WO2024104269A1

WO2024104269A1 - METHOD FOR PREPARING α-AMYLASE ENZYME ACTIVITY TEST SUBSTRATE PNPG7 OR OTHER OLIGOMALTOSIDES BY MEANS OF ENZYMATIC METHOD

Info

Publication number: WO2024104269A1
Application number: PCT/CN2023/130972
Authority: WO
Inventors: 阎振鑫; 祝长斌
Original assignee: 上海瀚诺威生物科技有限公司
Priority date: 2022-11-14
Filing date: 2023-11-10
Publication date: 2024-05-23
Also published as: CN118028404A

Abstract

A method for preparing an α-amylase enzyme activity test substrate pNPG7 or other oligomaltosides by means of an enzymatic method, which belongs to the field of biotechnology. The provided method effectively reduces the production cost of pNPG7 or other oligomaltosides. Maltoheptaose as a glycosyl donor in the route of the prior art is expensive and cannot be supplied in batches. The glycosyl donor required for the present technical route is α-cyclodextrin or β-cyclodextrin or γ-cyclodextrin, which has a price that is about one ten thousandth to one hundred thousandth of the price of maltoheptaose and can be supplied in batches. The provided method significantly improves the yield of pNPG7 or other oligomaltosides, and the yield of pNPG7 or other oligomaltosides reaches 86 mM (109 g/L), which is 15.6 times that reported in the literature, in a 0.2-20 L reaction system. The yield of the target product pNPG7 or other oligomaltosides is significantly improved, while the production cost is reduced, which opens up a brand-new path for promoting the industrial production of pNPG7 or other oligomaltosides.

Description

An enzymatic preparation method for α-amylase activity test substrate pNPG7 or other oligomeric maltosides

The present invention claims priority to a patent application filed on November 14, 2022, with application number 202211424950.8 and invention name “An enzymatic preparation method for an α-amylase activity test substrate pNPG7”.

Technical Field

The invention relates to an enzymatic preparation method of alpha-amylase activity test substrate pNPG7 or other oligomeric maltosides, belonging to the field of biotechnology.

Background technique

The α-amylase activity detection kit is mainly used in the diagnosis of acute pancreatitis. Acute pancreatitis is mainly caused by cholecystitis, which is mainly caused by obesity and a high-fat diet. Due to the current socioeconomic development, obesity and a high-fat diet have become increasingly common social problems. Therefore, the incidence of diseases such as cholecystitis and acute pancreatitis (currently 4.9-73.4/100,000) will increase year by year in the future, and the market demand for α-amylase activity detection will also increase year by year.

p-Nitrophenyl-α-maltoheptaglycoside (pNPG7) is the key substrate of the α-amylase activity test kit and is also the key intermediate for the synthesis of 4,6-ethylene-p-nitrophenyl-α-maltoheptaglycoside (EPS, another substrate of the α-amylase activity test kit). However, the synthesis of pNPG7 is extremely difficult. There are seven glycosidic bonds with specific stereo configurations in the molecular structure of pNPG7. The organic synthesis method is complicated, with low yield, high energy consumption, and serious pollution, making it difficult to achieve the goal of mass production. In 1990, Koichi OGAWA et al. ^[1] published an article introducing an enzymatic synthesis technology route for pNPG7. The article introduced a transglycosylation reaction of an α-amylase that can hydrolyze short-chain starch (DP=23) to produce maltohexaose. This α-amylase can use maltoheptaose as a glycosyl donor and p-nitrophenyl-α-glucoside (pNPG) as an acceptor to produce pNPG7. However, this enzymatic process for producing pNPG7 has the problems of expensive glycosyl donor maltoheptaose (McLean, 937 yuan/100 mg, purity 95%) and low pNPG7 yield (about 5.5 mM), which makes it impossible to produce on a large scale. Therefore, the present invention reports a low-cost and efficient enzymatic process for preparing pNPG7 or other oligomeric maltosides.

[1]. Koichi OGAWA, O.U.T.N., Enzymatic Synthesis of p-Nitrophenyl Maltoheptaoside by Transglycosylation of Maltohexaose forming Amylase. Agric. Bioi. Chern., 1990. 54(3): p.581-586.

Summary of the invention

In order to solve the above technical problems, the present invention has obtained a series of cyclodextrinases with transglycosylation activity through preliminary screening. The present invention further uses the screened cyclodextrinases with transglycosylation activity to prepare pNPG7 or other oligomaltosides, using cyclodextrin as a glycosyl donor and benzene rings, alcohols or glycosides as glycosyl acceptors to synthesize oligomaltosides with a degree of polymerization of 6-9 by transglycosylation in an enzymatic reaction system containing a cyclodextrinase with transglycosylation activity. Specifically, only α, Using β and γ cyclodextrin as glycoside donors and pNPG (p-nitrophenyl-α-glucoside), pNP (p-nitrophenol), other benzene rings (including but not limited to chromophores such as 2-chloro-4-nitrophenol and tetramethylumbelliferone), other glycosides (including but not limited to benzene ring glucosides, click chemistry glucosides, nucleosides, etc.) or alcohols as glycoside acceptors, it is possible to achieve a one-step transglycosylation synthesis of pNPG7 or other oligomaltosides.

The present invention provides a novel enzymatic preparation method of pNPG7 or other oligomeric maltosides. The method screens the transglycosylation reaction of multiple cyclodextrinases with transglycosylation activity, designs a reasonable reaction route, and uses α, β, and γ cyclodextrins as glycosyl donors, pNPG, pNP, other benzene rings (including but not limited to chromophores such as 2-chloro-4-nitrophenol and tetramethylumbelliferone), other glycosides (including but not limited to benzene ring glucosides, click chemistry glucosides, nucleosides, etc.) or alcohols as glycosyl acceptors to synthesize pNPG7 or other oligomeric maltosides by transglycosylation in one step (Figure 1). The starting raw materials of this process route are cheap, the conversion rate is high, and industrial production can be realized.

The present invention provides a cyclodextrinase with transglycosylation activity, wherein the cyclodextrinase is selected from one or more of the cyclodextrinase PpCDase derived from Palaeococcus pacificus DY20341, the cyclodextrinase BsCDase derived from Bacillus sphaericus, the cyclodextrinase Ps03CDase derived from Paenibacillus sp.MY03, the cyclodextrinase Ps92CDase derived from Paenibacillus sp.PAMC21692, the cyclodextrinase TsCDase derived from Thermococcus sp.B1001 and the cyclodextrinase PfCDase derived from Pyrococcus furiosus DSM 3638.

The gene sequence of the cyclodextrinase PpCDase is NCBI Reference Sequence: NZ_CP006019.1 (gene 875910-877907), and the amino acid sequence is NCBI Reference Sequence: WP_048164969.1; the gene sequence of the cyclodextrinase TsCDase is GenBank: AB034969.2 (gene 248-2230), and the amino acid sequence is GenBank: BAB18100.1; the gene sequence of the cyclodextrinase BsCDase is GenBank: X62576.1, and the amino acid sequence is UniProtKB/Swiss-Prot: Q08341.1; the gene sequence of the cyclodextrinase PfCDase is NCBI Reference Sequence: NC_003413.1 (gene 1790387-1792324), amino acid sequence NCBI Reference Sequence: WP_011013079.1; the gene sequence of the cyclodextrinase Ps03CDase NCBI Reference Sequence: NZ_MXQD01000009.1 (gene 101836-103611), amino acid sequence NCBI Reference Sequence: WP_087568083.1; the gene sequence of the cyclodextrinase Ps92CDase GenBank: CP060293.1 (gene 6259030-6260805), amino acid sequence NCBI Reference Sequence: WP_187105536.1.

The present invention also provides a method for preparing oligomeric maltosides with a degree of polymerization of 6-9 by enzymatic method, wherein cyclodextrin is used as a glycosyl donor, benzene rings, alcohols or glycosides are used as glycosyl acceptors, and oligomeric maltosides with a degree of polymerization of 6-9 are synthesized by transglycosylation in an enzymatic reaction system containing a cyclodextrinase having transglycosylation activity, especially the production of pNPG7. Specifically, the method comprises (a), (b) or (c):

(a) Using α-cyclodextrin as the glycosyl donor, p-nitrophenyl-α-glucoside, p-nitrophenol, other benzene rings, alcohol The glycosides or other glycosides are used as glycosyl acceptors, and pNPG7 or other oligomeric maltosides with a degree of polymerization of 6-7 are synthesized by transglycosylation in an enzymatic reaction system containing a cyclodextrinase having transglycosylation activity;

(b) using β-cyclodextrin as a glycosyl donor and p-nitrophenyl-α-glucoside, p-nitrophenol, other benzene rings, alcohols or other glycosides as glycosyl acceptors, and synthesizing pNPG7 or other oligomeric maltosides with a degree of polymerization of 7-8 by transglycosylation in an enzymatic reaction system containing a cyclodextrinase having transglycosylation activity;

(c) Using γ-cyclodextrin as a glycosyl donor and p-nitrophenyl-α-glucoside, p-nitrophenol, other benzene rings, alcohols or other glycosides as glycosyl acceptors, pNPG7 or other oligomaltosides with a degree of polymerization of 8-9 are synthesized by transglycosylation in an enzymatic reaction system containing a cyclodextrinase having transglycosylation activity. It should be noted that the glycosyl acceptors of other glycosides refer to glycoside glycosyl acceptors other than p-nitrophenyl-α-glucoside. The glycosyl acceptors of other benzene rings refer to benzene ring glycosyl acceptors other than p-nitrophenol.

In some embodiments, the glycosyl acceptor includes p-nitrophenyl-α-glucoside, p-nitrophenol, other benzene rings, alcohols or glycosides. Among them, the acceptor that does not contain a glycosyl group on the glycosyl acceptor itself includes benzene rings or alcohols; the acceptor that contains a glycosyl group on the glycosyl acceptor itself includes glycosides;

In some embodiments, the degree of polymerization of pNPG7 or other oligomaltoside synthesized by transglycosylation is 6-9. Specifically, when the glycosyl donor is α-cyclodextrin, the degree of polymerization of the obtained oligomaltoside is 6-7; when the glycosyl donor is β-cyclodextrin, the degree of polymerization of the obtained oligomaltoside is 7-8; when the glycosyl donor is γ-cyclodextrin, the degree of polymerization of the obtained oligomaltoside is 8-9.

In some embodiments, α-cyclodextrin (α-CD) is used as a glycosyl donor, p-nitrophenol (pNP) is used as a glycosyl acceptor, and pNPG6 is synthesized by one-step transglycosylation in an enzymatic reaction system containing the cyclodextrinase;

In some embodiments, β-cyclodextrin (β-CD) is used as a glycosyl donor, p-nitrophenyl-α-glucoside (pNPG) is used as a glycosyl acceptor, and pNPG8 is synthesized by one-step transglycosylation in an enzymatic reaction system containing the cyclodextrinase;

In some embodiments, γ-cyclodextrin (γ-CD) is used as a glycosyl donor and p-nitrophenol (pNP) is used as a glycosyl acceptor, and pNPG8 is synthesized by a one-step transglycosylation in an enzymatic reaction system containing the cyclodextrinase; or p-nitrophenyl-α-glucoside (pNPG) is used as a glycosyl acceptor, and pNPG9 is synthesized by a one-step transglycosylation in an enzymatic reaction system containing the cyclodextrinase.

In some embodiments, the glycoside is selected from one or more of phenyl ring glucoside, click chemistry glucoside, and nucleoside.

In some embodiments, the benzene ring glucoside is selected from one or more of chromophore-containing glucoside and benzene ring natural product glucoside.

In some embodiments, the chromophore-containing glucoside is selected from one or more of p-nitrobenzene-α/β-glucoside, 2-chloro-4-nitrobenzene-α/β-glucoside, and tetramethylumbelliferyl-α/β-glucoside, and the transglycosylation product is p-nitrobenzene-α/β-maltose. One or more of maltoheptaglycoside, p-nitrobenzene-α/β-maltooctaglycoside, p-nitrobenzene-α/β-maltononaglycoside, 2-chloro-4-nitrobenzene-α/β-maltoheptaglycoside, 2-chloro-4-nitrobenzene-α/β-maltooctaglycoside, 2-chloro-4-nitrobenzene-α/β-maltononaglycoside, tetramethylumbelliferyl-α/β-maltoheptaglycoside, tetramethylumbelliferyl-α/β-maltooctaglycoside, and tetramethylumbelliferyl-α/β-maltononaglycoside. It should be noted that p-nitrobenzene-α/β-glucoside is p-nitrobenzene-α-glucoside or p-nitrobenzene-β-glucoside, and the explanations for other α/β descriptions are the same as here.

In some embodiments, the p-nitrobenzene-α/β-glucoside is selected from p-nitrobenzene-α-D-glucoside or p-nitrobenzene-β-D-glucoside. The transglycosylation product is p-nitrobenzene-α-D-maltoheptaglycoside, p-nitrobenzene-α-D-maltooctaglycoside, p-nitrobenzene-α-D-maltononaglycoside, p-nitrobenzene-β-D-maltoheptaglycoside, p-nitrobenzene-β-D-maltooctaglycoside, and p-nitrobenzene-β-D-maltononaglycoside.

In some embodiments, the benzene ring natural product glucoside is selected from one or more of α/β-arbutin, salidroside, and indigoside, and the transglycosylation product is one or more of α/β-arbutin maltoheptaglycoside, α/β-arbutin maltooctaglycoside, α/β-arbutin maltononaglycoside, salidroside maltoheptaglycoside, salidroside maltooctaglycoside, salidroside maltononaglycoside, indigoside maltoheptaglycoside, indigoside maltooctaglycoside, and indigoside maltononaglycoside.

In some embodiments, the indigoside is selected from indolyl-β-glucoside, and the transglycosylation product is one or more of indolyl-β-maltoheptaside, indolyl-β-maltooctaside and indolyl-β-maltononaside.

In some embodiments, the click chemistry glucoside is selected from, for example, azido-PEGn-glucose, propargyl-PEGm-glucose (n, m are positive integers), etc., and the transglycosylation product is one or more of azido-PEGn-maltoheptaglycoside, azido-PEGn-maltooctaglycoside, azido-PEGn-maltononaglycoside, propargyl-PEGm-maltoheptaglycoside, propargyl-PEGm-maltooctaglycoside and propargyl-PEGm-maltononaglycoside.

In some embodiments, the nucleoside is selected from one or more of cytarabine and dooxyfluridine, and the transglycosylation product is one or more of cytarabine maltohexaosyl, cytarabine maltoheptaosyl, cytarabine maltooctaosyl, dooxyfluridine maltohexaosyl, dooxyfluridine maltoheptaosyl, and dooxyfluridine maltooctaosyl.

In some embodiments, the benzene ring glycosyl acceptor is selected from one or more of p-nitrophenol, 2-chloro-4-nitrophenol, and tetramethylumbelliferone, and the transglycosylation product is one or more of p-nitrophenyl-α-maltohexaosyl, p-nitrophenyl-α-maltoheptaosyl, and p-nitrophenyl-α-maltooctaosyl, 2-chloro-4-nitrobenzene-α-maltohexaosyl, 2-chloro-4-nitrobenzene-α-maltoheptaosyl, 2-chloro-4-nitrobenzene-α-maltooctaosyl, tetramethylumbelliferone-α-maltohexaosyl, tetramethylumbelliferone-α-maltoheptaosyl, and tetramethylumbelliferone-α-maltooctaosyl.

In some embodiments, the alcohol glycosyl acceptor is selected from one or more of methanol, ethanol, n-propanol, isopropanol, and n-butanol, and the transglycosylation product is one or more of the corresponding alkane maltohexaglycoside, alkane maltoheptaglycoside, and alkane maltooctaglycoside.

In some embodiments, the cyclodextrinase is an immobilized enzyme, a liquid enzyme, or a cell expressing the cyclodextrinase.

In some embodiments, the cell expressing the cyclodextrinase uses Escherichia coli, Bacillus subtilis, Pichia pastoris or Saccharomyces cerevisiae as a host cell.

In some embodiments, the cell expressing the cyclodextrinase uses the pET series as a vector.

In some embodiments, the cell expressing the cyclodextrinase uses pET28a or pET32a as a vector.

In some embodiments, the enzymatic reaction system further includes a pH regulator.

In some embodiments, the concentration of α-cyclodextrin (α-CD) in the enzymatic reaction system is 1-50%, the concentration of β-cyclodextrin (β-CD) is 1-30%, and the concentration of γ-cyclodextrin (γ-CD) is 1-30%. The present invention has found through experiments that the solubility of the substance can be improved by high temperature, ultrasound, etc. At high concentrations, the yield is further improved, so the concentration of the receptor is also further improved. Optionally, the concentration of α-cyclodextrin (α-CD) can be 1-20%, 20-40%, or 40-50%, etc. The concentration of β-cyclodextrin (β-CD) can be, for example, 1-5%, 5-10%, or 10-30%, etc. The concentration of γ-cyclodextrin (γ-CD) can be, for example, 1-5%, 5-10%, or 10-30%, etc. Preferably, the concentration of α-cyclodextrin is 20-40%, the concentration of β-cyclodextrin is 5-10%, and the concentration of γ-cyclodextrin is 5-10%.

In some embodiments, the concentration of the glycosyl acceptor in the enzymatic reaction system is 5-500mmol/L. Preferably, the concentration of the glycoside glycosyl acceptor in the enzymatic reaction system is 10-500mmol/L, the concentration of the benzene ring glycosyl acceptor is 5-500mmol/L, and the concentration of the alcohol is 5%-30% (w/v). Further preferably, the concentration of p-nitrophenyl-α-glucoside (pNPG) in the enzymatic reaction system is 10-500mmol/L, the concentration of p-nitrophenol (pNP) is 5-500mmol/L, the concentration of other glycosides is 5-500mmol/L, the concentration of other benzene rings is 5-500mmol/L, and the concentration of alcohol is 5%-30% (w/v). Optionally, the concentration of pNPG in the enzymatic reaction system can be, for example, 10-50mmol/L, 50-400mmol/L or 400-500mmol/L, etc. The concentration of pNP can be, for example, 5-20 mmol/L, 20-200 mmol/L, or 200-500 mmol/L, etc. The concentration of other glycosides can be, for example, 5-20 mmol/L, 20-200 mmol/L, or 200-500 mmol/L, etc. The concentration of other benzene rings can be 5-20 mmol/L, 20-200 mmol/L, or 200-500 mmol/L, etc. The concentration of alcohols can be, for example, 5%-10% (w/v), 10%-20% (w/v), or 20%-30% (w/v), etc.

In some embodiments, the pNPG concentration in the enzymatic reaction system is 50-400 mmol/L, and the pNP concentration is 20-200 mmol/L.

In some embodiments, the cyclodextrinase in the enzymatic reaction system includes a liquid enzyme or an immobilized enzyme with a concentration of 100-5000 KU/L. Alternatively, the concentration of the liquid enzyme or immobilized enzyme in the enzymatic reaction system may be, for example, 100-600 KU/L, 600-1600 KU/L or 1600-5000 KU/L.

In some embodiments, the concentration of the liquid enzyme or immobilized enzyme in the enzymatic reaction system is 600-1600 KU/L.

In some embodiments, the pH of the enzymatic reaction system is 6-8.

In some embodiments, when the cyclodextrinase is a mesophilic enzyme, the pH is 6; when the cyclodextrinase is a thermophilic enzyme, the pH is 7.5.

In some embodiments, the reaction temperature of the enzymatic reaction system is 20-95°C.

In some embodiments, when the cyclodextrinase is a mesophilic enzyme, the reaction temperature is 20-45°C; when the cyclodextrinase is a thermophilic enzyme, the reaction temperature is 70-95°C.

In some embodiments, when the cyclodextrinase is a mesophilic enzyme, the reaction temperature is 40°C; when the cyclodextrinase is a thermophilic enzyme, the reaction temperature is 85°C.

In some embodiments, the reaction time of the enzymatic reaction system is 2-30 hours.

In some embodiments, when the cyclodextrinase is a mesophilic enzyme, the reaction time is 6 hours; when the cyclodextrinase is a thermophilic enzyme, the reaction time is 24 hours.

The present invention provides the use of the cyclodextrinase or the method in preparing an oligomeric maltoside product containing a degree of polymerization of 6-9, in particular, the use of the product of pNPG7.

In some embodiments, the cyclodextrinase with transglycosylation activity is selected from one or more of the cyclodextrinase PpCDase derived from Palaeococcus pacificus DY20341, the cyclodextrinase BsCDase derived from Bacillus.sphaericus, the cyclodextrinase Ps03CDase derived from Paenibacillus sp.MY03, the cyclodextrinase Ps92CDase derived from Paenibacillus sp.PAMC21692, the cyclodextrinase TsCDase derived from Thermococcus sp.B1001, and the cyclodextrinase PfCDase derived from Pyrococcus furiosus DSM 3638.

The present invention also provides a method for detecting the transglycosylation activity of cyclodextrinase, which comprises adding a glycosyl donor and a glycosyl acceptor to a reaction system containing cyclodextrinase to carry out a transglycosylation reaction, and confirming the transglycosylation product.

In some embodiments, cyclodextrin is used as the glycosyl donor, and benzene rings, alcohols or glycosides are used as the glycosyl acceptors.

In some embodiments, α, β or γ-cyclodextrin is used as a glycosyl donor, and p-nitrophenyl-α-glucoside, p-nitrophenol, other glycosides or alcohols are used as glycosyl acceptors; specifically, α-cyclodextrin is used as a glycosyl donor, and p-nitrophenyl-α-glucoside, p-nitrophenol, other glycosides or alcohols are used as glycosyl acceptors; or, β-cyclodextrin is used as a glycosyl donor, and p-nitrophenyl-α-glucoside, p-nitrophenol, other glycosides or alcohols are used as glycosyl acceptors; or, γ-cyclodextrin is used as a glycosyl donor, and p-nitrophenyl-α-glucoside, p-nitrophenol, other glycosides or alcohols are used as glycosyl acceptors. Among them, the selection of other glycosides or alcohols is the same as described in the method for preparing pNPG7 or other oligomaltosides by enzymatic method of the present invention.

The concentration of the glycosyl donor is 5-10%, and the glycosyl donor can be any one of α-cyclodextrin, β-cyclodextrin or γ-cyclodextrin. The concentration of the glycosyl acceptor is greater than 5mmol/L.

In some embodiments, the cyclodextrinase has α-, β-, or γ-cyclodextrin hydrolysis activity.

The present invention also provides a method for preparing pNPG7 by enzymatic method, the method comprising (a) or (b):

(a) using α-cyclodextrin (α-CD) as a glycosyl donor and p-nitrophenyl-α-D-glucoside (pNPG) as a glycosyl acceptor, and synthesizing pNPG7 by one-step transglycosylation in an enzymatic reaction system containing the cyclodextrinase;

(b) using β-cyclodextrin (β-CD) as a glycosyl donor and p-nitrophenol (pNP) as a glycosyl acceptor, pNPG7 is synthesized by one-step transglycosylation in an enzymatic reaction system containing the cyclodextrinase.

In one embodiment, the cyclodextrinase is an immobilized enzyme, a liquid enzyme, or a cell expressing the cyclodextrinase.

In one embodiment, the cell expressing the cyclodextrinase uses Escherichia coli, Bacillus subtilis, Pichia pastoris or Saccharomyces cerevisiae as a host cell.

In one embodiment, the cell expressing the cyclodextrinase uses the pET series as a vector.

In one embodiment, the cell expressing the cyclodextrinase uses pET28a or pET32a as a vector.

In one embodiment, the enzymatic reaction system further includes a pH regulator.

In one embodiment, the concentration of α-CD in the enzymatic reaction system is 1-14%, and the concentration of β-CD is 1-3%.

In one embodiment, the concentration of p-nitrophenyl-α-D-glucoside (pNPG) in the enzymatic reaction system is 10-180 mmol/L, and the concentration of p-nitrophenol (pNP) is 5-100 mmol/L.

In one embodiment, the pNPG concentration in the enzymatic reaction system is 50-140 mmol/L, and the pNP concentration is 20-45 mmol/L.

In one embodiment, the concentration of the liquid enzyme or immobilized enzyme in the enzymatic reaction system is 100-5000 KU/L.

In one embodiment, the concentration of the liquid enzyme or immobilized enzyme in the enzymatic reaction system is 600-1600 KU/L.

In one embodiment, the pH of the enzymatic reaction system is 6-8.

In one embodiment, when the cyclodextrinase is a mesophilic enzyme, the pH is 6; when the cyclodextrinase is a thermophilic enzyme, the pH is 7.5.

In one embodiment, the reaction temperature of the enzymatic reaction system is 20-95°C.

In one embodiment, when the cyclodextrinase is a mesophilic enzyme, the reaction temperature is 20-45°C; when the cyclodextrinase is a thermophilic enzyme, the reaction temperature is 70-95°C.

In one embodiment, when the cyclodextrinase is a mesophilic enzyme, the reaction temperature is 40°C; when the cyclodextrinase is a thermophilic enzyme, the reaction temperature is 85°C.

In one embodiment, the reaction time of the enzymatic reaction system is 2-30 hours.

In one embodiment, when the cyclodextrinase is a mesophilic enzyme, the reaction time is 6 hours; when the cyclodextrinase is a thermophilic enzyme, the reaction time is 24 hours.

The present invention provides application of the cyclodextrinase or the method in preparing a product containing pNPG7.

In one embodiment, the cyclodextrinase having transglycosylation activity comprises a cyclodextrinase derived from Palaeococcus pacificus Any one of the cyclodextrinase PpCDase derived from DY20341, the cyclodextrinase TsCDase derived from Thermococcus sp. B1001, the cyclodextrinase BsCDase derived from Bacillus. sphaericus, the cyclodextrinase PfCDase derived from Pyrococcus furiosus DSM 3638, the cyclodextrinase Ps03CDase derived from Paenibacillus sp. MY03, or the cyclodextrinase Ps92CDase derived from Paenibacillus sp. PAMC21692.

The present invention also provides a method for detecting the transglycosylation activity of cyclodextrinase, wherein the method comprises adding a glycosyl donor and a glycosyl acceptor into a reaction system containing cyclodextrinase to carry out a transglycosylation reaction.

In one embodiment, the transglycosylation reaction uses α-cyclodextrin as a glycosyl donor and p-nitrophenyl-α-D-glucoside as a glycosyl acceptor;

Alternatively, the transglycosylation reaction uses β-cyclodextrin as a glycosyl donor and p-nitrophenol as a glycosyl acceptor.

In one embodiment, the concentration of p-nitrophenyl-α-D-glucoside is greater than 10 mmol/L, and the concentration of p-nitrophenol is greater than 5 mmol/L.

In one embodiment, the cyclodextrinase has α- or β-cyclodextrin hydrolyzing activity.

Beneficial effects:

The present invention, through understanding the reaction mechanism of cyclodextrinase, designs a new enzymatic synthesis route of pNPG7 or other oligomeric maltosides (Figure 1), greatly reduces the cost of raw materials, obtains cyclodextrinase with high transglycosylation activity through large-scale new enzyme screening, greatly improves the yield of pNPG7 or other oligomeric maltosides, and opens up a new path for the industrial production of pNPG7 or other oligomeric maltosides.

1. The present invention effectively reduces the production cost of pNPG7 or other oligomeric maltosides. The glycosyl donors of the existing technical route, such as maltoheptaose and maltopentaose, are expensive and cannot be supplied in bulk. The current market price of maltoheptaose with a purity of 95% is 937 yuan/100 mg for McLean and 900 yuan/100 mg for source leaves, and there is no bulk supplier. The glycosyl donors required for this technical route are three cyclodextrins (cyclic), specifically α-CD, β-CD and γ-CD, α-CD is about 200 yuan/kg, β-CD is about 30 yuan/kg, and its price is about one ten-thousandth to one hundred-thousandth of maltoheptaose, and it can be supplied in bulk, with a stable supply and a relatively low price.

2. The present invention significantly increases the yield of pNPG7 or other oligomaltosides. Existing literature reports that the reaction system is only 10mL, and the maximum pNPG7 yield is 5.5mM; the new process proposed in this patent (Example 6) has a yield of 86mM (109g/L) in a 0.2L reaction system, which is 15.6 times that reported in the literature. While reducing production costs, the present invention significantly increases the yield of the target product pNPG7, promotes the development of the pNPG7 industry, and promotes the optimization of the production capacity of pNPG7 derivatives and their downstream products.

3. It is known to those skilled in the art and the general consensus in the industry that the transglycosylation reaction can generally only achieve one reaction to transfer one sugar to the receptor, and the obtained sugar is a monosaccharide. If you want to obtain oligomaltosides such as pNPG7, you need to obtain pNPG1 first. Then pNPG2, pNPG3, pNPG4, pNPG5, pNPG6 are obtained in sequence, and then pNPG7 can be obtained. The present invention has found through a large number of experimental studies that at least 6 glucose units can be transferred to the glycosyl acceptor by using the specific glycosyl donors α, β, and γ cyclodextrin of the present invention, and for the first time, the one-time synthesis of oligomeric maltosides with a degree of polymerization of 6-9 is achieved using cyclodextrin as a glycosyl donor.

4. The starting glycosyl acceptor of the present invention may have one glycosyl unit or may have no glycosyl unit, that is, the acceptor of the present invention may be selected in a variety of ways, such as glycosides, benzene rings, alcohols, etc.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Reaction scheme of pNPG7 or other oligomaltosides.

Figure 2. TLC (A) and HPLC (B) detection of cyclodextrinase transglycosylation activity.

Figure 3. Optimal pH and pH stability of cyclodextrinase. A is the optimal pH of Ps03Cdase, B is the optimal pH of PpCDase, C is the pH stability of Ps03Cdase, and D is the pH stability of PpCDase.

Figure 4. Optimal temperature and temperature stability of cyclodextrinase. A is the optimal temperature of Ps03CDase, B is the optimal temperature of PpCDase, C is the temperature stability of Ps03CDase, and D is the temperature stability of PpCDase.

Figure 5. Optimal α-CD concentration (A) and optimal pNPG concentration (B) for cyclodextrin enzymatic transglycosylation reaction.

Figure 6. Cyclodextrin enzymatic transglycosylation reaction increases α-CD concentration (A) and pNPG concentration (B).

Figure 7. Optimal enzyme activity concentration (A) and reaction time (Ps03CDase, B and PpCDase, C) in transglycosylation reactions.

Figure 8. Enzyme activity concentration (A) and reaction time (Ps03CDase, B) in transglycosylation reactions.

Figure 9. Reaction scheme for enzymatic production of pNPG7.

Figure 10. HPLC analysis of the reaction solution of pNPG7 prepared by enzymatic method, P1 is the target product of pNPG7.

Figure 11. TLC analysis of pNPG7 after separation and purification, P1 is pNPG7 after separation and purification, and ST is the standard.

Figure 12. Mass spectrometry analysis of pNPG7. The molecular ion peak of 1296.5 is consistent with the molecular ion peak of pNPG7+Na ⁺ .

Figure 13. H-NMR analysis of pNPG7.

Figure 14. Reaction scheme for the enzymatic production of pNPG7 using pNP and β-cyclodextrin as substrates.

Figure 15. HPLC analysis of salidroside maltoheptaside produced by cyclodextrinase transglycosylation.

Figure 16. HPLC analysis of cyclodextrinase transglycosylation to produce arbutin maltoheptaglycoside, with α-arbutin as the receptor in Figure A and β-arbutin as the receptor in Figure B.

Figure 17. HPLC analysis of cyclodextrinase transglycosylation to produce indigoside maltoheptaglycoside.

Figure 18. HPLC analysis of CNPG7 produced by cyclodextrin enzymatic transglycosylation.

Figure 19. HPLC analysis of cyclodextrinase transglycosylation to produce 4-MUG7.

Figure 20. HPLC analysis of cyclodextrinase transglycosylation to produce ethyl maltohexaoside.

Figure 21. HPLC analysis of cyclodextrinase transglycosylation to produce azido-PEG4-β-glucose maltoheptaside.

Detailed ways

The epoxy resin involved in the present invention was purchased from Xi'an Lanxiao Technology New Materials Co., Ltd., the DNS reagent involved in the present invention was purchased from Solebow Biotechnology Co., Ltd., and common reagents such as LB culture medium, antibiotics and IPTG were purchased from Zishenggong (Shanghai) Bioengineering Co., Ltd., and all chemical reagents used were of analytical grade.

Enzyme activity assay method:

Standard enzyme activity assay system: The enzyme activity of cyclodextrinase is expressed by detecting the reducing power of oligosaccharides released by cyclodextrinase hydrolyzing α-CD using DNS reagent.

The specific reaction system is as follows: 10% α-CD is dissolved in 50 mM Na ₂ HPO ₄ /NaH ₂ PO ₄ buffer at pH 7.0, an appropriate amount of immobilized cyclodextrinase prepared in Example 3 is added, reacted at 37°C for 15 min, an appropriate amount of DNS solution is added, heated at 99°C for 5 min, and the absorbance is measured at a wavelength of 540 nm.

In the present invention, a compound whose name does not indicate a specific configuration includes all configurations of the compound, and a compound whose name contains a configuration specifically refers to the designated configuration.

In the present invention, cyclodextrin (CD) is a general term for a series of cyclic oligosaccharides usually containing 6 to 12 D-pyranose units. Molecules containing 6, 7, and 8 glucose units are respectively called α-, β-, and γ-cyclodextrin.

Phenyl ring sugar acceptors refer to compounds with a benzene ring or a benzene ring in parallel, and do not contain sugar groups themselves. The following are several examples of benzyl ring sugar acceptors: chromophores such as p-nitrophenol, 2-chloro-4-nitrophenol, and tetramethylumbelliferone;

Alcohol glycosyl acceptors refer to glycosyl acceptors that have short-chain alcohols that are water-soluble or partially water-soluble and do not contain glycosyl groups themselves. The following are several examples of alcohol glycosyl acceptors: methanol, ethanol, n-propanol, isopropanol, n-butanol, etc.

Glycoside glycoside acceptors refer to glycoside acceptors composed of monosaccharides and non-sugar parts connected by glycosidic bonds. The following are several examples of glycoside glycoside acceptors: benzene ring glucosides, in which the monosaccharide part is glucose and the non-sugar part is a compound having a benzene ring or a compound connected with benzene. The benzene ring glucosides include glucosides containing chromophores such as p-nitrophenol, 2-chloro-4-nitrophenol, and tetramethylumbelliferone, and also include benzene ring natural product glucosides such as arbutin, salidroside, and indigoside;

Another example of a glycoside sugar acceptor is a "click chemistry glucoside", in which the monosaccharide portion is glucose, and the non-sugar portion is an azido or alkynyl group, or an azido or alkynyl group with a linker, that is, a "click chemistry glucoside" refers to an azide- or alkynyl-modified glucoside; specific examples of "click chemistry glucoside" include azido-PEGn-glucose, propargyl-PEGm-glucose (n, m are positive integers), etc.

Another example of glycoside sugar acceptor is nucleoside, wherein the monosaccharide portion is ribose or deoxyribose, and the non-sugar portion is a purine or pyrimidine base. Specific examples of nucleoside include cytarabine, doxifluridine, and the like.

Oligomeric maltoside: refers to maltoside with a maltose polymerization degree of 6-9 produced by the transglycosylation reaction of the above-mentioned benzene ring, alcohol or glycoside sugar acceptors with cyclodextrin.

Cyclodextrinase CDase refers to an enzyme that uses cyclodextrin as a substrate, especially α, β, γ-cyclodextrin as a substrate, to catalyze the hydrolysis of glycosidic bonds, and similar enzymes with the same catalytic function.

The present invention provides a method for preparing pNPG7, pNPG8 and pNPG9 on a large scale by using α-, β- or γ-cyclodextrin as a glycosyl donor and pNPG as a glycosyl acceptor;

In the method provided by the present invention, the glycosyl acceptor can also be a benzene ring glucoside, such as 2-chloro-4-nitrobenzene-α/β-glucoside, α/β-arbutin, salidroside, indigoside, etc., and the transglycosylation product is 2-chloro-4-nitrobenzene-α/β-maltoheptaglycoside, 2-chloro-4-nitrobenzene-α/β-maltooctaglycoside, 2-chloro-4-nitrobenzene-α/β-maltononaglycoside, α/β-arbutin maltoheptaglycoside, α/β-arbutin maltooctaglycoside, α/β-arbutin maltononaglycoside, salidroside maltoheptaglycoside, salidroside maltooctaglycoside, salidroside maltononaglycoside, indigoside maltoheptaglycoside, indigoside maltooctaglycoside and indigoside maltononaglycoside. One or more. Furthermore, the transglycosylation products of indigoside, such as indolyl-β-glucoside, are indolyl-β-maltoheptaside, indolyl-β-maltooctaside and indolyl-β-maltononaside.

In the method provided by the present invention, the glycosyl acceptor can also be a click chemistry glucoside, for example, selected from azido-PEGn-glucose, propargyl-PEGm-glucose (n, m are positive integers), etc., and the transglycosylation product is azido-PEGn-maltoheptaglycoside, azido-PEGn-maltooctaglycoside and azido-PEGn-maltononaglycoside, propargyl-PEGm-maltoheptaglycoside, propargyl-PEGm-maltooctaglycoside and propargyl-PEGm-maltononaglycoside, etc.

In the method provided by the present invention, the glycosyl acceptor can also be a nucleoside, such as cytarabine, doxorubicin, etc., and the transglycosylation product is cytarabine maltoheptaglycoside, cytarabine maltooctaglycoside, cytarabine maltononaglycoside, doxorubicin maltoheptaglycoside, doxorubicin maltooctaglycoside, doxorubicin maltononaglycoside, etc.

In the method provided by the present invention, the glycosyl acceptor can also be alcohols, such as methanol, ethanol, n-propanol, isopropanol, n-butanol, etc., and the transglycosylation products are the corresponding alkane maltohexaglycoside, alkane maltoheptaglycoside and alkane maltooctaglycoside.

In the method provided by the present invention, the glycosyl acceptor can also be a benzene ring glycosyl acceptor, for example, one or more of p-nitrophenol, 2-chloro-4-nitrophenol, and tetramethylumbelliferone, and the transglycosylation product is one or more of p-nitrophenyl-α-maltohexaosyl, p-nitrophenyl-α-maltoheptaosyl, and p-nitrophenyl-α-maltooctaosyl, 2-chloro-4-nitrobenzene-α-maltohexaosyl, 2-chloro-4-nitrobenzene-α-maltoheptaosyl, 2-chloro-4-nitrobenzene-α-maltooctaosyl, tetramethylumbelliferone-α/β-maltohexaosyl, tetramethylumbelliferone-α/β-maltoheptaosyl, and tetramethylumbelliferone-α/β-maltooctaosyl.

Example 1: Screening of cyclodextrinase with transglycosylation activity

(1) Preparation of crude enzyme solution

The gene encoding cyclodextrinase was constructed into pET28a or pET32a prokaryotic expression vector and transformed into Escherichia coli A series of recombinant bacteria expressing cyclodextrinase were obtained from the strain BL21 (DE3), and the recombinant bacteria were inoculated into LB medium without antibiotics, and cultured for 2 hours at 37°C and 200rpm. When the OD ₆₀₀ was about 0.6, an IPTG (isopropylthiogalactoside) inducer with a final concentration of 0.1-1mM was added, and the temperature was lowered to 12-22°C, the speed was lowered to 150rpm, and the induction culture was carried out for 24 hours. Then, the bacterial precipitate was collected by centrifugation at 3000-10000rpm for 2-10 minutes. Resuspend the cells with _50mM _Na2HPO4 / _NaH2PO4 buffer at pH 6.0-8.0, then centrifuge at _{3000-10000rpm} for 2-10min to collect the bacterial precipitate again, resuspend the cells with an appropriate amount of 50mM _Na2HPO4 / _NaH2PO4 buffer at pH _6.0-8.0 , break the cells by ultrasonic or _homogenizer , centrifuge at 12000rpm for 20min, and collect the supernatant as the crude cyclodextrinase solution.

(2) Screening of cyclodextrinase with transglycosylation activity

The crude cyclodextrinase solution prepared in step (1) is used to detect the hydrolysis activity of the cyclodextrinase. The cyclodextrinase having α-, β- or γ-cyclodextrin hydrolysis activity can be used to further screen and identify the transglycosylation activity.

A suitable amount of active cyclodextrinase was tested for transglycosylation: in 100mM Tris-HCl at pH 7.5, 5% (w/v) α, β or γ cyclodextrin was used as the glycosyl donor, 100mM pNPG or 40mM pNP was used as the glycosyl acceptor, and the reaction was carried out at 40°C for 2-12h. The transglycosylation product was detected by TLC and HPLC. As shown in Figure 2, the product spot was detected in the transglycosylation product area of TLC, and the product peak was detected in the transglycosylation product area of HPLC. To further confirm the transglycosylation product, the molecular weight of the product must be detected by LC-MS to confirm that it is a transglycosylation product. Cyclodextrinase with transglycosylation activity can only be detected in an artificial environment with a high concentration of glycosyl acceptors. The higher the concentration of glycosyl acceptors, the more obvious its transglycosylation activity. It has been verified that the following cyclodextrinases have transglycosylation activity:

For example, the cyclodextrinase PpCDase from Palaeococcus pacificus DY20341, gene sequence NCBI Reference Sequence: NZ_CP006019.1 (gene 875910-877907), amino acid sequence NCBI Reference Sequence: WP_048164969.1; the cyclodextrinase BsCDase from Bacillus sphaericus, gene sequence GenBank: X62576.1, amino acid sequence UniProtKB/Swiss-Prot: Q08341.1; the cyclodextrinase Ps03CDase from Paenibacillus sp.MY03, gene sequence NCBI Reference Sequence: NZ_MXQD01000009.1 (gene 101836-103611), amino acid sequence NCBI Reference Sequence: WP_087568083.1; sp.PAMC21692 cyclodextrinase Ps92CDase, gene sequence GenBank: CP060293.1 (gene 6259030-6260805), amino acid sequence NCBI Reference Sequence: WP_187105536.1; cyclodextrinase TsCDase from Thermococcus sp.B1001, gene sequence GenBank: AB034969.2 (gene 248-2230), amino acid sequence GenBank: BAB18100.1; cyclodextrinase PfCDase from Pyrococcus furiosus DSM 3638, gene sequence NCBI Reference Sequence: NC_003413.1 (gene 1790387-1792324), amino acid sequence NCBI Reference Sequence: WP_011013079.1;

Example 2: Preparation of cyclodextrinase immobilized enzyme

The cyclodextrinase with transglycosylation activity screened in step (2) of Example 1 was used to prepare an immobilized enzyme.

First, a crude cyclodextrinase solution was prepared by the method of step (1) of Example 1, and then 50 g of epoxy resin was added to 100 mL of the crude cyclodextrinase solution, and adsorbed for 3-20 h at 15-35° C. and 100-200 rpm, and then the immobilized enzyme resin was filtered and eluted with a 50 mM Na ₂ HPO ₄ /NaH ₂ PO ₄ buffer solution at pH 6.0-8.0 to wash away the floating protein on the surface. The enzyme activity of the immobilized enzyme was determined by the DNS method, and the enzyme activity reached 100-500 KU/g.

Example 3: Analysis of enzymatic properties of cyclodextrinase

The immobilized enzyme prepared in Example 2 was subjected to enzymatic property analysis:

3.1 Optimal pH and pH stability of enzymes

The enzymatic activity of cyclodextrinase was determined using Na ₂ HPO ₄ /Citric Acid buffer at pH 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, and 8.0 and Tris-HCl buffer at pH 8.0, 8.5, and 9.0, respectively.

Using the above-mentioned buffer solutions of different pH values, the enzymatic activities of cyclodextrinase Ps03CDase and PpCDase were determined under the standard enzyme activity assay system, and the optimal pH values of the cyclodextrinase were 7.5 and 6.0, respectively, as shown in Figures 3A and 3B. After the cyclodextrinase was stored in the above-mentioned buffer solutions of different pH values at 4°C for 12 h, the enzymatic activities were determined in a pH 7.0 Na ₂ HPO ₄ /NaH ₂ PO ₄ buffer environment under standard enzyme activity assay conditions, and the pH stability of the cyclodextrinase Ps03CDase and PpCDase was detected. The enzymatic activity of the cyclodextrinase Ps03CDase was maintained above 80% at pH 5.5-9.0, and the enzymatic activity of the cyclodextrinase PpCDase was maintained above 80% at pH 5.5-7.5, as shown in Figures 3C and 3D.

3.2 Optimal reaction temperature and temperature stability of enzymes

The activity of cyclodextrinase Ps03CDase was measured at 20, 25, 30, 35, 40, 45, and 50°C according to the method of standard enzyme activity determination, and the optimal reaction temperature of cyclodextrinase Ps03CDase was 40°C, as shown in Figure 4A. The activity of cyclodextrinase PpCDase was measured at 70, 75, 80, 85, 90, and 95°C according to the method of standard enzyme activity determination, and the optimal reaction temperature of cyclodextrinase PpCDase was 85°C, as shown in Figure 4B.

After the cyclodextrinase was incubated at 20, 25, 30, 35, 40, 45, 50, and 55°C for 60 min, the enzyme activity of the cyclodextrinase Ps03CDase was determined according to the method in the standard enzyme activity assay, and the temperature stability of the cyclodextrinase Ps03CDase was detected. As shown in Figure 4C, the residual enzyme activity of the cyclodextrinase Ps03CDase reached more than 90% after being incubated at 20-45°C for 60 min. The cyclodextrinase was incubated at 70, 75, 80, 85, 90, and 95°C for 60 min, and the enzyme activity of the cyclodextrinase PpCDase was determined according to the method in the standard enzyme activity assay, and the temperature stability of the cyclodextrinase PpCDase was detected. As shown in Figure 4D, the residual enzyme activity was 85% after being incubated at 95°C for 60 min.

Example 4: Optimal Substrate Concentration in Transglycosylation Reaction

The relative enzyme activity of transglycosylation refers to the relative enzyme activity of transglycosylation determined by the peak area of the transglycosylation product after the reaction solution is inactivated and analyzed by HPLC under transglycosylation conditions.

4.1 Determination of the optimal α-cyclodextrin concentration in transglycosylation reactions

Preparation of pNPG: Use DMSO to prepare 2 mol/L pNPG stock solution, and dilute and prepare according to the requirements of different reaction concentrations.

In 0.5 mL of 50 mM Na ₂ HPO ₄ /NaH ₂ PO ₄ reaction system, pNPG (15 mg/0.5 mL) with a final concentration of 100 mmol/L and 1% (5 mg/0.5 mL), 2% (10 mg/0.5 mL), 3% (15 mg/0.5 mL), 4% (20 mg/0.5 mL), 5% (25 mg/0.5 mL), 6% (30 mg/0.5 mL), 7% (35 mg/0.5 mL), 8% (40 mg/0.5 mL), 9% (45 mg/0.5 mL), 10% (50 mg/0.5 mL), 15% (20 mg/0.5 mL), 20% (30 mg/0.5 mL), 25% (20 mg/0.5 mL), 30% (35 mg/0.5 mL), 40% (40 mg/0.5 mL), 5% (45 mg/0.5 mL), 6% (50 mg/0.5 mL), 7% (50 mg/0.5 mL), 8% (50 mg/0.5 mL), 10% (50 mg/0.5 mL), 15% (20 mg/0.5 mL), 20 ... 0% (50 mg/0.5 mL), 11% (55 mg/0.5 mL), 12% (60 mg/0.5 mL), 13% (65 mg/0.5 mL), and 14% (70 mg/0.5 mL) of α-cyclodextrin, and finally 1000 KU/L of immobilized cyclodextrin enzyme Ps03CDase were added. The reaction temperature was 40°C for 5 hours. HPLC analysis is shown in Figure 5A. The relative enzyme activity of the transglycosylation of cyclodextrin enzyme Ps03CDase increases with the increase of the concentration of α-cyclodextrin.

In 0.5 mL of 50 mM Na ₂ HPO ₄ /NaH ₂ PO ₄ reaction system, pNPG with a final concentration of 100 mmol/L and 1%, 2%, 3%, 4%, and 5% α-cyclodextrin were prepared, and finally immobilized cyclodextrinase PpCDase with a final concentration of 500 KU/L was added. The reaction temperature was 85°C and the reaction time was 16 h. HPLC analysis was shown in Figure 5A. The relative transglycosylase activity of the cyclodextrinase PpCDase increased with the increase of the concentration of α-cyclodextrin, and the relative transglycosylase activity reached the maximum at 3% α-cyclodextrin.

In 0.5mL of _50mM _Na2HPO4 / _NaH2PO4 reaction system, pNPG (15mg/ _0.5mL ) with a final concentration of 100mmol/L and 5% (25mg/0.5mL), 10% (50mg/0.5mL), 15% (75mg/0.5mL), 20% (100mg/0.5mL), 25% (125mg/0.5mL), 30% (150mg/0.5mL), 35% (175mg/0.5mL) were prepared. The solubility of α-cyclodextrin can be improved by high temperature, ultrasound and other methods known in the art. Finally, 1000KU/L of immobilized cyclodextrin enzyme Ps03CDase was added, the reaction temperature was 40℃, 5h, and HPLC analysis was shown in Figure 6A. The relative enzyme activity of cyclodextrin enzyme Ps03CDase increased with the increase of α-cyclodextrin concentration, reaching the maximum value at 30%.

4.2 Determination of the optimal pNPG concentration in transglycosylation reactions

In 0.5 mL of 50 mM Na ₂ HPO ₄ /NaH ₂ PO ₄ reaction system, pNPG with final concentrations of 40, 50, 60, 70, 80, 90, 100, 110, 120, and 130 mmol/L and 14% α-cyclodextrin were prepared, and finally 1000 KU/L of immobilized cyclodextrinase Ps03CDase was added. The reaction temperature was 40°C for 5 h. HPLC analysis showed in Figure 5B that the yield of pNPG7 increased with the increase of pNPG concentration, and the yield of pNPG7 reached the maximum when the pNPG concentration was 120 mMol/L.

In 0.5 mL of 50 mM Na ₂ HPO ₄ /NaH ₂ PO ₄ reaction system, pNPG with final concentrations of 40, 50, 60, 70, 80, 90, 100, 110, 120, 130 mmol/L and 3% α-cyclodextrin were prepared, and finally 1000 KU/L of fixed The cyclodextrinase PpCDase was used, the reaction temperature was 85°C, and the reaction time was 16 h. HPLC analysis was shown in Figure 5B. The yield of pNPG7 increased with the increase of pNPG concentration, and the yield of pNPG7 reached the maximum when the pNPG concentration was 130 mMol/L.

In 0.5 mL of 50 mM Na ₂ HPO ₄ /NaH ₂ PO ₄ reaction system, pNPG and 30% α-cyclodextrin were prepared with final concentrations of 50, 75, 100, 125, 150, 175, 200, 225, and 250 mmol/L, and finally 1000 KU/L of immobilized cyclodextrinase Ps03CDase was added. The reaction temperature was 40°C for 5 h. HPLC analysis showed in Figure 6B that the yield of pNPG7 increased with the increase of pNPG concentration, and the yield of pNPG7 reached the maximum when the pNPG concentration was 200 mMol/L.

Example 5: Optimal enzyme activity concentration and reaction time in transglycosylation reaction

5.1 Determination of the optimal enzyme activity concentration in transglycosylation reaction

In 0.5 mL of 50 mM Na ₂ HPO ₄ /NaH ₂ PO ₄ reaction system, pNPG with a final concentration of 120 mmol/L and 14% α-cyclodextrin were prepared, and finally different concentrations of immobilized cyclodextrinase Ps03CDase (1000, 1100, 1200, 1300, 1400, 1500, 1600 KU/L) were added respectively. The reaction temperature was 40°C for 5 h. HPLC analysis was shown in Figure 7A. When the activity concentration of cyclodextrinase Ps03CDase reached 1500 KU/L, the yield of pNPG7 reached the maximum.

In 0.5 mL of 50 mM Na ₂ HPO ₄ /NaH ₂ PO ₄ reaction system, pNPG with a final concentration of 120 mmol/L and 3% α-cyclodextrin were prepared, and finally different concentrations of immobilized cyclodextrinase PpCDase (600, 700, 800, 900, 1000, 1100 KU/L) were added respectively. The reaction temperature was 85°C for 18 h. HPLC analysis was shown in Figure 7A. When the activity concentration of cyclodextrinase PpCDase reached 1000 KU/L, the yield of pNPG7 reached the maximum.

In 0.5 mL of 50 mM Na ₂ HPO ₄ /NaH ₂ PO ₄ reaction system, pNPG with a final concentration of 200 mmol/L and 30% α-cyclodextrin were prepared, and finally different concentrations of immobilized cyclodextrinase Ps03CDase (1000, 1100, 1200, 1300, 1400, 1500, 1600 KU/L) were added respectively. The reaction temperature was 40°C for 5 h. HPLC analysis was shown in Figure 8A. When the activity concentration of cyclodextrinase Ps03CDase reached 1500 KU/L, the yield of pNPG7 reached the maximum.

5.2 Determination of the optimal reaction time in transglycosylation reactions

In 0.5 mL of 50 mM Na ₂ HPO ₄ /NaH ₂ PO ₄ reaction system, pNPG with a final concentration of 120 mmol/L and 14% α-cyclodextrin were prepared, and finally 1500 KU/L of immobilized cyclodextrinase Ps03CDase was added. The reaction temperature was 40°C, and the reaction time was 2, 3, 4, 5, 6, and 7 h. HPLC analysis is shown in Figure 7B. When the reaction lasted for 6 h, the yield of pNPG7 reached the maximum.

In 0.5 mL of 50 mM Na ₂ HPO ₄ /NaH ₂ PO ₄ reaction system, pNPG with a final concentration of 120 mmol/L and 3% α-cyclodextrin were prepared, and finally 1000 KU/L of immobilized cyclodextrinase PpCDase was added. The reaction temperature was 85°C, and the reaction time was 15, 18, 21, 24, 27, and 30 h. HPLC analysis is shown in Figure 7C. When the reaction lasted for 24 h, the yield of pNPG7 reached the maximum.

In 0.5 mL of 50 mM Na ₂ HPO ₄ /NaH ₂ PO ₄ reaction system, pNPG with a final concentration of 200 mmol/L and 30% α-cyclodextrin were prepared, and finally 1500 KU/L of immobilized cyclodextrinase Ps03CDase was added. The reaction temperature was 40°C, and the reaction time was 2, 3, 4, 5, 6, and 7 h. HPLC analysis is shown in Figure 8B. When the reaction lasted for 6 h, the yield of pNPG7 reached the maximum.

Example 6: Enzymatic production of pNPG7

14% α-cyclodextrin and 120 mmol/L pNPG were prepared with 0.2 L of 50 mM Na ₂ HPO ₄ /NaH ₂ PO ₄ buffer (pH 7.5), and finally 1500 KU/L of immobilized cyclodextrin enzyme Ps03CDase was added. The temperature was raised to 40°C, 200 rpm, and kept warm for 6 h. The immobilized enzyme was removed by filtration, and the reaction was terminated to obtain the crude product of pNPG7. The reaction route is shown in FIG9 .

HPLC analysis is shown in Figure 10, and the pNPG7 content produced by the reaction is high and the by-products are relatively small; TLC analysis after separation and purification is shown in Figure 11, and the purity after reaction purification is high. After purification and drying of the 0.2L reaction system, pNPG7 dry powder was obtained, which was 11g (43.2mmol/L) after weighing, and the total molar conversion rate of pNPG was 36%.

0.2L of 50mM Na ₂ HPO ₄ /NaH ₂ PO ₄ buffer (pH 7.5) was used to prepare 30% α-cyclodextrin and 200mmol/L pNPG, and finally 1500KU/L of immobilized cyclodextrin enzyme Ps03CDase was added, and the temperature was raised to 40°C, 200rpm, and kept warm for 6h. The immobilized enzyme was removed by suction filtration, and the reaction was terminated to obtain the crude product of pNPG7. According to HPLC analysis, the yield of pNPG7 was calculated to be 109.7g/L, and the molar conversion rate of pNPG was 43%. After purification and drying of the 0.2L reaction system, pNPG7 dry powder was obtained, which was 17.5g after weighing, and the purification yield was 80%.

The mass spectrometry analysis is shown in Figure 12, and the detected molecular ion peak of 1296.5 is consistent with the molecular ion peak of pNPG7+Na ⁺ ; the HNMR analysis is shown in Figure 13, and the ratio of benzene ring hydrogen: anomeric hydrogen: sugar ring hydrogen is about 4:7:42, and the coupling value of anomeric hydrogen is between 3-4, which is consistent with the hydrogen spectrum characteristics of pNPG7.

Example 7: Enzymatic production of pNPG7

0.2 L of 50 mM Na ₂ HPO ₄ /NaH ₂ PO ₄ buffer (pH 6) was used to prepare 3% α-cyclodextrin and 130 mmol/L pNPG, and finally 1000 KU/L immobilized cyclodextrinase PpCDase was added, the temperature was raised to 85°C, 200 rpm, and the temperature was kept for 24 hours. The immobilized enzyme was removed by filtration, and the reaction was terminated to obtain a crude product of pNPG7. The pNPG7 content produced by the reaction was high and the by-products were small. After 0.2 L of the reaction system was purified and dried, the pNPG7 dry powder was weighed to be 6 g (23.1 mmol/L), and the pNPG molar conversion rate was 19.3%.

Example 8: Enzymatic production of kilogram-scale pNPG7

Use 20 L of 50 mM Na ₂ HPO ₄ /NaH ₂ PO ₄ buffer (pH 7.5) to prepare 14% α-cyclodextrin and 120 mmol/L pNPG, and finally add 1500 KU/L immobilized cyclodextrin enzyme Ps03CDase. Raise the temperature to 40°C, 200 rpm, and keep warm for 6 h. Remove the immobilized enzyme by filtration, terminate the reaction, and obtain the crude product of pNPG7. After purification and drying of the 20 L reaction system, 1.185 kg of pNPG7 dry powder is obtained, and the total molar conversion rate of pNPG is 38%.

Use 20 L of 50 mM Na ₂ HPO ₄ /NaH ₂ PO ₄ buffer (pH 7.5) to prepare 30% α-cyclodextrin and 200 mmol/L pNPG, and finally add 1500 KU/L immobilized cyclodextrin enzyme Ps03CDase. Raise the temperature to 40°C, 200 rpm, and keep warm for 6 h. Filter and remove the immobilized enzyme, terminate the reaction, and obtain the crude product of pNPG7. After purification and drying of the 20 L reaction system, 1.88 kg of pNPG7 dry powder is obtained, and the total molar conversion rate of pNPG is 36%.

Example 9: Enzymatic production of pNPG7 using pNP and β-cyclodextrin as substrates

Use 0.2 L of 50 mM Na ₂ HPO ₄ /NaH ₂ PO ₄ buffer (pH 7.5) to prepare 2% (4 g/0.2 L) β-cyclodextrin and 40 mmol/L (1.112 g) pNP, and finally add 1200 KU/L immobilized cyclodextrin enzyme Ps92CDase. Heat to 45°C, 200 rpm, keep warm for 4 h, filter to remove the immobilized enzyme, terminate the reaction, and obtain the crude product of pNPG7; after purification and drying of the 0.2 L reaction system, about 3 g of pNPG7 dry powder was obtained, and the pNP molar conversion rate was 5.9%. The reaction route is shown in Figure 14.

Example 10: Using salidroside as a glycosyl acceptor and α, β, γ-cyclodextrin as a glycosyl donor to produce salidroside maltoheptaglycoside, salidroside maltooctaglycoside and salidroside maltononaglycoside

Use 0.2L of _50mM _Na2HPO4 / _NaH2PO4 buffer (pH 7.5) to prepare 30% α, β or γ- _cyclodextrin and 200mmol/L salidroside, and finally add 1500KU/L immobilized cyclodextrinase Ps03CDase, raise the temperature to 40°C, 200rpm, keep warm for 6h, filter to remove the immobilized enzyme, terminate the reaction, and obtain the crude product of salidroside maltoheptaglycoside, salidroside maltooctaglycoside or salidroside maltononaglycoside. Through HPLC analysis, it was calculated that the yield of salidroside maltoheptaglycoside was 99 g/L, and the molar conversion rate of salidroside was 38.9%; the yield of salidroside maltooctaglycoside was 117.6 g/L, and the molar conversion rate of salidroside was 41%; the yield of salidroside maltononaglycoside was 118.1 g/L, and the molar conversion rate of salidroside was 37%; as shown in Figure 15.

Example 11: Production of α or β-arbutin maltoheptaglycoside, α or β-arbutin maltooctaglycoside and α or β-arbutin maltononaglycoside using α or β-arbutin as glycosyl acceptor and α, β, γ-cyclodextrin as glycosyl donor

0.2 L of 50 mM Na ₂ HPO ₄ /NaH ₂ PO ₄ buffer (pH 7.5) was used to prepare 30% α, β or γ-cyclodextrin and 200 mmol/L α or β-arbutin, and finally 1500 KU/L of immobilized cyclodextrinase Ps03CDase was added, the temperature was raised to 40°C, 200 rpm, and the temperature was kept for 6 h. The immobilized enzyme was removed by filtration and the reaction was terminated to obtain α or β-arbutin maltoheptaglycoside or α or β- Crude product of arbutin maltooctaglycoside or α or β-arbutin maltononaglycoside. After HPLC analysis, the yield of α or β-arbutin maltoheptaglycoside was calculated to be 104.5 g/L, and the molar conversion rate of α or β-arbutin was 42%; the yield of α or β-arbutin maltooctaglycoside was 118.1 g/L, and the molar conversion rate of α or β-arbutin was 43%; the yield of α or β-arbutin maltononaglycoside was 125.6 g/L, and the molar conversion rate of α or β-arbutin was 40%; as shown in Figure 16.

Example 12: Production of indigoside maltoheptaglycoside, indigoside maltooctaglycoside and indigoside maltononaglycoside using indigoside as glycosyl acceptor and α, β, γ-cyclodextrin as glycosyl donor

0.2L of 50mM Na ₂ HPO ₄ /NaH ₂ PO ₄ buffer (pH 7.5) was used to prepare 30% α or β or γ-cyclodextrin, 200mmol/L indigoside, and finally 1500KU/L of immobilized cyclodextrinase Ps03CDase was added, the temperature was raised to 40°C, 200rpm, and the temperature was kept for 6h. The immobilized enzyme was removed by suction filtration, and the reaction was terminated to obtain the crude product of indigoside maltoheptaglycoside, indigoside maltooctaglycoside, or indigoside maltononaglycoside. According to HPLC analysis, the yield of indigoside maltoheptaglycoside was calculated to be 100.4g/L, and the molar conversion rate of indigoside was 39.6%; the yield of indigoside maltooctaglycoside was 115.2g/L, and the molar conversion rate of indigoside was 40.3%; the yield of indigoside maltononaglycoside was 121.9g/L, and the molar conversion rate of indigoside was 38.3%; as shown in Figure 17.

Example 13: Production of 2-chloro-4-nitrobenzene-α or β-maltoheptaglycoside (CNPG7), 2-chloro-4-nitrobenzene-α or β-maltooctaglycoside (CNPG8) and 2-chloro-4-nitrobenzene-α or β-maltononaglycoside (CNPG9) using 2-chloro-4-nitrobenzene-α or β-glucoside (CNPG) as a glycosyl acceptor and α, β, γ-cyclodextrin as a glycosyl donor

0.2L of _50mM _Na2HPO4 / _NaH2PO4 buffer (pH 7.5) was used to prepare 30% α, β or γ- _cyclodextrin , 200mmol/L CNPG, and finally 1500KU/L immobilized cyclodextrinase Ps03CDase was added, the temperature was raised to 40°C, 200rpm, and the temperature was kept for 6h. The immobilized enzyme was removed by filtration, and the reaction was terminated to obtain the crude product of CNPG7, CNPG8 or CNPG9. According to HPLC analysis, the yield of CNPG7 was calculated to be 99.4g/L, and the molar conversion rate of CNPG was 37.1%; the yield of CNPG8 was 115.9g/L, and the molar conversion rate of CNPG was 38.6%; the yield of CNPG9 was 119.7g/L, and the molar conversion rate of CNPG was 36.0%; as shown in Figure 18.

Example 14: Production of tetramethylumbelliferyl α/β-maltoheptaglycoside (4-MUG7), tetramethylumbelliferyl α/β-maltooctaglycoside (4-MUG8) and tetramethylumbelliferyl α/β-maltononaglycoside (4-MUG9) using tetramethylumbelliferyl α/β-glucoside (4-MUG) as glycosyl acceptor and α, β, γ-cyclodextrin as glycosyl donor

Use 0.2L of _50mM _Na2HPO4 / _NaH2PO4 buffer (pH 7.5) to prepare 30% α, β or γ- _cyclodextrin and 200mmol/L 4-MUG, and finally add 1500KU/L immobilized cyclodextrin enzyme Ps03CDase, raise the temperature to 40℃, 200rpm, keep warm for 6h, filter to remove the immobilized enzyme, terminate the reaction, and obtain the crude product of 4-MUG7, 4-MUG8 or 4-MUG9. Through HPLC analysis, it was calculated that the yield of 4-MUG7 was 81.7 g/L, and the molar conversion rate of 4-MUG was 31.2%; the yield of 4-MUG8 was 98 g/L, and the molar conversion rate of 4-MUG was 33.3%; the yield of 4-MUG9 was 95.4 g/L, and the molar conversion rate of 4-MUG was 29.2%; as shown in Figure 19.

Example 15: Production of ethyl maltohexaglycoside, ethyl maltoheptaglycoside and ethyl maltooctaglycoside using ethanol as glycosyl acceptor and α, β, γ-cyclodextrin as glycosyl donor

0.2L of _50mM _Na2HPO4 / _NaH2PO4 buffer (pH 7.5) was used to prepare 30% α, β or γ- _cyclodextrin and 15% (w/v) ethanol, and finally 1500KU/L of immobilized cyclodextrinase Ps03CDase was added, and the temperature was raised to 40°C, 200rpm, and the temperature was kept for 6h. The immobilized enzyme was removed by filtration, and the reaction was terminated to obtain the crude product of ethyl maltohexaglycoside, ethyl maltoheptaglycoside or ethyl maltooctaglycoside. According to HPLC-RID analysis, the yield of ethyl maltohexaglycoside was 34g/L, and the yield of ethyl maltoheptaglycoside was calculated to be 43g/L; the yield of ethyl maltooctaglycoside was 39g/L; as shown in Figure 20.

Example 16: Production of azido-PEG4-β-glucose maltoheptaglycoside, azido-PEG4-β-glucose maltooctaglycoside and azido-PEG4-β-glucose maltononaglycoside using azido-PEG4-β-glucose as glycosyl acceptor and α, β, γ-cyclodextrin as glycosyl donor

Use 0.2L of _50mM _Na2HPO4 / _NaH2PO4 buffer (pH 7.5) to prepare 30% α, β or γ- _cyclodextrin and 200mM azido-PEG4-β-glucose, and finally add 1500KU/L immobilized cyclodextrin enzyme Ps03CDase, raise the temperature to 40°C, 200rpm, keep warm for 6h, filter to remove the immobilized enzyme, terminate the reaction, and obtain the crude product of azido-PEG4-β-glucose maltoheptaglycoside, azido-PEG4-β-glucose maltooctaglycoside or azido-PEG4-β-glucose maltononaglycoside. According to HPLC-RID analysis, the yield of azido-PEG4-β-glucose maltoheptaglycoside was 48.7 g/L, and the molar conversion rate was 18%; the calculated yield of azido-PEG4-β-glucose maltooctaglycoside was 62.1 g/L, and the molar conversion rate was 20.5%; the yield of azido-PEG4-β-glucose maltononaglycoside was 58.7 g/L, and the molar conversion rate was 17.5%; as shown in Figure 21.

Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

A method for preparing oligomeric maltosides with a degree of polymerization of 6-9 by enzymatic method, characterized in that cyclodextrin is used as a glycosyl donor, benzene rings, alcohols or glycosides are used as glycosyl acceptors, and oligomeric maltosides with a degree of polymerization of 6-9 are synthesized by transglycosylation in an enzymatic reaction system containing a cyclodextrinase with transglycosylation activity.
The method according to claim 1, characterized in that the method comprises (a), (b) or (c):

(a) using α-cyclodextrin as a glycosyl donor and p-nitrophenyl-α-glucoside, p-nitrophenol, other benzene rings, alcohols or other glycosides as glycosyl acceptors, and synthesizing pNPG7 or other oligomeric maltosides with a degree of polymerization of 6-7 by transglycosylation in an enzymatic reaction system containing a cyclodextrinase having transglycosylation activity;

(b) using β-cyclodextrin as a glycosyl donor and p-nitrophenyl-α-glucoside, p-nitrophenol, other benzene rings, alcohols or other glycosides as glycosyl acceptors, and synthesizing pNPG7 or other oligomeric maltosides with a degree of polymerization of 7-8 by transglycosylation in an enzymatic reaction system containing a cyclodextrinase having transglycosylation activity;

(c) Using γ-cyclodextrin as a glycosyl donor and p-nitrophenyl-α-glucoside, p-nitrophenol, other benzene rings, alcohols or other glycosides as glycosyl acceptors, pNPG8 or other oligomeric maltosides with a degree of polymerization of 8-9 are synthesized by transglycosylation in an enzymatic reaction system containing a cyclodextrinase having transglycosylation activity.
The method according to claim 1, characterized in that the glycoside sugar acceptor is selected from one or more of phenylcyclic glucoside, click chemistry glucoside, and nucleoside;

Preferably, the benzene ring glucoside is selected from one or more of chromophore-containing glucoside and benzene ring natural product glucoside; more preferably, the chromophore-containing glucoside is selected from one or more of p-nitrobenzene-α/β-glucoside, 2-chloro-4-nitrobenzene-α/β-glucoside and tetramethylumbelliferyl glucoside, and the benzene ring natural product glucoside is selected from one or more of α/β-arbutin, salidroside and indigoside; further preferably, the indigoside is selected from indolyl-β-glucoside; the p-nitrobenzene-α/β-glucoside is selected from p-nitrobenzene-α-D-glucoside or p-nitrobenzene-β-D-glucoside;

Preferably, the click chemistry glucoside is selected from azido-PEGn-glucose and propargyl-PEGm-glucose, wherein n and m are positive integers;

Preferably, the nucleoside is selected from one or more of cytarabine and doxifluridine;

And/or, the benzene ring glycosyl acceptor is selected from one or more of p-nitrophenol, 2-chloro-4-nitrophenol, and tetramethylumbelliferone;

And/or, the alcohol glycosyl acceptor is selected from one or more of methanol, ethanol, n-propanol, isopropanol and n-butanol.
The method according to claim 1, characterized in that the cyclodextrinase having transglycosylation activity is selected from one or more of the cyclodextrinase PpCDase derived from Palaeococcus pacificus DY20341, the cyclodextrinase BsCDase derived from Bacillus.sphaericus, the cyclodextrinase Ps03CDase derived from Paenibacillus sp.MY03, the cyclodextrinase Ps92CDase derived from Paenibacillus sp.PAMC21692, the cyclodextrinase TsCDase derived from Thermococcus sp.B1001, and the cyclodextrinase PfCDase derived from Pyrococcus furiosus DSM 3638.
The method according to claim 2 is characterized in that the concentration of α-cyclodextrin in the enzymatic reaction system is 1-50%, the concentration of β-cyclodextrin is 1-30%, and the concentration of γ-cyclodextrin is 1-30%; preferably, the concentration of α-cyclodextrin is 20-40%, the concentration of β-cyclodextrin is 5-10%, and the concentration of γ-cyclodextrin is 5-10%.
The method according to claim 1 is characterized in that the concentration of the glycosyl acceptor in the enzymatic reaction system is 5-500mmol/L, preferably, the concentration of the glycoside glycosyl acceptor in the enzymatic reaction system is 10-500mmol/L, the concentration of the benzene ring glycosyl acceptor is 5-500mmol/L, and the concentration of the alcohol is 5%-30% (w/v).
The method according to claim 1 is characterized in that the cyclodextrinase in the enzymatic reaction system comprises a liquid enzyme or an immobilized enzyme with a concentration of 100-5000 KU/L.
The method according to claim 1 is characterized in that the pH of the enzymatic reaction system is 6-8, the reaction temperature is 20-95°C, and the reaction time is 2-30h.
Use of a cyclodextrinase having transglycosylation activity or the method according to any one of claims 1 to 8 in the preparation of oligomaltosides with a degree of polymerization of 6-9, especially pNPG7.
The use according to claim 9 is characterized in that the cyclodextrinase with transglycosylation activity is selected from one or more of the cyclodextrinase PpCDase derived from Palaeococcus pacificus DY20341, the cyclodextrinase BsCDase derived from Bacillus.sphaericus, the cyclodextrinase Ps03CDase derived from Paenibacillus sp.MY03, the cyclodextrinase Ps92CDase derived from Paenibacillus sp.PAMC21692, the cyclodextrinase TsCDase derived from Thermococcus sp.B1001, and the cyclodextrinase PfCDase derived from Pyrococcus furiosus DSM 3638.
A method for detecting the transglycosylation activity of cyclodextrinase, characterized in that the method comprises adding a glycosyl donor and a glycosyl acceptor to a reaction system containing cyclodextrinase to carry out a transglycosylation reaction, and confirming the transglycosylation product;

Cyclodextrin is used as the glycosyl donor, and benzene rings, alcohols or glycosides are used as the glycosyl acceptors.
The method according to claim 11, characterized in that α-cyclodextrin is used as a glycosyl donor, and p-nitrophenyl-α-glucoside, p-nitrophenol, other benzene rings, other glycosides or alcohols are used as glycosyl acceptors; or, β-cyclodextrin is used as a glycosyl donor, and p-nitrophenyl-α-glucoside, p-nitrophenol, other benzene rings, other glycosides or alcohols are used as glycosyl acceptors; or, γ-cyclodextrin is used as a glycosyl donor, and p-nitrophenyl-α-glucoside, p-nitrophenol, other benzene rings, other glycosides or alcohols are used as glycosyl acceptors;

The concentration of the glycosyl donor is 5-10%, and the concentration of the glycosyl acceptor is greater than 5 mmol/L.
The method according to claim 12, characterized in that the cyclodextrinase has α-, β- or γ-cyclodextrin hydrolysis activity.
A method for preparing pNPG7 by enzymatic method, characterized in that the method comprises (a) or (b):

(a) using α-cyclodextrin as a glycosyl donor and p-nitrophenyl-α-D-glucoside as a glycosyl acceptor, transglycosylation is performed to synthesize pNPG7 in an enzymatic reaction system containing a cyclodextrinase having transglycosylation activity;

(b) pNPG7 was synthesized by transglycosylation in an enzymatic reaction system containing cyclodextrinase having transglycosylation activity, using β-cyclodextrin as a glycosyl donor and p-nitrophenol as a glycosyl acceptor.
The method according to claim 14, characterized in that the cyclodextrinase having transglycosylation activity comprises a mesophilic enzyme or a thermophilic enzyme;

The high temperature enzymes include cyclodextrinase PpCDase derived from Palaeococcus pacificus DY20341 and cyclodextrinase TsCDase derived from Thermococcus sp. B1001;

The mesophilic enzymes include cyclodextrinase BsCDase derived from Bacillus sphaericus, cyclodextrinase PfCDase derived from Pyrococcus furiosus DSM 3638, cyclodextrinase Ps03CDase derived from Paenibacillus sp. MY03, and cyclodextrinase Ps92CDase derived from Paenibacillus sp. PAMC21692.
The method according to claim 14, characterized in that the concentration of α-cyclodextrin in the enzymatic reaction system is 1-14%, and the concentration of β-cyclodextrin is 1-3%.
The method according to claim 14, characterized in that the concentration of p-nitrobenzene-α-D-glucoside in the enzymatic reaction system is 10-180 mmol/L, and the concentration of p-nitrophenol is 5-100 mmol/L.
The method according to claim 14, characterized in that the concentration of the liquid enzyme or immobilized enzyme in the enzymatic reaction system is 100-5000 KU/L.
The method according to claim 14 is characterized in that the pH of the enzymatic reaction system is 6-8, the reaction temperature is 20-95°C, and the reaction time is 2-30h.
Use of a cyclodextrinase having transglycosylation activity or the method according to any one of claims 14 to 19 in the preparation of a product containing pNPG7.
The use according to claim 20 is characterized in that the cyclodextrinase with transglycosylation activity includes any one of the cyclodextrinase PpCDase derived from Palaeococcus pacificus DY20341, the cyclodextrinase TsCDase derived from Thermococcus sp. B1001, the cyclodextrinase BsCDase derived from Bacillus. sphaericus, the cyclodextrinase PfCDase derived from Pyrococcus furiosus DSM 3638, the cyclodextrinase Ps03CDase derived from Paenibacillus sp. MY03, and the cyclodextrinase Ps92CDase derived from Paenibacillus sp. PAMC21692.
A method for detecting the transglycosylation activity of cyclodextrinase, characterized in that the method comprises adding a glycosyl donor and a glycosyl acceptor to a reaction system containing cyclodextrinase to carry out a transglycosylation reaction;

α-cyclodextrin is used as a glycosyl donor and p-nitrophenyl-α-D-glucoside is used as a glycosyl acceptor; or β-cyclodextrin is used as a glycosyl donor and p-nitrophenol is used as a glycosyl acceptor;

The concentration of p-nitrobenzene-α-D-glucoside is greater than 10 mmol/L, and the concentration of p-nitrophenol is greater than 5 mmol/L.
The method according to claim 22, characterized in that the cyclodextrinase has α- or β-cyclodextrin hydrolysis activity.