WO2019076021A1 - 一种橙皮素的制备方法、橙皮素中间体的制备方法和用于制备橙皮素的生物酶 - Google Patents

一种橙皮素的制备方法、橙皮素中间体的制备方法和用于制备橙皮素的生物酶 Download PDF

Info

Publication number
WO2019076021A1
WO2019076021A1 PCT/CN2018/084377 CN2018084377W WO2019076021A1 WO 2019076021 A1 WO2019076021 A1 WO 2019076021A1 CN 2018084377 W CN2018084377 W CN 2018084377W WO 2019076021 A1 WO2019076021 A1 WO 2019076021A1
Authority
WO
WIPO (PCT)
Prior art keywords
rhamnosidase
reaction
hesperetin
glucosidase
hesperidin
Prior art date
Application number
PCT/CN2018/084377
Other languages
English (en)
French (fr)
Inventor
傅荣昭
刘立辉
刘滔滔
曹磊
郭杏林
Original Assignee
邦泰生物工程(深圳)有限公司
江西邦泰绿色生物合成生态产业园发展有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 邦泰生物工程(深圳)有限公司, 江西邦泰绿色生物合成生态产业园发展有限公司 filed Critical 邦泰生物工程(深圳)有限公司
Priority to PCT/CN2018/084377 priority Critical patent/WO2019076021A1/zh
Priority to CN201880001976.7A priority patent/CN109312375B/zh
Publication of WO2019076021A1 publication Critical patent/WO2019076021A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/02—Oxygen as only ring hetero atoms
    • C12P17/06—Oxygen as only ring hetero atoms containing a six-membered hetero ring, e.g. fluorescein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
    • C12N9/2445—Beta-glucosidase (3.2.1.21)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/16—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing two or more hetero rings
    • C12P17/162—Heterorings having oxygen atoms as the only ring heteroatoms, e.g. Lasalocid
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/0104—Alpha-L-rhamnosidase (3.2.1.40)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01043—Beta-L-rhamnosidase (3.2.1.43)

Definitions

  • the invention relates to the technical field of biomedicine, in particular to a preparation method of hesperetin, a preparation method of hesperetin intermediate and a biological enzyme for preparing hesperetin.
  • Hesperetin has a molecular formula of C 16 H 14 O 6 and a melting point of 227.5 ° C. It has poor water solubility, is almost insoluble in water, is insoluble in chloroform and pretty, and is easily soluble in ethanol. It is mostly in the form of hesperidin in nature. Studies have shown that hesperetin is a kind of traditional Chinese medicine extract, mainly found in traditional Chinese medicines such as dried tangerine peel, green skin and clam shell. It is their main active ingredient and has anti-tumor, anti-oxidation, anti-inflammatory and anti-atherosclerosis. A variety of effects.
  • acid hydrolysis method is used to hydrolyze neohesperidin or hesperidin to obtain hesperetin.
  • hesperetin there are many reports on the preparation of hesperetin such as aqueous phase-ethanol method, methanol method and cyclohexanol method.
  • these methods are cumbersome, have serious pollution, and are difficult to purify the product.
  • the enzymatic hydrolysis method has the characteristics of lower hydrolysis temperature and better control of hydrolysis reaction, but the yield of enzyme produced by fermentation is not high, the price is expensive, and the hydrolysis rate is low. If the enzyme system contains various enzymes, The resulting reaction product is still complex, making separation and purification difficult.
  • the present invention provides a preparation method of hesperetin, a preparation method of hesperetin intermediate, and a biological enzyme for preparing hesperetin; the preparation method of the invention has simple process and high yield and Safe and environmentally friendly.
  • the present invention provides a method for preparing hesperetin, comprising:
  • the substrate preparation solution is added to a buffer containing ⁇ -L-rhamnosidase and ⁇ -glucosidase at a rate of 0.1-100 mL/min by a bottom stream addition reaction to obtain a reaction solution, and the reaction solution is added. After completion, the reaction is continued for 0.5-1 h, wherein the pH of the reaction solution is 6.0-7.0, the reaction temperature is maintained at 45-65 ° C, and the ⁇ -L-rhamnosidase is derived from Streptomyces, the ⁇ - Glucosidase is derived from the genus Thermotoxin;
  • the pH of the reaction mixture was adjusted to 3.0 to 5.0 to completely precipitate the solid product, and the solid product was collected, which was hesperetin.
  • the process employs a biological enzymatic method, and the hesperidin is represented by the formula (I), and the hesperetin is represented by the formula (III).
  • formula (II) shows Hesperitin-7-O-glucoside, also known as hesperidin monoglucoside, molecular formula C 22 H 24 O 11 , relative molecular mass.
  • 464 which is an intermediate in the process of hydrolyzing hesperidin to produce hesperetin, is a product obtained by removing hemoside from hesperidin, and is collectively referred to as hesperetin intermediate in the present invention.
  • the content of the hesperetin intermediate is very low, which is negligible compared to the content of hesperetin.
  • the chemical structural formula of the novel hesperidin is as shown in (VI), and the hesperetin can also be obtained by the same as the above-mentioned bioenzymatic orange.
  • the gene coding sequence of the ⁇ -L-rhamnosidase comprises the nucleotide sequence shown in SEQ ID NO: 1, and the gene coding sequence of the ⁇ -glucosidase comprises SEQ ID NO: The nucleotide sequence shown in 2.
  • Alpha-L-rhamnosidase and ⁇ -glucosidase are widely available and are currently found in animal tissues, plants and microorganisms, but there are relatively few reports on animal tissues and plant sources.
  • the enzymatic properties of ⁇ -L-rhamnosidase or ⁇ -glucosidase from different microbial sources vary, including the specific activity of the enzyme, the substrate range of the enzyme, the optimum pH, the optimum temperature, the action time and the enzyme. Stability and other aspects.
  • the optimum pH of the bacterial-derived ⁇ -L-rhamnosidase is neutral or alkaline
  • the optimum pH of the ⁇ -L-rhamnosidase secreted by the fungus is generally in the acidic range.
  • ⁇ -L-rhamnosidase has hydrolysis ability to hesperidin or neohesperidin; the ⁇ -L-rhamnosidase of the present invention is derived from Streptomyces, through hydrolysis The activity size of the product (hesperidin or neohesperidin) is screened to obtain a highly viable gene, and the ⁇ -L-rhamnosidase of the present invention is expressed, and the ⁇ -L-rhamnosidase can be Efficient and specific hydrolysis of hesperidin or neohesperidin and obtain hesperetin-7-O-glucoside.
  • the ⁇ -glucosidase of the present invention belongs to the genus Thermotoga petrophila RKU-1, which is obtained by the same method, and the ⁇ -glucosidase can efficiently and specifically hydrolyze hesperetin-7-O- Glucoside gives hesperetin.
  • the ⁇ -L-rhamnosidase and the ⁇ -glucosidase are produced by co-expression of E. coli.
  • the ⁇ -L-rhamnosidase of the present invention and the ⁇ -glucosidase are produced by co-expression of E. coli Rosetta (DE3).
  • E. coli Rosetta E. coli Rosetta
  • the ⁇ -L-rhamnosidase and the ⁇ -glucosidase are two separate protein molecules.
  • the ⁇ -L-rhamnosidase and the ⁇ -glucosidase participate in the reaction in the form of a crude enzyme solution.
  • the mass ratio of the ⁇ -L-rhamnosidase to the ⁇ -glucosidase is 1: (0.1-10).
  • the crude enzyme solution is obtained by inducing expression of the ⁇ -L-rhamnosidase and the ⁇ -glucosidase by Escherichia coli Rosetta (DE3), and then centrifuging and cleaning the Escherichia coli Rosetta (DE3) Collected after breaking the cell.
  • the invention adopts a method for obtaining ⁇ -L-rhamnosidase and ⁇ -glucosidase crude enzyme liquid hydrolyzed hesperidin or neohesperidin and preparing hesperetin by using a co-expression form, which has the advantages of simple process, cost saving and high yield. And green.
  • the ⁇ -L-rhamnosidase is produced by expression in E.
  • the ⁇ -glucosidase is produced by expression in E. coli.
  • the ⁇ -L-rhamnosidase can be adjusted in a certain range in the mass concentration of the crude enzyme solution.
  • the ⁇ -L-rhamnosidase has a mass concentration in the crude enzyme solution ranging from 0.2 to 3%.
  • the carboxy terminus of the amino acid sequence of the ⁇ -L-rhamnosidase contains a His tag.
  • the His tag facilitates the isolation and purification of the expressed protein, as well as analysis and tracking in experiments, such as for analysis in immunoblot experiments.
  • the specific process for preparing the substrate preparation solution comprises suspending hesperidin or neohesperidin in water, and adding 5-10 mol/L sodium hydroxide solution to the hesperidin or the new The hesperidin is completely dissolved to obtain a substrate preparation solution.
  • the final concentration of the sodium hydroxide in the substrate formulation is from 0.25 to 0.5 mol/L.
  • the sodium hydroxide solution of the concentration can effectively increase the solubility of hesperidin. Accordingly, the sodium hydroxide solution of the concentration can effectively increase the solubility of neohesperidin.
  • the substrate preparation solution is added to a buffer containing ⁇ -L-rhamnosidase and ⁇ -glucosidase at a rate of 0.1-100 mL/min by a bottom stream addition reaction.
  • the addition time of the substrate preparation liquid is 4-6 hours.
  • the substrate preparation solution has a running time of 4.5 to 5.5 h.
  • the addition time refers to the time during which the substrate preparation liquid is completely added to the reaction liquid, and the flow rate of the substrate preparation liquid in the addition of the bottom stream is performed according to the addition time. Adjustment.
  • the hesperidin or the new hesperidin has a mass solubility in the reaction solution of 2.5 to 10%. Further, optionally, the hesperidin or the new hesperidin has a mass solubility of 5-10% in the reaction solution. For example, the hesperidin or the new hesperidin has a mass solubility in the reaction solution of 5%, or 8%, or 10%.
  • the pH of the reaction solution is from 6.0 to 7.0, and the reaction temperature is maintained at 45 to 65 °C. Further, optionally, the pH of the reaction liquid is 6.5-7.0. Alternatively, the reaction temperature is maintained at 55-65 °C.
  • the ⁇ -L-rhamnosidase has a mass concentration in the reaction solution of 0.05-0.5%. Further optionally, the ⁇ -L-rhamnosidase has a mass concentration in the reaction solution of 0.1 to 0.5%.
  • the buffer of the buffer comprises phosphate, Tris buffer or other buffer.
  • the buffer comprises a phosphate buffer or a Tris buffer.
  • the buffer comprises a phosphate buffer.
  • the buffer includes a sodium phosphate buffer.
  • the sodium phosphate buffer and the sodium hydroxide solution have the same cation, which can reduce the addition of other impurity cations and prevent other impurity cations from reducing the enzyme (including ⁇ -L-rhamnosidase or ⁇ -glucosidase).
  • Biological activity The pH of the buffer is 6.0-7.0.
  • the concentration of the buffer is 80-110 mmol/L.
  • the concentration of the buffer is 70-100 mmol/L.
  • the concentration of the buffer is from 90 to 110 mmol/L.
  • the concentration of the buffer is 70 mmol/L, or 80 mmol/L, or 100 mmol/L.
  • the process of adjusting the pH of the reaction solution to 3.0-5.0 to completely precipitate the solid product comprises adding an acid solution to the reaction solution and adjusting the pH of the reaction solution to 3.0-5.0, and stirring the reaction 0.5- After 1 h, the solid product was completely precipitated.
  • the concentration of the acid solution is 2-6 mol/L. Further, the concentration of the acid solution is 4-6 mol/L.
  • the acid solution includes hydrochloric acid.
  • the collecting process comprises the steps of filtering, washing, drying and recrystallizing the reaction solution.
  • the drying process comprises vacuum drying at a temperature of 50-60 °C.
  • the recrystallization process comprises: completely dissolving the dried solid in methanol at a temperature of 40-60 ° C, filtering to obtain a filtrate, concentrating the filtrate to 5-10% of the original volume, and adding water to the The hesperetin crystal solids are all precipitated, and the operation is repeated to obtain a fine orange hesper.
  • the preparation method of the hesperetin provided by the first aspect of the invention has the advantages of simple process, low cost and environmental protection; and the hesperetin obtained by the preparation method has an extremely high yield.
  • the invention utilizes the bottom stream addition method to add the hesperidin or neohesperidin bottom stream to the reaction system, and the final concentration of the substrate can reach 10%, which is far greater than the prior art, compared with the existing preparations reported.
  • the process has been improved by about 5,000 times, and the production capacity has been greatly improved to adapt to large-scale industrial production.
  • the present invention provides a method for preparing an hesperetin intermediate, comprising:
  • the substrate preparation solution is added to a buffer containing ⁇ -L-rhamnosidase at a rate of 0.1-100 mL/min by a bottom stream addition reaction to obtain a reaction solution, and the reaction is continued after the completion of the addition.
  • 1h wherein the pH of the reaction solution is 6.0-7.0, the reaction temperature is maintained at 45-65 ° C, and the ⁇ -L-rhamnosidase is derived from Streptomyces;
  • the solid product precipitated in the reaction liquid was collected, and the solid product was an hesperetin intermediate.
  • the collecting process comprises a process of filtering, washing, drying, and recrystallizing the reaction solution.
  • the recrystallization process comprises: completely dissolving the dried solid in an acetone solution at a temperature of 30-40 ° C, filtering to obtain a filtrate, and concentrating the filtrate to 5-10% of the original volume, and then adding water to the solution.
  • the hesperidin intermediate crystal solids are all precipitated, and the operation is repeated to obtain a fine product of the hesperidin intermediate.
  • the acetone solution means an aqueous solution having an acetone volume ratio of 60 to 80%.
  • the solid product of the hesperetin intermediate is hardly further hydrolyzed in the reaction system, that is, the content of the hesperetin in the reaction system is extremely small.
  • the hesperetin solid product can be obtained by subjecting the collected hesperetin intermediate to the bottom stream addition and adding the ⁇ -glucosidase.
  • the preparation method of the hesperetin intermediate provided by the second aspect of the invention provides the preparation of the hesperetin intermediate by using ⁇ -L-rhamnosidase and the bottom stream addition method, the method is simple, the yield is High, green and environmentally friendly; and the specificity of ⁇ -L-rhamnosidase is good, which can effectively reduce the further hydrolysis of the hesperidin intermediate.
  • the present invention provides a biological enzyme for preparing hesperetin, which comprises ⁇ -L-rhamnosidase and ⁇ -glucosidase, the gene of ⁇ -L-rhamnosidase
  • the coding sequence includes the nucleotide sequence set forth in SEQ ID NO: 1
  • the gene coding sequence of the ⁇ -glucosidase includes the nucleotide sequence set forth in SEQ ID NO: 2.
  • amino acid sequence of the ⁇ -L-rhamnosidase comprises the amino acid sequence set forth in SEQ ID NO: 3.
  • the gene coding sequence of the ⁇ -glucosidase includes the nucleotide sequence as shown in SEQ ID NO: 4.
  • the gene coding sequence of the amino acid sequence shown in SEQ ID NO: 3 is represented by SEQ ID NO: 1; alternatively, the gene coding sequence of the amino acid sequence of the ⁇ -L-rhamnosidase should be Considering a degenerate base, ie, the coding gene of the amino acid sequence set forth in SEQ ID NO: 3 includes the nucleotide sequence set forth in SEQ ID NO: 1, and the protective range should also protect the base having SEQ ID NO: 1 A nucleotide sequence of degenerate nature, the amino acid sequence corresponding to these nucleotide sequences is still SEQ ID NO: 3. Similarly, for the ⁇ -glucosidase, the gene encoding the amino acid sequence shown in SEQ ID NO: 4 should also consider degenerate bases.
  • the ⁇ -L-rhamnosidase can efficiently hydrolyze hesperidin or neohesperidin and obtain hesperetin-7-O-glucoside.
  • the ⁇ -glucosidase can efficiently hydrolyze hesperetin-7-O-glucoside to obtain hesperetin.
  • the hesperidin or the new hesperidin is hydrolyzed by ⁇ -L-rhamnosidase to obtain hesperetin-7-O-glucoside (hesperetin intermediate), which does not hydrolyze to form other
  • the intermediate product, the hesperetin-7-O-glucoside is also not decomposed by the alpha-L-rhamnosidase.
  • the ⁇ -L-rhamnosidase of the present invention is derived from Streptomyces, and the gene sequence of the ⁇ -L-rhamnosidase is obtained by an optimized screening experiment.
  • the ⁇ -glucosidase of the present invention is derived from the genus Thermotoxin of the sea, and the gene sequence of the ⁇ -glucosidase is obtained by an optimized screening experiment.
  • the ⁇ -L-rhamnosidase is produced by expression in E. coli.
  • the ⁇ -glucosidase is produced by expression by E. coli.
  • the ⁇ -L-rhamnosidase and the ⁇ -glucosidase are heterologously expressed in the system in which the E. coli expression is expressed.
  • the present invention preferably has an E. coli expression system which is simple and feasible, has a short culture period, low fermentation cost, and high protein yield.
  • the ⁇ -L-rhamnosidase and ⁇ -glucosidase have good biological activity and high purity, and can be widely used in the fields of biopharmaceuticals, protein production and the like.
  • Preferred alpha-L-rhamnosidases and beta-glucosidases of the invention have higher yields and are more biologically active than conventional fermentation systems. Both ⁇ -L-rhamnosidase and ⁇ -glucosidase have good specificity.
  • the ⁇ -L-rhamnosidase and ⁇ -glucosidase are heterologously expressed in E. coli by constructing a recombinant plasmid comprising the ⁇ -L-rhamnosidase and/or The gene coding sequence of the ⁇ -glucosidase.
  • the gene coding sequence of the ⁇ -L-rhamnosidase is 5
  • the RBS sequence was inserted in front of the end, and the RBS sequence was inserted before the 5' end of the gene coding sequence of the ⁇ -glucosidase.
  • the nucleotide sequence of the RBS sequence is set forth in SEQ ID NO: 5.
  • the RBS sequence of the present invention is a ribosome binding site (RBS) sequence, which can effectively promote independent transcription of two sets of protein genes ( ⁇ -L-rhamnosidase gene and ⁇ -glucosidase gene). And translation.
  • RBS ribosome binding site
  • the vector plasmid of the recombinant plasmid is a pET22b(+) plasmid. Inserting the gene coding sequence of the ⁇ -L-rhamnosidase and/or the ⁇ -glucosidase into the pET22b(+) plasmid to obtain a recombinant plasmid, which can be efficiently and productively produced in the large intestine Expression in Bacillus gives ⁇ -L-rhamnosidase and/or the ⁇ -glucosidase.
  • the ⁇ -L-rhamnosidase and/or the gene coding sequence of the ⁇ -glucosidase when the ⁇ -L-rhamnosidase and/or the gene coding sequence of the ⁇ -glucosidase is inserted into the pET22b(+) plasmid, the ⁇ -L-rhamnosidase and/or The 5' end of the gene coding sequence of ⁇ -glucosidase may be added with a start codon (such as ATG), and the ⁇ -L-rhamnosidase and/or the gene coding sequence of the ⁇ -glucosidase A stop codon (such as TAA) can be added to the 3' end.
  • the 5' end of the ⁇ -L-rhamnosidase and/or the gene coding sequence of the ⁇ -glucosidase is inserted into the RBS sequence after the start codon is added.
  • the nucleotide sequence of the His-tag (histidine tag) is added to the gene fragment of the ⁇ -L-rhamnosidase, and the expressed protein is tagged with His tag, and the His tag is favorable for expression. Isolation and purification of post-proteins, as well as analysis and tracing in experiments, such as for immunoblot experiments.
  • the His tag can be used to isolate the ⁇ -L-rhamnosidase and ⁇ -glucosidase.
  • the present invention also provides a recombinant plasmid comprising one or both of a gene coding sequence of ⁇ -L-rhamnosidase and a gene coding sequence of the ⁇ -glucosidase, the ⁇
  • the gene coding sequence of the -L-rhamnosidase comprises the nucleotide sequence set forth in SEQ ID NO: 1
  • the gene coding sequence of the ⁇ -glucosidase comprises the nucleotide set forth in SEQ ID NO: sequence.
  • the invention also provides a preparation method of a recombinant plasmid, comprising:
  • the RBS sequence is inserted before the 5' end of the ⁇ -glucosidase gene fragment.
  • the RBS sequence is inserted before the 5' end of the ⁇ -L-rhamnosidase gene fragment.
  • the enzyme cleavage site of the double digestion reaction may be Nde I endonuclease and Xho I endonuclease.
  • the preparation method according to the present invention adopts a biological enzymatic method (including ⁇ -L-rhamnosidase and ⁇ -glucosidase), and the reaction is carried out by using a bottom stream addition method, and the final concentration of the substrate can reach 10%. Compared with the traditional process, it has increased by about 5,000 times;
  • the preparation method of the invention does not use organic reagents in the preparation process, adopts the biological enzymatic method, is efficient and simple, has low cost, is environmentally friendly, and can be widely applied to industrial scale production;
  • the hesperetin and the intermediate thereof prepared by the preparation method of the invention have high purity and can be widely used in the pharmaceutical field or the biomedical field;
  • the invention utilizes E. coli heterologous expression of ⁇ -L-rhamnosidase and ⁇ -glucosidase, and has high expression level of enzyme, good hydrolysis activity, strong specificity and single hydrolysis product.
  • FIG. 1 is a plasmid map of a recombinant plasmid pET22b-BGL04-Rha01 according to an embodiment of the present invention
  • FIG. 2 is a plasmid map of a recombinant plasmid pET22b-Rha01 according to an embodiment of the present invention
  • FIG. 3 is a gel electrophoresis diagram of a BGL04-Rha01 crude enzyme solution according to an embodiment of the present invention
  • FIG. 4 is a gel electrophoresis diagram of a Rha01 crude enzyme solution according to an embodiment of the present invention.
  • Figure 6 is a high performance liquid chromatogram of hesperetin-7-O-glucoside provided by an embodiment of the present invention.
  • the gene coding sequences of ⁇ -L-rhamnosidase (Rha01) and ⁇ -glucosidase (BGL04) were obtained by PCR amplification experiments.
  • the gene coding sequence of ⁇ -L-rhamnosidase comprises the nucleotide sequence shown in SEQ ID NO: 1
  • the gene coding sequence of ⁇ -glucosidase comprises the nucleoside as shown in SEQ ID NO: 2.
  • the base sequence of the upstream primer corresponding to the ⁇ -L-rhamnosidase is shown in SEQ ID NO: 6, and the base sequence of the downstream primer is shown in SEQ ID NO: 7.
  • the base sequence of the upstream primer corresponding to the ⁇ -glucosidase is shown in SEQ ID NO: 8
  • the base sequence of the downstream primer is shown in SEQ ID NO: 9. Inserting the RBS sequence before the 5' end of the Rha01 and the BGL04 gene, the RBS sequence is set forth in SEQ ID NO: 5, and inserting it into the plasmid pET22b(+) by the upstream and downstream primers and constructing pET22b- BGL04-Rha01 recombinant plasmid.
  • ⁇ -L-rhamnosidase (Rha01) gene coding sequence
  • the gene coding sequence of the ⁇ -L-rhamnosidase comprising SEQ ID NO:
  • the nucleotide sequence shown in Figure 1 the base sequence of the upstream primer is shown in SEQ ID NO: 10
  • the base sequence of the downstream primer is shown in SEQ ID NO: 11.
  • the gene of ⁇ -L-rhamnosidase was inserted into the plasmid pET22b(+) by the upstream and downstream primers and the pET22b-BGL04-Rha01 recombinant plasmid was constructed.
  • the PCR amplification system was carried out by the corresponding upstream primer and downstream primer, respectively, and the amplification system was configured as follows:
  • the PCR amplification procedure was: predenaturation at 98 ° C for 2 min; denaturation at 98 ° C for 10 s; annealing at 58 ° C for 15 s; extension at 72 ° C for 3 min; after 30 cycles, extension at 72 ° C for 10 min.
  • the PCR products were purified by gel recovery kit and digested with restriction endonucleases Nde I and EcoR I, EcoR I and Xho I respectively. After digestion, they were ligated with T4 ligase and digested with Nde I and Xho I. Plasmid pET22b(+).
  • the ligation product was transferred into Escherichia coli DH5 ⁇ , and after screening with ampicillin resistance (Amp+), the colonies were picked and sequenced. After sequencing, the recombinant plasmid pET22b-co-expressing ⁇ -L-rhamnosidase and ⁇ -glucosidase was obtained. BGL04-Rha01 and pET22b-Rha01.
  • the plasmid map of the recombinant plasmid pET22b-BGL04-Rha01 is shown in Figure 1; the plasmid map of the recombinant plasmid pET22b-Rha01 is shown in Figure 2.
  • One or more of the constructed recombinant plasmids pET22b-Rha01, pET22b-BGL04 and pET22b-BGL04-Rha01 were transferred into E. coli Rosetta (DE3), and inoculated with 1% of the inoculum to LB medium containing 10 mL.
  • ⁇ -L-rhamnosidase (Rha01) crude enzyme solution ⁇ -glucosidase (BGL04) crude enzyme solution
  • BGL04-Rha01 crude enzyme solution contains ⁇ -L-rhamnosidase (Rha01) and ⁇ -glucosidase (BGL04).
  • FIG. 1 is a gel electrophoresis diagram of the BGL04-Rha01 crude enzyme solution, wherein the lane Marker is a molecular weight step (Thermo Scientific PageRuler), the lane BSA is a bovine serum albumin at a concentration of 1 mg/mL, and the lane A is a bacterial cell.
  • the lane Marker is a molecular weight step (Thermo Scientific PageRuler)
  • the lane BSA is a bovine serum albumin at a concentration of 1 mg/mL
  • the lane A is a bacterial cell.
  • FIG. 4 is a gel electrophoresis diagram of the crude Rha01 enzyme solution, including Marker (Thermo Scientific PageRuler) and BSA lanes, Lane 1 is the supernatant after cell disruption, and Lane 2 is the total protein after cell disruption. Wherein the molecular weight of Rha01 is about 87 kDa.
  • the molecular sizes of the ⁇ -L-rhamnosidase (Rha01) and ⁇ -glucosidase (BGL04) are similar to the theoretical calculation values of the corresponding proteins.
  • a method for preparing hesperetin comprising:
  • hesperidin 100 g was suspended in 300 mL of water, and a 10 mol/L sodium hydroxide solution was added until the hesperidin was completely dissolved to obtain a substrate preparation liquid.
  • the substrate preparation solution was added to a sodium phosphate buffer (300 mL) containing a crude enzyme solution (400 mL) co-expressed with ⁇ -L-rhamnosidase and ⁇ -glucosidase at a rate of 1 mL/min. , stirring reaction in 100 mM) to obtain a reaction solution; adding about 5 h until the completion of the substrate preparation liquid addition, the reaction is continued for 1 h, the reaction pH is controlled to 6.0, and the temperature is maintained at 55 ° C;
  • the reaction solution was diluted 50 times with methanol at regular intervals, and the result of liquid phase analysis was carried out by micropore filtration.
  • Liquid phase detection using Yuexu Xtimate C18, 5 ⁇ m ⁇ 250 ⁇ 4.6mm for the analytical column, 5mL acetic acid was added to 1000mL of aqueous solution: acetonitrile 70:30, column temperature was 30 ° C, detection wavelength was UV208nm, flow rate was 1.0mL / Min.
  • Figure 5 is a high performance liquid chromatogram of hesperetin detected during the experiment, and the conversion was 99.2%.
  • the reaction temperature in the bottom stream addition is in the range of 45-65 ° C
  • the orange peel is separately selected when the reaction temperature is constant at 45 ° C, 50 ° C, 55 ° C, 60 ° C, 65 ° C.
  • the preparation of the primes and the conversion were calculated to obtain the conversion of hesperetin at different reaction temperatures, as shown in Table 1.
  • the mass concentration of the reaction substrate hesperidin was adjusted, and the mass concentration of the hesperidin substrate was selected to be 3%, 4%, 5%, 6%, 7%, 8%, and 9
  • the preparation of hesperetin was carried out at % and 10%, respectively, and the conversion was calculated to obtain the conversion rate of hesperetin at a mass concentration of different hesperidin substrates, as shown in Table 2.
  • the speed of the addition in the bottom stream addition is adjusted, and the substrate preparation liquid is continuously added within 4-9 hours, and the extraction time is 4h, 5h, 6h, 7h respectively.
  • the preparation of hesperetin was carried out at 8h and 9h, and the conversion rate was calculated to obtain the conversion rate of hesperetin at different addition times, as shown in Table 3.
  • Table 1 shows the conversion (%) of hesperetin at different reaction temperatures:
  • the hesperidin in the preparation method of the hesperetin, can be efficiently hydrolyzed to obtain hesperetin at a reaction temperature of 45-65 ° C, and the hesperetin
  • the conversion rate is ⁇ 89%; in particular, when the reaction temperature is 55-65 ° C, the conversion of hesperetin is ⁇ 98%, which is far greater than the conversion of 20-50% in the conventional process.
  • Table 2 shows the conversion rate (%) of hesperetin at different mass concentrations of hesperidin substrate:
  • Hesperidin substrate mass concentration (%) Quality of hesperidin (g) Quality of hesperetin (g) Conversion rate(%) 3 30 14.8 99.8 4 40 19.7 99.5 5 50 24.6 99.3 6 60 29.5 99.3 7 70 33.9 98.0 8 80 38.3 96.7 9 90 44.2 95.1 10 100 44.6 90.0
  • Table 3 shows the conversion (%) of hesperetin at different reaction temperatures:
  • the conversion rate of the hesperetin is ⁇ 75%;
  • the conversion rate of the hesperetin is ⁇ 92%, which is far greater than 20-50% in the conventional process. Conversion rate.
  • the addition time of the substrate preparation liquid is 4-6 hours, which ensures both the aging and the high conversion rate.
  • a method for preparing hesperetin comprising:
  • hesperidin 100 g was suspended in 300 mL of water, and a 10 mol/L sodium hydroxide solution was added until the hesperidin was completely dissolved to obtain a substrate preparation liquid.
  • the substrate preparation solution was added to a sodium phosphate buffer (300 mL) containing a crude enzyme solution (400 mL) co-expressed with ⁇ -L-rhamnosidase and ⁇ -glucosidase at a rate of 1 mL/min. , 100 mM) was stirred to obtain a reaction solution; adding about 5 h until the completion of the substrate preparation solution was continued, the reaction was continued for 1 h, the reaction pH was controlled at 7.0, and the temperature was maintained at 55 ° C;
  • the reaction solution was diluted 50 times with methanol at regular intervals, and the results of liquid phase analysis were carried out by micropore filtration, and the conversion was 99.1%.
  • a method for preparing hesperetin comprising:
  • hesperidin 100 g was suspended in 300 mL of water, and a 10 mol/L sodium hydroxide solution was added until the hesperidin was completely dissolved to obtain a substrate preparation liquid.
  • the substrate preparation solution was added to a sodium phosphate buffer (300 mL) containing a crude enzyme solution (400 mL) co-expressed with ⁇ -L-rhamnosidase and ⁇ -glucosidase at a rate of 1 mL/min. , 100 mM) was stirred to obtain a reaction solution; adding about 5 h until the completion of the substrate preparation solution was continued, the reaction was continued for 1 h, the reaction pH was controlled at 6.5, and the temperature was maintained at 55 ° C;
  • the reaction solution was diluted 50 times with methanol at regular intervals, and the results of liquid phase analysis were carried out by micropore filtration, and the conversion was 99.4%.
  • a method for preparing hesperetin comprising:
  • neohesperidin 100 g was suspended in 300 mL of water, and a 10 mol/L sodium hydroxide solution was added until the new hesperidin was completely dissolved to obtain a substrate preparation liquid.
  • the substrate preparation solution was added to a sodium phosphate buffer (300 mL) containing a crude enzyme solution (400 mL) co-expressed with ⁇ -L-rhamnosidase and ⁇ -glucosidase at a rate of 1 mL/min. ,) stirring reaction to obtain a reaction solution; adding about 5h until the substrate preparation liquid is added, the reaction is continued for 1 hour, the reaction pH is controlled to 6.5, and the temperature is maintained at 55 ° C;
  • the reaction solution was diluted 50 times with methanol at regular intervals, and the results of liquid phase analysis were carried out by micropore filtration, and the conversion was 99.0%.
  • a method for preparing an hesperetin intermediate comprising:
  • hesperidin 100 g was suspended in 300 mL of water, and 10 mol/L sodium hydroxide solution was added until the hesperidin was completely dissolved to obtain a substrate preparation liquid;
  • the substrate preparation solution was added to a sodium phosphate buffer solution (300 mL, 100 mM) containing ⁇ -L-rhamnosidase crude enzyme solution (300 mL) at a rate of 1 mL/min for stirring reaction. After the completion of the addition, the reaction was continued for 4 h, the reaction pH was controlled to 6.5, and the temperature was maintained at 55 ° C;
  • the reaction solution was filtered, and the filter cake was washed with 0.5 volume of deionized water, and the filter cake was dried at 50 ° C to obtain a crude product of the hesperidin intermediate.
  • the crude product was dissolved in 100 mL of 80% acetone, stirred at 30 ° C until completely dissolved, and the filtrate was filtered.
  • the filtrate was concentrated in vacuo to 10% of the original volume, and then 4 times volume of deionized water was added to precipitate a solid.
  • the filter cake was filtered, and the filter cake was rinsed with a small amount of deionized water, and the filter cake was dried to obtain a hesperidin intermediate product.
  • the reaction solution was diluted 50 times with methanol at regular intervals, and the results of liquid phase analysis were carried out by micropore filtration, and the conversion was 99.2%.
  • a method for preparing hesperetin comprising:
  • the filter cake After washing the filter cake with deionized water, the filter cake is dried at 60 ° C to obtain a fine product of hesperetin.
  • the reaction solution is diluted 50 times with methanol at regular intervals, and the liquid phase is injected after microfiltration. The reaction results were analyzed, and the conversion of the reaction was found to be 10%.
  • a method for preparing hesperetin comprising:
  • hesperidin 100 g was suspended in 300 mL of water, and a 10 mol/L sodium hydroxide solution was added until the hesperidin was completely dissolved to obtain a substrate preparation liquid.
  • the substrate preparation solution was added to a sodium phosphate buffer (300 mL) containing a crude enzyme solution (400 mL) co-expressed with ⁇ -L-rhamnosidase and ⁇ -glucosidase at a rate of 1 mL/min.
  • the stirring reaction is carried out to obtain a reaction liquid; the addition is carried out for about 5 hours until the completion of the addition of the substrate preparation liquid, and the reaction is continued for 1 hour, the reaction pH is controlled to be 6.0, and the temperature is maintained at 30 ° C;
  • a method for preparing hesperetin comprising:
  • hesperidin 100 g was suspended in 300 mL of water, and a 10 mol/L sodium hydroxide solution was added until the hesperidin was completely dissolved to obtain a substrate preparation liquid.
  • the substrate preparation solution was added to a sodium phosphate buffer (300 mL) containing a crude enzyme solution (400 mL) co-expressed with ⁇ -L-rhamnosidase and ⁇ -glucosidase at a rate of 1 mL/min.
  • the stirring reaction is carried out to obtain a reaction liquid; the addition is carried out for about 5 hours until the completion of the addition of the substrate preparation liquid, the reaction is continued for 1 hour, the reaction pH is controlled to 9.0, and the temperature is maintained at 55 ° C;

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

本发明提供了一种橙皮素的制备方法，包括：将橙皮苷或新橙皮苷悬浮于水中，并加入氢氧化钠溶液至所述橙皮苷或所述新橙皮苷完全溶解后得到底物配制液；采用底物流加法将所述底物配制液以0.1-1mL/min的速度加入至含有α-L-鼠李糖苷酶和β-葡萄糖苷酶的缓冲液中进行搅拌反应并得到反应液，流加完成后继续反应0.5-1h，所述反应液的pH为6.0-7.0，反应温度维持在45-65℃，所述α-L-鼠李糖苷酶来源于链霉菌属，所述β-葡萄糖苷酶来源于海栖热袍菌属；调节所述反应液的pH至3.0-5.0使固体产物完全析出，收集所述固体产物，制得橙皮素。该方法高效简便，绿色环保，可以适用于工业化大规模生产。

Description

一种橙皮素的制备方法、橙皮素中间体的制备方法和用于制备橙皮素的生物酶 技术领域
本发明涉及生物医药技术领域，特别涉及一种橙皮素的制备方法、橙皮素中间体的制备方法和用于制备橙皮素的生物酶。
背景技术
橙皮素(Hesperetin)，分子式为C 16H 14O 6，熔点为227.5℃，其水溶性很差，几乎不溶于水，也不溶于氯仿和笨，易溶于乙醇等。在自然界中多以橙皮苷(Hesperidin)的形式存在。研究表明，橙皮素是一种中药提取物，主要存在于陈皮、青皮、枳壳等中药材中，是他们的主要起效成分，具有抗肿瘤、抗氧化、抗炎症、防止动脉粥样硬化等多种功效。
目前常用酸水解法将新橙皮苷(Neohesperidin)或橙皮苷水解得到橙皮素，此外还有很多文献报道的如水相-乙醇法、甲醇法、环己醇法等制备橙皮素方法，但这些方法工艺繁琐、污染严重，并且产物纯化困难等。近来发展的酶水解法有水解温度较低，水解反应较好控制等特点，但是经发酵产酶的产率并不高，价格昂贵，而且水解率较低，若酶系中含有多种酶，得到的反应产物仍然复杂，使得分离纯化困难。
因此，发展一种工艺简单、成本低、产率高且绿色环保的橙皮素制备方法具有重要意义。
发明内容
为了解决上述技术问题，本发明提供了一种橙皮素的制备方法、橙皮素中间体的制备方法和用于制备橙皮素的生物酶；本发明所述制备方法工艺简单、产量高和安全环保。
第一方面，本发明提供了一种橙皮素的制备方法，包括：
将橙皮苷或新橙皮苷悬浮于水中，并加入氢氧化钠溶液至所述橙皮苷或所述新橙皮苷完全溶解后得到底物配制液；
采用底物流加法将所述底物配制液以0.1-100mL/min的速度加入至含有α-L-鼠李糖苷酶和β-葡萄糖苷酶的缓冲液中进行搅拌反应并得到反应液，流加完成后继续反应0.5-1h，其中，所述反应液的pH为6.0-7.0，反应温度维持在45-65℃，所述α-L-鼠李糖苷酶来源于链霉菌属，所述β-葡萄糖苷酶来源于海栖热袍菌属；
调节所述反应液的pH至3.0-5.0使固体产物完全析出，收集所述固体产物，所述固体产物即为橙皮素。
本发明中，所述橙皮素的制备方法的具体工艺路线如下所示：
Figure PCTCN2018084377-appb-000001
其中，所述工艺采用生物酶法，所述橙皮苷如式(Ⅰ)所示，所述橙皮素如式(Ⅲ)所 示。图中，式(Ⅱ)所示为橙皮素-7-O-葡萄糖苷(Hesperitin-7-O-glucoside)，也称橙皮素单葡萄糖苷，分子式C 22H 24O 11，相对分子质量464，是在生物酶水解橙皮苷生成橙皮素过程中的中间体，是由橙皮苷脱掉一个鼠李糖之后的产物，在本发明中统称为橙皮素中间体。所述橙皮素中间体的含量很低，相较于橙皮素的含量可以忽略不计。所述新橙皮苷的化学结构式如(Ⅵ)所示，将新橙皮苷按照上述生物酶法橙同样可以制得所述橙皮素。
Figure PCTCN2018084377-appb-000002
可选地，所述α-L-鼠李糖苷酶的基因编码序列包括如SEQ ID NO：1所示的核苷酸序列，所述β-葡萄糖苷酶的基因编码序列包括如SEQ ID NO：2所示的核苷酸序列。
α-L-鼠李糖苷酶和β-葡萄糖苷酶的来源广泛，目前在动物组织、植物和微生物中均有发现，然而关于动物组织和植物来源的报道相对较少。不同微生物来源的α-L-鼠李糖苷酶或β-葡萄糖苷酶的酶学性质存在差异，包括酶的比活性、酶作用的底物范围、最适pH、最适温度、作用时间和酶的稳定性等方面。例如，细菌来源的α-L-鼠李糖苷酶最适pH呈中性或碱性，而真菌分泌的α-L-鼠李糖苷酶的最适pH一般在酸性范围内。并非所有的α-L-鼠李糖苷酶均对橙皮苷或新橙皮苷都有水解能力；本发明所述α-L-鼠李糖苷酶来源于链霉菌属(Streptomyces)，通过水解底物(橙皮苷或新橙皮苷)的活力大小筛选获得活力较高的基因，经表达得到本发明所述的α-L-鼠李糖苷酶，所述α-L-鼠李糖苷酶可以高效专一的水解橙皮苷或新橙皮苷并得到橙皮素-7-O-葡萄糖苷。本发明所述β-葡萄糖苷酶属于海栖热袍菌属(Thermotoga petrophila RKU-1)，采用同样方法获得，所述β-葡萄糖苷酶可以高效专一的水解橙皮素-7-O-葡萄糖苷得到橙皮素。
可选地，所述α-L-鼠李糖苷酶和所述β-葡萄糖苷酶通过大肠杆菌共表达产生。可选地，本发明所述α-L-鼠李糖苷酶和所述β-葡萄糖苷酶通过大肠杆菌Rosetta(DE3)共表达产生。其中所述α-L-鼠李糖苷酶和所述β-葡萄糖苷酶是两个独立的蛋白分子。所述α-L-鼠李糖苷酶和所述β-葡萄糖苷酶以粗酶液的形式参与反应。可选地，所述α-L-鼠李糖苷酶和所述β-葡萄糖苷酶的质量比为1：(0.1-10)。所述所述粗酶液是由大肠杆菌Rosetta(DE3)诱导表达所述α-L-鼠李糖苷酶和所述β-葡萄糖苷酶后，对所述大肠杆菌Rosetta(DE3)进行离心、清洗、破胞后收集得到。本发明通过采用共表达形式得到α-L-鼠李糖苷酶和β-葡萄糖苷酶粗酶液水解橙皮苷或新橙皮苷并制备橙皮素的方法，工艺精简，节约成本，产量高，而且绿色环保。可选地，所述α-L-鼠李糖苷酶通过大肠杆菌表达产生；所述β-葡萄糖苷酶通过大肠杆菌表达产生。所述α-L-鼠李糖苷酶在所述粗酶液的质量浓度可以进行一定范围的调节。可选地，所述α-L-鼠李糖苷酶在所述粗酶液的质量浓度范围为0.2-3％。
可选地，所述α-L-鼠李糖苷酶的氨基酸序列的羧基端含有His标签。所述His标签有利于表达后蛋白的分离纯化，及在实验中的分析和追踪，比如用于免疫印迹实验时的分析。当所述α-L-鼠李糖苷酶和所述β-葡萄糖苷酶通过大肠杆菌共表达产生时，所述α-L-鼠李糖苷酶和所述 β-葡萄糖苷酶之间可以分别分离及提纯。
本发明中，所述底物配制液制备的具体过程包括，将橙皮苷或新橙皮苷悬浮于水中，并加入5-10mol/L氢氧化钠溶液至所述橙皮苷或所述新橙皮苷完全溶解后得到底物配制液。可选地，所述氢氧化钠在底物配制液中的终浓度为0.25-0.5mol/L。所述浓度的氢氧化钠溶液可以有效提高橙皮苷的溶解性。相应地，所述浓度的氢氧化钠溶液可以有效提高新橙皮苷的溶解性。
本发明中，所述采用底物流加法将所述底物配制液以0.1-100mL/min的速度加入至含有α-L-鼠李糖苷酶和β-葡萄糖苷酶的缓冲液中进行搅拌反应并得到反应液的过程中，所述底物配制液的流加时间为4-6h。进一步地，可选地，所述底物配制液的流加时间为4.5-5.5h。所述流加时间是指所述底物配制液完全流加至所述反应液中的时间，所述底物配制液在所述底物流加法中的流加的速度根据所述流加时间进行调节。
可选地，所述橙皮苷或所述新橙皮苷在所述反应液的质量溶度为2.5-10％。进一步地，可选地，所述橙皮苷或所述新橙皮苷在所述反应液的质量溶度为5-10％。例如，所述橙皮苷或所述新橙皮苷在所述反应液的质量溶度为5％，或为8％，或为10％。可选地，所述反应液的pH为6.0-7.0，反应温度维持在45-65℃。进一步地，可选地，所述反应液的pH为6.5-7.0。可选地，所述反应温度维持在55-65℃。
可选地，所述α-L-鼠李糖苷酶在所述反应液中的质量浓度为0.05-0.5％。进一步可选地，所述α-L-鼠李糖苷酶在所述反应液中的质量浓度为0.1-0.5％。
可选地，所述缓冲液的缓冲剂包括磷酸盐、Tris缓冲剂或其他缓冲剂。优选地，所述缓冲液包括磷酸盐缓冲液或Tris缓冲液。进一步地，优选地，所述缓冲液包括磷酸盐缓冲液。具体地，所述缓冲液包括磷酸钠缓冲液。所述磷酸钠缓冲液中和所述氢氧化钠溶液具有相同的阳离子，可以减少其他杂质阳离子的添加，防止其他杂质阳离子降低酶(包括α-L-鼠李糖苷酶或β-葡萄糖苷酶)的生物活性。所述缓冲液的pH为6.0-7.0。可选地，所述缓冲液的浓度为80-110mmol/L。进一步可选地，所述缓冲液的浓度为70-100mmol/L。优选地，所述缓冲液的浓度为90-110mmol/L。例如，所述缓冲液的浓度为70mmol/L，或为80mmol/L，或为100mmol/L。
可选地，所述调节所述反应液的pH至3.0-5.0使固体产物完全析出的过程包括向所述反应液加入酸溶液并调节所述反应液的pH至3.0-5.0，搅拌反应0.5-1h后使固体产物完全析出。可选地，所述酸溶液的浓度为2-6mol/L。进一步地，所述酸溶液的浓度为4-6mol/L。所述酸溶液包括盐酸。
可选地，所述收集的过程包括将对所述反应液进行过滤、洗涤、干燥和重结晶过程。其中，所述干燥过程包括采用真空干燥，温度为50-60℃。所述重结晶过程包括：将干燥后的所述固体完全溶解于甲醇中，温度在40-60℃，过滤得到滤液，对所述滤液进行浓缩至原体积的5-10％后加水至所述橙皮素结晶固体全部析出，重复操作得到橙皮素的精品。
本发明第一方面所提供的橙皮素的制备方法，所述制备方法工艺简单，成本低廉、绿色环保；由所述制备方法制得的橙皮素具有极高的产率。本发明利用底物流加法，将含有橙皮苷或新橙皮苷底物流加到反应体系中，底物终浓度可高到达10％，远远大于现有技术，相较与已报道现有制备工艺提高了5000倍左右，产能得到大幅度提高，适应大规模工业化生产。
第二方面，本发明提供了一种橙皮素中间体的制备方法，包括：
将橙皮苷或新橙皮苷悬浮于水中，并加入氢氧化钠溶液至所述橙皮苷或所述新橙皮苷完全溶解后得到底物配制液；
采用底物流加法将所述底物配制液以0.1-100mL/min的速度加入至含有α-L-鼠李糖苷酶的缓冲液中进行搅拌反应并得到反应液，流加完成后继续反应0.5-1h，其中，所述反应液的pH为6.0-7.0，反应温度维持在45-65℃，所述α-L-鼠李糖苷酶来源于链霉菌属；
收集所述反应液中析出的固体产物，所述固体产物即为橙皮素中间体。
可选地，所述收集的过程包括对所述反应液进行过滤、洗涤、干燥和重结晶过程。所述重结晶过程包括：将干燥后的所述固体完全溶解于丙酮溶液中，温度在30-40℃，过滤得到滤液，对所述滤液进行浓缩至原体积的5-10％后加水至所述橙皮素中间体结晶固体全部析出，重复操作得到橙皮素中间体的精品。所述丙酮溶液是指丙酮体积比为60-80％的水溶液。
可选地，所述橙皮素中间体的制备方法中，所述橙皮素中间体的固体产物在反应体系几乎不进行进一步的水解，即所述橙皮素在反应体系中的含量特别微小。将所述收集得到的橙皮素中间体再次采用底物流加法，并加入所述β-葡萄糖苷酶，可以制得所述橙皮素固体产物。
本发明第二方面提供的一种橙皮素中间体的制备方法，利用α-L-鼠李糖苷酶，并采用底物流加法实现对橙皮素中间体的制备，该方法工艺简单，产率高，绿色环保；并且α-L-鼠李糖苷酶的专一性好，可以有效较少橙皮素中间体的进一步水解。
第三方面，本发明提供了用于制备橙皮素的生物酶，所述生物酶包括α-L-鼠李糖苷酶和β-葡萄糖苷酶，所述α-L-鼠李糖苷酶的基因编码序列包括如SEQ ID NO：1所示的核苷酸序列，所述β-葡萄糖苷酶的基因编码序列包括如SEQ ID NO：2所示的核苷酸序列。
可选地，所述α-L-鼠李糖苷酶的氨基酸序列包括如SEQ ID NO：3所示的氨基酸序列。所述β-葡萄糖苷酶的基因编码序列包括如SEQ ID NO：4所示的核苷酸序列。
其中，所述如SEQ ID NO：3所示的氨基酸序列的基因编码序列如SEQ ID NO：1所示；可选的，所述α-L-鼠李糖苷酶的氨基酸序列的基因编码序列应该考虑简并碱基，即如SEQ ID NO:3所示的氨基酸序列的编码基因包括如SEQ ID NO:1所示的核苷酸序列，保护范围还应该保护与SEQ ID NO:1具有碱基简并性质的核苷酸序列，这些核苷酸序列对应的氨基酸序列仍然为SEQ ID NO:3。同样的，对于所述β-葡萄糖苷酶，所述如SEQ ID NO：4所示的氨基酸序列的编码基因同样应该考虑简并碱基。
可选地，所述α-L-鼠李糖苷酶可以高效专一的水解橙皮苷或新橙皮苷并得到橙皮素-7-O-葡萄糖苷。所述β-葡萄糖苷酶可以高效专一的水解橙皮素-7-O-葡萄糖苷得到橙皮素。所述橙皮苷或所述新橙皮苷经α-L-鼠李糖苷酶水解后得到橙皮素-7-O-葡萄糖苷(橙皮素中间体)过程中，并不会水解生成其他中间体产物，所述橙皮素-7-O-葡萄糖苷也不会被所述α-L-鼠李糖苷酶继续分解。本发明所述α-L-鼠李糖苷酶源于链霉菌属，所述α-L-鼠李糖苷酶的基因序列经优化筛选实验获得。本发明所述β-葡萄糖苷酶源于海栖热袍菌属，所述β-葡萄糖苷酶的基因序列经优化筛选实验获得。
可选地，所述α-L-鼠李糖苷酶通过大肠杆菌表达生成。所述β-葡萄糖苷酶通过通过大肠杆菌表达生成。所述α-L-鼠李糖苷酶和所述β-葡萄糖苷酶在所述大肠杆菌表达生成的体系中均属于异源表达。本发明优选地大肠杆菌表达系统，简单可行，培养周期短，发酵成本低，蛋白产量高。所述α-L-鼠李糖苷酶和β-葡萄糖苷酶具有很好的生物活性，纯度高，可广泛应用于生物制药、蛋白生产等领域。相比于传统其他发酵体系，本发明优选的α-L-鼠李糖苷酶和β-葡萄糖苷酶具有更高的产率，并且具有更强的生物活性。所述α-L-鼠李糖苷酶和β-葡萄糖苷酶都具有良好的专一性。
可选地，所述α-L-鼠李糖苷酶和β-葡萄糖苷酶通过构建重组质粒在大肠杆菌中异源表达； 所述重组质粒包括所述α-L-鼠李糖苷酶和/或所述β-葡萄糖苷酶的基因编码序列。进一步地，当所述重组质粒同时包括所述α-L-鼠李糖苷酶和所述β-葡萄糖苷酶的基因编码序列时，所述α-L-鼠李糖苷酶的基因编码序列的5’端前插入RBS序列，所述β-葡萄糖苷酶的基因编码序列的5’端前插入RBS序列。所述RBS序列的核苷酸序列如SEQ ID NO：5所示。本发明所述RBS序列为核糖体结合位点(ribosome binding site，RBS)序列，可以有效促进两组蛋白基因(α-L-鼠李糖苷酶基因和β-葡萄糖苷酶基因)进行独立的转录及翻译。
本发明中，所述重组质粒的载体质粒为pET22b(+)质粒。将所述α-L-鼠李糖苷酶和/或所述β-葡萄糖苷酶的基因编码序列插入至所述pET22b(+)质粒中得到重组质粒，所述重组质粒可以高效、高产的在大肠杆菌中的表达得到α-L-鼠李糖苷酶和/或所述β-葡萄糖苷酶。
可选地，所述α-L-鼠李糖苷酶和/或所述β-葡萄糖苷酶的基因编码序列插入pET22b(+)质粒时，所述α-L-鼠李糖苷酶和/或所述β-葡萄糖苷酶的基因编码序列的5’端可加入起始密码子(如ATG)，所述α-L-鼠李糖苷酶和/或所述β-葡萄糖苷酶的基因编码序列的3’端可加入终止密码子(如TAA)。可选地，所述α-L-鼠李糖苷酶和/或所述β-葡萄糖苷酶的基因编码序列的5’端在加入始密码子之后再插入RBS序列。
可选地，所述α-L-鼠李糖苷酶的基因片段的上增设His标签(组氨酸标签)的核苷酸序列，能使表达后的蛋白带上His标签，His标签有利于表达后蛋白的分离纯化，及在实验中的分析和追踪，比如用于免疫印迹实验时的分析。所述His标签可以用于分离所述α-L-鼠李糖苷酶和β-葡萄糖苷酶。
本发明还提供了一种重组质粒，所述重组质粒包括α-L-鼠李糖苷酶的基因编码序列和所述β-葡萄糖苷酶的基因编码序列中的一种或两种，所述α-L-鼠李糖苷酶的基因编码序列包括如SEQ ID NO：1所示的核苷酸序列，所述β-葡萄糖苷酶的基因编码序列包括如SEQ ID NO：2所示的核苷酸序列。
本发明还提供了一种重组质粒的制备方法，包括：
(1)提供上游引物和下游引物，所述上游引物和下游引物的碱基序列分别如SEQ ID NO:6-SEQ ID NO:11所示；
(2)提供或制备α-L-鼠李糖苷酶和/或β-葡萄糖苷酶基因模板，并以步骤(1)所得上游引物和下游引物为PCR引物，扩增所述α-L-鼠李糖苷酶和/或所述β-葡萄糖苷酶基因片段；
(3)取pET22b(+)质粒，将步骤(2)扩增得到的所述α-L-鼠李糖苷酶和/或所述β-葡萄糖苷酶基因片段和所述pET22b(+)质粒利用相同的内切酶分别进行双酶切反应，纯化回收后进行连接，得到所述重组质粒。
可选地，所述步骤(3)中，在所述β-葡萄糖苷酶基因片段的5’端前插入RBS序列。可选地，所述步骤(3)中，在所述α-L-鼠李糖苷酶基因片段的5’端前插入RBS序列。可选地，所述步骤(3)中，所示双酶切反应的酶切位点可以为Nde I内切酶和Xho I内切酶。
本发明的有益效果包括以下几个方面：
1、本发明所述的制备方法，采用生物酶法(包括α-L-鼠李糖苷酶和β-葡萄糖苷酶)，并通过采用底物流加法进行反应，底物终浓度可高到达10％，相比于传统工艺提高了5000倍左右；
2、本发明所述的制备方法，制备过程中未使用有机试剂，采用生物酶法，高效简便，成本低，绿色环保，可以广泛适用于工业化规模生产；
3、由本发明所述的制备方法制备的橙皮素及其中间体，纯度高，可在制药领域或生物医学领域中有广泛的应用；
4、本发明利用大肠杆菌异源表达α-L-鼠李糖苷酶和β-葡萄糖苷酶，酶的表达水平高，水解活力好，特异性强，水解产物单一。
附图说明
图1为本发明一实施例提供的pET22b-BGL04-Rha01重组质粒的质粒图谱；
图2为本发明一实施例提供的pET22b-Rha01重组质粒的质粒图谱；
图3为本发明一实施例提供的BGL04-Rha01粗酶液的凝胶电泳图；
图4为本发明一实施例提供的Rha01粗酶液的凝胶电泳图；
图5为本发明一实施例提供的橙皮素的高效液相色谱图；
图6为本发明一实施例提供的橙皮素-7-O-葡萄糖苷的高效液相色谱图。
具体实施方式
以下所述是本发明实施例的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明实施例原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也视为本发明实施例的保护范围。
若无特别说明，本发明实施例所采用的原料及其它化学试剂皆为市售商品。
(1)构建pET22b-BGL04-Rha01、pET22b-Rha01重组质粒
a)提供上游引物和下游引物，采用PCR扩增实验得到α-L-鼠李糖苷酶(Rha01)和β-葡萄糖苷酶(BGL04)的基因编码序列。其中，α-L-鼠李糖苷酶的基因编码序列包括如SEQ ID NO：1所示的核苷酸序列，β-葡萄糖苷酶的基因编码序列包括如SEQ ID NO：2所示的核苷酸序列。所述α-L-鼠李糖苷酶对应的上游引物的碱基序列如SEQ ID NO:6所示，下游引物的碱基序列如SEQ ID NO:7所示。所述β-葡萄糖苷酶对应的上游引物的碱基序列如SEQ ID NO:8所示，下游引物的碱基序列如SEQ ID NO:9所示。在所述Rha01和所述BGL04基因的5’端前插入RBS序列，所述RBS序列如SEQ ID NO:5所示，并通过上下游引物将其插入至质粒pET22b(+)中并构建pET22b-BGL04-Rha01重组质粒。
b)提供上游引物和下游引物，采用PCR扩增实验得到α-L-鼠李糖苷酶(Rha01)基因编码序列，所述α-L-鼠李糖苷酶的基因编码序列包括如SEQ ID NO：1所示的核苷酸序列，所述上游引物的碱基序列如SEQ ID NO:10所示，下游引物的碱基序列如SEQ ID NO:11所示。通过上下游引物进行将α-L-鼠李糖苷酶的基因插入至质粒pET22b(+)中并构建pET22b-BGL04-Rha01重组质粒。
通过对应的上游引物和下游引物分别进行PCR扩增体系，配置扩增体系如下：
Figure PCTCN2018084377-appb-000003
PCR扩增程序为：98℃预变性2min；98℃变性10s；58℃退火15s；72℃延伸3min；30个循环后，72℃延伸10min。PCR产物经胶回收试剂盒纯化后，分别利用限制性内切酶Nde I和EcoR I、EcoR I和Xho I进行酶切，酶切后用T4连接酶连接已用Nde I和Xho I酶切处理的质粒 pET22b(+)。连接产物转入大肠杆菌DH5α，经过氨苄抗性(Amp+)的筛选后挑取菌落送测序，测序成功后即得到α-L-鼠李糖苷酶和β-葡萄糖苷酶共表达的重组质粒pET22b-BGL04-Rha01和pET22b-Rha01。所述重组质粒pET22b-BGL04-Rha01的质粒图谱如图1所示；所述重组质粒pET22b-Rha01的质粒图谱如图2所示。
(2)表达α-L-鼠李糖苷酶和β-葡萄糖苷酶
将构建的重组质粒pET22b-Rha01、pET22b-BGL04和pET22b-BGL04-Rha01中的一种或多种转入大肠杆菌Rosetta(DE3)中，并以1％的接种量接种至含有10mL的LB培养基(100μg/mL氨苄青霉素(Amp))的50mL三角瓶中，维持恒定的37℃，200rpm的摇晃速率，过夜培养后，将菌液以1％的接种量转接到含有1L的LB培养基(100μg/mL Amp)的2L三角瓶中，继续37℃恒温培养至培养基中的OD600值达到0.6左右，加入终浓度为0.5mM左右的诱导剂IPTG，在37℃条件培养10小时后离心收集菌体。将菌体分别用4倍质量的100mM的PBS缓冲液(pH＝6.0)进行重悬并超声破碎20min，得到重组酶粗酶液，包括α-L-鼠李糖苷酶(Rha01)粗酶液、β-葡萄糖苷酶(BGL04)粗酶液以及BGL04-Rha01粗酶液。其中BGL04-Rha01粗酶液中含有α-L-鼠李糖苷酶(Rha01)和β-葡萄糖苷酶(BGL04)。
将表达获得的α-L-鼠李糖苷酶(Rha01)粗酶液、β-葡萄糖苷酶(BGL04)粗酶液和BGL04-Rha01粗酶液。分别进行SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)鉴定。图3为所述BGL04-Rha01粗酶液的凝胶电泳图，其中泳道Marker为分子量标阶梯(Thermo Scientific PageRuler)，泳道BSA为质量浓度为1mg/mL的牛血清白蛋白，泳道A为菌体破胞后BGL04-Rha01粗酶液上清液，泳道B为菌体破胞后Rha01粗酶液上清液，泳道C为菌体破胞后BGL04粗酶液上清液，Rha01的分子量为87kDa，其中BGL04的分子量为72kDa。图4为所述Rha01粗酶液的凝胶电泳图，其中包括Marker(Thermo Scientific PageRuler)和BSA泳道，泳道1为菌体破胞后上清液，泳道2为菌体破胞后总蛋白，其中Rha01的分子量约为87kDa。所述α-L-鼠李糖苷酶(Rha01)和β-葡萄糖苷酶(BGL04)的分子大小均于相应蛋白的理论计算值相近。
实施例1
一种橙皮素的制备方法，包括：
将100g橙皮苷悬浮于300mL水中，并加入10mol/L氢氧化钠溶液至所述橙皮苷完全溶解后得到底物配制液。
采用底物流加法将所述底物配制液以1mL/min的速度加入至含有α-L-鼠李糖苷酶和β-葡萄糖苷酶共表达的粗酶液(400mL)的磷酸钠缓冲液(300mL,100mM)中进行搅拌反应得到反应液；流加约5h至底物配制液流加完成后继续反应1h，控制反应pH为6.0，温度维持在55℃；
在上述反应液中缓慢加入2mol/L HCl，加至pH到5.0搅拌0.5h，过滤得到橙皮素的固体，烘干得到橙皮素的粗品，确保水分不大于5％；将粗品溶解在1000mL的甲醇中，60℃至完全溶解后过滤除去滤饼得滤液，将滤液进行旋蒸至原体积的10％后加入4倍体积的去离子水，搅拌0.5h后，过滤得滤饼，用50mL去离子水冲洗滤饼后，将滤饼在60℃进行干燥得橙皮素的精品，确保水分不大于1％。
本实施例中，每隔一定时间取反应液用甲醇稀释50倍，微孔过滤后进样进行液相分析反应结果。液相检测使用月旭Xtimate C18，5μm×250×4.6mm为分析柱，取5mL乙酸加入到1000mL的水溶液：乙腈＝70：30，柱温为30℃，检测波长为UV208nm，流速为1.0mL/min。图5是实验过程中检测的橙皮素的高效液相色谱图，实验测得转化率为99.2％。
按照本实施例1的实验参数，在底物流加法中的反应温度在45-65℃区间内，选取在反应 温度恒定在45℃、50℃、55℃、60℃、65℃时分别进行橙皮素的制备，并计算转化率，得到在不同反应温度下橙皮素的转化率，如表1所示。
按照本实施例1的实验参数，调节反应底物橙皮苷的质量浓度，选取在橙皮苷底物的质量浓度在3％、4％、5％、6％、7％、8％、9％和10％时分别进行橙皮素的制备，并计算转化率，得到在不同橙皮苷底物的质量浓度下橙皮素的转化率，如表2所示。
按照本实施例1的实验参数，调节底物流加法中的流加的速度，将所述底物配制液分别在4-9h内流加完成，选取流加时间分别为4h、5h、6h、7h、8h和9h下进行橙皮素的制备，并计算转化率，得到在不同流加时间下的橙皮素的转化率，如表3所示。
表1为在不同反应温度下橙皮素的转化率(％)：
反应温度(℃) 橙皮苷的质量(g) 橙皮素的质量(g) 转化率(％)
45 100 44.1 89.1
50 100 47.2 95.3
55 100 49.1 99.2
60 100 49.0 99.0
65 100 48.8 98.5
从表1中可以看出，本实施方式中，所述橙皮素的制备方法中在反应温度为45-65℃时，可以高效的水解橙皮苷得到橙皮素，所述橙皮素的转化率≥89％；特别地，当反应温度为55-65℃时，所述橙皮素的转化率≥98％，远远大于传统工艺中的20-50％的转化率。
表2为在不同橙皮苷底物的质量浓度下橙皮素的转化率(％)：
橙皮苷底物质量浓度(％) 橙皮苷的质量(g) 橙皮素的质量(g) 转化率(％)
3 30 14.8 99.8
4 40 19.7 99.5
5 50 24.6 99.3
6 60 29.5 99.3
7 70 33.9 98.0
8 80 38.3 96.7
9 90 44.2 95.1
10 100 44.6 90.0
从表2中可以看出，本实施方式中，所述橙皮素的制备方法中在所述橙皮苷底物的质量浓度在3-10％时，所述橙皮素的转化率≥90％；特别地，当橙皮苷底物的质量浓度在3-7％时，所述橙皮素的转化率≥98％，远远大于传统工艺中的20-50％的转化率。
表3为在不同反应温度下橙皮素的转化率(％)：
流加时间(h) 流加的速度(mL/min) 橙皮苷的质量(g) 橙皮素的质量(g) 转化率(％)
3 1.67 100 37.1 75.0
4 1.25 100 45.5 92.0
5 1.00 100 49.0 99.1
6 0.83 100 49.1 99.2
7 0.71 100 49.1 99.2
8 0.63 100 49.2 99.4
9 0.56 100 49.2 99.3
从表3中可以看出，本实施方式中，所述橙皮素的制备方法中在底物配制液分别在3-9h内 流加完成时，所述橙皮素的转化率≥75％；特别地，当橙皮素的制备方法中在底物配制液分别在4-9h内流加完成时，所述橙皮素的转化率≥92％，远远大于传统工艺中的20-50％的转化率。本发明所述橙皮素的制备方法中，优选地，底物配制液的流加时间为4-6h，这样既保证的时效，又具有较高的转化率。当所述底物配制液的流加时间小于3h，所述橙皮素的转化率将低于50％。
实施例2
一种橙皮素的制备方法，包括：
将100g橙皮苷悬浮于300mL水中，并加入10mol/L氢氧化钠溶液至所述橙皮苷完全溶解后得到底物配制液。
采用底物流加法将所述底物配制液以1mL/min的速度加入至含有α-L-鼠李糖苷酶和β-葡萄糖苷酶共表达的粗酶液(400mL)的磷酸钠缓冲液(300mL,100mM)中进行搅拌反应得到反应液；流加约5h至底物配制液流加完成后继续反应1h，控制反应pH为7.0，温度维持在55℃；
在上述反应液中缓慢加入2mol/L HCl，加至pH到5.0搅拌0.5h，过滤得到橙皮素的固体，烘干得到橙皮素的粗品，确保水分不大于5％；将粗品溶解在1000mL的甲醇中，60℃至完全溶解后过滤除去滤饼得滤液，将滤液进行旋蒸至原体积的10％后加入4倍体积的去离子水，搅拌0.5h后，过滤得滤饼，用50mL去离子水冲洗滤饼后，将滤饼在60℃进行干燥得橙皮素的精品，确保水分不大于1％。
本实施例中，每隔一定时间取反应液用甲醇稀释50倍，微孔过滤后进样进行液相分析反应结果，实验测得转化率为99.1％。
实施例3
一种橙皮素的制备方法，包括：
将100g橙皮苷悬浮于300mL水中，并加入10mol/L氢氧化钠溶液至所述橙皮苷完全溶解后得到底物配制液。
采用底物流加法将所述底物配制液以1mL/min的速度加入至含有α-L-鼠李糖苷酶和β-葡萄糖苷酶共表达的粗酶液(400mL)的磷酸钠缓冲液(300mL,100mM)中进行搅拌反应得到反应液；流加约5h至底物配制液流加完成后继续反应1h，控制反应pH为6.5，温度维持在55℃；
在上述反应液中缓慢加入2mol/L HCl，加至pH到5.0搅拌0.5h，过滤得到橙皮素的固体，烘干得到橙皮素的粗品，确保水分不大于5％；将粗品溶解在1000mL的甲醇中，60℃至完全溶解后过滤除去滤饼得滤液，将滤液进行旋蒸至原体积的10％后加入4倍体积的去离子水，搅拌0.5h后，过滤得滤饼，用50mL去离子水冲洗滤饼后，将滤饼在60℃进行干燥得橙皮素的精品，确保水分不大于1％。
本实施例中，每隔一定时间取反应液用甲醇稀释50倍，微孔过滤后进样进行液相分析反应结果，实验测得转化率为99.4％。
实施例4
一种橙皮素的制备方法，包括：
将100g新橙皮苷悬浮于300mL水中，并加入10mol/L氢氧化钠溶液至新橙皮苷完全溶解后得到底物配制液。
采用底物流加法将所述底物配制液以1mL/min的速度加入至含有α-L-鼠李糖苷酶和β-葡萄糖苷酶共表达的粗酶液(400mL)的磷酸钠缓冲液(300mL,)中进行搅拌反应得到反应液；流加约5h至底物配制液流加完成后继续反应1h，控制反应pH为6.5，温度维持在55℃；
在上述反应液中缓慢加入4mol/L HCl，加至pH到3.0搅拌0.5h，过滤得到橙皮素的固体，烘干得到橙皮素的粗品，确保水分不大于5％；将粗品溶解在1000mL的甲醇中，60℃至完全溶解后过滤除去滤饼得滤液，将滤液进行旋蒸至原体积的10％后加入4倍体积的去离子水，搅拌0.5h后，过滤得滤饼，用50mL去离子水冲洗滤饼后，将滤饼在60℃进行干燥得橙皮素的精品，确保水分不大于1％。
本实施例中，每隔一定时间取反应液用甲醇稀释50倍，微孔过滤后进样进行液相分析反应结果，实验测得转化率为99.0％。
实施例5
一种橙皮素中间体的制备方法，包括：
将100g橙皮苷悬浮于300mL水中，并加入10mol/L氢氧化钠溶液至所述橙皮苷完全溶解后得到底物配制液；
采用底物流加法将所述底物配制液以1mL/min的速度加入至含有α-L-鼠李糖苷酶粗酶液(300mL)的磷酸钠缓冲液(300mL,100mM)中进行搅拌反应，流加完成后继续反应4h，控制反应pH为6.5，温度维持在55℃；
将上述反应液进行过滤，滤饼通过0.5倍体积的去离子水冲洗，取滤饼50℃烘干得到橙皮素中间体的粗品。将粗品溶解在100mL的80％的丙酮中，30℃搅拌至完全溶解后过滤得滤液，将滤液进行真空浓缩至原体积的10％后加入4倍体积的去离子水，有固体析出。过滤得滤饼，用少量去离子水冲洗滤饼后，将滤饼进行干燥得到橙皮素中间体精品。
本实施例中，每隔一定时间取反应液用甲醇稀释50倍，微孔过滤后进样进行液相分析反应结果，实验测得转化率为99.2％。
对比例1
一种橙皮素的制备方法，包括：
在磷酸钠缓冲液(600mL,100mM)中加入100g橙皮苷、并加入至含有α-L-鼠李糖苷酶和β-葡萄糖苷酶共表达的粗酶液(400mL)进行搅拌反应6h得到反应液；控制反应pH为6.0，温度维持在55℃；
在上述反应液中缓慢加入2mol/L HCl，加至pH到5.0搅拌0.5h，过滤得到橙皮素的固体，烘干得到橙皮素的粗品，确保水分不大于5％；将粗品溶解在1000mL的甲醇中，60℃至完全溶解后过滤除去滤饼得滤液，将滤液进行旋蒸至原体积的10％后加入4倍体积的去离子水，搅拌0.5h后，过滤得滤饼，用50mL去离子水冲洗滤饼后，将滤饼在60℃进行干燥得橙皮素的精品，本实施例中，每隔一定时间取反应液用甲醇稀释50倍，微孔过滤后进样进行液相分析反应结果，实验测得反应的转化率为10％。
对比例2
一种橙皮素的制备方法，包括：
将100g橙皮苷悬浮于300mL水中，并加入10mol/L氢氧化钠溶液至所述橙皮苷完全溶解后得到底物配制液。
采用底物流加法将所述底物配制液以1mL/min的速度加入至含有α-L-鼠李糖苷酶和β-葡萄糖苷酶共表达的粗酶液(400mL)的磷酸钠缓冲液(300mL)中进行搅拌反应得到反应液；流加约5h至底物配制液流加完成后继续反应1h，控制反应pH为6.0，温度维持在30℃；
在上述反应液中缓慢加入2mol/L HCl，加至pH到5.0搅拌0.5h，过滤得到橙皮素的固体，烘干得到橙皮素的粗品，确保水分不大于5％；将粗品溶解在1000mL的甲醇中，60℃至完全 溶解后过滤除去滤饼得滤液，将滤液进行旋蒸至原体积的10％后加入4倍体积的去离子水，搅拌0.5h后，过滤得滤饼，用50mL去离子水冲洗滤饼后，将滤饼在60℃进行干燥得橙皮素的精品；实验测得橙皮素的转化率为58％。
对比例3
一种橙皮素的制备方法，包括：
将100g橙皮苷悬浮于300mL水中，并加入10mol/L氢氧化钠溶液至所述橙皮苷完全溶解后得到底物配制液。
采用底物流加法将所述底物配制液以1mL/min的速度加入至含有α-L-鼠李糖苷酶和β-葡萄糖苷酶共表达的粗酶液(400mL)的磷酸钠缓冲液(300mL)中进行搅拌反应得到反应液；流加约5h至底物配制液流加完成后继续反应1h，控制反应pH为9.0，温度维持在55℃；
在上述反应液中缓慢加入2mol/L HCl，加至pH到5.0搅拌0.5h，过滤得到橙皮素的固体，烘干得到橙皮素的粗品，确保水分不大于5％；将粗品溶解在1000mL的甲醇中，60℃至完全溶解后过滤除去滤饼得滤液，将滤液进行旋蒸至原体积的10％后加入4倍体积的去离子水，搅拌0.5h后，过滤得滤饼，用50mL去离子水冲洗滤饼后，将滤饼在60℃进行干燥得橙皮素的精品，实验测得橙皮素的转化率为30％。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对本发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。

Claims (10)

  1. 一种橙皮素的制备方法，其中，包括：
    将橙皮苷或新橙皮苷悬浮于水中，并加入氢氧化钠溶液至所述橙皮苷或所述新橙皮苷完全溶解后得到底物配制液；
    采用底物流加法将所述底物配制液以0.1-1mL/min的速度加入至含有α-L-鼠李糖苷酶和β-葡萄糖苷酶的缓冲液中进行搅拌反应并得到反应液，流加完成后继续反应0.5-1h，其中，所述反应液的pH为6.0-7.0，反应温度维持在45-65℃，所述α-L-鼠李糖苷酶来源于链霉菌属，所述β-葡萄糖苷酶来源于海栖热袍菌属；
    调节所述反应液的pH至3.0-5.0使固体产物完全析出，收集所述固体产物，所述固体产物即为橙皮素。
  2. 如权利要求1所述的制备方法，其中，所述α-L-鼠李糖苷酶的基因编码序列包括如SEQ ID NO：1所示的核苷酸序列，所述β-葡萄糖苷酶的基因编码序列包括如SEQ ID NO：2所示的核苷酸序列。
  3. 如权利要求1或2所述的制备方法，其中，所述α-L-鼠李糖苷酶和所述β-葡萄糖苷酶通过大肠杆菌共表达产生。
  4. 如权利要求1的制备方法，其中，所述α-L-鼠李糖苷酶的氨基酸序列的羧基端含有His标签。
  5. 如权利要求1的制备方法，其中，所述采用底物流加法将所述底物配制液以0.1-1mL/min的速度加入至含有α-L-鼠李糖苷酶和β-葡萄糖苷酶的缓冲液中进行搅拌反应并得到反应液的工程中，所述底物配制液的流加时间为4-6h。
  6. 如权利要求1所述的制备方法，其中，所述氢氧化钠在底物配制液中的终浓度为0.25-0.5mol/L。
  7. 如权利要求1所述的制备方法，其中，所述收集的过程包括对所述反应液进行过滤、洗涤、干燥和重结晶操作。
  8. 一种橙皮素中间体的制备方法，其中，包括：
    将橙皮苷或新橙皮苷悬浮于水中，并加入氢氧化钠溶液至所述橙皮苷或所述新橙皮苷完全溶解后得到底物配制液；
    采用底物流加法将所述底物配制液以0.1-1mL/min的速度加入至含有α-L-鼠李糖苷酶的缓冲液中进行搅拌反应并得到反应液，流加完成后继续反应0.5-1h，其中，所述反应液的pH为6.0-7.0，反应温度维持在45-65℃，所述α-L-鼠李糖苷酶来源于链霉菌属；
    收集所述反应液中析出的固体产物，所述固体产物即为橙皮素中间体。
  9. 用于制备橙皮素的生物酶，其中，所述生物酶包括α-L-鼠李糖苷酶和β-葡萄糖苷酶，所述α-L-鼠李糖苷酶的基因编码序列包括如SEQ ID NO：1所示的核苷酸序列，所述β-葡萄糖苷酶的基因编码序列包括如SEQ ID NO：2所示的核苷酸序列；所述α-L-鼠李糖苷酶来源于链霉菌属，所述β-葡萄糖苷酶来源于海栖热袍菌属。
  10. 如权利要求9所述的生物酶，其中，所述α-L-鼠李糖苷酶和所述β-葡萄糖苷酶通过构建重组质粒在大肠杆菌中异源表达；所述重组质粒包括所述α-L-鼠李糖苷酶和/或所述β-葡萄糖苷酶的基因编码序列。
PCT/CN2018/084377 2018-04-25 2018-04-25 一种橙皮素的制备方法、橙皮素中间体的制备方法和用于制备橙皮素的生物酶 WO2019076021A1 (zh)

Priority Applications (2)

Application Number Priority Date Filing Date Title
PCT/CN2018/084377 WO2019076021A1 (zh) 2018-04-25 2018-04-25 一种橙皮素的制备方法、橙皮素中间体的制备方法和用于制备橙皮素的生物酶
CN201880001976.7A CN109312375B (zh) 2018-04-25 2018-04-25 一种橙皮素的制备方法、橙皮素中间体的制备方法和用于制备橙皮素的生物酶

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2018/084377 WO2019076021A1 (zh) 2018-04-25 2018-04-25 一种橙皮素的制备方法、橙皮素中间体的制备方法和用于制备橙皮素的生物酶

Publications (1)

Publication Number Publication Date
WO2019076021A1 true WO2019076021A1 (zh) 2019-04-25

Family

ID=65221773

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2018/084377 WO2019076021A1 (zh) 2018-04-25 2018-04-25 一种橙皮素的制备方法、橙皮素中间体的制备方法和用于制备橙皮素的生物酶

Country Status (2)

Country Link
CN (1) CN109312375B (zh)
WO (1) WO2019076021A1 (zh)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113881659A (zh) * 2021-09-24 2022-01-04 华南理工大学 一种固定化酶法制备橙皮素二氢查耳酮葡萄糖苷的方法
CN114231545A (zh) * 2020-09-09 2022-03-25 中国中医科学院中药研究所 一种鼠李糖转移酶基因、制备方法及其表达和应用
CN115896201A (zh) * 2023-01-09 2023-04-04 成都欧康医药股份有限公司 一种4-甲氧基-3,5`,7`-三羟基黄烷酮的制备方法

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3924498A1 (fr) 2019-02-11 2021-12-22 Les Laboratoires Servier Methode de biosynthese de la diosmine et/ou de l'hesperidine dans un microorganisme
CN112226395B (zh) * 2020-09-10 2022-07-05 广西大学 一株大肠杆菌工程菌及其全细胞催化产淫羊藿苷元的方法
CN114164244B (zh) * 2021-11-11 2024-03-01 华南理工大学 一种制备橙皮素-7-o-葡萄糖苷和橙皮素的方法
CN114164161B (zh) * 2022-02-15 2022-05-13 佛山市汇腾生物技术有限公司 一种生产新橙皮苷的双酶共表达菌株及其构建方法和应用

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103224968A (zh) * 2013-05-06 2013-07-31 佛山市金骏康健康科技有限公司 一种酶法制备新橙皮苷的方法
CN103374593A (zh) * 2012-04-13 2013-10-30 浙江震元制药有限公司 微生物发酵将棘白菌素b转化为棘白菌素b母核的方法
CN105886484A (zh) * 2016-04-25 2016-08-24 中国农业科学院饲料研究所 一种嗜热纤维素酶及其编码基因和应用
CN106119268A (zh) * 2016-08-05 2016-11-16 集美大学 一种提高α‑L‑鼠李糖苷酶r‑Rha1热稳定性的方法
CN106148446A (zh) * 2015-04-22 2016-11-23 中国科学院大连化学物理研究所 一种通过酶促水解新橙皮苷或橙皮苷制备橙皮素的方法

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3583894A (en) * 1968-03-29 1971-06-08 Us Agriculture Enzyme preparation of hesperetin dihydrochalcone glucoside
MY177500A (en) * 2014-03-03 2020-09-16 Hayashibara Co Glycosyl hesperetin and process for producing the same and uses thereof
CN106318957B (zh) * 2016-10-26 2019-05-07 南京林业大学 土曲霉CCF 3059 α-L-鼠李糖苷酶突变体及其应用
CN107245484B (zh) * 2016-12-23 2020-10-09 枣庄市杰诺生物酶有限公司 一种腈水解酶及其应用
CN107641621B (zh) * 2017-06-14 2021-07-23 江苏康缘药业股份有限公司 一种糖苷酶组合物及酶法制备淫羊藿苷元的方法

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103374593A (zh) * 2012-04-13 2013-10-30 浙江震元制药有限公司 微生物发酵将棘白菌素b转化为棘白菌素b母核的方法
CN103224968A (zh) * 2013-05-06 2013-07-31 佛山市金骏康健康科技有限公司 一种酶法制备新橙皮苷的方法
CN106148446A (zh) * 2015-04-22 2016-11-23 中国科学院大连化学物理研究所 一种通过酶促水解新橙皮苷或橙皮苷制备橙皮素的方法
CN105886484A (zh) * 2016-04-25 2016-08-24 中国农业科学院饲料研究所 一种嗜热纤维素酶及其编码基因和应用
CN106119268A (zh) * 2016-08-05 2016-11-16 集美大学 一种提高α‑L‑鼠李糖苷酶r‑Rha1热稳定性的方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
SHEN, LIJING: "Study on Preparation and Catalytic Propertie of the Hesperidinase", ZHEJIANG UNIVERSITY OF TECHNOLOGY FOR MASTER'S DEGREE, 31 December 2006 (2006-12-31), pages 69 - 77 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114231545A (zh) * 2020-09-09 2022-03-25 中国中医科学院中药研究所 一种鼠李糖转移酶基因、制备方法及其表达和应用
CN114231545B (zh) * 2020-09-09 2023-06-20 中国中医科学院中药研究所 一种鼠李糖转移酶基因、制备方法及其表达和应用
CN113881659A (zh) * 2021-09-24 2022-01-04 华南理工大学 一种固定化酶法制备橙皮素二氢查耳酮葡萄糖苷的方法
CN113881659B (zh) * 2021-09-24 2024-02-20 华南理工大学 一种固定化酶法制备橙皮素二氢查耳酮葡萄糖苷的方法
CN115896201A (zh) * 2023-01-09 2023-04-04 成都欧康医药股份有限公司 一种4-甲氧基-3,5`,7`-三羟基黄烷酮的制备方法
CN115896201B (zh) * 2023-01-09 2023-05-26 成都欧康医药股份有限公司 一种4-甲氧基-3,5`,7`-三羟基黄烷酮的制备方法

Also Published As

Publication number Publication date
CN109312375A (zh) 2019-02-05
CN109312375B (zh) 2022-06-17

Similar Documents

Publication Publication Date Title
WO2019076021A1 (zh) 一种橙皮素的制备方法、橙皮素中间体的制备方法和用于制备橙皮素的生物酶
JP4561010B2 (ja) Ｄ−ヒダントインハイドロラーゼをコードするｄｎａ、ｎ−カルバミル−ｄ−アミノ酸ハイドロラーゼをコードするｄｎａ、該遺伝子を含む組み換えｄｎａ、該組み換えｄｎａにより形質転換された細胞、該形質転換細胞を用いるタンパク質の製造方法、および、ｄ−アミノ酸の製造方法
CN109762832B (zh) 羧酸酯酶基因、重组质粒、重组工程菌及编码蛋白和应用
WO2015055127A1 (zh) 一种左旋吡喹酮的制备方法
WO2014166326A1 (zh) 一种β−葡萄糖苷酶及其表达基因与应用
CN113930404B (zh) 一种酶法合成手性枸橼酸托法替布中间体的方法
US20220135960A1 (en) Polypeptide tag, highly soluble recombinant nitrilase and application thereof in synthesis of pharmaceutical chemicals
WO2019100676A1 (zh) 一种酶改质甜菊糖的制备方法和制备用酶及应用
WO2010041269A1 (en) Process for preparation of mycophenolic acid, its salt and ester derivatives
JP2862870B2 (ja) 新規ペプチド
JP4561009B2 (ja) Ｄ−ヒダントインハイドロラーゼをコードするｄｎａ、ｎ−カルバミル−ｄ−アミノ酸ハイドロラーゼをコードするｄｎａ、該遺伝子を含む組み換えｄｎａ、該組み換えｄｎａにより形質転換された細胞、該形質転換細胞を用いるタンパク質の製造方法、および、ｄ−アミノ酸の製造方法
CN112645952B (zh) 一种(r)-(+)-9-(2-羟丙基)腺嘌呤的合成方法
RU2453604C1 (ru) Гибридный белок (варианты), штамм бактерий escherichia coli - продуцент гибридного белка (варианты) и способ получения безметионинового интерферона альфа-2 человека
CN113249348B (zh) 羰基还原酶、其基因、含有该基因的重组表达转化体及其应用
JPS626679A (ja) ヒトリゾチ−ムの微生物学的製造法
CN114875003B (zh) 一种短链脱氢酶的突变体、编码基因及编码基因获得方法、突变体的应用
CN116004575B (zh) 一种β-葡萄糖醛酸酶及其应用
CN114150036B (zh) 一种用于制备光学纯的6-氟-色满-2-羧酸的连续双相分批拆分工艺
CN113564195B (zh) 一种果糖胺脱糖酶毕赤酵母表达载体、基因工程菌及构建方法和蛋白表达方法
CN116478963A (zh) 一种耐热α-L-鼠李糖苷酶及其制备方法和应用
JPH11113594A (ja) 光学活性な１−（４−ｔ−ブチルフェニル）−５−オキソ−３−ピロリジンカルボン酸および／またはその鏡像対掌体エステルの製造方法
CN113046338A (zh) 里氏木霉来源的高选择性脂肪酶及其应用
JP2570651B2 (ja) Ｍｅｔ−インスリン様成長因子Ｉの製造方法
CN117778435A (zh) 一种无可溶标签制备鼠源rna酶抑制剂的工艺
JPS61225199A (ja) 新規ヒトインタ−ロイキン２ポリペチド誘導体

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 18867826

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 18867826

Country of ref document: EP

Kind code of ref document: A1