A method for synthesizing gamma-cyclodextrin

Abstract

The present invention relates to a method for synthesizing γ-cyclodextrin, and belongs to the field of enzyme application technology. The present invention uses cellulose widely present in nature as a substrate, first uses cellulase as a catalyst, uses a raw material containing cellulose as a substrate, and obtains a raw material containing cellobiose; then γ-cyclodextrin with a wide range of application value is synthesized by a multi-enzyme cascade one-pot catalytic reaction such as cellobiose phosphorylase (CBP), α-glucan phosphorylase (AGP), γ-cyclodextrin glycosyltransferase (CGT), polyphosphate glucokinase (PPGK), phosphoglucomutase (PGM), etc. The preparation method of γ-cyclodextrin provided by the present invention not only expands the application range of cellulose biomass, but also provides a certain basis for the high-value utilization of biomass, so that its added value is increased by more than 10 times.

Description

Technical Field

[0001] The invention provides a novel biosynthesis method of gamma-cyclodextrin, belonging to the technical field of enzyme application. Background Art

[0002] Cyclodextrin (CD) is a general term for cyclic oligosaccharides composed of six or more glucoses connected by α-1,4 glycosidic bonds. Among them, α-cyclodextrin (α-CD), β-cyclodextrin (β-CD) and γ-cyclodextrin (γ-CD) are the most common. Compared with α-CD and β-CD, γ-CD has: (1) a larger cavity than β-cyclodextrin, which can include a wider range of guest molecules; (2) better water solubility. At room temperature 25°C, the solubility of γ-cyclodextrin is 25.6g / 100ml, while the solubility of α-CD is 12.7g / 100ml water and the solubility of β-CD is only 1.88g / 100ml water. Therefore, γ-CD has greater demand and broader application prospects in many fields such as analytical chemistry, medicine, food, and cosmetics. It is reported that α-CD, β-CD and γ-CD account for 15%, 70% and 5% of the global cyclodextrin production, respectively. The low production volume and excellent performance lead to the high price of γ-CD.

[0003] In the early 1970s, with the emergence of cyclodextrin glycosyltransferase (CGTase, EC 2.4.1.19), cyclodextrin began to enter industrial trial production, but the output and quality could not meet the market demand. Therefore, researchers have been exploring and improving the synthesis technology of γ-CD. At present, the international γ-CD market is basically monopolized by foreign manufacturers. The synthesis methods of γ-CD include fermentation, enzyme reaction and chemical synthesis. Since the chemical synthesis method is too difficult to achieve and the fermentation method has the problem of strain safety, the enzyme reaction method is often used in actual production.

[0004] Chinese patent CN202010236866.8 discloses a crude enzyme powder of γ-cyclodextrin glucosyltransferase for converting cassava starch to prepare γ-CD; Chinese patent CN201510176752.8 provides a method for synthesizing γ-CD from starch by catalyzing it with two enzymes (4αGTase and CGTase); CN201910354232.X discovered a CGTase with high temperature stability, which is not easy to retrograde starch and can improve the conversion rate of starch to synthesize γ-CD. CN202010257965.4 utilizes a reaction system containing starch solution, γ-CGTase, imidazole ionic liquid and organic solvent (methanol, ethanol or acetone), and controls the temperature at 50-80°C to react and generate γ-CD. This patent can significantly increase the hydrophobicity of γ-CD by adding imidazole ionic liquid and in combination with a specific buffer, thereby promoting the co-precipitation of γ-cyclodextrin and the organic solvent. The conversion rate of γ-cyclodextrin is increased from 34.69%-50.35% to 59.52% in the best embodiment.

[0005] The existing enzyme-catalyzed synthesis of γ-CD processes mostly use amylose as a substrate during the reaction process. So far, no relevant reports have been found on the synthesis of γ-CD without starch as a substrate. To date, γ-CD has not been produced on a large scale in China, because the high price caused by low conversion rate limits its application and promotion. Due to the need for iteration of γ-CD production process, it is urgent to develop a simple, efficient and large-scale industrialized γ-CD synthesis technology. Summary of the invention

[0006] Purpose of the invention: To provide an economical and practical γ-cyclodextrin synthesis process to meet the needs of various industries for γ-cyclodextrin (γ-CD).

[0007] Technical solution: The present invention achieves a technological breakthrough in synthesizing γ-cyclodextrin from non-food biomass through a multi-enzyme cascade one-pot enzyme reaction, while expanding the application prospects of agricultural waste such as straw.

[0008] Microcrystalline cellulose (MCC) is mainly composed of linear polysaccharides bonded by β-1,4-glucosidic bonds, which are obtained by dilute acid hydrolysis of natural cellulose. In general plant fibers, microcrystalline cellulose accounts for about 70%, and the other 30% is amorphous.

[0009] The technology of the present invention uses abundant microcrystalline cellulose as raw material and obtains gamma-cyclodextrin through a series of process treatments.

[0010] The present invention provides a method for preparing γ-cyclodextrin, which comprises the following steps:

[0011] Step 1. Using cellulase as a catalyst and a raw material containing cellulose as a substrate to prepare a raw material containing cellobiose;

[0012] Preferably, the raw material containing cellulose includes but is not limited to cellulose phosphate (RAC).

[0013] Step 2. Using a raw material containing cellobiose as a substrate to carry out a multi-enzyme cascade reaction to synthesize γ-cyclodextrin. Using cellobiose as a substrate to carry out a multi-enzyme cascade reaction to synthesize γ-cyclodextrin, the multi-enzyme cascade reaction solution includes but is not limited to: cellobiose phosphorylase (CBP), α-glucan phosphorylase (AGP), γ-cyclodextrin glycosyltransferase (CGT), polyphosphate glucokinase (PPGK), and phosphoglucomutase (PGM).

[0014] The cellulose-containing raw material described in step 1, such as phosphoric acid-swollen cellulose, can be purchased from commercial channels; it can also be prepared according to existing public literature, such as the method disclosed in Chinese Patent 201611056999.7 (Invention Name: An extracellular aldonic acid lactonase PoALAC and its application), or 200980153709.2 (Invention Name: Yeast expressing cellulase for simultaneous saccharification and fermentation using cellulose), or 202011015802.1 (Invention Name: A method for preparing oligosaccharides from cellulose by composite enzyme catalysis and its application); or microcrystalline cellulose (MCC) is mixed with concentrated phosphoric acid to prepare phosphoric acid-swollen cellulose.

[0015] Preferably, the cellulase in step 1 includes but is not limited to: cellulose exohydrolase (CBH), which acts on a raw material containing cellulose (such as phosphoric acid-swollen cellulose) to synthesize cellobiose;

[0016] Preferably, the cellulase exo-tase CBH in step 1 is also called: exoglucanase. Its sources include but are not limited to: wild-type or genetically modified cells such as Trichoderma reesei, Yarrowia lipolytica cells, Talaromyces emersonii, Neosartorya fischeri, Humicola grisea, Chaetomium thermophilum, filamentous fungi and Cellulomonas fimi; preferably, the cellulase exo-tase is derived from Trichoderma reesei, referred to as CBH I.

[0017] Preferably, the β-glucosidase (BGL) in step 1 is derived from, but not limited to, wild-type or genetically modified cells of Trichoderma sp., such as Trichoderma reesei or Trichoderma viride, Penicillium piceum, Aspergillus niger, etc. Preferably, β-glucosidase (β-glucosidase) EC3.2.1.21.

[0018] Preferably, the source of the cellobiose phosphorylase (CBP) in step 2 includes, but is not limited to, wild-type or genetically modified cells such as Clostridium thermocellum, Cellvibrio gilvus, Cellulomonas sp., Clostridium stercorarium, Thermotoga neapolitana, Cellulomonas uda and Ruminococcus albus; preferably, the cellobiose phosphorylase is selected from EC: 2.4.1.20, derived from Clostridium thermocellum, with an amino acid sequence as shown in SEQ ID NO: 1, and a microorganism having an amino acid sequence similarity of more than 60% to SEQ ID NO: 1.

[0019] Preferably, the source of α-glucan phosphorylase (AGP) in step 2 includes, but is not limited to: Hordeum vulgaresub sp., Hungateiclostridium thermocellum, Thermus thermophilus, Pyrococcus sp., Pyrococcus horikoshii ) , Pyrococcus furiosus, Thermococcus barophilus, Thermococcus kodakarensis, Thermotoga maritima and other wild-type or genetically modified cells; preferably, the α-glucan phosphorylase (AGP) (Alpha-1,4glucan phosphorylase) is selected from EC: 2.4.1.1, derived from Hordeum vulgare sub sp., the amino acid sequence is shown in SEQ ID NO: 2, and contains a microorganism having an amino acid sequence similarity of more than 60% to SEQ ID NO: 2.

[0020] Preferably, the source of the γ-cyclodextrin glycosyltransferase (CGT) in step 2 includes but is not limited to: wild-type or genetically modified cells such as Evansella clarkii, Escherichia coli, Alkaliphilic Bacillus, Bacillus clausii, and Bacillus clarkii 7364; Preferably, the γ-cyclodextrin glycosyltransferase (Cyclodextrin glucanotransferase) is selected from EC: 2.4.1.19, derived from Evansella clarkii, the amino acid sequence is shown in SEQ ID NO: 3, and contains a microorganism having an amino acid sequence similarity of more than 60% to SEQ ID NO: 3.

[0021] Preferably, the polyphosphate glucokinase (PPGK) in step 2 is selected from phosphate glucokinase EC: 5.4.2.2, derived from Arthrobacter sp., with an amino acid sequence as shown in SEQ ID NO: 4, and a microorganism having an amino acid sequence similarity of more than 60% to SEQ ID NO: 4.

[0022] Preferably, the phosphoglucomutase (PGM) source in step 2 includes but is not limited to wild-type or genetically modified cells such as Acetobacter xylinum, Pyrococcus horikoshii, Thermococcus kodakarensis, Arabidopsis, and Oryctolagus cuniculus; preferably, the phosphoglucomutase (PGM) is selected from

[0023] EC: 2.7.1.63, derived from Thermococcus kodakarensis, the amino acid sequence is shown in SEQ ID NO: 5, and microorganisms containing an amino acid sequence having more than 60% similarity to SEQ ID NO: 5.

[0024] The various enzymes described in the technical solution of the present application may be used in crude enzyme solution or in purified enzyme solution.

[0025] Preferably, the reaction solution in step 2 further comprises: cellulose and / or cellobiose and / or maltodextrin and / or glucose and / or cyclodextrin.

[0026] Preferably, in the preparation method of γ-cyclodextrin, the reaction solution in step 2 further comprises: sodium hexametaphosphate [(NaPO3)6], a metal magnesium salt (such as magnesium chloride, Mg 2+ );

[0027] Preferably, the reaction temperature in step 2 is 40-60°C, preferably 50°C.

[0028] Beneficial Effects

[0029] Cellulose is the most abundant natural polymer in nature and one of the most promising polymer resources. It is renewable, biodegradable and biocompatible. The technology of the present invention uses the abundant phosphoric acid swollen cellulose as a substrate to obtain cellobiose through an enzyme reaction, and then uses cellobiose as a substrate through a multi-enzyme cascade reaction to synthesize γ-cyclodextrin in one pot, achieving a technological breakthrough in synthesizing γ-cyclodextrin from non-food biomass. On the other hand, the present application provides a glucose-6-phosphate and inorganic phosphorus ion co-regeneration module based on PPGK and PGM. Through the enzyme-linked reaction of PPGK and PGM, the inhibition of the catalytic activity of CGT by cellobiose and glucose is successfully released, while the conversion rate of cellobiose is improved.

[0030] The preparation method of the present invention uses microcrystalline cellulose as a raw material, which is rich in sources, has a fast reaction speed, mild conditions, is easy to industrialize and scale up, and does not require the addition of ATP or other relatively expensive materials, greatly saving time and production costs. Therefore, the production cost of γ-cyclodextrin is much lower than the production cost of starch or maltodextrin as raw materials. The technology of the present invention increases the added value of cellulose by more than 10 times and expands the application prospects of agricultural waste such as straw. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 .Route diagram of the method for preparing γ-cyclodextrin of the present invention.

[0032] Figure 2 .This is the SDS-PAGE gel analysis pattern of the purified CBP, AGP, CGT, PPGK and PGM proteins in Example 1.

[0033] Figure 3 . HPLC spectra of γ-cyclodextrin standard, reaction solution of Example 5 without enzyme, reaction solution of Example 5 with enzyme, and reaction solution of Example 5 with enzyme after purification. DETAILED DESCRIPTION

[0034] The experimental methods used in the following examples are conventional methods unless otherwise specified; all materials, reagents, etc., are commercially available unless otherwise specified.

[0035] The present invention is further described in detail below through examples. It should be understood that the specific implementation methods described herein are only used to explain the present invention and are not used to limit the present invention. Modifications or replacements to the details and forms of the technical solution without departing from the structural concept and scope of use of the present invention all fall within the protection scope of the present invention.

[0036] Unless otherwise defined, all technical terms and scientific terms used in this specification have the same meanings as those commonly understood by those skilled in the art. In general, the nomenclature used in this specification and the experimental methods described below are well known and commonly used in the art.

[0037] A method for biosynthesizing γ-cyclodextrin, comprising:

[0038] Step 1. Using cellulase as a catalyst and a raw material containing cellulose as a substrate to prepare a raw material containing cellobiose;

[0039] Preferably, the raw materials containing cellobiose include but are not limited to phosphoric acid swollen cellulose;

[0040] Step 2. Using the raw material containing cellobiose as a substrate, synthesizing γ-cyclodextrin by a multi-enzyme cascade reaction;

[0041] The multi-enzyme cascade reaction solution includes: cellobiose phosphorylase, glucan phosphorylase, γ-cyclodextrin glycosyltransferase, polyphosphate glucose kinase, and phosphoglucomutase.

[0042] Preferably, the reaction solution in step 2 further comprises: cellulose and / or cellobiose and / or maltodextrin and / or glucose and / or cyclodextrin.

[0043] In some embodiments, the preparation method of phosphoric acid swollen cellulose is as follows: microcrystalline cellulose is mixed with concentrated phosphoric acid to react, and after the reaction is completed, acetone is added to precipitate cellulose, and the precipitate is filtered and collected; the precipitate is washed with an alkaline solution until the pH of the washing liquid is neutral, and then washed with deionized water for multiple times, and freeze-dried to obtain phosphoric acid swollen cellulose powder. The alkaline solution is a commonly used alkaline solution for adjusting the pH of the solution to neutral, and there is no particular restriction on its variety and concentration. Its variety is an alkaline solution such as sodium bicarbonate, sodium carbonate, sodium hydroxide, etc., and its concentration is such as a solution with a mass percentage of 1-5%. The concentrated phosphoric acid is commercially available concentrated phosphoric acid.

[0044] In some embodiments, the method for preparing cellobiose is: using phosphoric acid-swollen cellulose as a substrate, and obtaining cellobiose by cellobiohydrolase catalysis.

[0045] The various enzymes mentioned above can be purchased commercially or prepared according to known methods.

[0046] In some embodiments, an E. coli expression system is used to express cellobiose phosphorylase, glucan phosphorylase, γ-cyclodextrin glycosyltransferase, polyphosphate glucokinase, and phosphoglucomutase.

[0047] In some embodiments, the reaction system described in step 2 further includes: (NaPO3)6, a metal magnesium salt (such as magnesium chloride, Mg 2+ ), HEPES buffer. Preferably, the concentration of (NaPO3)6 is in the range of 20-50mM, preferably in the range of 25-45mM; Mg 2+ The concentration is in the range of 40-60 mM, preferably in the range of 45-53 mM. Preferably, the reaction solution also includes primer maltodextrin; preferably, the amount of primer maltodextrin is in the range of 0.3-0.7 g / L, preferably 0.4-0.5 g / L.

[0048] In some embodiments, the enzyme-linked reaction is carried out at 40-60° C. for 3-10 h, preferably at 50° C. for 6 h.

[0049] In the following examples, the solutions required for the experiments were prepared as follows:

[0050] LB medium: Weigh 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L sodium chloride (NaCl) and prepare with ddH2O. Sterilize with high pressure steam for later use. In the experiment, if necessary, add 50 mg / L kanamycin sulfate to the medium for resistance selection.

[0051] PBS buffer (20 mM imidazole): 2.46 g NaH2PO4·2H2O, 12.24 g Na2HPO4·12H2O, 11.688 g NaCl, 1.36 g imidazole, add about 800 mL of ddH2O to dissolve, adjust the pH to 7.4, and then add ddH2O to make up to 1 L.

[0052] PBS buffer (without imidazole): 2.46 g NaH2PO4·2H2O, 12.24 g Na2HPO4·12H2O, 11.688 g NaCl, add about 800 mL of ddH2O to dissolve, adjust the pH to 7.4, and then add ddH2O to make up to 1 L.

[0053] Bacteria preservation solution: Glycerol: 50% (m / v), prepared with ddH2O, sterilized at high temperature for use.

[0054] The present invention will be further described in detail below in conjunction with specific embodiments. It should be noted that the specific embodiments are intended to explain the present invention rather than to limit it.

[0055] In the following examples, CBP enzyme, the amino acid sequence is shown in SEQ ID NO: 1, and is derived from Clostridium thermocellum; AGP enzyme, the amino acid sequence is shown in SEQ ID NO: 2, and is derived from Hordeum vulgare sub sp.; CGT enzyme, the amino acid sequence is shown in SEQ ID NO: 3, and is derived from Evansella clarkii; PPGK enzyme, the amino acid sequence is shown in SEQ ID NO: 4, and is derived from Arthrobacter sp.; PGM enzyme, the amino acid sequence is shown in SEQ ID NO: 5, and is derived from Thermococcus kodakarensis.

[0056] The cellulase exo-CBH I is derived from Trichoderma reesei.

[0057] HPLC detection conditions of γ-cyclodextrin solution: Sugar-D amino column (COSMOSIL Sugar-D 4.6ID×250mm), differential detector, mobile phase of 65% acetonitrile, column temperature of 35°C, flow rate of 0.5mL / min, retention time of 30min.

[0058] The γ-cyclodextrin standard (C6086γ-cyclodextrin) was purchased from MacLean; the HPLC peak time (rt) was 18.37 min.

[0059] Microcrystalline cellulose (Avicel PH-101) was purchased from Sigma.

[0060] In the examples and control examples, conversion rate = product concentration / starting substrate concentration x 100%

[0061] The purification method of γ-cyclodextrin described in the embodiment of the present application can be purified according to the existing known purification method of γ-cyclodextrin. For example, the solution after the enzyme reaction is subjected to nanofiltration treatment, and then pure water is added, and the secondary nanofiltration treatment is continued to obtain a nanofiltration solution; (2) the concentrated solution after nanofiltration is used as a raw material for simulated moving bed purification, and the stationary phase of the simulated moving bed chromatography is dextran gel G-15, and the mobile phase is a mixed solution of ethanol and water; the operating temperature of the simulated moving bed chromatography system is 20±2°C, the injection liquid flow rate is 15-36mL / min, and the eluent flow rate is 2865mL / min, (3) the effluent is collected, and the effluent is concentrated by nanofiltration at 40-60°C, the pressure is 0.8-1.0MPa, and the nanofiltration membrane is 300-500, and its volume is concentrated to 1 / 4-1 / 6 of the original solution volume; after drying, the purified γ-cyclodextrin product is obtained.

[0062] Example 1. Expression of CBP, AGP, CGT, PPGK and PGM using E. coli expression system.

[0063] Step 1. Transformation of PET28a-CBP, PET28a-CBP and PET28a-CDP plasmids:

[0064] PET28a-CBP, PET28a-AGP, PET28a-CGTP, PET28a-PGMP, and PET28a-PPGK plasmids were synthesized by Genentech.

[0065] Take out the competent cells of Escherichia coli Rosetta (DE3) from -80℃ and place them in centrifuge tubes respectively, and quickly insert them into ice. After 5 minutes, wait for the bacterial block to melt, add the target plasmids respectively, and gently mix them by hand at the bottom of the EP tube (avoid using a gun to suck), and let it stand in ice for 25 minutes. Heat shock in a 42℃ water bath for 45 seconds, quickly put it back on ice and let it stand for 2 minutes. Add 700μL of sterile LB medium without antibiotics to the centrifuge tube, mix it well, and resuscitate it at 37℃, 200rpm for 60 minutes. Centrifuge at 5000rpm for 1 minute to collect the bacteria, retain about 100μL of supernatant, gently blow and resuspend the bacterial block and spread it on a plate containing LB medium with kanamycin resistance, and invert the plate in a 37℃ incubator for overnight culture.

[0066] Step 2. Colony verification:

[0067] Single colonies were picked from the overnight culture plates and placed in EP tubes containing 1.5 mL of LB liquid culture medium with kanamycin resistance, and cultured at 37°C, 200 rpm for 3 hours until turbidity, and then PCR was performed on the bacterial solution. Then agarose gel electrophoresis and detection were performed to obtain bands of the same size as the CBP, AGP, CGT, PPGK and PGM genes. The plasmids that were successfully connected to the PCR fragments and introduced were selected for the next step of protein expression.

[0068] Step 3. Expression and purification of CBP, AGP, CGT, PPGK and PGM:

[0069] (a) Prepare LB liquid culture medium and sterilize it by high-temperature steam sterilization at 121°C for 30 min.

[0070] (b) After the above solution is cooled, take out the E. coli Rosetta (DE3) strain stored at -20℃ and 50mg / mL kanamycin solution, shake them after they are completely dissolved, first add 50μL of kanamycin solution to a 300mL conical flask, shake them well; add 50μL of bacterial culture storage solution, shake them well. Incubate at 37℃, 220rpm overnight for activation. After activation, the color can be seen to change significantly to an opaque egg yolk color.

[0071] (c) First, take out about 5 mL of liquid from the 2 L conical flask as a reference for the subsequent absorbance measurement. Add 1 mL of kanamycin solution to each 2 L conical flask, shake well, and then add 10 mL of activated E. coli culture medium to each flask and culture at 37°C and 220 rpm.

[0072] (d) After about 4-5 hours, take about 3 mL of liquid from one bottle of culture medium and measure the absorbance at 600 nm. If the OD value is not between 0.4-0.6, continue culturing for a while and measure again until the OD value reaches 0.4-0.6. Then, add 1 mL of completely melted 1M IPTG solution to each conical flask, lower the shaker temperature to 16°C, and culture at 220 rpm for about 16 hours (or overnight).

[0073] (e) Pour the E. coli culture into a 250 mL centrifuge cup, balance it, and centrifuge it at 4000×g for 10 min (4°C). After centrifugation, pour out the supernatant directly, pour the precipitate into a 50 mL centrifuge tube, add a small amount of PBS solution containing 20 mM imidazole (containing 10% glycerol), vortex and resuspend the cells, and then store them at -20°C.

[0074] (f) The bacterial solution was disrupted by high-pressure disruptor and then ultracentrifuged at 13,000 × g at 4°C for 50 min.

[0075] (g) The proteins were purified using AKTA and Ni columns and desalted after purification to obtain relatively pure CBP, AGP, CGT, PPGK and PGM enzyme proteins.

[0076] The purified CBP, AGP, CGT, PPGK and PGM were analyzed by SDS-PAGE gel. Figure 2 shown.

[0077] Example 2. Using microcrystalline cellulose as raw material, phosphoric acid swollen cellulose was prepared.

[0078] Weigh 1g of microcrystalline cellulose (Avicel PH-101), add a little ddH2O to moisten, slowly add 30mL of concentrated phosphoric acid solution (concentration of about 86%) precooled to 0-5℃, cool to 0℃, stir for 60min, and add 20mL of acetone precooled to 0-5℃ and continue stirring for 10min after it becomes completely transparent, to produce a white flocculent precipitate. Centrifuge, wash with 100mL of deionized water 5 times, wash the precipitate with 5% sodium carbonate solution until the pH of the filtrate is in the range of 5.0-7.0, freeze-dry the treated sample to obtain phosphoric acid swollen cellulose.

[0079] Example 3. Preparation of cellobiose.

[0080] Cellobiose is synthesized by exocellulase using phosphate-swollen cellulose as substrate.

[0081] The reaction conditions are as follows: 10 g / mL phosphoric acid swollen cellulose, 20 U / g cellulose exocellulase (CBH I), constant volume with citric acid buffer (pH 4.8), reaction at 45°C for 24 hours. The reaction solution containing cellobiose is obtained for use in the following examples.

[0082] The concentration of cellobiose in the reaction solution was detected by HPLC. The HPLC analysis conditions were as follows: the chromatographic column was Aminex HPX-87H (300×7.8 mm), the mobile phase was 5 mM dilute sulfuric acid, the detector was RI detector, the flow rate was 0.5 mL / min, the column temperature was 45°C, and the retention time was 30 min.

[0083] Example 4. One-pot synthesis of γ-cyclodextrin.

[0084] The reaction conditions are as follows: 10 mg / mL cellobiose, 20 mM (NaPO3)6, 60 mM MgCl2, 0.2 mg / mL CBP, 0.2 mg / mL AGP, 0.2 mg / mL CGT, 0.2 mg / mL PPGK, 0.4 mg / mL PGM, HEPES buffer (pH 8.0) supplemented to 1 mL, 60 ° C for 6 hours. A no enzyme control was also performed. The HPLC method detected that the concentration of γ-cyclodextrin in the multi-enzyme cascade reaction solution was 3.14 mg / mL, and the cellobiose conversion rate was 31.4%. No γ-cyclodextrin was produced in the reaction solution without enzyme.

[0085] Example 5. One-pot synthesis of γ-cyclodextrin.

[0086] The reaction conditions are as follows: 10 mg / mL cellobiose, 30 mM (NaPO3)6, 50 mM magnesium chloride, 0.3 mg / mL CBP, 0.3 mg / mL AGP, 0.3 mg / mL CGT, 0.3 mg / mL PPGK, 0.6 mg / mL PGM, HEPES buffer (pH 8.0) supplemented to 1 mL, 50 ° C for 6 hours. At the same time, a no enzyme control was performed. The HPLC method detected that the concentration of γ-cyclodextrin in the reaction solution was 3.43 mg / mL, and the cellobiose conversion rate was 34.3%. No γ-cyclodextrin was produced in the reaction solution without enzyme.

[0087] Example 6. One-pot synthesis of γ-cyclodextrin.

[0088] The reaction conditions are as follows: 10 mg / mL cellobiose, 50 mM (NaPO3)6, 40 mM magnesium chloride, 0.6 mg / mL CBP, 0.6 mg / mL AGP, 0.6 mg / mL CGT, 0.6 mg / mL PPGK, 0.6 mg / mL PGM, HEPES buffer (pH 8.0) supplemented to 1 mL, 40 ° C for 6 hours. At the same time, a no enzyme control was performed. The γ-cyclodextrin concentration in the reaction solution was 3.67 mg / mL by HPLC, and the cellobiose conversion rate was 36.7%. No γ-cyclodextrin was produced in the reaction solution without enzyme.

[0089] Example 7. The reaction conditions are as follows: 10 mg / mL cellobiose, 30 mM (NaPO3)6, 50 mM magnesium chloride, 0.3 mg / mL CBP, 0.3 mg / mL AGP, 0.3 mg / mL CGT, 0.3 mg / mL PPGK, 0.6 mg / mL PGM, 0.5 mg / mL maltodextrin, HEPES buffer (pH 8.0) supplemented to 1 mL, 50°C for 6 h. The concentration of γ-cyclodextrin in the reaction solution was 4.09 mg / mL by HPLC, and the cellobiose conversion rate was 40.9%.

[0090] Example 8. The reaction conditions are as follows: 10 mg / mL cellobiose, 30 mM (NaPO3)6, 50 mM magnesium chloride, 0.3 mg / mL CBP, 0.3 mg / mL AGP, 0.3 mg / mL CGT, 0.3 mg / mL PPGK, 0.6 mg / mL PGM, 0.3 mg / mL maltodextrin, HEPES buffer (pH 8.0) supplemented to 1 mL, 50°C for 6 h. The concentration of γ-cyclodextrin in the reaction solution was 4.24 mg / mL by HPLC, and the cellobiose conversion rate was 42.4%.

[0091] Comparative Example 1. Referring to the technical scheme of Example 5, ATP was added to the reaction system to conduct γ-cyclodextrin synthesis research.

[0092] The reaction conditions are as follows: 10 mg / mL cellobiose, 30 mM (NaPO3)6, 50 mM magnesium chloride, 0.3 mg / mL CBP, 0.3 mg / mL AGP, 0.3 mg / mL CGT, 0.3 mg / mL PPGK, 0.6 mg / mL PGM, 8 mM ATP, HEPES buffer (pH 8.0) supplemented to 1 mL, 50°C for 6 hours, and a no enzyme control was performed. The HPLC method detected that the concentration of γ-cyclodextrin in the reaction solution was 3.41 mg / mL, and the cellobiose conversion rate was 34.1%.

[0093] The results of Control Example 1 and Example 5 show that the addition of ATP has no effect on the synthesis of γ-cyclodextrin.

[0094] Comparative Example 2-8. Referring to the technical solution of Example 5, the synthesis of γ-cyclodextrin was studied according to the enzyme addition amount described in Table 1.

[0095] The reaction conditions were as follows: 10 mg / mL cellobiose, 30 mM (NaPO3)6, 50 mM magnesium chloride, the amounts of CBP, AGP, CGT, PPGK, and PGM enzymes are shown in Table 1, HEPES buffer (pH 8.0) was added to 1 mL, and the reaction was performed at 50°C for 6 h. The concentration of γ-cyclodextrin in the reaction solution was detected by HPLC, and the results are recorded in Table 1.

[0096] Table 1

[0097] Table 1 Data Description:

[0098] (1) Using cellobiose as substrate, γ-cyclodextrin cannot be synthesized through the CBP-AGP enzyme cascade reaction.

[0099] (2) Using cellobiose as substrate, no cyclodextrin synthesis was detected by the CBP, AGP and CGT enzyme cascade reaction.

[0100] Comparative Example 9. Glucose-6-phosphate 10 mg / mL, 30 mM (NaPO3)6, 50 mM magnesium chloride, 0.3 mg / mL AGP, 0.3 mg / mL CGT, HEPES buffer (pH 8.0) supplemented to 1 mL, reacted at 50°C for 6 hours. The concentration of γ-cyclodextrin in the reaction solution was 1.2 mg / mL as determined by HPLC.

[0101] It was shown that γ-cyclodextrin (γ-CD) was synthesized by the enzymatic reaction of AGP and CGT using glucose-6-phosphate as substrate.

[0102] It is speculated that in Control Example 8, the substrate cellobiose or the intermediate reaction product glucose may have an inhibitory effect on CGT.

[0103] Comparative Example 10. In the reaction system of Comparative Example 9, glucose was added.

[0104] Glucose-6-phosphate 10 mg / mL, glucose 10 mg / mL, 30 mM (NaPO3)6, 50 mM magnesium chloride, 0.3 mg / mL AGP, 0.3 mg / mL CGT, HEPES buffer (pH 8.0) supplemented to 1 mL, reacted at 50°C for 6 hours. No γ-cyclodextrin was produced in the reaction solution by HPLC.

[0105] Control Example 11. In the reaction system of Control Example 9, cellobiose was added as follows:

[0106] Glucose-6-phosphate 10 mg / mL, cellobiose 10 mg / mL, 30 mM (NaPO3)6, 50 mM magnesium chloride, 0.3 mg / mL AGP, 0.3 mg / mL CGT, HEPES buffer (pH 8.0) supplemented to 1 mL, reacted at 50°C for 6 hours. No γ-cyclodextrin was generated in the reaction solution by HPLC.

[0107] Control Examples 9-11 illustrate that when glucose-6-phosphate is used as a substrate in the AGP and CGT enzyme-linked reaction system, after adding glucose or cellobiose, the reaction system loses the synthetic activity of γ-cyclodextrin, which indicates that cellobiose and glucose do have an inhibitory effect on CGT.

[0108] We have invented a glucose-6-phosphate and inorganic phosphate co-regeneration module based on PPGK and PGM. PPGK from Arthrobacter sp. can synthesize glucose-6-phosphate (G-6-P) using glucose and inorganic phosphate as substrates. PGM from Thermococcus kodakaraensis can synthesize glucose-1-phosphate (G-1-P) using G-6-P as substrate. Figure 1Through the enzyme-linked reaction of PPGK and PGM, the inhibition of cellobiose and glucose on the catalytic activity of CGT was successfully released, and the conversion rate of cellobiose was improved.

[0109] Comparative Examples 12-14. Referring to the technical solution of Example 5, the metal salt was added in the amount described in Table 2 to carry out the synthesis study of γ-cyclodextrin.

[0110] The reaction conditions were as follows: 10 mg / mL cellobiose, (NaPO3)6, Mg 2+ The added amounts are shown in Table 2, 0.3 mg / mL CBP, 0.3 mg / mL AGP, 0.3 mg / mL CGT, 0.3 mg / mL PPGK, 0.6 mg / mL PGM, HEPES buffer (pH 8.0) is added to 1 mL, and the reaction is carried out at 50°C for 6 hours. The concentration of γ-cyclodextrin in the reaction solution is detected by HPLC, and the results are recorded in Table 2.

[0111] Table 2

[0112]

[0113]

[0114] Table 2 Data Description:

[0115] (1) Compared with Examples 4-6 of the present invention, when Mg 2+ When the concentration is lower than 30mM, the yield of γ-cyclodextrin obtained by catalysis is low; when the concentration is higher than 70mM, the yield of γ-cyclodextrin obtained by catalysis does not change much;

[0116] (2) Compared with Examples 4-6 of the present invention, when the concentration of (NaPO3)6 is lower than 10 mM, the yield of γ-cyclodextrin obtained by catalysis is low; when the concentration of (NaPO3)6 is higher than 60 mM, the yield of γ-cyclodextrin obtained by catalysis does not change much.

[0117] The above description is only an embodiment of the present invention. All equivalent changes made according to the scope of the patent application of the present invention, as well as the research and selection of process parameters such as the temperature of the catalytic reaction, the amount of enzyme added, the reaction time, and the pH value, shall fall within the scope of the present invention.

[0118] It should be noted that the above examples are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention is described in detail with reference to the given examples, those skilled in the art may modify or replace the technical solution of the present invention as needed without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. A method for preparing γ-cyclodextrin, characterized in that: The steps include: Step 1. Using cellulase as a catalyst and a raw material containing cellulose as a substrate to prepare a raw material containing cellobiose; Step 2. Using the raw material containing cellobiose as a substrate to carry out a multi-enzyme cascade reaction to synthesize γ-cyclodextrin, the reaction solution of the multi-enzyme cascade reaction includes: cellobiose phosphorylase, α-glucan phosphorylase, γ-cyclodextrin glycosyltransferase, polyphosphate glucose kinase, phosphoglucomutase, sodium hexametaphosphate, Mg 2+ and HEPES buffer, wherein the sodium hexametaphosphate concentration is 20-50 mM, the Mg 2+ The concentration is 40-60mM.

2. The method for preparing γ-cyclodextrin according to claim 1, characterized in that: The cellulase in step 1 is selected from cellulase exonuclease and β-glucosidase.

3. The method for preparing γ-cyclodextrin according to claim 1, characterized in that: The source of the cellulase in step 1 is selected from wild-type or genetically modified cells of Trichoderma reesei, Yarrowia lipolytica cells, Talaromyces emersonii, Neosartorya fischeri, Humicola grisea, Chaetomium thermophilum, filamentous fungi and Cellulomonas fimi.

4. The method for preparing γ-cyclodextrin according to claim 2, characterized in that: The cellulase exoenzyme is derived from wild-type or genetically modified cells of Trichoderma reesei.

5. The method for preparing γ-cyclodextrin as claimed in claim 2, characterized in that: The source of the β-glucosidase is selected from wild-type or genetically modified cells of Trichoderma reesei, Trichoderma viride, Penicillium piceum, and Aspergillus niger.

6. The method for preparing γ-cyclodextrin according to claim 1, characterized in that: In step 2, the source of the cellobiose phosphorylase is selected from wild-type or genetically modified cells of Clostridium thermocellu, Cellvibrio gilvus, Cellulomonas sp., Clostridium stercorarium, Thermotoga neapolitana, Cellulomonas uda and Ruminococcus albus.

7. The method for preparing γ-cyclodextrin according to claim 1, characterized in that: The amino acid sequence of the cellobiose phosphorylase in step 2 is shown in SEQ ID NO:

1.

8. The method for preparing γ-cyclodextrin according to claim 1, characterized in that: The source of the α-glucan phosphorylase in step 2 is selected from: Hordeum vulgare sub sp., Hungateiclostridium thermocellum, Thermus thermophilus, Pyrococcus sp., Hyperthermophilic Archaea ( Pyrococcushorikoshii ) , Pyrococcus furiosus, Thermococcus barophilus, Thermococcus kodakarensis, and Thermotoga maritima.

9. The method for preparing γ-cyclodextrin according to claim 1, characterized in that: The amino acid sequence of the α-glucan phosphorylase (Alpha-1,4glucan phosphorylase) in step 2 is shown in SEQ ID NO:

2.

10. The method for preparing γ-cyclodextrin according to claim 1, characterized in that: The source of the γ-cyclodextrin glycosyltransferase in step 2 is selected from: wild-type or genetically modified cells of Evansella clarkii, Escherichia coli, Alkaliphilic Bacillus, and Bacillus clarkii.

11. The method for preparing γ-cyclodextrin according to claim 1, characterized in that: The amino acid sequence of the γ-cyclodextrin glycosyltransferase in step 2 is shown in SEQ ID NO:

3.

12. The method for preparing γ-cyclodextrin according to claim 1, characterized in that: The amino acid sequence of the polyphosphate glucokinase described in step 2 is shown in SEQ ID NO:

4.

13. The method for preparing γ-cyclodextrin according to claim 1, characterized in that: The source of the phosphoglucomutase in step 2 is selected from wild-type or genetically modified cells of Acetobacter xylinum, Pyrococcus horikoshii, Thermococcus kodakarensis, Arabidopsis, and Oryctolagus cuniculus.

14. The method for preparing γ-cyclodextrin according to claim 1, characterized in that: The phosphoglucomutase in step 2 is derived from Thermococcus kodakarensis, and its amino acid sequence is shown in SEQ ID NO:

5.

15. The method for preparing γ-cyclodextrin according to claim 1, characterized in that: The reaction temperature in step 2 is 40-60°C.

16. The method for preparing γ-cyclodextrin according to claim 1, characterized in that: The reaction temperature in step 2 is 50°C.

17. The method for preparing γ-cyclodextrin according to claim 1, characterized in that: The concentration of sodium hexametaphosphate in the reaction solution in step 2 is 25-45 mM.

18. The method for preparing γ-cyclodextrin according to claim 1, characterized in that: Mg in the reaction solution of step 2 2 + The concentration ranged from 40-60 mM.

19. The method for preparing γ-cyclodextrin according to claim 1, characterized in that: Mg in the reaction solution of step 2 2 + The concentration is 45-53mM.

20. The method for preparing γ-cyclodextrin according to claim 1, characterized in that: Step 2: The reaction solution also includes maltodextrin, the concentration of which is 0 . 3-0 . 7g / L.

21. The method for preparing γ-cyclodextrin according to claim 1, characterized in that: Step 2: The reaction solution further comprises maltodextrin, wherein the amount of the maltodextrin is 0 . 4-0 . 5g / L.

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