Preparation method of phosphatase mutant and monosaccharide

Abstract

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

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a preparation method of a phosphatase mutant and a monosaccharide. Background Art

[0002] Monosaccharides refer to carbohydrates that cannot be broken down into smaller sugar molecules. They are the basic units of oligosaccharides and polysaccharides, and are also important substances for energy metabolism in organisms. According to the scarcity of monosaccharides, they can be divided into common monosaccharides and rare monosaccharides; common monosaccharides include fructose, glucose, mannose and other sugars that exist in large quantities in nature and are easily utilized; rare monosaccharides include tagatose, allulose and sorbitol, which are rare in nature. Rare monosaccharides have become the main force of the new generation of sweeteners with their low calories, biological value and high sweetness. Among them, allulose has excellent performance in reducing blood sugar response, inhibiting liver lipase activity, and scavenging free radicals, making it not only a sweetener, but also has application prospects in the field of biomedicine. Under the condition of having the same sweetness as sucrose, tagatose has only half the calories of sucrose, which makes it advantageous 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] At present, the methods for biosynthesis of monosaccharides include one-step enzyme catalysis and multi-step enzyme catalysis. In the one-step enzyme catalysis method, a specific isomerase is used to catalyze the isomerization of the substrate to obtain the corresponding target monosaccharide, which has the advantages of simple reaction process and easy control, but there are problems such as expensive substrates and high production costs, which limit its application in large-scale industrial production. The multi-step enzyme catalysis method uses cheap polysaccharides such as starch as raw materials, and uses enzyme-catalyzed hydrolysis, isomerization, phosphorylation and dephosphorylation processes to obtain the corresponding target monosaccharide. Among them, the core of the multi-step enzyme catalysis method is 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 and low-cost bioproduction of monosaccharides. Summary of the invention

[0005] The inventors of the present invention have creatively found that the amino acid residues at one or more positions of the 18th, 46th, 111th, 116th, 134th, 166th, 170th and 188th positions of the wild-type phosphatase ST6P are subjected to site-directed mutagenesis based on the key amino acid residues of the catalytic active center region and the substrate channel of the amino acid sequence as shown in SEQ ID NO: 1 and their influence on the structure and catalytic activity, thereby improving the specific binding ability and catalytic activity of the specific monosaccharide-6-phosphate, thereby obtaining a phosphatase with high catalytic activity and specificity, and finally realizing the large-scale and low-cost preparation of monosaccharides of specific structures. Based on this, the technical scheme of the present invention is obtained.

[0006] In a first aspect, the present invention provides a phosphatase mutant, the phosphatase mutant comprising an amino acid fragment as shown in SEQ ID NO:3; wherein, the amino acid residue positions are encoded with reference to the amino acid fragment as shown in SEQ ID NO:3, 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; the 188th amino acid residue of the phosphatase mutant is asparagine, aspartic acid, serine, alanine or histidine.

[0007] Furthermore, the phosphatase mutant includes one or more segments of the amino acid fragments shown in SEQ ID NOs: 4-13.

[0008] In a second aspect, the present invention provides a method for constructing the above-mentioned phosphatase mutant, which comprises: performing PCR site-directed mutagenesis based 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 a host cell to obtain an engineered strain; and inducing expression of the engineered strain to obtain the phosphatase mutant.

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

[0010] In a third aspect, the present invention provides an engineered strain, wherein the engineered strain expresses the above-mentioned phosphatase mutant.

[0011] In a fourth aspect, the present invention provides the use of the above-mentioned phosphatase mutant in the production of monosaccharides.

[0012] Furthermore, when the monosaccharide is tagatose, the phosphatase mutant comprises an amino acid fragment having 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 having 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 having a sequence as shown in SEQ ID NO: 8 or 9.

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

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

[0017] In a fifth aspect, the present invention provides a method for preparing a monosaccharide, the method comprising: using the above-mentioned phosphatase mutant to perform an enzyme-catalyzed reaction on a reaction solution including a sugar substrate to obtain the monosaccharide.

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

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

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

[0021] Furthermore, the temperature of the enzyme catalytic reaction is 50-70°C and the time is 1-48h.

[0022] Beneficial effects:

[0023] The present invention provides a phosphatase mutant including an amino acid fragment with a sequence as shown in SEQ ID NO: 3, and through specific selection of amino acid residues obtained by mutation of one or more positions among positions 18, 46, 111, 116, 134, 166, 170 and 188, the phosphatase mutant is improved in catalytic activity and endowed with excellent specific binding ability for specific monosaccharide-6-phosphate, so as to obtain a phosphatase with both high catalytic activity and specificity, which is of great significance for realizing large-scale and low-cost biological preparation of monosaccharides with specific structures.

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

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

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

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

[0028] In some specific embodiments, when the phosphatase mutant preferably includes an amino acid fragment as shown in SEQ ID NO: 12 or 13, the phosphatase mutant has excellent catalytic activity and specificity for mannose-6-phosphate, can better catalyze the reaction process of converting mannose-6-phosphate into mannose, and shows great application prospects in the field of mannose production. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a schematic diagram of the structure of the recombinant plasmid pET28a-ST6P provided in Example 1 of the present invention. DETAILED DESCRIPTION

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

[0031] Table 1.

[0032]

[0033]

[0034]

[0035] The phosphatase mutant provided by the present invention is based on the wild-type phosphatase ST6P including the amino acid fragment of the sequence as shown in SEQ ID NO: 1, and a specific amino acid substitution mutation is performed on one or more positions of the 18th, 46th, 111th, 116th, 134th, 166th, 170th and 188th positions in the amino acid fragment of SEQ ID NO: 1. That is, the phosphatase mutant specifically includes the amino acid fragment of the sequence as shown in SEQ ID NO: 3, and the similarity between the amino acid fragment of the sequence as shown in SEQ ID NO: 3 and the amino acid fragment of the sequence as shown in SEQ ID NO: 1 is not 100%.

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

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

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

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

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

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

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

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

[0044] In the present invention, compared with the wild-type phosphatase ST6P, the phosphatase mutant preferably includes the above-mentioned E18D and N188H mutations, which specifically include an amino acid fragment with a sequence as shown in SEQ ID NO: 4. In this case, the phosphatase mutant has excellent catalytic activity and specificity for tagatose-6-phosphate.

[0045] In the present invention, compared with the wild-type phosphatase ST6P, the phosphatase mutant preferably includes the above-mentioned E18K and N188H mutations, which specifically include an amino acid fragment with a sequence as shown in SEQ ID NO: 5. In this case, the phosphatase mutant has excellent catalytic activity and specificity for tagatose-6-phosphate.

[0046] In the present invention, compared with the wild-type phosphatase ST6P, the phosphatase mutant preferably includes the above-mentioned S111I and F166I mutations, which specifically include an amino acid fragment with a sequence as shown in SEQ ID NO: 6. In this case, the phosphatase mutant has excellent catalytic activity and specificity for fructose-6-phosphate.

[0047] In the present invention, compared with the wild-type phosphatase ST6P, the phosphatase mutant preferably includes the above-mentioned L116I and N188A mutations, which specifically include an amino acid fragment with a sequence as shown in SEQ ID NO: 7. In this case, the phosphatase mutant has excellent catalytic activity and specificity for fructose-6-phosphate.

[0048] In the present invention, compared with the wild-type phosphatase ST6P, the phosphatase mutant preferably includes the above-mentioned S111C and L116I mutations, which specifically include an amino acid fragment with a sequence as shown in SEQ ID NO: 8. In this case, the phosphatase mutant has excellent catalytic activity and specificity for glucose-6-phosphate.

[0049] In the present invention, compared with the wild-type phosphatase ST6P, the phosphatase mutant preferably includes the above-mentioned S111C and N188A mutations, which specifically include an amino acid fragment whose sequence is shown in SEQ ID NO: 9. In this case, the phosphatase mutant has excellent catalytic activity and specificity for glucose-6-phosphate.

[0050] In the present invention, compared with the wild-type phosphatase ST6P, the phosphatase mutant preferably includes the above-mentioned V46T and L116R mutations, which specifically include an amino acid fragment with a sequence as shown in SEQ ID NO: 10. In this case, the phosphatase mutant has excellent catalytic activity and specificity for psicose-6-phosphate.

[0051] In the present invention, compared with the wild-type phosphatase ST6P, the phosphatase mutant preferably includes the above-mentioned L116R and T170H mutations, which specifically include an amino acid fragment with a sequence as shown in SEQ ID NO: 11. In this case, the phosphatase mutant has excellent catalytic activity and specificity for psicose-6-phosphate.

[0052] In the present invention, compared with the wild-type phosphatase ST6P, the phosphatase mutant preferably includes the above-mentioned V46G and S111A mutations, which specifically include an amino acid fragment with a sequence as shown in SEQ ID NO: 12. In this case, the phosphatase mutant has excellent catalytic activity and specificity for mannose-6-phosphate.

[0053] In the present invention, compared with the wild-type phosphatase ST6P, the phosphatase mutant preferably includes the above-mentioned V46G and T170H mutations, which specifically include an amino acid fragment with a sequence as shown in SEQ ID NO: 13. In this case, the phosphatase mutant has excellent catalytic activity and specificity for mannose-6-phosphate.

[0054] Based on the purpose of obtaining the above-mentioned phosphatase mutant, the present invention also provides a method for constructing the above-mentioned phosphatase mutant. The construction method specifically comprises: performing PCR site-directed mutagenesis based on the coding gene of the wild-type phosphatase ST6P to obtain the coding gene of the phosphatase mutant; taking the coding gene of the phosphatase mutant to transform the host cell to obtain an engineered strain; taking the engineered strain to induce expression to obtain the phosphatase mutant.

[0055] In the present 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 encoding gene of the wild-type phosphatase ST6P or the phosphatase mutant, and thus the present invention does not impose any particular limitation thereto.

[0056] In the present invention, the PCR site-directed mutagenesis is a technical means commonly used in the field of genetic engineering. Those skilled in the art can design mutation primers used for PCR site-directed mutagenesis according to the above mutations, and adjust the conditions used for PCR site-directed mutagenesis. Therefore, the present invention does not impose any particular limitation on it.

[0057] In the present invention, the host cell is a type of cell conventionally used in the field of genetic engineering, and specific examples thereof include but are not limited to Escherichia coli and / or yeast. Those skilled in the art can make adaptive selections of host cells according to actual needs, and thus the present invention does not impose any particular limitation thereto.

[0058] In the present invention, the method of "transforming the host cell with the coding gene of the phosphatase mutant" is a technical means conventionally used in the field of genetic engineering. Those skilled in the art can make adaptive choices according to actual needs, and therefore the present invention does not impose any particular limitation on it.

[0059] In the present invention, the induced expression is a technical means commonly used in the field of genetic engineering, and those skilled in the art can make adaptive choices according to actual needs, so the present invention does not impose any particular limitation on it.

[0060] Based on the purpose of obtaining the above phosphatase mutant, the present invention also provides an engineered strain that can express the above phosphatase mutant.

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

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

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

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

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

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

[0067] Based on the excellent application potential of the above phosphatase mutant in monosaccharide production, the present invention also provides a method for preparing monosaccharide. The preparation method specifically comprises: taking the above phosphatase mutant to perform an enzyme catalytic reaction on a reaction solution including a sugar substrate to obtain the monosaccharide. Specific examples of the sugar substrate include but are not limited to one or more of monosaccharide-6-phosphate, starch, maltodextrin, sucrose and fructose.

[0068] In the present invention, the sugar substrate is preferably monosaccharide-6-phosphate, and examples of the monosaccharide-6-phosphate include one or more of tagatose-6-phosphate, fructose-6-phosphate, glucose-6-phosphate, psicose-6-phosphate and mannose-6-phosphate. At this time, the desired monosaccharide can be prepared by only using the corresponding phosphatase mutant to perform an enzyme-catalyzed reaction on the monosaccharide-6-phosphate.

[0069] In some specific embodiments, when the carbohydrate substrate is preferably monosaccharide-6-phosphate, the added concentration of the 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 therebetween.

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

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

[0072] In the present invention, when the sugar substrate is preferably one or more of starch, maltodextrin and sucrose, the phosphatase mutant needs to be used in combination with enzyme molecules related to the sugar metabolic pathway such as glucan phosphorylase, glucose phosphomutase, glucose phosphoisomerase, sucrose phosphorylase, isoamylase and glucanotransferase to convert the sugar 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 the technical means conventionally used in the Izumoring biosynthesis method, and those skilled in the art can obtain them from the relevant patent literature or non-patent literature of the Izumoring biosynthesis method, so the present invention does not impose any special limitation on them. In some more preferred embodiments, those skilled in the art can design the reagents and conditions used in the enzyme-catalyzed reaction with reference to the document Whole-cell biosynthesis of D-tagatose from maltodextrin by engineered Escherichia coli with multi-enzyme co-expression system to achieve a good monosaccharide preparation effect.

[0074] Embodiments of the present invention are described in detail below, and the examples of the embodiments are intended to be used to explain the present invention, but should not be construed as limiting the present invention. In the embodiments, if specific techniques or conditions are not specified, the techniques or conditions described in the literature in this area or the product specifications are used. If the manufacturer is not specified for reagents or instruments used, they are all conventional products that can be obtained commercially.

[0075] The reagents used in the following examples and the methods for obtaining them 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] Tagatose-6-phosphate: isolated from D-tagatose (Sigma-Aldrich, catalog number T2751) by tagatose kinase catalysis (KEGG reaction formula: R02927);

[0081] Mannose-6-phosphate: separated from mannose by mannokinase catalysis (KEGG reaction formula R01326);

[0082] Allulose-6-phosphate: It is separated from fructose-6-phosphate by the catalytic reaction of psicose-6-phosphate-3-isomerase (KEGG reaction formula R09031).

[0083] Example 1

[0084] This example is used to illustrate the preparation of a wild-type phosphatase ST6P, which specifically includes:

[0085] (1) According to the amino acid sequence of the phosphatase ST6P from Thermococcus thermophilus (as shown in SEQ ID NO: 1), the Escherichia coli codon was optimized and synthesized to obtain the coding gene of the phosphatase ST6P (as shown in SEQ ID NO: 2), and the coding gene of the phosphatase ST6P was loaded on the pET28a vector to obtain the following: Figure 1 The recombinant plasmid pET28a-ST6P shown was transformed into the deposited strain Escherichia coli DH5a.

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

[0087] (3) The wild-type phosphatase ST6P expression strain was inoculated into 50 mL of LB medium containing 50 mg / L kanamycin, and cultured at 37°C and 200 rpm overnight to obtain a seed solution; the seed solution was inoculated into 200 mL of TB fermentation medium containing 50 mg / L kanamycin at an inoculum size of 2%, and cultured at 37°C and 200 rpm until the OD 600=0.8, add IPTG with a final concentration of 0.2mM, induce at 37℃ for 48h and then collect the bacteria by centrifugation; suspend the bacteria in PBS buffer (10mM, pH=7.2), disrupt the bacteria by ultrasonic disruption, 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 Shenggong), dialysis desalting and ultrafiltration concentration to obtain the wild-type phosphatase ST6P pure enzyme solution.

[0088] Example 2

[0089] This example is based on the wild-type phosphatase ST6P provided in Example 1, and constructs a wild-type phosphatase ST6P mutant library, specifically including:

[0090] 1. Obtaining mutation sites: The amino acid sequence and structure of the wild-type phosphatase mutant were subjected to bioinformatics analysis to determine the key amino acid residues in the catalytic active center of the enzyme, and the amino acid residues near the substrate channel were analyzed. Finally, 8 sites that may affect the catalytic activity and substrate-specific binding ability were screened, including 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 the mutants in the phosphatase ST6P mutant library finally constructed are shown in SEQ ID NO: 3.

[0091] 2. Obtaining positive clone transformants: Saturation mutation was performed on positions 18, 46, 111, 116, 134, 166, 170 and 188, respectively, and mutation primers for the specified mutation sites were designed; using the recombinant plasmid pET28a-ST6P provided in Example 1 as a template, each mutation primer was used to perform whole-plasmid PCR amplification on the recombinant plasmid pET28a-ST6P in the presence of high-fidelity Taq enzyme to obtain a linearized plasmid of the specified mutation site, and the linearized plasmid was recovered by electrophoresis and connected using a seamless cloning kit to obtain a recombinant plasmid of the specified mutation site; the recombinant plasmid of the specified mutation site was transformed into Escherichia coli BL21 (DE3) competent cells, coated 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 mutants: Randomly select 3 positive clone transformants from each LB plate for sequencing, and perform activity test after the corresponding mutation is verified by sequencing; take the positive clone transformants and inoculate them into a 96-deep-well plate containing TB fermentation medium at a 2% inoculation rate, and culture them at 37°C and 200 rpm until OD 600=0.8, IPTG was added at a final concentration of 0.2 mM, and the cells were collected by centrifugation after induction at 37°C for 24 h; the cells were suspended in PBS buffer (10 mM, pH = 7.2), and the cells were broken by freeze-thaw three times, and the supernatant was collected by centrifugation to obtain the crude enzyme solution of the mutant.

[0093] 4. Screening of mutants: Use PBS buffer to adjust the OD of each crude enzyme solution. 280 =10, add the adjusted crude enzyme solution to the monosaccharide 6-phosphate mixed reaction solution at 10% (v / v), react at 50°C for 2h, and use high performance liquid chromatography (HPLC) to determine the yield and content of each component in the product. The total catalytic activity of the wild-type crude enzyme solution (i.e., the sum of all monosaccharides generated by catalysis) is recorded as 100%, and the proportion of different monosaccharides generated in the sum of all products is used to characterize the catalytic specificity of the enzyme, and screen out mutants with improved catalytic activity or significantly changed specificity, as shown in Table 2.

[0094] Table 2.

[0095]

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

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

[0098] Example 3

[0099] This example is based on the mutants with improved activity or significantly changed specificity obtained in Example 2, and combined mutations are performed to obtain mutants with improved catalytic activity and specificity, specifically including:

[0100] 1. Obtaining crude enzyme solutions of mutants: The mutation sites of the mutants shown in Table 2 were combined, and crude enzyme solutions corresponding to the mutants were prepared according to the method provided in Example 2.

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

[0102] 3. Screening of mutants: The pure enzyme solution corresponding to each mutant was tested according to the method provided in Example 2. 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 proportion of different monosaccharides produced in the total of all products was used to characterize the catalytic specificity of the enzyme, and mutants with improved catalytic activity and specificity were screened out, as shown in Table 3.

[0103] Table 3.

[0104]

[0105]

[0106] From the results shown in Table 3, it can be seen that phosphatase mutant 6 (having both E18D and N188H mutations, the sequence is shown in SEQ ID NO: 4) and phosphatase mutant 12 (having both E18K and N188H mutations, the sequence is shown in SEQ ID NO: 5) have excellent catalytic activity and specificity for tagatose-6-phosphate.

[0107] Phosphatase mutant 25 (having both S111I and F166I mutations, the sequence of which is shown in SEQ ID NO: 6) and phosphatase mutant 28 (having both L116I and N188A mutations, the sequence of which is shown in SEQ ID NO: 7) have excellent catalytic activity and specificity for fructose-6-phosphate.

[0108] Phosphatase mutant 30 (having both S111C and L116I mutations, the sequence of which is shown in SEQ ID NO:8) and phosphatase mutant 32 (having both S111C and N188A mutations, the sequence of which is shown in SEQ ID NO:9) have excellent catalytic activity and specificity for glucose-6-phosphate.

[0109] Phosphatase mutant 34 (having both V46T and L116R mutations, the sequence of which is shown in SEQ ID NO: 10) and phosphatase mutant 36 (having both L116R and T170H mutations, the sequence of which is shown in SEQ ID NO: 11) have excellent catalytic activity and specificity for psicose-6-phosphate.

[0110] Phosphatase mutant 37 (having both V46G and S111A mutations, the sequence of which is shown in SEQ ID NO: 12) and phosphatase mutant 40 (having both V46G and T170H mutations, the sequence of which is shown in SEQ ID NO: 13) have excellent catalytic activity and specificity for mannose-6-phosphate.

[0111] Example 4

[0112] This example is used to illustrate the ability of the phosphatase mutant 6 and the phosphatase mutant 12 provided in Example 3 to synthesize tagatose using tagatose-6-phosphate as a substrate, specifically comprising: adding the pure enzyme solutions of the phosphatase mutant 6 and the phosphatase mutant 12 to the tagatose-6-phosphate reaction solution at a final concentration of 10 g / L, reacting at 60° C. for 12 h, and then adding 5 mM H2SO4 to terminate the reaction, determining the tagatose content in the product by the method provided in Example 2, and calculating the tagatose molar conversion rate, 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 Tagatose molar conversion rate (%) Wild-type phosphatase ST6P 56.3 Phosphatase mutant 6 93.1 Phosphatase mutant 12 88.5

[0116] From the results shown in Table 4, it can be seen that compared with the wild-type phosphatase ST6P, the phosphatase mutant 6 and the phosphatase mutant 12 showed higher catalytic efficiency in catalyzing the conversion of tagatose-6-phosphate into tagatose.

[0117] Example 5

[0118] This example is used to illustrate the ability of the phosphatase mutant 25 and the phosphatase mutant 28 provided in Example 3 to synthesize fructose using fructose-6-phosphate as a substrate, specifically comprising: adding the pure enzyme solutions of the phosphatase mutant 25 and the phosphatase mutant 28 to the fructose-6-phosphate reaction solution at a final concentration of 10 g / L, reacting at 60° C. for 12 h, and then adding 5 mM H2SO4 to terminate the reaction, determining the fructose content in the product by the method provided in Example 2, and calculating the fructose molar conversion rate. 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] From the results shown in Table 5, it can be seen that compared with the wild-type phosphatase ST6P, the phosphatase mutant 25 and the phosphatase mutant 28 showed higher catalytic efficiency in catalyzing the conversion of fructose-6-phosphate to fructose.

[0123] Example 6

[0124] This example is used to illustrate the ability of the phosphatase mutant 30 and the phosphatase mutant 32 provided in Example 3 to synthesize glucose using glucose-6-phosphate as a substrate, specifically comprising: adding the pure enzyme solutions of the phosphatase mutant 30 and the phosphatase mutant 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 adding 5 mM H2SO4 to terminate the reaction, determining the glucose content in the product by the method provided in Example 2, and calculating the glucose molar conversion rate. 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] From the results shown in Table 6, it can be seen that compared with the wild-type phosphatase ST6P, the phosphatase mutant 30 and the phosphatase mutant 32 exhibited higher catalytic efficiency in catalyzing the conversion of glucose-6-phosphate into glucose.

[0129] Example 7

[0130] This example is used to illustrate the ability of phosphatase mutant 34 and phosphatase mutant 36 provided in Example 3 to synthesize psicose using psicose-6-phosphate as a substrate, specifically comprising: adding pure enzyme solutions of phosphatase mutant 34 and phosphatase mutant 36 to the psicose-6-phosphate reaction solution at a final concentration of 10 g / L, reacting at 60° C. for 12 h, and then adding 5 mM H2SO4 to terminate the reaction, determining the psicose content in the product using the method provided in Example 2, and calculating the psicose molar conversion rate. The results are shown in Table 7.

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

[0132] Table 7.

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

[0134] From the results shown in Table 7, it can be seen that compared with the wild-type phosphatase ST6P, the phosphatase mutant 34 and the phosphatase mutant 36 showed higher catalytic efficiency in catalyzing the conversion of psicose-6-phosphate to psicose.

[0135] Example 8

[0136] This example is used to illustrate the ability of the phosphatase mutant 37 and the phosphatase mutant 40 provided in Example 3 to synthesize mannose using mannose-6-phosphate as a substrate, specifically comprising: adding the pure enzyme solutions of the phosphatase mutant 37 and the phosphatase mutant 40 to the mannose-6-phosphate reaction solution at a final concentration of 10 g / L, reacting at 60° C. for 12 h, and then adding 5 mM H2SO4 to terminate the reaction, determining the mannose content in the product by the method provided in Example 2, and calculating the mannose molar conversion rate. 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] From the results shown in Table 8, it can be seen that compared with the wild-type phosphatase ST6P, the phosphatase mutant 37 and the phosphatase mutant 40 exhibited higher catalytic efficiency in catalyzing the conversion of mannose-6-phosphate to mannose.

[0141] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention without departing from the principles and purpose of the present invention.

Claims

1. A phosphatase mutant, characterized in that: The phosphatase mutant includes an amino acid fragment as shown in SEQ ID NO: 3; Wherein, the amino acid residue positions are encoded with reference to the amino acid fragment shown in SEQ ID NO: 3, 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; the 188th amino acid residue of the phosphatase mutant is asparagine, aspartic acid, serine, alanine or histidine.

2. The phosphatase mutant according to claim 1, characterized in that The phosphatase mutant includes one or more segments of the amino acid fragments shown in SEQ ID NOs: 4-13.

3. The method for constructing a phosphatase mutant according to claim 1 or 2, characterized in that: The construction method comprises: performing PCR site-directed 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 a host cell to obtain an engineered strain; and inducing expression of the engineered strain to obtain the phosphatase mutant.

4. The method for constructing a phosphatase mutant according to claim 3, characterized in that: The wild-type phosphatase ST6P includes an amino acid fragment whose sequence is shown in SEQ ID NO:

1.

5. An engineered strain, characterized in that: The engineered strain expresses the phosphatase mutant according to claim 1 or 2.

6. Use of the phosphatase mutant according to claim 1 or 2 in monosaccharide production.

7. Use of the phosphatase mutant according to claim 6 in monosaccharide production, characterized in that: The monosaccharide is selected from one or more of tagatose, fructose, glucose, psicose and mannose.

8. Use of the phosphatase mutant according to claim 7 in monosaccharide production, characterized in that: When the monosaccharide is tagatose, the phosphatase mutant comprises an amino acid fragment as shown in SEQ ID NO: 4 or 5; Optionally, when the monosaccharide is fructose, the phosphatase mutant comprises an amino acid fragment having a sequence as shown in SEQ ID NO: 6 or 7; Optionally, when the monosaccharide is glucose, the phosphatase mutant comprises an amino acid fragment having a sequence as shown in SEQ ID NO: 8 or 9; Optionally, when the monosaccharide is psicose, the phosphatase mutant comprises an amino acid fragment having a sequence as shown in SEQ ID NO: 10 or 11; Optionally, when the monosaccharide is mannose, the phosphatase mutant comprises an amino acid fragment having a sequence as shown in SEQ ID NO: 12 or 13.

9. A method for preparing monosaccharide, characterized in that: The preparation method comprises: taking the phosphatase mutant according to claim 1 or 2 to perform an enzyme-catalyzed reaction on a reaction solution including a sugar substrate to obtain the monosaccharide.

10. The method for preparing monosaccharide according to claim 9, characterized in that: The sugar substrate is selected from one or more of tagatose-6-phosphate, fructose-6-phosphate, glucose-6-phosphate, psicose-6-phosphate and mannose-6-phosphate; Optionally, the phosphatase mutant is added at a concentration of 1 to 25 g / L; Optionally, the reaction solution includes 100-200 mM of a carbohydrate substrate, 1-20 mM of Mg 2+ and 50-200 mM HEPES buffer at pH 6.5-7.5; Optionally, the temperature of the enzyme catalyzed reaction is 50-70° C., and the time is 1-48 hours.

Images