary of the Invention

[0005] Based on a profound understanding of the key amino acid residues in the catalytic active center region and substrate channel of the wild-type phosphatase ST6P (as shown in SEQ ID NO:1) and their influence on its structure and catalytic activity, the inventors of this invention, through extensive and in-depth research and numerous experiments, creatively discovered that site-directed mutagenesis at one or more of the following positions (18, 46, 111, 116, 134, 166, 170, and 188) of the wild-type phosphatase ST6P can optimize the structure of the catalytic active center region and substrate channel, improving the specific binding ability and catalytic activity to specific monosaccharides (-6-phosphate). This results in a phosphatase with high catalytic activity and specificity, ultimately enabling the large-scale, low-cost preparation of monosaccharides with specific structures. Based on this, the technical solution of this invention is derived.

[0006] In a first aspect, the present invention provides a phosphatase mutant comprising an amino acid fragment with the sequence shown in SEQ ID NO:3; wherein, the amino acid residue positions of the phosphatase mutant are encoded with reference to the amino acid fragment shown in SEQ ID NO:3, wherein the 18th amino acid residue of the phosphatase mutant is glutamic acid, aspartic acid, or lysine; the 46th amino acid residue of the phosphatase mutant is valine, threonine, or glycine; the 111th amino acid residue of the phosphatase mutant is serine, isoleucine, cysteine, or alanine; the 116th amino acid residue of the phosphatase mutant is leucine, isoleucine, or arginine; the 134th amino acid residue of the phosphatase mutant is threonine, phenylalanine, or alanine; the 166th amino acid residue of the phosphatase mutant is phenylalanine, isoleucine, leucine, or valine; the 170th amino acid residue of the phosphatase mutant is threonine, histidine, or tryptophan; and the 188th amino acid residue of the phosphatase mutant is asparagine, aspartic acid, serine, alanine, or histidine.

[0007] Furthermore, the phosphatase mutant comprises one or more segments of amino acid fragments with sequences as shown in SEQ ID NO:4-13.

[0008] Secondly, the present invention provides a method for constructing the above-mentioned phosphatase mutant, the method comprising: performing site-directed PCR mutagenesis based on the coding gene of wild-type phosphatase ST6P to obtain the coding gene of the phosphatase mutant; transforming the coding gene of the phosphatase mutant into host cells to obtain an engineered strain; and inducing expression of the engineered strain to obtain the phosphatase mutant.

[0009] Furthermore, the wild-type phosphatase ST6P comprises an amino acid fragment with the sequence shown in SEQ ID NO:1.

[0010] Thirdly, the present invention provides an engineered strain that expresses the above-mentioned phosphatase mutant.

[0011] Fourthly, the present invention provides the application of the above-mentioned phosphatase mutant in monosaccharide production.

[0012] Furthermore, when the monosaccharide is tagatose, the phosphatase mutant comprises an amino acid fragment with a sequence as shown in SEQ ID NO:4 or 5.

[0013] Furthermore, when the monosaccharide is fructose, the phosphatase mutant comprises an amino acid fragment with a sequence as shown in SEQ ID NO:6 or 7.

[0014] Furthermore, when the monosaccharide is glucose, the phosphatase mutant comprises an amino acid fragment with a sequence as shown in SEQ ID NO:8 or 9.

[0015] Furthermore, when the monosaccharide is allulose, the phosphatase mutant comprises an amino acid fragment with a sequence as shown in SEQ ID NO:10 or 11.

[0016] Furthermore, when the monosaccharide is mannose, the phosphatase mutant includes an amino acid fragment with a sequence as shown in SEQ ID NO:12 or 13.

[0017] Fifthly, the present invention provides a method for preparing a monosaccharide, the method comprising: taking the above-mentioned phosphatase mutant and subjecting it to an enzymatic reaction in a reaction solution containing a carbohydrate substrate to obtain the monosaccharide.

[0018] Furthermore, the sugar substrate is selected from one or more of tagatose-6-phosphate, fructose-6-phosphate, glucose-6-phosphate, allulose-6-phosphate, and mannose-6-phosphate.

[0019] Furthermore, the concentration of the added phosphatase mutant is 1–25 g / L.

[0020] Furthermore, the reaction solution comprises 100–200 mM of a carbohydrate substrate and 1–20 mM of Mg. 2+ And 50–200 mM HEPES buffer at pH 6.5–7.5.

[0021] Furthermore, the enzyme-catalyzed reaction is carried out at a temperature of 50–70°C for a time of 1–48 h.

[0022] Beneficial effects:

[0023] This invention provides a phosphatase mutant comprising an amino acid fragment with the sequence shown in SEQ ID NO:3. Through specific selection of amino acid residues obtained by mutating one or more sites at positions 18, 46, 111, 116, 134, 166, 170, and 188, the phosphatase mutant can enhance its catalytic activity while simultaneously endowing it with excellent specific binding ability to specific monosaccharides (-6-phosphate). This results in a phosphatase possessing both high catalytic activity and specificity, which is of great significance for the large-scale, low-cost bioprocessing of monosaccharides with specific structures.

[0024] In some specific embodiments, when the phosphatase mutant preferably includes an amino acid fragment with a sequence such as SEQ ID NO:4 or 5, the phosphatase mutant has excellent catalytic activity and specificity for tagatose-6-phosphate, and can better catalyze the reaction process of converting tagatose-6-phosphate to tagatose, showing great application prospects in the field of tagatose production.

[0025] In some specific embodiments, when the phosphatase mutant preferably includes an amino acid fragment with a sequence such as SEQ ID NO:6 or 7, the phosphatase mutant has excellent catalytic activity and specificity for fructose-6-phosphate, and can better catalyze the reaction process of fructose-6-phosphate to fructose, showing great application prospects in the field of fructose production.

[0026] In some specific embodiments, when the phosphatase mutant preferably includes an amino acid fragment with a sequence as shown in SEQ ID NO:8 or 9, the phosphatase mutant has excellent catalytic activity and specificity for glucose-6-phosphate, and can better catalyze the reaction process of glucose-6-phosphate to glucose, showing great application prospects in the field of glucose production.

[0027] In some specific embodiments, when the phosphatase mutant preferably includes an amino acid fragment with a sequence as shown in SEQ ID NO:10 or 11, the phosphatase mutant has excellent catalytic activity and specificity for allulose-6-phosphate, and can better catalyze the reaction process of allulose-6-phosphate to allulose, showing great application prospects in the production of allulose.

[0028] In some specific embodiments, when the phosphatase mutant preferably includes an amino acid fragment with a sequence as shown in SEQ ID NO:12 or 13, the phosphatase mutant has excellent catalytic activity and specificity for mannose-6-phosphate, and can better catalyze the reaction process of converting mannose-6-phosphate to mannose, showing great application prospects in the field of mannose production. Attached Figure Description

[0029] Figure 1 This is a schematic diagram of the structure of the recombinant plasmid pET28a-ST6P provided in Example 1 of this invention. Detailed Implementation

[0030] The amino acid and nucleotide sequences involved in this invention are specifically shown in Table 1.

[0031] Table 1.

[0032]

[0033]

[0034]

[0035] The phosphatase mutant provided by this invention is based on wild-type phosphatase ST6P, which includes the amino acid fragment shown in SEQ ID NO:1. Specific amino acid substitution mutations are performed at one or more sites within the amino acid fragment shown in SEQ ID NO:1, specifically at positions 18, 46, 111, 116, 134, 166, 170, and 188. That is, the phosphatase mutant specifically includes the amino acid fragment shown in SEQ ID NO:3, and the similarity between the amino acid fragment shown in SEQ ID NO:3 and the amino acid fragment shown in SEQ ID NO:1 is not 100%.

[0036] In this invention, the 18th amino acid residue X of the phosphatase mutant specifically refers to glutamic acid, aspartic acid, or lysine. Specifically, when the 18th amino acid residue is glutamic acid, it indicates that the phosphatase mutant has not undergone a mutation at the 18th amino acid residue compared to the wild-type phosphatase ST6P. When the 18th amino acid residue is aspartic acid, it indicates that the phosphatase mutant has undergone a glutamic acid-to-aspartic acid substitution mutation (E18D mutation) at the 18th amino acid residue compared to the wild-type phosphatase ST6P. When the 18th amino acid residue is lysine, it indicates that the phosphatase mutant has undergone a glutamic acid-to-lysine substitution mutation (E18K mutation) at the 18th amino acid residue compared to the wild-type phosphatase ST6P.

[0037] In this invention, the 46th amino acid residue X of the phosphatase mutant specifically refers to valine, threonine, or glycine. Specifically, when the 46th amino acid residue is valine, it indicates that the phosphatase mutant has not undergone a mutation at the 46th amino acid residue compared to the wild-type phosphatase ST6P. When the 46th amino acid residue is threonine, it indicates that the phosphatase mutant has undergone a valine-to-threonine substitution mutation (V46T mutation) at the 46th amino acid residue compared to the wild-type phosphatase ST6P. When the 46th amino acid residue is glycine, it indicates that the phosphatase mutant has undergone a valine-to-glycine substitution mutation (V46G mutation) at the 46th amino acid residue compared to the wild-type phosphatase ST6P.

[0038] In this invention, the 111th amino acid residue X of the phosphatase mutant specifically refers to serine, isoleucine, cysteine, or alanine. Specifically, when the 111th amino acid residue is serine, it indicates that the phosphatase mutant has not undergone a mutation at the 111th amino acid residue compared to the wild-type phosphatase ST6P. When the 111th amino acid residue is isoleucine, it indicates that the phosphatase mutant has undergone a serine-to-isoleucine substitution mutation (S111I mutation) at the 111th amino acid residue compared to the wild-type phosphatase ST6P. When the 111th amino acid residue is cysteine, it indicates that the phosphatase mutant has undergone a serine-to-cysteine ​​substitution mutation (S111C mutation) at the 111th amino acid residue compared to the wild-type phosphatase ST6P. When the 111th amino acid residue is alanine, it indicates that the phosphatase mutant has undergone a serine-to-alanine substitution mutation (S111A mutation) at the 111th amino acid residue compared to the wild-type phosphatase ST6P.

[0039] In this invention, the 116th amino acid residue X of the phosphatase mutant specifically refers to leucine, isoleucine, or arginine. Specifically, when the 116th amino acid residue is leucine, it indicates that the phosphatase mutant has not undergone a mutation at the 116th amino acid residue compared to the wild-type phosphatase ST6P. When the 116th amino acid residue is isoleucine, it indicates that the phosphatase mutant has undergone a leucine-to-isoleucine substitution mutation (L116I mutation) at the 116th amino acid residue compared to the wild-type phosphatase ST6P. When the 116th amino acid residue is arginine, it indicates that the phosphatase mutant has undergone a leucine-to-arginine substitution mutation (L116R mutation) at the 116th amino acid residue compared to the wild-type phosphatase ST6P.

[0040] In this invention, the 134th amino acid residue X of the phosphatase mutant specifically refers to threonine, phenylalanine, or alanine. Specifically, when the 134th amino acid residue is threonine, it indicates that the phosphatase mutant has not undergone a mutation at the 134th amino acid residue compared to the wild-type phosphatase ST6P. When the 134th amino acid residue is phenylalanine, it indicates that the phosphatase mutant has undergone a threonine-to-phenylalanine substitution mutation (T134F mutation) at the 134th amino acid residue compared to the wild-type phosphatase ST6P. When the 134th amino acid residue is alanine, it indicates that the phosphatase mutant has undergone a threonine-to-alanine substitution mutation (T134A mutation) at the 134th amino acid residue compared to the wild-type phosphatase ST6P.

[0041] In this invention, the 166th amino acid residue X of the phosphatase mutant specifically refers to phenylalanine, isoleucine, leucine, or valine. Specifically, when the 166th amino acid residue is phenylalanine, it indicates that the phosphatase mutant has not undergone a mutation at the 166th amino acid residue compared to the wild-type phosphatase ST6P. When the 166th amino acid residue is isoleucine, it indicates that the phosphatase mutant has undergone a phenylalanine-to-isoleucine substitution mutation (F166I mutation) compared to the wild-type phosphatase ST6P. When the 166th amino acid residue is leucine, it indicates that the phosphatase mutant has undergone a phenylalanine-to-leucine substitution mutation (F166L mutation) compared to the wild-type phosphatase ST6P. When the 166th amino acid residue is valine, it indicates that the phosphatase mutant has undergone a phenylalanine-to-valine substitution mutation (F166V mutation) compared to the wild-type phosphatase ST6P.

[0042] In this invention, the 170th amino acid residue X of the phosphatase mutant specifically refers to threonine, histidine, or tryptophan. Specifically, when the 170th amino acid residue is threonine, it indicates that the phosphatase mutant has not undergone a mutation at the 170th amino acid residue compared to the wild-type phosphatase ST6P. When the 170th amino acid residue is histidine, it indicates that the phosphatase mutant has undergone a threonine-to-histidine substitution mutation (T170H mutation) at the 170th amino acid residue compared to the wild-type phosphatase ST6P. When the 170th amino acid residue is tryptophan, it indicates that the phosphatase mutant has undergone a threonine-to-tryptophan substitution mutation (T170W mutation) at the 170th amino acid residue compared to the wild-type phosphatase ST6P.

[0043] In this invention, the 188th amino acid residue X of the phosphatase mutant specifically refers to asparagine, aspartic acid, serine, alanine, or histidine. Specifically, when the 188th amino acid residue is asparagine, it indicates that the phosphatase mutant has not undergone a mutation at the 188th amino acid residue compared to the wild-type phosphatase ST6P. When the 188th amino acid residue is aspartic acid, it indicates that the phosphatase mutant has undergone a substitution mutation from asparagine to aspartic acid at the 188th amino acid residue compared to the wild-type phosphatase ST6P (N188D mutation). When the 188th amino acid residue is serine, it indicates that the phosphatase mutant has undergone a substitution mutation from asparagine to serine at the 188th amino acid residue compared to the wild-type phosphatase ST6P (N188S mutation). When the 188th amino acid residue is alanine, it indicates that the phosphatase mutant has undergone a substitution mutation from asparagine to alanine at the 188th amino acid residue compared to the wild-type phosphatase ST6P (N188A mutation). When the 188th amino acid residue is histidine, it means that the phosphatase mutant has undergone an asparagine-to-histidine substitution mutation (N188H mutation) at the 188th amino acid residue compared to the wild-type phosphatase ST6P.

[0044] In this invention, compared to the wild-type phosphatase ST6P, the phosphatase mutant preferably includes the aforementioned E18D and N188H mutations, specifically comprising the amino acid fragment shown in SEQ ID NO:4. In this case, the phosphatase mutant exhibits excellent catalytic activity and specificity for tagatose-6-phosphate.

[0045] In this invention, compared to the wild-type phosphatase ST6P, the phosphatase mutant preferably includes the aforementioned E18K and N188H mutations, specifically comprising the amino acid fragment shown in SEQ ID NO:5. In this case, the phosphatase mutant exhibits excellent catalytic activity and specificity for tagatose-6-phosphate.

[0046] In this invention, compared to the wild-type phosphatase ST6P, the phosphatase mutant preferably includes the aforementioned S111I and F166I mutations, specifically comprising the amino acid fragments shown in SEQ ID NO:6. In this case, the phosphatase mutant exhibits excellent catalytic activity and specificity for fructose-6-phosphate.

[0047] In this invention, compared to the wild-type phosphatase ST6P, the phosphatase mutant preferably includes the aforementioned L116I and N188A mutations, specifically comprising the amino acid fragment shown in SEQ ID NO:7. In this case, the phosphatase mutant exhibits excellent catalytic activity and specificity for fructose-6-phosphate.

[0048] In this invention, compared to the wild-type phosphatase ST6P, the phosphatase mutant preferably includes the aforementioned S111C and L116I mutations, specifically comprising the amino acid fragment shown in SEQ ID NO:8. In this case, the phosphatase mutant exhibits excellent catalytic activity and specificity for glucose-6-phosphate.

[0049] In this invention, compared to the wild-type phosphatase ST6P, the phosphatase mutant preferably includes the aforementioned S111C and N188A mutations, specifically comprising the amino acid fragment shown in SEQ ID NO:9. In this case, the phosphatase mutant exhibits excellent catalytic activity and specificity for glucose-6-phosphate.

[0050] In this invention, compared to the wild-type phosphatase ST6P, the phosphatase mutant preferably includes the aforementioned V46T and L116R mutations, specifically comprising the amino acid fragment shown in SEQ ID NO:10. In this case, the phosphatase mutant exhibits excellent catalytic activity and specificity for allulose-6-phosphate.

[0051] In this invention, compared to the wild-type phosphatase ST6P, the phosphatase mutant preferably includes the aforementioned L116R and T170H mutations, specifically comprising the amino acid fragment shown in SEQ ID NO:11. In this case, the phosphatase mutant exhibits excellent catalytic activity and specificity for allulose-6-phosphate.

[0052] In this invention, compared to the wild-type phosphatase ST6P, the phosphatase mutant preferably includes the aforementioned V46G and S111A mutations, specifically comprising the amino acid fragment shown in SEQ ID NO:12. In this case, the phosphatase mutant exhibits excellent catalytic activity and specificity for mannose-6-phosphate.

[0053] In this invention, compared to the wild-type phosphatase ST6P, the phosphatase mutant preferably includes the aforementioned V46G and T170H mutations, specifically comprising the amino acid fragment shown in SEQ ID NO:13. In this case, the phosphatase mutant exhibits excellent catalytic activity and specificity for mannose-6-phosphate.

[0054] To obtain the aforementioned phosphatase mutant, this invention also provides a method for constructing the phosphatase mutant. The method specifically includes: performing site-directed PCR mutagenesis on the coding gene of the wild-type phosphatase ST6P to obtain the coding gene of the phosphatase mutant; transforming the coding gene of the phosphatase mutant into host cells to obtain an engineered strain; and inducing expression of the engineered strain to obtain the phosphatase mutant.

[0055] In this invention, based on the amino acid sequence of the wild-type phosphatase ST6P or the phosphatase mutant, those skilled in the art can know the specific nucleotide sequence information of the coding gene of the wild-type phosphatase ST6P or the phosphatase mutant, and therefore this invention does not impose any particular limitation on it.

[0056] In this invention, the PCR site-directed mutagenesis is a commonly used technique in the field of genetic engineering. Those skilled in the art can design the mutation primers used for PCR site-directed mutagenesis based on the above-mentioned mutations, and adjust the conditions used for PCR site-directed mutagenesis. Therefore, this invention does not impose any particular limitations on it.

[0057] In this invention, the host cell is a type of cell commonly used in the field of genetic engineering. Specific examples include, but are not limited to, Escherichia coli and / or yeast. Those skilled in the art can make adaptive selections of the host cell according to actual needs, and therefore this invention does not impose any particular limitations on it.

[0058] In this invention, the method used to "transform host cells by taking the coding gene of the phosphatase mutant" is a conventional technique used in the field of genetic engineering. Those skilled in the art can make adaptive choices according to actual needs, and therefore this invention does not impose any particular limitations on it.

[0059] In this invention, the induced expression is a commonly used technique in the field of genetic engineering. Those skilled in the art can make adaptive choices according to actual needs, and therefore this invention does not impose any particular limitations on it.

[0060] With the aim of obtaining the above-mentioned phosphatase mutant, the present invention also provides an engineered strain. This engineered strain is capable of expressing the above-mentioned phosphatase mutant.

[0061] Based on the excellent catalytic activity and specificity of the aforementioned phosphatase mutants for specific monosaccharide-6-phosphate, this invention also provides the application of these phosphatase mutants in monosaccharide production. In this application, based on the excellent catalytic activity and specificity of the different phosphatase mutants for monosaccharide-6-phosphate, examples of the monosaccharide specifically include one or more of tagatose, fructose, glucose, allulose, and mannose.

[0062] In this invention, when the monosaccharide to be produced is tagatose, the phosphatase mutant preferably includes an amino acid fragment with a sequence as shown in SEQ ID NO:4 or 5.

[0063] In this invention, when the monosaccharide to be produced is fructose, the phosphatase mutant preferably includes an amino acid fragment with a sequence as shown in SEQ ID NO:6 or 7.

[0064] In this invention, when the monosaccharide to be produced is glucose, the phosphatase mutant preferably includes an amino acid fragment with a sequence as shown in SEQ ID NO:8 or 9.

[0065] In this invention, when the monosaccharide to be produced is allulose, the phosphatase mutant preferably includes an amino acid fragment with a sequence as shown in SEQ ID NO:10 or 11.

[0066] In this invention, when the monosaccharide to be produced is mannose, the phosphatase mutant preferably includes an amino acid fragment with a sequence as shown in SEQ ID NO:12 or 13.

[0067] Based on the excellent application potential of the above-mentioned phosphatase mutant in monosaccharide production, this invention also provides a method for preparing a monosaccharide. The method specifically includes: subjecting a reaction solution containing a carbohydrate substrate to an enzymatic reaction using the above-mentioned phosphatase mutant to obtain the monosaccharide. Specific examples of the carbohydrate substrate include, but are not limited to, one or more of the following: monosaccharide-6-phosphate, starch, maltodextrin, sucrose, and fructose.

[0068] In this invention, the preferred carbohydrate substrate is monosaccharide-6-phosphate, specifically including one or more of tagatose-6-phosphate, fructose-6-phosphate, glucose-6-phosphate, allulose-6-phosphate, and mannose-6-phosphate. In this case, the desired monosaccharide can be prepared simply by using a corresponding phosphatase mutant to catalyze the monosaccharide-6-phosphate reaction.

[0069] In some specific embodiments, when the sugar substrate is preferably a monosaccharide-6-phosphate, the concentration of the added phosphatase mutant is preferably 1 to 25 g / L, such as 1 g / L, 3 g / L, 5 g / L, 7 g / L, 9 g / L, 10 g / L, 15 g / L, 20 g / L, 25 g / L or any value between them.

[0070] In some specific embodiments, when the carbohydrate substrate is preferably a monosaccharide-6-phosphate, the reaction solution preferably includes 100-200 mM of the carbohydrate substrate, such as 100 mM, 150 mM, 180 mM, 200 mM, or any value between them; and 1-20 mM of Mg. 2+ Such as 1mM, 3mM, 5mM, 7mM, 9mM, 10mM, 15mM, 20mM or any value between them; 50-200mM HEPES buffer solution with pH 6.5-7.5, such as 50mM, 70mM, 100mM, 150mM, 200mM or any value between them.

[0071] In some specific embodiments, when the carbohydrate substrate is preferably a monosaccharide-6-phosphate, the conditions for the enzyme-catalyzed reaction include a temperature preferably of 50–70°C, such as 50°C, 53°C, 58°C, 60°C, 65°C, 69°C, 70°C or any value between them; and a time preferably of 1–48 h, such as 1 h, 6 h, 12 h, 24 h, 36 h, 48 h or any value between them.

[0072] In this invention, the carbohydrate substrate is preferably one or more of starch, maltodextrin, and sucrose. In this case, the phosphatase mutant needs to be used in conjunction with enzymes related to sugar metabolism pathways, such as dextran phosphorylase, glucose phosphate mutase, glucose phosphate isomerase, sucrase phosphorylase, isoamylase, and dextran transferase, to convert the carbohydrate substrate into monosaccharides using the Izumoring biosynthesis method.

[0073] In some specific embodiments, when the carbohydrate substrate is preferably one or more of starch, maltodextrin, and sucrose, the reagents and conditions used in the enzyme-catalyzed reaction are conventional techniques used in the Izumoring biosynthesis method, which can be obtained by those skilled in the art from relevant patent or non-patent literature on the Izumoring biosynthesis method. Therefore, this invention does not impose any particular limitations on them. In some more preferred embodiments, those skilled in the art can refer to the literature "Whole-cell biosynthesis of D-tagatose from maltodextrin by engineered Escherichia coli with multi-enzyme co-expression system" to design the reagents and conditions used in the enzyme-catalyzed reaction to achieve good monosaccharide preparation results.

[0074] The embodiments of the present invention are described in detail below. These embodiments are intended to explain the present invention and should not be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0075] The reagents used and their acquisition methods in the following examples specifically include:

[0076] TB fermentation medium (Sigma-Aldrich, catalog number T5574);

[0077] LB medium (Sigma-Aldrich, catalog number 71753-M);

[0078] Glucose-6-phosphate (Sigma-Aldrich, catalog number G7879);

[0079] Fructose-6-phosphate (Sigma-Aldrich, catalog number F1502);

[0080] Tagase-6-phosphate: obtained by separating D-tagase (Sigma-Aldrich, catalog number T2751) via a tagase kinase-catalyzed reaction (KEGG reaction formula: R02927);

[0081] Mannose-6-phosphate: obtained by isolating mannose via a mannokinase-catalyzed reaction (KEGG reaction formula R01326);

[0082] Allulose-6-phosphate: obtained by separating fructose-6-phosphate from allulose-6-phosphate via an allulose-6-phosphate-3-isomerase catalytic reaction (KEGG reaction formula R09031).

[0083] Example 1

[0084] This embodiment illustrates the preparation of a wild-type phosphatase ST6P, specifically including:

[0085] (1) Based on the amino acid sequence of ST6P phosphatase derived from *Thermococcus thermophilus* (as shown in SEQ ID NO:1), codon optimization and synthesis were performed on *Escherichia coli* to obtain the coding gene for ST6P phosphatase (as shown in SEQ ID NO:2). This coding gene for ST6P phosphatase was then loaded onto the pET28a vector, resulting in the following... Figure 1 The recombinant plasmid pET28a-ST6P was shown, and the recombinant plasmid pET28a-ST6P was transformed into the preserved strain Escherichia coli DH5a.

[0086] (2) The recombinant plasmid pET28a-ST6P was transformed into Escherichia coli BL21(DE3) competent cells (purchased from TransGen Biotech) to obtain wild-type phosphatase ST6P expressing strain;

[0087] (3) The wild-type ST6P phosphatase expression strain was inoculated into 50 mL of LB medium containing 50 mg / L kanamycin and cultured overnight at 37 °C and 200 rpm with shaking to obtain seed culture; the seed culture was then inoculated into 200 mL of TB fermentation medium containing 50 mg / L kanamycin at an inoculation rate of 2% and cultured at 37 °C and 200 rpm until OD600 reached. 600=0.8, add IPTG to a final concentration of 0.2 mM, induce at 37℃ for 48 h, and then collect the bacterial cells by centrifugation; suspend the bacterial cells in PBS buffer (10 mM, pH=7.2), and disrupt the bacterial cells by sonication. Collect the supernatant by centrifugation to obtain the wild-type crude enzyme solution. Take the wild-type crude enzyme solution for nickel column affinity chromatography (purchased from Shanghai Sangon Biotech), dialysis to remove salt and ultrafiltration concentration to obtain the wild-type phosphatase ST6P pure enzyme solution.

[0088] Example 2

[0089] This embodiment uses the wild-type phosphatase ST6P provided in Example 1 as a basis to construct a wild-type phosphatase ST6P mutant library, specifically including:

[0090] 1. Acquisition of mutation sites: Bioinformatics analysis was performed on the amino acid sequence and structure of the wild-type phosphatase mutant to identify key amino acid residues in the catalytic activity center region of the enzyme. The amino acid residues near the substrate channel were also analyzed, and eight sites that may affect catalytic activity and substrate-specific binding ability were finally screened out: glutamic acid at position 18, valine at position 46, serine at position 111, leucine at position 116, threonine at position 134, phenylalanine at position 166, threonine at position 170, and asparagine at position 188. The amino acid sequences of each mutant in the final constructed phosphatase ST6P mutant library are shown in SEQ ID NO:3.

[0091] 2. Obtaining positive clone transformants: Saturation mutations were performed at positions 18, 46, 111, 116, 134, 166, 170, and 188, respectively, to design mutation primers for the specified mutation sites. Using the recombinant plasmid pET28a-ST6P provided in Example 1 as a template, the recombinant plasmid pET28a-ST6P was amplified by full plasmid PCR in the presence of high-fidelity Taq enzyme using each mutation primer to obtain linearized plasmids with the specified mutation sites. The linearized plasmids were recovered by electrophoresis and ligated using a seamless cloning kit to obtain recombinant plasmids with the specified mutation sites. The recombinant plasmids with the specified mutation sites were transformed into Escherichia coli BL21(DE3) competent cells, plated on LB plates containing 50 mg / L kanamycin, and cultured overnight at 37°C to obtain positive clone transformants.

[0092] 3. Obtaining the crude enzyme solution of the mutant: Three positive clone transformants were randomly selected from each LB plate for sequencing. After the sequencing confirmed the presence of the corresponding mutation, activity testing was performed. The positive clone transformants were inoculated into 96-well plates containing TB fermentation medium at a 2% inoculum and cultured at 37℃ and 200 rpm until OD. 600When the concentration of the enzyme was 0.8, IPTG was added to a final concentration of 0.2 mM. After induction at 37°C for 24 h, the bacterial cells were collected by centrifugation. The bacterial cells were suspended in PBS buffer (10 mM, pH = 7.2), and the bacterial cells were disrupted by freeze-thaw cycles. The supernatant was collected by centrifugation to obtain the crude enzyme solution of the mutant.

[0093] 4. Screening of mutants: The OD values ​​of each crude enzyme solution were adjusted using PBS buffer. 280 =10, and the adjusted crude enzyme solution was added to the monosaccharide-6-phosphate mixed reaction solution at an addition rate of 10% (v / v). After reacting at 50℃ for 2 h, the yield and content of each component in the product were determined by high performance liquid chromatography (HPLC). The total catalytic activity of the wild-type crude enzyme solution (i.e., the sum of all monosaccharides produced by catalysis) was recorded as 100%. The catalytic specificity of the enzyme was characterized by the proportion of different monosaccharides produced in the total product. Mutants with improved catalytic activity or significantly changed specificity were screened, as shown in Table 2.

[0094] Table 2.

[0095]

[0096] The monosaccharide 6-phosphate mixture reaction solution includes 10 mM glucose-6-phosphate (G6P), 10 mM fructose-6-phosphate (F6P), 10 mM allulose-6-phosphate (A6P), 10 mM tagatose-6-phosphate (T6P), 10 mM mannose-6-phosphate (M6P), and 5 mM Mn 2+ And 50mM HEPES buffer.

[0097] The HPLC analysis conditions included: an Agilent 1260 high-performance liquid chromatograph with a RID detector, a Hi-Plex Ca column, a column temperature of 85℃, pure water as the mobile phase, a flow rate of 1 mL / min, and an injection volume of 10 μL.

[0098] Example 3

[0099] This embodiment uses the mutants with enhanced activity or significantly altered specificity obtained in Example 2 as a basis, and performs combined mutations to obtain mutants with both enhanced catalytic activity and specificity, specifically including:

[0100] 1. Obtaining crude enzyme solution of mutants: Combine the mutation sites of the mutants shown in Table 2 and prepare the crude enzyme solution corresponding to each mutant according to the method provided in Example 2.

[0101] 2. Obtaining pure enzyme solutions of mutants: The crude enzyme solution was subjected to nickel column affinity chromatography, dialysis for desalting, and ultrafiltration concentration to obtain pure enzyme solutions corresponding to each mutant.

[0102] 3. Screening of mutants: The pure enzyme solutions corresponding to each mutant were tested according to the method provided in Example 2. The total catalytic activity of the wild-type crude enzyme solution (i.e., the total amount of all monosaccharides produced by catalysis) was recorded as 100%. The proportion of different monosaccharides produced in the total amount of all products was used to characterize the catalytic specificity of the enzyme. Mutants with improved catalytic activity and specificity were screened out, as shown in Table 3.

[0103] Table 3.

[0104]

[0105]

[0106] As shown in Table 3, phosphatase mutant 6 (with both E18D and N188H mutations, as shown in SEQ ID NO:4) and phosphatase mutant 12 (with both E18K and N188H mutations, as shown in SEQ ID NO:5) exhibit excellent catalytic activity and specificity for tagatose-6-phosphate.

[0107] Phosphatase mutant 25 (with both S111I and F166I mutations, as shown in SEQ ID NO:6) and phosphatase mutant 28 (with both L116I and N188A mutations, as shown in SEQ ID NO:7) exhibit excellent catalytic activity and specificity for fructose-6-phosphate.

[0108] Phosphatase mutant 30 (with both S111C and L116I mutations, as shown in SEQ ID NO:8) and phosphatase mutant 32 (with both S111C and N188A mutations, as shown in SEQ ID NO:9) exhibit excellent catalytic activity and specificity for glucose-6-phosphate.

[0109] Phosphatase mutant 34 (with both V46T and L116R mutations, as shown in SEQ ID NO:10) and phosphatase mutant 36 (with both L116R and T170H mutations, as shown in SEQ ID NO:11) exhibit excellent catalytic activity and specificity for allulose-6-phosphate.

[0110] Phosphatase mutant 37 (with both V46G and S111A mutations, as shown in SEQ ID NO:12) and phosphatase mutant 40 (with both V46G and T170H mutations, as shown in SEQ ID NO:13) exhibit excellent catalytic activity and specificity for mannose-6-phosphate.

[0111] Example 4

[0112] This embodiment illustrates the ability of phosphatase mutants 6 and phosphatase mutant 12 provided in Example 3 to synthesize tagatose using tagatose-6-phosphate as a substrate. Specifically, the pure enzyme solutions of phosphatase mutant 6 and phosphatase mutant 12 were added to the tagatose-6-phosphate reaction solution at a final concentration of 10 g / L. After reacting at 60°C for 12 h, 5 mM H2SO4 was added to terminate the reaction. The tagatose content in the product was determined using the method provided in Example 2, and the molar conversion rate of tagatose was calculated. The results are shown in Table 4.

[0113] The tagatose-6-phosphate reaction solution includes 100 mM tagatose-6-phosphate, 10 mM MgSO4, and 100 mM HEPES buffer (pH = 7.0).

[0114] Table 4.

[0115] phosphatase Tag sugar molar conversion rate (%) Wild-type phosphatase ST6P 56.3 Phosphatase mutant 6 93.1 Phosphatase mutant 12 88.5

[0116] As shown in Table 4, phosphatase mutants 6 and 12 exhibit higher catalytic efficiency in the conversion of tagatose-6-phosphate to tagatose compared to wild-type phosphatase ST6P.

[0117] Example 5

[0118] This embodiment illustrates the ability of phosphatase mutants 25 and 28 provided in Example 3 to synthesize fructose using fructose-6-phosphate as a substrate. Specifically, the pure enzyme solutions of phosphatase mutants 25 and 28 were added to the fructose-6-phosphate reaction solution at a final concentration of 10 g / L. After reacting at 60°C for 12 h, 5 mM H2SO4 was added to terminate the reaction. The fructose content in the product was determined using the method provided in Example 2, and the fructose molar conversion rate was calculated. The results are shown in Table 5.

[0119] The fructose-6-phosphate reaction solution includes 100 mM fructose-6-phosphate, 10 mM MgSO4, and 100 mM HEPES buffer (pH = 7.0).

[0120] Table 5.

[0121] phosphatase Fructose molar conversion rate (%) Wild-type phosphatase ST6P 25.2 Phosphatase mutant 25 67.2 Phosphatase mutant 28 66.3

[0122] As shown in Table 5, phosphatase mutants 25 and 28 exhibit higher catalytic efficiency in the conversion of fructose-6-phosphate to fructose compared to wild-type phosphatase ST6P.

[0123] Example 6

[0124] This embodiment illustrates the ability of phosphatase mutants 30 and 32 provided in Example 3 to synthesize glucose using glucose-6-phosphate as a substrate. Specifically, it includes adding pure enzyme solutions of phosphatase mutants 30 and 32 to the glucose-6-phosphate reaction solution at a final concentration of 10 g / L, reacting at 60°C for 12 h, and then terminating the reaction with 5 mM H2SO4. The glucose content in the product is determined using the method provided in Example 2, and the glucose molar conversion rate is calculated. The results are shown in Table 6.

[0125] The glucose-6-phosphate reaction solution includes 100 mM glucose-6-phosphate, 10 mM MgSO4, and 100 mM HEPES buffer (pH = 7.0).

[0126] Table 6.

[0127] phosphatase Glucose molar conversion rate (%) Wild-type phosphatase ST6P 17.6 Phosphatase mutant 30 63.2 Phosphatase mutant 32 55.7

[0128] As shown in Table 6, compared with wild-type phosphatase ST6P, phosphatase mutants 30 and 32 exhibit higher catalytic efficiency in the conversion of glucose-6-phosphate to glucose.

[0129] Example 7

[0130] This embodiment illustrates the ability of phosphatase mutants 34 and 36 provided in Example 3 to synthesize allulose using allulose-6-phosphate as a substrate. Specifically, the pure enzyme solutions of phosphatase mutants 34 and 36 were added to the allulose-6-phosphate reaction solution at a final concentration of 10 g / L. After reacting at 60°C for 12 h, 5 mM H2SO4 was added to terminate the reaction. The allulose content in the product was determined using the method provided in Example 2, and the allulose molar conversion rate was calculated. The results are shown in Table 7.

[0131] The allulose-6-phosphate reaction solution includes 100 mM allulose-6-phosphate, 10 mM MgSO4, and 100 mM HEPES buffer (pH = 7.0).

[0132] Table 7.

[0133] phosphatase Allulose molar conversion rate (%) Wild-type phosphatase ST6P 32.9 Phosphatase mutant 34 61.1 Phosphatase mutant 36 70.3

[0134] As shown in Table 7, phosphatase mutants 34 and 36 exhibit higher catalytic efficiency in the conversion of allulose-6-phosphate to allulose compared to wild-type phosphatase ST6P.

[0135] Example 8

[0136] This embodiment illustrates the ability of phosphatase mutants 37 and 40 provided in Example 3 to synthesize mannose using mannose-6-phosphate as a substrate. Specifically, the pure enzyme solutions of phosphatase mutants 37 and 40 were added to the mannose-6-phosphate reaction solution at a final concentration of 10 g / L. After reacting at 60°C for 12 h, 5 mM H2SO4 was added to terminate the reaction. The mannose content in the product was determined using the method provided in Example 2, and the mannose molar conversion rate was calculated. The results are shown in Table 8.

[0137] The mannose-6-phosphate reaction solution includes 100 mM mannose-6-phosphate, 10 mM MgSO4, and 100 mM HEPES buffer (pH = 7.0).

[0138] Table 8.

[0139] phosphatase Mannose molar conversion rate (%) Wild-type phosphatase ST6P 33.8 Phosphatase mutant 37 62.3 Phosphatase mutant 40 51.1

[0140] As shown in Table 8, phosphatase mutants 37 and 40 exhibit higher catalytic efficiency in the conversion of mannose-6-phosphate to mannose compared to wild-type phosphatase ST6P.

[0141] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention without departing from the principles and spirit of the present invention.

Claims

1. A phosphatase mutant, characterized in that, The phosphatase mutant was obtained by mutating the wild-type phosphatase ST6P with the amino acid sequence shown in SEQ ID NO:

1. The mutation is N188H mutation, E18D+N188H mutation, E18K+N188H mutation, F166V+N188H mutation, T170H+N188H mutation, or T170W+N188H mutation.

2. The phosphatase mutant according to claim 1, characterized in that, The amino acid sequence of the phosphatase mutant is shown in SEQ ID NO:4 or 5.

3. The method for constructing the phosphatase mutant according to claim 1 or 2, characterized in that, The construction method includes: performing site-directed PCR mutagenesis on the coding gene of wild-type phosphatase ST6P to obtain the coding gene of the phosphatase mutant; transforming the coding gene of the phosphatase mutant into host cells to obtain an engineered strain; and inducing expression of the engineered strain to obtain the phosphatase mutant.

4. An engineered bacterial strain, characterized in that, The engineered strain expresses the phosphatase mutant as described in claim 1 or 2.

5. The application of the phosphatase mutant according to claim 1 or 2 in tagatose production.

6. The application of the phosphatase mutant according to claim 5 in tagatose production, characterized in that, The amino acid sequence of the phosphatase mutant is shown in SEQ ID NO:4 or 5.

7. A method for preparing tagatose, characterized in that, The preparation method includes: taking the phosphatase mutant of claim 1 or 2 and subjecting it to an enzyme-catalyzed reaction with a reaction solution containing tagatose-6-phosphate to obtain the tagatose.

8. The method for preparing tagatose according to claim 7, characterized in that, The concentration of the added phosphatase mutant is 1~25 g / L.

9. The method for preparing tagatose according to claim 7, characterized in that, The reaction solution includes 100-200 mM tagatose-6-phosphate and 1-20 mM Mg. 2+ And 50-200 mM HEPES buffer at pH 6.5-7.

5.

10. The method for preparing tagatose according to claim 7, characterized in that, The enzyme-catalyzed reaction is carried out at a temperature of 50-70°C for a time of 1-48 hours.

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