Cyclodextrinase mutants and their use in glycosylation

Abstract

The application discloses a cyclodextrinase mutant and application thereof in glycosylation, and belongs to the technical field of genetic engineering. The cyclodextrinase and parent cyclodextrinase with more than 60% homology have one or more amino acid mutations in the peptide regions located at 45-52, 107-113, 163-166, 355-360, 374-376 and 465-468, or amino acid mutations occur at the equivalent positions of the parent cyclodextrinase, and a series of cyclodextrinase mutants with improved performance are provided. The mutant has at least any of the following improved performances: improved enzyme activity, improved product specificity, and reduced product hydrolysis rate, and can meet various requirements of practical application and research.

Description

Technical Field

[0001] This invention relates to cyclodextrinase mutants and their application in glycosylation, belonging to the field of genetic engineering technology. Background Technology

[0002] p-Nitrophenyl-α-D-maltoheptaglycoside (pNPG7) is a key substrate in α-amylase activity assay kits and also a crucial intermediate in the synthesis of 4,6-ethylidene-p-nitrophenyl-α-D-maltoheptaglycoside (EPS, another substrate in α-amylase activity assay kits). However, the synthesis of pNPG7 presents significant challenges. The pNPG7 molecule contains seven glycosidic bonds with specific stereoconfigurations, making organic synthesis methods complex, yielding low yields, consuming high energy, and causing severe pollution, thus hindering mass production.

[0003] Cyclodextrinase (CDase, EC 3.2.1.54) catalyzes the hydrolysis of α / β / γ-cyclodextrins and belongs to the glycoside hydrolase family 13. Cyclodextrinases can also hydrolyze starch and pullulan, but their activity is strongest against cyclodextrins. The amino acid sequence similarity of cyclodextrinases specific to different substrates ranges from 40% to 60%. A small number of microbial cyclodextrinases possess strong transglycosylation capabilities, thus these enzymes can be used as tools for compound glycosylation.

[0004] Previous work has shown that cyclodextrinase BsCDase from Bacillus sphaericus, cyclodextrinase Ps92CDase from Paenibacillus sp. PAMC21692, and cyclodextrinase Ps03CDase from Paenibacillus sp. MY03 can be used to produce pNPG7 via glycosylation. However, these cyclodextrinases have problems such as the existence of many isomers in the glycosylation products, hydrolysis of glycosylation products, and low glycosylation yield. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides a series of performance-enhanced cyclodextrinase mutants, which possess at least one of the following performance enhancements: increased enzyme activity, improved product specificity, and reduced product hydrolysis rate, thereby meeting various needs in practical applications and research.

[0006] Improving enzyme activity refers to increasing product yield; for example, when using α-cyclodextrin and p-nitrobenzene-α-D-glucoside as substrates, under the same conditions (enzyme dosage, substrate dosage, and reaction time), the yield of p-nitrobenzene-α-D-maltoheptacoside can be increased.

[0007] Improving product specificity refers to increasing the proportion of the target product in the product and reducing the content of isomers. For example, in the synthesis of p-nitrobenzene-α-D-maltoheptaglycoside, there are two isomers, which means reducing the proportion of isomers. The degree of improvement in product specificity can be assessed by the purity of the target product. The calculation method is: purity (%) = target product yield / (target product yield + isomer yield) × 100%.

[0008] Reducing the product hydrolysis rate refers to decreasing the hydrolysis rate of the product by cyclodextrinase. For example, in the synthesis of p-nitrobenzene-α-D-maltoheptaside, cyclodextrinase has the problem of hydrolyzing p-nitrobenzene-α-D-maltoheptaside into p-nitrobenzene-α-D-maltohexaside. In this example, the method for calculating the reduced product hydrolysis rate is: Product hydrolysis capacity (%) = p-nitrobenzene-α-D-maltohexaside (pNPG6) yield / p-nitrobenzene-α-D-maltoheptaside (pNPG7) yield × 100%.

[0009] The first objective of this invention is to provide a performance-enhanced cyclodextrinase mutant, wherein the cyclodextrinase mutant has one or more amino acid mutations at any amino acid residue in the peptide region located at 45-52, 107-113, 163-166, 355-360, 374-376, 465-468, based on the amino acid sequence corresponding to the parental cyclodextrinase, or has one or more amino acid mutations at any amino acid residue in the parental cyclodextrinase corresponding to the above-mentioned peptide region equivalent positions; the amino acid sequence of the parental cyclodextrinase has at least 60% identity with the amino acid shown in SEQ ID NO.1 and has cyclodextrinase activity.

[0010] In one embodiment, the performance is selected from one or more of enzyme activity, improved product specificity, and reduced product hydrolysis rate.

[0011] In one embodiment, the parental cyclodextrinase is a cyclodextrinase with the amino acid sequence shown in SEQ ID NO.1.

[0012] In one embodiment, the amino acid represented by SEQ ID NO.1 is a cyclodextrinase Ps03CDase-WT derived from Paenibacillus sp.MY03, and its nucleotide sequence is shown in SEQ ID NO.7.

[0013] In one embodiment, the parental cyclodextrinase having at least 60% identity with the amino acid shown in SEQ ID NO.1 comes from different strains, including but not limited to cyclodextrinase BsCDase-WT from Bacillus sphaericus, cyclodextrinase Ps92CDase-WT from Paenibacillus sp. PAMC21692, cyclodextrinase Bh76CDase-WT from Bacillus horti, cyclodextrinase Po68CDase-WT from Paenibacillus odorifer, and cyclodextrinase Ps61CDase-WT from Paenibacillus sp. 32O-W.

[0014] In one embodiment, the amino acid sequence of BsCDase-WT is shown in SEQ ID NO.2; the amino acid sequence of Ps92CDase-WT is shown in SEQ ID NO.3; the amino acid sequence of Bh76CDase-WT is shown in SEQ ID NO.4; the amino acid sequence of Po68CDase-WT is shown in SEQ ID NO.5; and the amino acid sequence of Ps61CDase-WT is shown in SEQ ID NO.6. Wherein, positions 358, 376, and 467 of the above-mentioned cyclodextrinase parents are conserved sequences, meaning that the wild-type cyclodextrinases from different sources have the same amino acid sequence at positions 358, 376, and 467 as in SEQ ID NO.1.

[0015] In one embodiment, the nucleotide sequence of BsCDase-WT is shown in SEQ ID NO.8; the nucleotide sequence of Ps92CDase-WT is shown in SEQ ID NO.9; the nucleotide sequence of Bh76CDase-WT is shown in SEQ ID NO.10; the nucleotide sequence of Po68CDase-WT is shown in SEQ ID NO.11; and the nucleotide sequence of Ps61CDase-WT is shown in SEQ ID NO.12.

[0016] In one embodiment, the catalytic pocket of Ps03CDase-WT is L-shaped, wherein the long side of the L-shape binds cyclodextrin as a glycosyl donor, and the short side of the L-shape can bind various types of glycosyl acceptors; the entire catalytic pocket is regulated by six loops: loops at positions 45-52 and 107-113 of Chain A, and loops at positions 163-166, 355-360, 374-376, and 465-468 of Chain B; the mutation sites at positions 48, 49, 111, and 165 belong to the donor-binding domain of the cyclodextrinase; and positions 358, 376, 466, and 467 belong to the acceptor-binding domain of the cyclodextrinase.

[0017] In one implementation, the active pockets of BsCDase-WT, Ps92CDase-WT, Bh76CDase-WT, Po68CDase-WT, and Ps61CDase-WT are all regulated by the above six loops, and their overall three-dimensional structures are highly similar to those of Ps03CDase-WT.

[0018] In one embodiment, by mutating the amino acids in the six loop regions of the above-mentioned cyclodextrin enzyme parent, the entry point of the catalytic pocket is reduced, the internal space of the catalytic pocket is expanded, and the polarity of the receptor binding domain is reduced (reducing the entry of water molecules).

[0019] In one embodiment, the cyclodextrinase mutant has one or more amino acid mutations at positions 48, 49, 111, 165, 358, 376, 466, and 467, based on the amino acid sequence corresponding to the parental cyclodextrinase, or has one or more amino acid mutations at amino acid residues at equivalent positions of the parental cyclodextrinase; the amino acid sequence of the parental cyclodextrinase has at least 60% identity with the amino acid shown in SEQ ID NO.1 and has cyclodextrinase activity.

[0020] In one embodiment, the amino acid sequence corresponding to SEQ ID NO.1 has one or more of the following amino acid mutations:

[0021] In the peptide region of 45-52, the 48th amino acid is mutated to W; when the amino acid sequence of the parental cyclodextrinase is SEQ ID NO.1, it is named Ps03CDase-S48W.

[0022] Alternatively, the 49th amino acid may be mutated to K or R; when the amino acid sequence of the parental cyclodextrinase is SEQ ID NO.1, it may be named Ps03CDase-E49K and Ps03CDase-E49R, respectively.

[0023] In the peptide region of 107-113, the amino acid at position 111 is mutated to K or R; when the amino acid sequence of the parental cyclodextrinase is SEQ ID NO.1, it is named Ps03CDase-E111K and Ps03CDase-E111R, respectively.

[0024] In the peptide region of 163-166, the amino acid at position 165 is mutated to F; when the amino acid sequence of the parental cyclodextrinase is SEQ ID NO.1, it is named Ps03CDase-A165F;

[0025] In the 355-360 peptide region, the amino acid at position 358 is mutated to F, Y or I; when the amino acid sequence of the parental cyclodextrinase is SEQ ID NO.1, it is named Ps03CDase-M358F, Ps03CDase-M358F and Ps03CDase-M358I, respectively.

[0026] In the peptide region of 374-376, the amino acid at position 376 is mutated to S or A; when the amino acid sequence of the parental cyclodextrinase is SEQ ID NO.1, it is named Ps03CDase-Y376S and Ps03CDase-Y376A, respectively.

[0027] In the peptide region of 465-468, the amino acid at position 466 is mutated to K or R; when the amino acid sequence of the parental cyclodextrinase is SEQ ID NO.1, it is named Ps03CDase-H466K and Ps03CDase-H466R, respectively.

[0028] Alternatively, the amino acid at position 467 may be mutated to E; when the amino acid sequence of the parental cyclodextrinase is SEQ ID NO.1, it may be named Ps03CDase-D467E.

[0029] In one embodiment, the amino acid sequence of Ps03CDase-E49K is shown in SEQ ID NO. 13; the amino acid sequence of Ps03CDase-E49R is shown in SEQ ID NO. 14; the amino acid sequence of Ps03CDase-E111K is shown in SEQ ID NO. 15; the amino acid sequence of Ps03CDase-E111R is shown in SEQ ID NO. 16; the amino acid sequence of Ps03CDase-A165F is shown in SEQ ID NO. 17; the amino acid sequence of Ps03CDase-M358F is shown in SEQ ID NO. 18; the amino acid sequence of Ps03CDase-M358I is shown in SEQ ID NO. 19; the amino acid sequence of Ps03CDase-M358Y is shown in SEQ ID NO. 45; the amino acid sequence of Ps03CDase-Y376S is shown in SEQ ID NO. 20; and the amino acid sequence of Ps03CDase-Y376A is shown in SEQ ID NO. 13. The amino acid sequence of Ps03CDase-H466K is shown in SEQ ID NO.21; the amino acid sequence of Ps03CDase-H466R is shown in SEQ ID NO.23; and the amino acid sequence of Ps03CDase-D467E is shown in SEQ ID NO.24.

[0030] In one embodiment, the cyclodextrinase mutant has at least the following mutations that are equivalent to or correspond to the parental cyclodextrinase mutant: E111K and M358F; when the amino acid sequence of the parental cyclodextrinase is SEQ ID NO.1, it is named Ps03CDase-E111K / M358F, and the amino acid sequence is shown in SEQ ID NO.25.

[0031] In one embodiment, the cyclodextrinase mutant has at least the following mutations that are equivalent to or correspond to the parental cyclodextrinase mutant: D467E and M358F; when the amino acid sequence of the parental cyclodextrinase is SEQ ID NO.1, it is named Ps03CDase-D467E / M358F, and the amino acid sequence is shown in SEQ ID NO.26.

[0032] In one embodiment, the cyclodextrinase mutant has at least the following mutations that are equivalent to or correspond to the parental cyclodextrinase mutant: D467E and M358Y; when the amino acid sequence of the parental cyclodextrinase is SEQ ID NO.1, it is named Ps03CDase-D467E / M358Y, and the amino acid sequence is shown in SEQ ID NO.27.

[0033] In one embodiment, the cyclodextrinase mutant has at least the following mutations that are equivalent to or correspond to the parental cyclodextrinase mutant: H466K and M358F; when the amino acid sequence of the parental cyclodextrinase is SEQ ID NO.1, it is named Ps03CDase-H466K / M358F, and the amino acid sequence is shown in SEQ ID NO.28.

[0034] In one embodiment, the cyclodextrinase mutant has at least the following mutations equivalent to or corresponding to the parental cyclodextrinase mutant: H466K and M358Y; when the amino acid sequence of the parental cyclodextrinase is SEQ ID NO.1, it is named Ps03CDase-H466K / M358Y, and the amino acid sequence is shown in SEQ ID NO.29.

[0035] In one embodiment, the cyclodextrinase mutant has at least the following mutations equivalent to or corresponding to the parental cyclodextrinase mutant: H466R and M358F; when the amino acid sequence of the parental cyclodextrinase is SEQ ID NO.1, it is named Ps03CDase-H466R / M358F, and the amino acid sequence is shown in SEQ ID NO.30.

[0036] In one embodiment, the cyclodextrinase mutant contains at least the following mutations equivalent to or corresponding to the parental cyclodextrinase mutant: E111R, H466K, and M358F; when the amino acid sequence of the parental cyclodextrinase is SEQ ID NO.1, it is named Ps03CDase-E111R / H466K / M358F, and the amino acid sequence is shown in SEQ ID NO.31.

[0037] In one embodiment, the cyclodextrinase mutant contains at least the following mutations equivalent to or corresponding to the parental cyclodextrinase mutant: E111R, H466R, and M358F; when the amino acid sequence of the parental cyclodextrinase is SEQ ID NO.1, it is named Ps03CDase-E111R / H466R / M358F, and the amino acid sequence is shown in SEQ ID NO.32.

[0038] In one embodiment, the cyclodextrinase mutant contains at least the following mutations equivalent to or corresponding to the parental cyclodextrinase mutant: E111K, H466K, and M358Y; when the amino acid sequence of the parental cyclodextrinase is SEQ ID NO.1, it is named Ps03CDase-E111K / H466K / M358Y, and the amino acid sequence is shown in SEQ ID NO.33.

[0039] In one embodiment, the cyclodextrinase mutant contains at least the following mutations equivalent to or corresponding to the parental cyclodextrinase mutant: E49K, D467E, and M358F; when the amino acid sequence of the parental cyclodextrinase is SEQ ID NO.1, it is named Ps03CDase-E49K / D467E / M358F, and the amino acid sequence is shown in SEQ ID NO.34.

[0040] In one embodiment, the cyclodextrinase mutant contains at least the following mutations equivalent to or corresponding to the parental cyclodextrinase mutant: S48W, D467E, and M358F; when the amino acid sequence of the parental cyclodextrinase is SEQ ID NO.1, it is named Ps03CDase-S48W / D467E / M358F, and the amino acid sequence is shown in SEQ ID NO.35.

[0041] In one embodiment, BsCDase-WT is used as the parental cyclodextrinase, and at least simultaneously, the following mutations, which are equivalent to or correspond to the parental cyclodextrinase mutant, are present: D467E and M358F, named BsCDase-D467E / M358F, with the amino acid sequence shown in SEQ ID NO.36.

[0042] In one embodiment, BsCDase-WT is used as the parental cyclodextrinase, and at least simultaneously, the following mutations, which are equivalent to or correspond to the parental cyclodextrinase mutant, are present: E49K, D467E, and M358F, named BsCDase-E49K / D467E / M358F, with the amino acid sequence shown in SEQ ID NO.37.

[0043] In one embodiment, Ps92CDase-WT is used as the parental cyclodextrinase, and at least simultaneously, the following mutations, which are equivalent to or correspond to the parental cyclodextrinase mutant, are present: D467E and M358F, named Ps92CDase-D467E / M358F, with the amino acid sequence shown in SEQ ID NO.38.

[0044] In one embodiment, Ps92CDase-WT is used as the parental cyclodextrinase, and at least simultaneously, the following mutations, which are equivalent to or correspond to the parental cyclodextrinase mutant, are present: E49K, D467E, and M358F, named Ps92CDase-E49K / D467E / M358F, with the amino acid sequence shown in SEQ ID NO.39.

[0045] In one embodiment, Bh76CDas-WT is used as the parental cyclodextrinase, and at least simultaneously, the following mutations, which are equivalent to or correspond to the parental cyclodextrinase mutant, are present: D467E and M358F, named Bh76CDas-D467E / M358F, with the amino acid sequence shown in SEQ ID NO.40.

[0046] In one embodiment, Bh76CDas-WT is used as the parental cyclodextrinase, and at least simultaneously, the following mutations, which are equivalent to or correspond to the parental cyclodextrinase mutant, are present: E49K, D467E, and M358F, named Bh76CDas-E49K / D467E / M358F, with the amino acid sequence shown in SEQ ID NO.41.

[0047] In one embodiment, Po68CDase-WT is used as the parental cyclodextrinase, and at least simultaneously, the following mutations, which are equivalent to or correspond to the parental cyclodextrinase mutant, are present: D468E and M359F, named Po68CDase-D468E / M359F, with the amino acid sequence shown in SEQ ID NO.42.

[0048] In one embodiment, Po68CDase-WT is used as the parental cyclodextrinase, and at least simultaneously, the following mutation, which is equivalent to or corresponds to the parental cyclodextrinase mutant, is present: E112K, named Po68CDase-E112K, with the amino acid sequence shown in SEQ ID NO.43.

[0049] In one embodiment, Ps61CDase-WT is used as the parental cyclodextrinase, and at least simultaneously, the following mutations, which are equivalent to or correspond to the parental cyclodextrinase mutant, are present: D467E and M358F, named Ps61CDase-D465E / M356F, with the amino acid sequence shown in SEQ ID NO.44.

[0050] In one embodiment, the above-mentioned cyclodextrinase mutant, compared with the parental cyclodextrinase, has improved enzyme activity and product specificity, while also having a lower product hydrolysis rate.

[0051] A second object of the present invention is to provide a polynucleotide encoding any of the above-described cyclodextrinase mutants.

[0052] A third objective of this invention is to provide a plasmid vector carrying the aforementioned polynucleotides.

[0053] A fourth object of the present invention is to provide cells expressing any of the above-described cyclodextrinase mutants or the above-described plasmid vectors.

[0054] In one embodiment, the cell can be any cell useful in the recombination production of mutants, such as prokaryotes or eukaryotes.

[0055] Alternatively, the prokaryotic host cell can be any Gram-positive or Gram-negative bacterium, and the eukaryote can be a cell of a mammal, insect, plant, or fungus.

[0056] A fifth object of the present invention is to provide a method for synthesizing oligomaltodextrin using any of the above-described cyclodextrinase mutants; said oligomaltodextrin is synthesized by means of a glycosyl donor and a glycosyl acceptor via cyclodextrinase mutant catalysis.

[0057] In one embodiment, the method includes: using cyclodextrin as a glycosyl donor and benzene rings, alcohols, or glycosides as glycosyl acceptors, in an enzymatic reaction system containing a cyclodextrin enzyme mutant of the present invention with transglycosylation activity to synthesize oligomaltodextrin with a degree of polymerization of 6-9 (i.e., containing 6-9 glucose molecules).

[0058] Optionally, the oligomaltodextrin contains 6 to 9 glucose units.

[0059] Optionally, the glycosyl donor includes α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin.

[0060] Optionally, the glycosyl receptor includes: p-nitrophenyl-α-glucoside, p-nitrophenol, other benzene rings, alcohols, or other glycosides; wherein, receptors that do not contain a glycosyl group on their own include benzene rings or alcohols; receptors that contain one glycosyl group on their own include glycosides.

[0061] In some embodiments, the glycoside receptor includes, but is not limited to, one or more of benzene ring glucosides, click chemo glucosides, and nucleosides.

[0062] In some embodiments, the benzene ring glucosides include, but are not limited to, glucosides containing chromophores, benzene ring natural product glucosides, or one or more of these.

[0063] In some embodiments, clickable chemical glucosides include, but are not limited to, azide-PEGn-glucose, propargyl-PEGm-glucose (n, m are positive integers), etc.

[0064] In some embodiments, nucleosides include, but are not limited to, one or more of cytarabine and deoxyfluorouridine.

[0065] In some embodiments, the benzene ring glycosyl acceptor includes, but is not limited to, one or more of p-nitrophenol, 2-chloro-4-nitrophenol, and tetramethylumbelliferone.

[0066] In some embodiments, the alcohol glycosyl acceptor includes, but is not limited to, one or more of methanol, ethanol, n-propanol, isopropanol, and n-butanol.

[0067] Optionally, the glycosyl receptor includes: 2-chloro-4-nitrophenyl-α / β-glucoside, α / β-arbutin, and rhodioloside.

[0068] In one embodiment, the method includes:

[0069] (a) Using α-cyclodextrin as the glycosyl donor and p-nitrophenyl-α-glucoside or other glycosides (including but not limited to benzene ring glucosides, click chemical glucosides, nucleosides, etc.), p-nitrophenol, other benzene rings (including but not limited to 2-chloro-4-nitrophenol and tetramethylumbelliferone and other chromophores), alcohols, etc. as glycosyl acceptors, transglycosylation is performed in the enzymatic reaction system of the above-mentioned cyclodextrin enzyme mutant to synthesize pNPG7 or other oligomaltodextrin glycosides with a degree of polymerization of 6-7; or,

[0070] (b) Using β-cyclodextrin as the glycosyl donor and p-nitrophenyl-α-glucoside or other glycosides (including but not limited to benzene ring glucosides, click chemical glucosides, nucleosides, etc.), p-nitrophenol, other benzene rings (including but not limited to 2-chloro-4-nitrophenol and tetramethylumbelliferone and other chromophores), alcohols, etc. as glycosyl acceptors, transglycosylation is performed in the enzymatic reaction system of the above-mentioned cyclodextrin enzyme mutant to synthesize pNPG7 or other oligomaltodextrin glycosides with a degree of polymerization of 7-8; or,

[0071] (c) Using γ-cyclodextrin as the glycosyl donor and p-nitrophenyl-α-glucoside or other glycosides (including but not limited to benzene ring glucosides, click chemical glucosides, nucleosides, etc.), p-nitrophenol, other benzene rings (including but not limited to 2-chloro-4-nitrophenol and tetramethylumbelliferone and other chromophores), alcohols, etc. as glycosyl acceptors, pNPG8 or other oligomaltodextrin with a degree of polymerization of 8-9 is synthesized by transglycosylation in the enzymatic reaction system of the above-mentioned cyclodextrin enzyme mutant.

[0072] It should be noted that "other glycoside receptors" refers to glycoside receptors other than p-nitrophenyl-α-glucoside. "Other benzene ring receptors" refers to benzene ring receptors other than p-nitrophenol.

[0073] In one embodiment, the oligomaltodextrins with a degree of polymerization of 6-9 includes, but is not limited to, pNPG6, pNPG7, pNPG8, pNPG9, 2-chloro-4-nitrophenyl-α / β-maltoheptaside (CNPG7), 2-chloro-4-nitrophenyl-α / β-maltooctaside (CNPG8), 2-chloro-4-nitrophenyl-α / β-maltononaside (CNPG9), α / β-arbutin maltoheptaside, and α / β-arbutin maltooctaside. Glycosides, α / β-arbutin, Rhodiola rosea maltheptaside, Rhodiola rosea maltoctaside, Rhodiola rosea maltheptaside, azido-PEG4-β-D-maltheptaside, azido-PEG4-β-D-maltoctaside, azido-PEG4-β-D-maltheptaside, propargyl-PEG3-β-D-maltheptaside, propargyl-PEG3-β-D-maltoctaside, propargyl-PEG3-β-D-maltheptaside, propargyl-PEG3-β-D-maltoctaside, propargyl-PEG3-β-D-maltheptaside, etc.

[0074] In one embodiment, the cyclodextrinase mutant of the present invention uses pNPG as a glycosyl acceptor (the acceptor can also be 2-chloro-4-nitrophenyl-α / β-glucoside, α / β-arbutin, rhodioloside, indigoside, etc.) and α-cyclodextrin as a glycosyl donor (the donor can also be β-cyclodextrin and γ-cyclodextrin). It can produce high yields of pNPG7 with a low isomer ratio and oligomaltodextrin oligosaccharides with a degree of polymerization of 6-9 produced by the aforementioned donors and acceptors, and has broad prospects for industrial application.

[0075] In one embodiment, the cyclodextrinase mutant is used to prepare pNPG7, which can produce high yields of pNPG7 with low isomers.

[0076] A sixth object of the present invention is to provide the use of any of the above-described cyclodextrinase mutants in the preparation of p-nitrophenyl-α-D-maltoheptaglycoside.

[0077] The present invention also provides a method for preparing the above-mentioned cyclodextrinase mutant, the method comprising:

[0078] The host cells described in this invention are cultured under conditions suitable for cyclodextrinase expression, and the cyclodextrinase is isolated from the culture system.

[0079] A seventh object of the present invention is to provide a method for improving at least one property of a cyclodextrinase, wherein, based on the amino acid sequence corresponding to the parental cyclodextrinase, there is one or more amino acid mutations at any amino acid residues in the peptide region located at 45-52, 107-113, 163-166, 355-360, 374-376, 465-468, or one or more amino acid mutations occur at any amino acid residues in the parental cyclodextrinase corresponding to the equivalent positions of the above-mentioned peptide regions; the amino acid sequence of the parental cyclodextrinase has at least 60% identity with the amino acid shown in SEQ ID NO.1 and has cyclodextrinase activity;

[0080] The improvement of at least one performance includes increasing enzyme activity, increasing product specificity, and reducing the hydrolysis rate of the product.

[0081] An eighth object of the present invention is to provide the use of any of the above-described cyclodextrinase mutants, polynucleotides, plasmid vectors, or cells in the food, biological, pharmaceutical, or cosmetic fields.

[0082] Optionally, in the food sector, this includes: preparing modified starch and improving bread quality.

[0083] Optionally, the biological field includes: preparing enzyme preparations and preparing reagent kit materials.

[0084] Optionally, the pharmaceutical field includes: the preparation of drug carriers.

[0085] Optionally, the cosmetics field includes: the preparation of creams, lotions, lotions, facial cleansers, shower gels, and shampoos.

[0086] Beneficial effects of the present invention

[0087] This invention provides a series of cyclodextrinase mutants with improved performance, which have at least the following performance improvements: increased enzyme activity, increased product specificity, and reduced product hydrolysis rate, thus meeting various needs for practical applications and research.

[0088] Specifically:

[0089] (1) Under the conditions of α-cyclodextrin dosage of 303mM (30% w / v), pNPG dosage of 300mM, enzyme concentration of 1U, and reaction time of 150min, the pNPG7 yield of an optional cyclodextrin enzyme mutant of the present invention reached the highest level of 235.4g / L, which is significantly higher than that of the parental cyclodextrin enzyme.

[0090] (2) The pNPG7 prepared by an optional cyclodextrinase mutant of the present invention has high purity, up to 95%, and its isomer content is only 5%, which is significantly better than the parental cyclodextrinase.

[0091] (3) An optional cyclodextrinase mutant of the present invention has a low hydrolysis rate of the product, with a minimum of only 10%, which is significantly better than the parental cyclodextrinase.

[0092] (4) The cyclodextrinase mutant of the present invention has more than 60% identity with different parental cyclodextrinases, that is, the mutation site of the present invention has good universality. Attached Figure Description

[0093] Figure 1 The standard track for pNPG7;

[0094] Figure 2 The reaction formula for the cyclodextrinase-catalyzed production of pNPG7;

[0095] Figure 3 HPLC analysis chromatogram for the production of pNPG7 from Ps03CDase-WT;

[0096] Figure 4 HPLC analysis chromatogram for the production of pNPG7 using Ps03CDase-D467E-M358F;

[0097] Figure 5 The sequence alignment of the cyclodextrinases studied in this patent is shown below. In section A, the sites marked with pentagrams are mutation sites; section B is a sequence similarity tree diagram; section C is a sequence similarity matrix diagram; Ps03CDase.pro is the parental cyclodextrinase Ps03CDase-WT; Ps92CDase.pro is the parental cyclodextrinase Ps92CDase-WT; BsCDase.pro is the parental cyclodextrinase BsCDase-WT; Bh76CDase.pro is the parental cyclodextrinase Bh76CDase-WT; Po68CDase.pro is the parental cyclodextrinase Po68CDase-WT; and Ps6lCDase.pro is the parental cyclodextrinase Ps61CDase-WT.

[0098] Figure 6 This is a three-dimensional structural diagram of cyclodextrinase; the green part is the loop that constitutes the catalytic active center, and the yellow part is the maltotetraose co-crystallized with cyclodextrinase.

[0099] Figure 7 The cyclodextrinase mutant was used in the reaction to prepare oligomaltodextrin with a degree of polymerization of 6-9.

[0100] Figure 8 HPLC analysis for the production of rhodioloside maltoheptacoside by cyclodextrin enzyme transglycosylation;

[0101] Figure 9HPLC analysis of arbutin maltoheptaside produced by cyclodextrin enzyme transglycosylation; where A represents α-arbutin as the acceptor and B represents β-arbutin as the acceptor.

[0102] Figure 10 HPLC analysis for the production of indigo maltoheptaglycoside by cyclodextrin enzymatic transglycosylation;

[0103] Figure 11 HPLC analysis of CNPG7 produced by cyclodextrin enzyme transglycosylation. Detailed Implementation

[0104] The technical solution of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0105] Some definitions or terms involved in this invention:

[0106] Improved properties: The term "improved properties" refers to characteristics associated with variants that are improved relative to their parents. Such improved properties include, but are not limited to, increased product specificity, reduced hydrolytic activity, and enhanced transglycosylation activity.

[0107] Corresponding to, equivalent to: As used herein, the terms “corresponding to” and “equivalent to” refer to the manner in which a particular amino acid in a sequence is identified (where a particular amino acid sequence is referenced). For example, for the purposes of this invention, when referring to a particular amino acid position, a person skilled in the art can compare another amino acid sequence with the already referenced amino acid sequence to determine which particular amino acid might be of interest in the other amino acid sequence. The comparison of another amino acid sequence with, for example, sequences shown in SEQ ID NOs 1-6 or any other sequences listed herein has been described elsewhere herein. Alternative comparison methods may be used, and such methods are well known to those skilled in the art.

[0108] Mutant: As used herein, when referring to variations of the invention, the terms "mutant," "peptide variant," "peptide," or "cyclodextrinase mutant" mean a polypeptide having cyclodextrinase activity and containing alterations (i.e., substitutions, insertions, and / or deletions) at one or more (e.g., several) positions relative to the "parent" cyclodextrinase. Substitution means replacing an amino acid occupying a position with a different amino acid; deletion means removing an amino acid occupying a position; and insertion means adding an amino acid adjacent to and immediately following the amino acid occupying a position.

[0109] The cyclodextrinase mutant of the present invention has one or more amino acid mutations at any amino acid residue in the peptide regions corresponding to SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, or SEQ ID NO.6, specifically at positions 45-52, 107-113, 163-166, 355-360, 374-376, and 465-468, or at any amino acid residue in the parental cyclodextrinase corresponding to the equivalent positions of the aforementioned peptide regions. The sequence of the cyclodextrinase mutant is more than 60% identical to the amino acid sequence of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, or SEQ ID NO.6, and all of them contain the aforementioned six peptide regions in their active pockets.

[0110] In the variations describing the invention, the nomenclature described below has been adapted for ease of reference. Accepted IUPAC single-letter or three-letter amino acid abbreviations are used. Substitution: For amino acid substitutions, the following nomenclature is used: original amino acid, position, substituted amino acid. Thus, the substitution of glutamic acid at position 49 with arginine is represented as "E49R". Multiple mutations are separated by a symbol (" / "), for example, "E49R / M358F" represents the substitution of glutamic acid (E) and methionine (M) at positions 49 and 358 with arginine (R) and phenylalanine (F), respectively.

[0111] Specifically, for example, “Ps03CDase-E111R / H466R / M358F” represents Ps03CDase-WT as the parent (amino acid sequence as shown in SEQ ID NO.1), with the glutamic acid at position 111 (E) mutated to arginine (R), the histidine at position 466 (H) mutated to (R), and the methionine at position 358 (M) mutated to phenylalanine (F).

[0112] Parent or parental cyclodextrinase or cyclodextrinase parent: As used herein, the term "parental" cyclodextrinase refers to a cyclodextrinase modified to produce a cyclodextrinase mutant of the present invention. The term also refers to a polypeptide to which the mutant of the present invention is compared. The parent can be a naturally occurring (wild-type) polypeptide, or it can be, or even a variant thereof, prepared by any suitable means. For example, the parental protein can be a variant of a naturally occurring polypeptide whose amino acid sequence has been modified or altered. Thus, the parental cyclodextrinase may have one or more (or one or more) amino acid substitutions, deletions, and / or insertions. Therefore, the parental cyclodextrinase can be a variant of the parental cyclodextrinase. The parent can also be an allelic variant, which is a polypeptide encoded by any of two or more alternative forms of a gene occupying the same chromosomal locus. As used herein, the term "parent" or "parental cyclodextrinase" refers to a cyclodextrinase with an amino acid sequence such as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, or SEQ ID NO.6, or any cyclodextrinase whose amino acid sequence has at least 60% sequence identity with any of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, or SEQ ID NO.6. A parental cyclodextrinase may also be a polypeptide comprising a fragment of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, or SEQ ID NO.6.

[0113] Wild-type enzyme: When referring to an amino acid or nucleic acid sequence, the term "wild-type" means that the amino acid or nucleic acid sequence is a naturally occurring or naturally occurring sequence. As used herein, the term "naturally occurring" refers to any substance found in nature (e.g., a protein, amino acid, or nucleic acid sequence). Conversely, the term "non-naturally occurring" refers to any substance not found in nature (e.g., recombinant nucleic acid and protein sequences produced in a laboratory, or modifications of wild-type sequences). When the parent enzyme is not a variant enzyme, the terms "wild-type enzyme" and "parent enzyme" are used interchangeably.

[0114] Sequence identity: The degree of association between two amino acid sequences or two nucleotide sequences is described by the parameter "sequence identity".

[0115] For the purposes of this invention, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. [Journal of Molecular Biology] 48:443-453) is used to determine sequence identity between two amino acid sequences. This algorithm is implemented in the Needle program of the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. [Trends in Genetics] 16:276-277) (preferably version 5.0.0 or later). The parameters used can be a vacancy opening penalty of 10, a vacancy extension penalty of 0.5, and an EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The Needle-labeled "longest identity" output (obtained using the -nobrief option) is used as the identity percentage and calculated as follows:

[0116] (identical residues x 100) / (alignment length - total number of vacancies in the alignment);

[0117] Optionally, the DNAMAN8 software (default parameters) can be used to compare the amino acid sequences of different parents.

[0118] Alternatively, the parameters used can be a vacancy open penalty of 10, a vacancy extension penalty of 0.5, and EDNAFULL (the EMBOSS version of NCBI NUC4.4) to replace the matrix. The output of "Longest Identity" marked with Needle (obtained using the -nobrief option) is used as the identity percentage and calculated as follows:

[0119] (identical deoxyribonucleotides × 100) / (alignment length - total number of vacancies in the alignment)

[0120] Expression: As used in this article, “expression” refers to any step involving variant generation, including but not limited to transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0121] Expression vector: As used herein, the term “expression vector” refers to a linear or circular DNA molecule that contains a polynucleotide encoding a variant and is operatively linked to a control sequence that provides for its expression.

[0122] Host cell: The term "host cell" means any cell type that is readily transformed, transfected, transduced, etc., using nucleic acid constructs or expression vectors containing the polynucleotides of the present invention. The term "host cell" encompasses any offspring of a parent cell that differs from the parent cell due to mutations occurring during replication, along with recombinant host cells, isolated host cells (e.g., isolated recombinant host cells), and heterologous host cells.

[0123] Recombination: When used to refer to cells, nucleic acids, proteins, or vectors, the term "recombination" means that the cell has been modified from its natural state. Thus, for example, recombinant cells express genes not found in the natural (non-recombinant) form of the cell, or express natural genes at different levels or under different conditions compared to those found in nature. The difference between recombinant nucleic acids and their natural sequences lies in the operative linking of one or more nucleotides and / or a foreign sequence (e.g., a foreign promoter in an expression vector). The difference between recombinant proteins and their natural sequences may lie in the fusion of one or more amino acids and / or a foreign sequence. A vector containing nucleic acids encoding a polypeptide is a recombinant vector. The term "recombination" is synonymous with "genetically modified" and "transgenic."

[0124] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0125] WT represents wild-type cyclodextrinase. For example, Ps03CDase-WT represents the wild-type of Ps03CDase, and its corresponding gene is represented by italicized Ps03CDase-WT.

[0126] Ps03CDase-S48W / E111R represents a double-mutated cyclodextrinase based on the Ps03CDase-WT amino acid sequence, where amino acid position 48 is mutated from serine to tryptophan and amino acid position 111 is mutated from glutamic acid to arginine. Other mutations follow the same pattern, and their genes are indicated in italics.

[0127] Example 1: Generation and purification of parental cyclodextrinase and cyclodextrinase mutants

[0128] 1. Production and purification of parental cyclodextrinase:

[0129] Example of preparation and purification of parental cyclodextrinase Ps03CDase-WT with an amino acid sequence as shown in SEQ ID NO.1:

[0130] (1) Molecular cloning of Ps03CDase and construction of expression strains

[0131] The gene sequence of Ps03CDase (nucleotide sequence as shown in SEQ ID NO.7) was artificially synthesized and constructed into a suitable expression plasmid. The expression plasmids include, but are not limited to, pET32, pET28a, pET22a, pET21a, ppic3.5k and pMA5. Taking pET28a as an example, the gene sequence of Ps03CDase was inserted between the restriction endonucleases EcoRI and XhoI to construct the plasmid vector, named pET28a-Ps03CDase-WT.

[0132] The constructed pET28a-Ps03CDase-WT was transformed into an expression strain, which includes, but is not limited to, Escherichia coli BL21(DE3), Bacillus subtilis WB600 / WB800, Pichia pastoris x33 / DM71 / GS115, etc. Taking Escherichia coli BL21(DE3) as an example, the expression strain was constructed and named E.coli-BL21(DE3)-pET28a-Ps03CDase-WT.

[0133] (2) Induction of PsO3CDase expression and preparation of enzyme solution

[0134] Successfully transformed single colonies of E. coli-BL21(DE3)-pET28a-Ps03CDase-WT were picked from the transformation plate and inoculated into LB liquid medium containing 50 μg / mL kanamycin for overnight culture. The overnight culture was then inoculated into 500 mL of LB medium containing 50 μg / mL kana at a 1% v / v inoculation rate and cultured at 37°C and 200 rpm until the bacterial growth rate reached OD. 600 Once the concentration reaches 0.5, add IPTG to a final concentration of 0.1 mmol / L and incubate at 16°C and 150 rpm for 24 h.

[0135] After cultivation, the fermentation broth was centrifuged at 4℃ and 8000rpm for 10min, the bacterial sludge was collected, resuspended in PBS and then sonicated. After disruption, it was centrifuged at 4℃ and 8000rpm for 15min, and the supernatant was collected to obtain the crude enzyme solution.

[0136] (3) Purification of PsO3CDase

[0137] After adding imidazole to a final concentration of 5 mM to the crude enzyme solution, the solution was passed through a nickel ion affinity chromatography column at a flow rate of 2 mL / min. Two column volumes were washed with wash buffer (PBS + 40 mM imidazole) to fully elute any contaminating proteins; then 2-4 column volumes were washed with elution buffer (PBS + 200 mM) to completely elute PsO3CDase. The eluted PsO3CDase was dialyzed with PBS to reduce the imidazole concentration and then purified to obtain pure PsO3CDase-WT enzyme solution.

[0138] 2. Preparation of cyclodextrinase mutants

[0139] The cyclodextrinase mutant of the present invention is prepared by a standard procedure, which can be summarized as follows: random and / or site-directed mutations are introduced into a gene, the mutated gene is ligated into an expression vector, then host cells are transformed, the transformed host cells are fermented, and the cyclodextrinase mutant is obtained from the fermentation broth. For example, by introducing a site-directed mutation into the parental cyclodextrinase gene, the cyclodextrinase mutant can be obtained by referring to the above-described method for generating and purifying the parental cyclodextrinase.

[0140] The preparation of cyclodextrinase mutants, taking the mutant Ps03CDase-E49K as an example, is as follows:

[0141] The plasmid vector pET28a-Ps03CDase-WT was prepared from "1. Generation and purification of parental cyclodextrinase". Primers were designed to mutate the nucleotide corresponding to glutamic acid at position 49 of the Ps03CDase-WT amino acid sequence in the plasmid to a nucleotide that can be translated into lysine, and the mutant plasmid vector was constructed and named pET28a-Ps03CDase-E49K.

[0142] The mutant plasmid vector was transformed into the expression strain E.coli-BL21(DE3), and the successfully transformed strain was screened and named E.coli-BL21(DE3)-pET28a-Ps03CDase-E49K. The pure enzyme solution of Ps03CDase-E49K was obtained by expression and purification.

[0143] Other cyclodextrinase mutants can be constructed according to the above method. Among them, double-mutant or multi-mutant cyclodextrinase mutants can be prepared by multiple mutations based on the plasmid vector pET28a-Ps03CDase-WT.

[0144] Cyclodextrinase mutants were prepared and named according to the mutation sites as follows: Ps03CDase-E49K (SEQ ID NO.13), Ps03CDase-E49R (SEQ ID NO.14), Ps03CDase-E111K (SEQ ID NO.15), Ps03CDase-E111R (SEQ ID NO.16), Ps03CDase-A165F (SEQ ID NO.17), Ps03CDase-M358F (SEQ ID NO.18), Ps03CDase-M358I (SEQ ID NO.19), Ps03CDase-M358Y (SEQ ID NO.45), Ps03CDase-Y376S (SEQ ID NO.20), Ps03CDase-Y376A (SEQ ID NO.21), and Ps03CDase-H466K (SEQ ID NO.13). NO.22), Ps03CDase-H466R (SEQ ID NO.23), Ps03CDase-D467E (SEQ ID NO.24), Ps03CDase-E111K / M358F (SEQ ID NO.25), Ps03CDase-D467E / M358F (SEQ ID NO.26), Ps03CDase-D467E / M358Y (SEQ ID NO.27), Ps03CDase-H466K / M358F (SEQ ID NO.28), Ps03CDase-H466K / M358Y (SEQ ID NO.29), Ps03CDase-H466R / M358F (SEQ ID NO.30), Ps03CDase-E111R / H466K / M358F (SEQ ID NO.31), Ps03CDase-E111R / H466R / M358F (SEQ ID NO.32), Ps03CDase-E111K / H466K / M358Y (SEQ ID NO.33), Ps03CDase-E49K / D467E / M358F (SEQ ID NO.34), Ps03CDase-S48W / D467E / M358F (SEQ ID NO.35).

[0145] Example 2: Performance determination method for cyclodextrinase mutants

[0146] The parental cyclodextrinase and cyclodextrinase mutant prepared in Example 1 were used to determine the performance of pNPG7 (p-nitrophenyl-α-D-maltoheptaside) synthesized using pNPG (p-nitrobenzene-α-D-glucoside) and α-CD. Specifically:

[0147] 1. Establishment of the pNPG7 standard curve

[0148] Standard solutions of pNPG7 with concentration gradients of 2000 μM, 1000 μM, 500 μM, 250 μM, and 125 μM were prepared using pure water. The standard solutions of pNPG7 were analyzed using the conditions in Table 1. A standard curve was plotted with the standard solution concentration as the ordinate (y-value, in μM) and the HPLC peak area as the abscissa (x-value), as shown below. Figure 1 As shown, the standard curve formula is y = 0.1958x - 38.386, R0 2 =0.9989, with a linear range of 125–2000 μM.

[0149] Table 1. HPLC detection methods for enzyme-catalyzed reactions

[0150]

[0151]

[0152] 2. Enzyme-catalyzed reaction

[0153] The ability of cyclodextrinase to catalyze the production of pNPG7 before and after mutation was analyzed using enzyme-catalyzed reactions. The enzyme catalytic principle of cyclodextrinase-catalyzed production of pNPG7 is as follows: Figure 2 As shown. The enzyme-catalyzed reaction conditions are as follows:

[0154] Buffer system: 200 mM Tris-HCl buffer solution at pH 7.5;

[0155] Glycosyl donor: α-CD concentration was 303 mM;

[0156] Glycosyl receptor: pNPG concentration was 300 mM;

[0157] Enzyme concentration: 1 U cyclodextrinase;

[0158] Reaction temperature: 40℃;

[0159] Stirring speed: 500 rpm;

[0160] Reaction time: 150 min;

[0161] After the enzyme-catalyzed reaction was completed, the enzyme-catalyzed product was inactivated with methanol, diluted 100 times, and the product was detected by HPLC to analyze the catalytic performance of the parental cyclodextrinase or the cyclodextrinase mutant.

[0162] The liquid chromatography analysis results of Ps03CDase-WT are as follows: Figure 3As shown, the results indicate that the peak times of the isomers are relatively concentrated, and the product contains one or more isomers (P1, P2). Based on the peak times comparison with the pNPG7 standard, matrix spectroscopy, and 1H NMR analysis, P3 was identified as the target product pNPG7, with the chemical structure shown below. Figure 2 As shown in the image.

[0163] 3. Analyze the performance of mutants

[0164] The performance indicators for mutants include:

[0165] (1) P3 yield (g / L, i.e., the combined performance of the mutant's transglycosylation activity and its activity on the hydrolysis of transglycosylation products); the higher the P3 yield, the stronger the mutant's activity in catalyzing the formation of substrate product P3 (pNPG7).

[0166] (2) P3 purity (the proportion of P3 in the total isomers, i.e., the product specificity of the mutant), is calculated as follows: P3 purity (%) = P3 / (P1+P2+P3)×100%; the higher the P3 purity, the stronger the product specificity of the mutant in catalyzing the substrate to generate P3 (pNPG7).

[0167] (3) The hydrolysis ability of P3 (product) (i.e., the hydrolysis activity of the mutant on P3) is calculated as follows: hydrolysis ability of P3 (%) = pNPG6 / P3 × 100%; the higher the index, the easier it is for the mutant to hydrolyze P3 (pNPG7), and the worse the stability of P3 after it is generated.

[0168] Table 2. Performance of Ps03CDase-WT and its mutants

[0169]

[0170]

[0171] The results are shown in Table 2. The results show that the P3 yield of Ps03CDase-WT is 72.82 g / L, the proportion of P3 isomers is only 47%, and there is still a serious problem of hydrolysis of transglycosylation products. The hydrolysis capacity of Ps03CDase-WT for P3 is 44%.

[0172] The HPLC chromatogram of the mutant Ps03CDase-S48W / D467E / M358F is shown below. Figure 4 As shown, the results indicate that:

[0173] (1) In the reaction of mutant Ps03CDase-S48W / D467E-M358F, the P1 isomer was significantly reduced and almost completely eliminated, the P2 isomer was partially reduced, and the P3 isomer accounted for 95% of all isomers.

[0174] (2) The mutant Ps03CDase-S48W / D467E-M358F has a reduced hydrolytic capacity for P3 to 22%, which is significantly better than the wild-type Ps03CDase-WT's 44%.

[0175] (3) The P3 yield reached 235.9 g / L, and the molar conversion rate of pNPG (the number of moles of pNPG7 produced / the number of moles of pNPG fed × 100%) reached 58%, which is 3.2 times that of wild-type Ps03CDase-WT.

[0176] In addition, the other mutants, compared with the wild type, have at least one of the following performance improvements: increased P3 production, increased proportion of P3 isomers, and reduced ability to hydrolyze P3.

[0177] Example 3: Effects of wild-type mutants of cyclodextrinase from other parents

[0178] 1. Comparison of cyclodextrinase sequences from different parents

[0179] The amino acid sequences of six cyclodextrinases were aligned using the default parameters of DNAMAN8. The alignment results are as follows: Figure 5 As shown in the figure. The results indicate that the amino acid sequence similarities of Ps03CDase-WT (amino acid sequence as shown in SEQ ID NO.1), BsCDase-WT (amino acid sequence as shown in SEQ ID NO.2), Ps92CDase-WT (amino acid sequence as shown in SEQ ID NO.3), BH76CDase-WT (amino acid sequence as shown in SEQ ID NO.4), Po68CDase-WT (amino acid sequence as shown in SEQ ID NO.5), and Ps61CDase-WT (amino acid sequence as shown in SEQ ID NO.6) are 97%, 92%, 76%, 68%, and 60%, respectively.

[0180] The three-dimensional structure diagram of Ps03CDase-WT is as follows: Figure 6As shown, the green part is the loop that constitutes the catalytic active center, and the yellow part is the maltotetrasaccharide co-crystallized with cyclodextrinase. It can be seen that the catalytic pocket of Ps03CDase-WT is L-shaped, in which the long side of the L-shape binds cyclodextrin as a glycosyl donor, and the short side of the L-shape can bind various types of glycosyl acceptors. The entire catalytic pocket is regulated by six loops: loops at positions 45-52 and 107-113 of Chain A, and loops at positions 163-166, 355-360, 374-376, and 465-468 of Chain B. The mutation sites at positions 48, 49, 111, and 165 belong to the donor-binding domain of cyclodextrinase; and positions 358, 376, 466, and 467 belong to the acceptor-binding domain of cyclodextrinase.

[0181] Comparison of BsCDase-WT, Ps92CDase-WT, Bh76CDase-WT, Po68CDase-WT and Ps61CDase-WT reveals that the active pockets of the aforementioned parental cyclodextrinases are all composed of the above 6 loops, and their overall three-dimensional structure is highly similar to that of Ps03CDase-WT.

[0182] 2. Testing the performance of cyclodextrinase mutants from different parents

[0183] Following the methods of Examples 1 and 2, the parental cyclodextrinase (Ps03CDase) was modified to wild-type cyclodextrinases (BsCDase, Ps92CDase, Bh76CDase, Po68CDase, and Ps61CDase) with a sequence similarity of more than 60% to Ps03CDase. Combinatorial mutations were performed at positions 49, 467, and 358, which are equivalent to the parental cyclodextrinase (Ps03CDase), to prepare different cyclodextrinase mutants. The amino acid sequences are shown in SEQ ID NO. 36-44, respectively, and their performance in producing pNPG7 was tested.

[0184] Table 3. Performance of BsCDase, Ps92CDase, Bh76CDase, Po68CDase, Ps61CDase-WT and their mutants

[0185]

[0186]

[0187] The results are shown in Table 3. Combination mutations at positions 49, 467, and 358, based on the wild-type cyclodextrinases from other parents, resulted in cyclodextrinase mutants that all improved the effective production of pNPG7 (increasing yield and purity, and reducing the hydrolytic ability of the product). Among them, the Ps92CDase mutants (Ps92CDase-D467E / M358F and Ps92CDase-E49K / D467E / M358F) showed the highest yield and the highest proportion of P3 isomers compared to other mutants. The other mutants also exhibited at least one of the following performance improvements compared to the wild type: increased P3 yield, increased proportion of P3 isomers, and reduced hydrolytic ability of P3.

[0188] It is evident that all six cyclodextrinase sequences exhibit good transglycosylation activity and can effectively produce pNPG7. In particular, Ps61CDase, which shares approximately 60% similarity with the other five sequences, produces a higher pNPG7 yield than BsCDase, which shares 92.1% similarity with Ps03CDase.

[0189] Example 4: Application of cyclodextrinase mutant in the preparation of oligomaltodextrin with a degree of polymerization of 6-9

[0190] The mutant Ps03CDase-S48W / D467E / M358F from Example 2 was used to test its effect on preparing 6-9 oligomaltodextrins.

[0191] Based on Example 2, the glycosyl acceptors were changed to 2-chloro-4-nitrophenyl-α / β-glucoside, α / β-arbutin, and rhodioloside; the glycosyl donors were changed to α, β, and γ-cyclodextrins; and the remaining steps were the same as in Example 2 to prepare maltoheptaglycoside, maltooctaglycoside, and maltononaglycoside. The reaction process is as follows: Figure 7 As shown in the figure, the results are shown in Table 4.

[0192] Table 4. Production performance detection after alteration of glycosyl receptors.

[0193] Yield (g / L) Molar conversion rate (%) Rhodioloside and maltoheptacoside 125g / L 32.8％ Rhodioloside and maltodextrin 136g / L 31.6％ Rhodioloside and maltodextrin 159g / L 33.2％ α or β-arbutin maltoheptaside 145g / L 38.8％ α or β-arbutin maltooctaglycoside 167g / L 39.6％ α or β-arbutin maltodextrin 177g / L 37.6％ Indigo maltoheptaglycoside 134g / L 35.3％ Indigo maltodextrin 159g / L 37.1％ Indigo maltodextrin 171g / L 35.8％ CNPG7 139g / L 34.6％ CNPG8 165g / L 36.6％ CNPG9 186g / L 37.3％

[0194] The liquid chromatography results for rhodioloside maltoheptaglycoside (glycosyl acceptor: rhodioloside; glycosyl donors: α, β, γ-cyclodextrin) are as follows: Figure 8 As shown, the results indicated that the yield of rhodioloside maltoheptacoside was 125 g / L, with a molar conversion rate of 32.8%. The conversion effects of the cyclodextrinase mutant on rhodioloside maltooctacoside and rhodioloside maltononacoside were also investigated. The results showed that the yield of rhodioloside maltooctacoside was 136 g / L, with a molar conversion rate of 31.6%; the yield of rhodioloside maltononacoside was 159 g / L, with a molar conversion rate of 33.2%.

[0195] Liquid chromatography results for α- or β-arbutin maltoheptaside (glycosyl acceptor is α / β-arbutin; glycosyl donor is α, β, γ-cyclodextrin) are as follows: Figure 9 As shown, the results indicated that the yield of α- or β-arbutin maltoheptacoside was 145 g / L, with a molar conversion rate of 38.8%. The conversion effects of the cyclodextrinase mutant on α- or β-arbutin maltooctacoside and α- or β-arbutin maltononacoside were also examined. The results showed that the yield of α- or β-arbutin maltooctacoside was 167 g / L, with a molar conversion rate of 39.6%; the yield of α- or β-arbutin maltononacoside was 177 g / L, with a molar conversion rate of 37.6%.

[0196] The liquid chromatography results for indoside maltoheptaglycoside (glycosyl acceptor is indoside; glycosyl donors are α, β, and γ-cyclodextrin) are as follows: Figure 10 As shown, the results indicated that the yield of indoside maltoheptacoside was 134 g / L, with a molar conversion rate of 35.3%. The conversion effects of the cyclodextrinase mutant on indoside maltoside octacoside and indoside maltoside nonacoside were examined. The results showed that the yield of indoside maltoside octacoside was 159 g / L, with a molar conversion rate of 37.1%; the yield of indoside maltoside nonacoside was 171 g / L, with a molar conversion rate of 35.8%.

[0197] The liquid chromatography results for CNPG7 (glycosyl acceptor is 2-chloro-4-nitrophenyl-α / β-glucoside; glycosyl donor is α, β, γ-cyclodextrin) are as follows: Figure 11 As shown, the results indicated that the yield of CNPG7 was 139 g / L, and the molar conversion rate of CNPG7 was 34.6%. The conversion effects of the cyclodextrinase mutant on CNPG8 and CNPG9 were also tested. The results showed that the yield of CNPG8 was 165 g / L, and the molar conversion rate of CNPG8 was 36.6%; the yield of CNPG9 was 186 g / L, and the molar conversion rate of CNPG was 37.3%. Example 5: Application of the mutant in the preparation of enzyme preparations

[0198] The cyclodextrinase mutants obtained in Examples 1 and 3 were used to prepare enzyme preparations. Taking the pure enzyme solution of Ps03CDase-WT in Example 1 as an example, the enzyme preparation was prepared.

[0199] 1. Solid enzyme preparations

[0200] The preparation method of freeze-dried enzyme powder is as follows:

[0201] (1) The pure enzyme solution of Ps03CDase-WT was mixed with sucrose, trehalose, and calcium ions to obtain a lyophilized solution (in which 1.5% w / v sucrose, 1.5% w / v trehalose, and 5 mM Ca2+ were added). 2+Ps03CDase-WT 10U / mL); Pour the lyophilized solution into a clean, sterile glass petri dish with a glass bottom diameter of 14cm, ensuring that the liquid level does not exceed 1cm.

[0202] (2) Freeze-dry at -80℃ for 24 hours to obtain freeze-dried enzyme powder.

[0203] 2. Liquid enzyme preparations

[0204] The preparation method of liquid enzyme preparations includes the following steps:

[0205] The pure enzyme solution of Ps03CDase-WT was mixed with glycerol, sodium bisulfite, sodium D-isoascorbate, dipotassium hydrogen phosphate, and sodium benzoate to prepare a liquid enzyme preparation (Ps03CDase-WT 1U / mL, glycerol 60v / v, sodium bisulfite 0.02%v / v, sodium D-isoascorbate 0.5%v / v, sodium benzoate 0.25%v / v, water to 100%).

[0206] All other cyclodextrinase mutants can be prepared into enzyme preparations using the above method.

[0207] Example 6: Application of mutants in the preparation of reagent kits

[0208] 1. Preparation of reagent kit

[0209] The enzyme preparation (solid or liquid) obtained in Example 5 is used to compose the kit, which includes:

[0210] Enzyme preparation, buffer solution (20mM Tris-HCl, pH 7.4, 100mM NaCl), substrate solution (a solution containing cyclodextrin compounds such as β-cyclodextrin, α-cyclodextrin, etc.), standard solution (glucose standard), color indicator (3,5-dinitrosalicylic acid).

[0211] Other buffer solutions (e.g., PBS), standard solutions (e.g., maltose), or color indicators (e.g., Fehling's reagent, Benedict's reagent, tetrazolium blue) may also be used in the above kits without affecting the detection results.

[0212] 2. Reagent kit detection method

[0213] The degradation rate of the substrate was detected using the above-mentioned kit, and the detection method is as follows:

[0214] (1) Prepare standard solutions of different concentrations and detect absorbance at 570 nm to construct standard curves;

[0215] (2) Mix the enzyme preparation and buffer solution to prepare a 0.1 U / mL cyclodextrinase mutant solution, mix it with the substrate solution to be tested (100 μL, 10 mM) at a volume ratio of 1:1, incubate the reaction system at 37 °C for 30 min, detect the absorbance at 570 nm, and calculate the degradation rate of the substrate to be tested based on the standard curve.

[0216] The sequence involved in this invention:

[0217] SEQ ID NO.1 Ps03CDase-WT amino acid sequence

[0218] MIMLEAVYHRMGQNWSYAYNDSTLHIRIRTKRNNVPRIDLHCGDKYNSEKYKETISMERLASDGLFDYWQAAVQPRFRRLVYYFALHSDNGEILYFLEKGFFDQPPKVMYEGLFDFPYLNPQDVHMPPAWVKDAVFYQIFPERFANG DPSNDPEGVQEWGGTPSAGNFFGGDLQGVIDHLDYLSELGINALYFNPLFAATTNHKYDTADYMRIDPHFGTNEKLRELVDACHARGIRVLLDAVFNHCGHTFPPFVDVLNNGPDSRYADWFHIREWPLRVVDGIPTYGTFAFEPIMP KLNTANEEVKAYLLNVGRFWLEEMGLDGWRLDVANEVDHQFWREFRSEIKRINPSAYILGEIMHDSMPWLQGDQFDAVMNYPFTNILLGFFARRLTNAAEFAQAIGTQLAGYPQQVTEVSFNLLGSHDTTRLLTLCGGNVERMKLATL FQLTYLGAPCIYYGDEIGMDGEHDPLNRKCMEWDKSKQNTELLSFFRSMISLRKAHPALRGSGLRFLPVPEHPQLLVYERWDDNERFLFMLNNEDAPAVAAAIPAAHPAASWRTVNSEPYAVVEESAIQVALPPYGYTILHAPAAGTAG

[0219] SEQ ID NO.2 BsCDase-WT amino acid sequence

[0220] MIMLEAVYHRMGQNWSYAYNDSTLHIRIRTKRDNVPRIDLHCGEKYDPEKYKETIPMERMASDGLFDYWQAAVQPRYRRLVYYFALHSDNGDAVYFMEKGFFDQPPKVMYEGLFDFPYLNRQDVHTPPAWVKEAIFYQIFPERFANGDPSNDPEGVQEWGGTPSAGNFFGGDLQGVIDHLDYLSDLGVNALYFNPLFAATTNHKYDTADYMKIDPQFGTNEKLKELVDACHARGMRVLLDAVFNHCGHTFPPFVDVLNNGLNSRYADWFHVREWPLRVVDGIPTYDTFAFEPIMPKLNTGNEEVKAYLLNVGRYWLEEMGLDGWRLDVANEVDHQFWREFRSEIKRINPSAYILGEIMHDSMPWLQGDQFDAVMNYPFTNILLNFFARRLTNAAEFAQAIGTQLAGYPQQVTEVSFNLLGSHDTTRLLTLCSGNVERMKLATLFQLTYQGTPCIYYGDEIGMDGEYDPLNRKCMEWDKSKQNTELLAFFRSMISLRKAHPALRGSGLRFLPVLEHPQLLVYERWDDNERFLIMLNNEDAPVNVVIPAAQPGASWRTVNGEPCAVVEESSIQAALPPYGYAILHAPIAGTAE

[0221] SEQ ID NO.3 Amino acid sequence of Ps92CDase-WT

[0222] MIMLEAVYHRMGQNWSYAYNDSTLHIRIRTKRNNVSHIDLHCGDKYNPEKYKVTISMERLASDELFDYWQAAVQPRFRRLVYYFALHSDNGEILYFMEKGFFDQPPKVMYEGLFDFPYLNPQDVHMPPAWVKDAVFYQIFPERFANGDPSNDPEGVQEWGGTPSAGNFFGGDLQGVIDHLDYLSELGINALYFNPLFAATTNHKYDTADYLRIDPHFGTNEKLKELVDACHARGIRVLLDGVFNHCGHTFPPFVDVLNNGPDSRYADWFHIREWPLRVVDGIPTYGTFAFEPIMPKLNTANEEVKAYLLNVGRFWLEEMGLDGWRLDVANEVDHQFWREFRSEIKRINPSAYILGEIMHDSMPWLQGDQFDAVMNYPFTNILLNFFARRLTNATEFAQAIGTQLAGYPQQVTEVSFNLLGSHDTTRLLTLCGGNVERMKLATLFQLTYQGTPCIYYGDEIGMDGEYDPLNRKCMEWDKSKQNTELLSFFRSMISLRKAHPALRGSGLRFLPTPEHPQLLVYERWDANERFLFMLNNEDAPVVAAIPAALPAASWRTVNSEPYATVEESAIQVTLPPYGYTILHASAAGTAG

[0223] SEQ ID NO.4Bh76CDase-WT Amino Acid Sequence

[0224] MIMLEAVYHRIGQNWSYAYSSKTLHIRIRTKRGNVSRIELICGDKYSWDKFNEVIEMDLLSSDGLFDYWQAAVQPAFRRLVYYFVLYSDHVEPVYYLEKGFYNDPPTVTYEGLFDFPFLNSEDVHMPPEWVKDTVFYQIFPERFANGDLSNDPENTLEWGGTPSPSNFFGGDLQGIMDHLDYLSELGINAIYFNPLFAATTNHKYDTMDYFNVDPHFGTNEKLKELIIACHARGIRVLLDGVFNHCGHTFPPFVDVLANGQSSRYADWFHVHKWPLEVVNGTPTYDTFGFEPIMPKLNTGNDEVKEYLISVGRYWIEEIGIDGWRLDVANEVDHQFWREFRREIKKSNPSAYILGEIMHDSMPWLQGDQFDAVMNYPFTNILLNFFAHRRTNANQFAQAINTQLVSYPQQITEVSFNLLGSHDTVRLLTLCGGNVQRMKLATLFQLTFQGTPCIYYGDEIGLIGQHDPDNRKCMEWDTRKQNLDLFAFFRQMINLRKSHAALRGSGLKFLPLPEHTQILAYERWDDRDRFLFVLNNEDASVSIVIPALQPSKWRSVESPATIHFIDGEVHMSLPPYGYAILHTHEEDYRA

[0225] SEQ ID NO.5 Amino acid sequence of Po68CDase-WT

[0226] MSMIQPEAVYHRMGQNWSYAYDESTLHIRIRTKRDNVSQIDLFCGDKFGWDTTHEVIPMQRLASDGLFDYWQASVRPVYRRLAYYFALYDKDEVLYFLEKGFYPEPPAVVYEGLFDFPFLNREDVHSPPSWVKDAVFYQIFPERFANGDPTNDPTDVLEWGGTPSPRNFFGGDLQGVLDHLDYLCELGINAIYFNPLFAATTNHKYDTKDYYLVDPHFGTNTLLKELVDSCHSKGIRVILDGVFNHCGHTFPPFVDLLANGQSSRYADWFHVREWPPQVIDGIPTYDTFAFEPIMPKFNTENPEVIEYLLNAGRYWIEEIGIDGWRLDVANEVSHSFWRQFCSTIKSINPEAYLVGEIMHDSLPWLLGDQFDAVMNYPLTNIQINFFAHGQINAAQFSQSVATMLANYPQQITEAAFNLLDSHDTVRFLTLCGGDIRKLKLAVLFQMTFQGAPCVYYGDEIGMDGGYDPDNRKCMEWDSQKQNQELFEYYRWTISLRKSHPAFRSSGFKALEIPGYPKLLVYERWDKQSRFIIVLNNEDVMAEVIIPVTGADTIWVNLKTQKRTETSTSQLHLNLPEFGYAVLQAVND

[0227] SEQ ID NO.6 Ps61CDase-WT Amino Acid Sequence

[0228] MNLQAVYHRIKQNWSYAYDAETLHIRLRTARGDVQRVRLLCGDKYDWKRTNMEIDMERGVSDALFDYWEARVRPPYKRLIYRFALHDGEKTVYYSEAGFDTELTPQYYKGAFDFPYLHETDRIAPPEWVKDAVFYQIFPERFANG DPSNDPDGTEPWGGEPTPKNFFGGDLQGVIDRLDYLTELGVNAIYFTPVFEATTNHKYDTSDYFRVDPHFGTNETLKTLVRECHARGIRVLLDAVFNHSGRQFAPFVDVIEKGEESPYKDWFHVRKWPLRIEDGVPTYETFAFEPI MPKLNTSNPEVRDYLLKVASYWMDEIGIDGWRLDVANEVDHQFWREFRQTVKKANPDAYILGEIMHDAMPWLQGDQFDAVMNYPFTNTVIDFFASQEIDSAGFAAAIGALLASYPAQVNEASFNLVGSHDTPRLLTRCGGDRRLAR LAVLFQFTFVGVPCVYYGDEVGLDGGYDPGCRKCMEWDEARQDREQLAFYRALIRLRLSHRALRTGGFRFLQAEPGSSTLAYERWDERERFVILINRGGRQAGFRLPAAGTEWTDALSGEAVAAAGGNLVCSLPPYGYKVLRSIRV

[0229] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A performance-enhanced cyclodextrinase mutant, characterized in that, The cyclodextrinase mutant is based on the amino acid sequence corresponding to the parental cyclodextrinase, with one or more amino acid mutations at any amino acid residue in the peptide region located at 45-52, 107-113, 163-166, 355-360, 374-376, 465-468, or with one or more amino acid mutations at any amino acid residue in the parental cyclodextrinase corresponding to the above-mentioned peptide region equivalent positions; the amino acid sequence of the parental cyclodextrinase has at least 60% identity with the amino acid shown in SEQ ID NO.1 and has cyclodextrinase activity.

2. The cyclodextrinase mutant according to claim 1, characterized in that, Corresponding to the amino acid sequence shown in SEQ ID NO.1, it has one or more of the following amino acid mutations: In the peptide region of 45-52, the 48th amino acid is mutated to W; or, the 49th amino acid is mutated to K or R; In the peptide region of 107-113, the amino acid at position 111 is mutated to K or R; In the peptide region of 163-166, the amino acid at position 165 is mutated to F; In the 355-360 peptide region, the amino acid at position 358 is mutated to F, Y, or I; In the peptide region of 374-376, the amino acid at position 376 is mutated to S or A; In the peptide region of 465-468, the amino acid at position 466 is mutated to K or R; or, the amino acid at position 467 is mutated to E.

3. The cyclodextrinase mutant according to any one of claims 1 to 2, characterized in that, The cyclodextrinase mutant contains at least the following mutations equivalent to or corresponding to the parental cyclodextrinase: E111K and M358F; Alternatively, the cyclodextrinase mutant may simultaneously contain at least the following mutations equivalent to or corresponding to the parental cyclodextrinase: D467E and M358F; Alternatively, the cyclodextrinase mutant may simultaneously contain at least the following mutations equivalent to or corresponding to the parental cyclodextrinase: D467E and M358Y; Alternatively, the cyclodextrinase mutant may simultaneously possess at least the following mutations equivalent to or corresponding to the parental cyclodextrinase: H466K and M358F; Alternatively, the cyclodextrinase mutant may simultaneously contain at least the following mutations equivalent to or corresponding to the parental cyclodextrinase: H466K and M358Y; Alternatively, the cyclodextrinase mutant may simultaneously possess at least the following mutations equivalent to or corresponding to the parental cyclodextrinase: H466R and M358F; Alternatively, the cyclodextrinase mutant may simultaneously possess at least the following mutations equivalent to or corresponding to the parental cyclodextrinase: E111R, H466K, and M358F; Alternatively, the cyclodextrinase mutant may simultaneously possess at least the following mutations equivalent to or corresponding to the parental cyclodextrinase: E111R, H466R, and M358F; Alternatively, the cyclodextrinase mutant may simultaneously possess at least the following mutations equivalent to or corresponding to the parental cyclodextrinase: E111K, H466K, and M358Y; Alternatively, the cyclodextrinase mutant may simultaneously possess at least the following mutations equivalent to or corresponding to the parental cyclodextrinase: E49K, D467E, and M358F; Alternatively, the cyclodextrinase mutant may simultaneously possess at least the following mutations equivalent to or corresponding to the parental cyclodextrinase: S48W, D467E, and M358F.

4. A polynucleotide encoding a cyclodextrinase mutant according to any one of claims 1 to 3.

5. A recombinant vector carrying the polynucleotide of claim 4.

6. Cells expressing any of the cyclodextrinase mutants of claims 1 to 3 or the recombinant vector of claim 5.

7. A method for synthesizing oligomaltodextrin, characterized in that, The cyclodextrinase mutant according to any one of claims 1 to 3 is used; the oligomaltodextrin is synthesized by glycosyl donor and glycosyl acceptor via cyclodextrinase mutant catalysis. Optionally, the oligomaltodextrin contains 6 to 9 glucose units; Optionally, the glycosyl donor includes: α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin; Optionally, the glycosyl receptor includes: p-nitrophenyl-α-D-glucoside, 2-chloro-4-nitrophenyl-α / β-glucoside, α / β-arbutin, and rhodioloside.

8. The use of the cyclodextrinase mutant according to any one of claims 1 to 3 in the preparation of p-nitrophenyl-α-D-maltoheptaglycoside.

9. A method for improving at least one property of cyclodextrinase, characterized in that, Based on the amino acid sequence corresponding to the parental cyclodextrinase, one or more amino acid mutations are present at any amino acid residue in the peptide regions located at 45–52, 107–113, 163–166, 355–360, 374–376, and 465–468, or one or more amino acid mutations occur at any amino acid residue in the parental cyclodextrinase corresponding to the equivalent positions of the above-mentioned peptide regions; the amino acid sequence of the parental cyclodextrinase has at least 60% identity with the amino acid shown in SEQ ID NO.1 and has cyclodextrinase activity; The improvement of at least one performance includes increasing enzyme activity, increasing product specificity, and reducing the hydrolysis rate of the product.

10. The use of the cyclodextrinase mutant according to any one of claims 1 to 3, the polynucleotide according to claim 4, the plasmid vector according to claim 5, or the cell according to claim 6 in the fields of food, biology, medicine, or cosmetics; Optionally, the food sector includes: To prepare modified starch and improve bread quality; Optionally, the biological field includes: preparing enzyme preparations and preparing reagent kit raw materials; Optionally, the pharmaceutical field includes: preparing drug carriers; Optionally, the cosmetics field includes: the preparation of creams, lotions, lotions, facial cleansers, shower gels, and shampoos.

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