Glycosidase and application thereof in preparation of ginsenoside Rd

Abstract

The invention discloses glycosidase and application of the glycosidase in preparation of ginsenoside Rd. The amino acid sequence of the glycosidase comprises a sequence as shown in SEQ ID NO. 1. The glycosidase catalyzes a complex feed liquid containing ginsenoside Rb1, the ginsenoside Rd is prepared through conversion in a neutral non-buffer system, the enzymatic activity is good, the process is simple, the reaction condition is mild, the conversion rate of the ginsenoside Rd can reach 71% or above, and the glycosidase is suitable for large-scale preparation and wide in application prospect.

Description

Technical Field

[0001] The invention relates to the technical field of enzyme catalysis, and in particular to a glycosidase with high activity and application thereof in the preparation of ginsenoside Rd. Background Art

[0002] Glycosyltransferases have important application prospects in improving the drugability of natural products and catalytic transformation. Studies have found that rare ginsenosides can be obtained by hydrolyzing and removing the sugar groups from saponin molecules with abundant sources. Enzymatic conversion has the advantages of being environmentally friendly, mild conditions, high selectivity, and controllable conversion process, and is an ideal means for preparing rare ginsenosides. Enzymes of different properties act on glycosidic bonds of different configurations and compositions, thereby forming the target product in a directional manner. Using β-galactosidase from Aspergillus oryzae and lactase crude enzyme solution from Penicillium, a triol saponin mixture can be hydrolyzed to produce ginsenosides Rg2 and Rh1. Using naringinase crude enzyme solution, a triol saponin mixture can be hydrolyzed to produce intestinal bacterial metabolites F1 and 20(S)-PPT. A diol saponin mixture can be converted into F2, CK, and Rd by lactase, β-galactosidase, and cellulase crude enzyme solution, respectively, and can be converted into Rd, Rg3, and CK by lactase crude enzyme solution. R-Rg3 and Rf can be converted into PPD(S, R) and 20(S)-PPT respectively by using crude glycosidase from Aspergillus niger, with conversion rates of 100% and 90.4% respectively. Rh2 can be produced from ginsenoside β-glucosidase with a conversion rate of more than 60%, a purity of 90% and an enzyme recovery rate of 60% after the reaction.

[0003] The prior art enzymatic conversion to prepare rare ginsenosides usually needs to be carried out in a buffer system that deviates from neutrality, and the use of buffer salts causes difficulties in subsequent treatment and environmental problems. The present invention separates a glycosidase from the fermentation product of Aspergillus niger and performs protein sequencing, which is used for the conversion and preparation of ginsenoside Rd. It can be carried out in a neutral non-buffered system, has the advantages of good enzyme activity, simple process, mild reaction conditions, low cost, high conversion rate of ginsenoside Rd, and is suitable for large-scale preparation. Summary of the invention

[0004] The purpose of the present invention is to provide a glycosidase, and use the glycosidase to prepare ginsenoside Rd, solving the problems of using a large amount of buffer salt, complicated steps, and small liquid processing capacity in the current ginsenoside enzyme catalytic conversion preparation process. The glycosidase of the present invention and its preparation method have outstanding substantive characteristics and significant beneficial effects compared with the prior art:

[0005] The present invention first discovered a glycosidase capable of catalyzing the efficient conversion of ginsenoside Rb1 into ginsenoside Rd in a neutral non-buffered system from the fermentation product of Aspergillus niger (CICC strain collection number 2238), with a conversion rate of more than 71%.

[0006] The present invention discloses a glycosidase and its application in the preparation of ginsenoside Rd. The amino acid sequence of the enzyme includes: (1) the sequence shown in SEQ ID NO.1, or (2) an enzyme obtained by replacing, deleting or adding one or at least two amino acid residues to the sequence shown in SEQ ID NO.1 using the general technical means in the art, and having an amino acid sequence with the same or similar function as the sequence described in (1).

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] A glycosidase and its application in the preparation of ginsenoside Rd. The amino acid sequence of the glycosidase is shown as SEQ ID NO.1.

[0009] SEQ ID NO.1:

[0010] DSQLKSSQNGVGVKLTAYPECSSDTLSTYPAALSAVVANLDSTVSDSENGVVGKLVRPDG

[0011] VGGVHLDNHKWTSSSVLGNPTVFNSATECYATSCTFSGSEGASSASKYGSPSCSTIVLSDV

[0012] AVPSGTTLDLTDLNDGTHVIFEGETTFNYKPSGPLVSVSGTDITVTGADGAYLNGDGSRW

[0013] WDGEGSNGGKTKPKFFYAHDLTSSTISGIYIQNSPVQVFSIDGSTYTMEDITIDNGPALVGA

[0014] ANTDGFDIGDSTYITITGANVYNQDDCVAVNSGENIYFSGGVCSGGHGLSIGSVGGRSDNT

[0015] VKNVTFYDSEIKSSQNGVRIKTIYGDTGSVSEVTYKEITLSDITDYGIVVEQNYDDTSKSPTD

[0016] GITIEDFVLDNVQGSVESSGTNIYIVCGSDSCTDWTWTDVDVSGGKTSSDCENVPDDISC

[0017] A glycosidase and its application in the preparation of ginsenoside Rd, characterized in that ginsenoside Rd is prepared by catalytic conversion using ginseng extract as raw material.

[0018] Furthermore, the catalytic conversion preparation comprises the following steps:

[0019] Add a certain concentration of ginseng extract aqueous solution to the reaction container, add glycosidase solution according to the feed-enzyme ratio, and convert for a certain time under certain temperature conditions. After the conversion is completed, add it to the pre-treated D101 macroporous resin column, wash with water, recover the enzyme solution, elute with 70% aqueous ethanol, concentrate the eluate, and dry it.

[0020] Furthermore, the concentration of ginseng extract in the catalytic conversion step is 1 to 36.36 mg / ml;

[0021] Furthermore, in the catalytic conversion step, the feed-enzyme ratio is 1:1 to 1:2.5;

[0022] Furthermore, the temperature in the catalytic conversion step is 45 to 60°C;

[0023] Furthermore, the conversion time in the catalytic conversion step is 1.5 to 6 hours;

[0024] Furthermore, the conversion rate of ginsenoside Rd is 37.75-71.2%.

[0025] The present invention discloses a glycosidase and its application in the preparation of ginsenoside Rd. The amino acid sequence of the glycosidase includes the sequence shown in SEQ ID NO.1, or an enzyme having the same or similar function as the amino acid sequence of the sequence shown in SEQ ID NO.1 after substitution, deletion or addition of one or at least two amino acid residues. The glycosidase catalyzes a complex liquid containing the substrate ginsenoside Rb1 to transform and prepare ginsenoside Rd in a neutral non-buffered system, has good enzyme activity, simple process, mild reaction conditions, and a ginsenoside Rd conversion rate of more than 71%, is suitable for large-scale preparation, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the secondary mass spectrum of the labeled peptide obtained by chymotrypsin digestion in Example 2;

[0027] Figure 2 HPLC spectrum of the substrate and catalytic product samples of Example 5. DETAILED DESCRIPTION

[0028] The present invention will be further described in detail below through specific implementation examples, but it should not be understood that the present invention is limited to the following examples, and all technologies realized based on the above content of the present invention belong to the scope of the present invention.

[0029] Ginseng root extract (pure root), specification: 60% total saponin (UV detection), Shaanxi Kepler Biotechnology Co., Ltd. D-101 macroporous resin, pre-treated by soaking in 95% ethanol for one day and night, then wet-packed, washed with distilled water until there is no alcohol smell, and set aside.

[0030] Example 1

[0031] Glycosidase screening: Add saturated ammonium sulfate solution dropwise to the enzyme solution from Aspergillus niger until the enzyme is completely precipitated. Centrifuge at 6000rpm for 10min, discard the supernatant, add an appropriate amount of saturated salt solution, mix well, and centrifuge again. Repeat several times until the supernatant is colorless or light yellow. Finally, a mixed enzyme precipitate containing sulfate is obtained and stored in the refrigerator for later use.

[0032] Dialysis: Boil the dialysis bag 7000 in a large amount of 2% (w / v) sodium bicarbonate solution for 10 minutes, then rinse it with pure water, and refrigerate it in the presence of pure water for later use. Add a small amount of aqueous solution to the enzyme precipitate obtained by salting out to just dissolve the enzyme, pour the solution into the treated dialysis bag, place it in iced pure water, and stir it magnetically. Take out an appropriate amount of dialysate every half an hour, add barium chloride solution, observe the precipitation formation, and replace the dialysate until the amount of precipitation no longer increases. Repeat the above steps until there is basically no precipitation, and finally obtain a mixed enzyme solution.

[0033] Ultrafiltration centrifugation: Ultrafiltration at 70, 60, 50, 40, 30, 20KD, centrifugation, enzyme solution ultrafiltration concentrate. Screening conditions: Use ginsenoside Rb1 as substrate to screen the enzyme solution ultrafiltration concentrate, and detect the results of catalytic generation of ginsenoside Rd by neutral non-buffered system. Panax notoginseng extract containing 1.0 mg / mL ginsenoside Rb1, enzyme solution ultrafiltration concentrate, reaction temperature of 40°C, reaction time of 1.5h.

[0034] Enzyme molecule determination: Accurately weigh the mixed enzyme solid obtained by freeze drying, add water to dissolve to 12.5mg / ml, use an ultrafiltration centrifuge tube with a pore size of 40KDa, and centrifuge at 8000rpm for 10min. Take out the permeate and concentrate, and add pure water to the same volume. The theoretical concentrations of the two enzyme solutions are 15.15mg / ml respectively. The two enzyme solutions are used to catalyze the conversion of two ginsenoside extract solutions under the same suitable conditions for 1.5h, and the catalytic products are analyzed by high performance liquid chromatography.

[0035] The results showed that the 40KD ultrafiltration concentrate exhibited significant selectivity for ginsenoside Rd product.

[0036] Freeze drying: Pour the mixed enzyme solution into a plastic petri dish, freeze it in a refrigerator for 1 hour, and then use a freeze dryer to dry it for about 24 hours to obtain dry glycosidase solid.

[0037] Example 2

[0038] The protein sample was enzymatically digested by using trypsin, chymotrypsin, elastase, proteinase K, and Glu-C + trypsin to digest the purified glycosidase solid according to the following steps, and finally 5 enzyme-digested peptide samples were obtained.

[0039] 1) Enzyme digestion: Take an enzyme with a concentration of 0.025μg / μL (diluted with 25mmol / L NH4HCO3), add the enzyme solution at a volume of 7-10μL / tube, put it in a 4℃ refrigerator for incubation for 40min, take it out and add 5-10μL 25mmol / L NH4HCO3 solution to each tube, seal it and place it in a 37℃ water bath for enzymatic digestion for 16h. 2) Peptide extraction: Add 100μL / tube of extraction solution (5% TFA-50% ACN-45% water), bathe it in a 37℃ water bath for 1h, ultrasonicate it for 5min, centrifuge it for 5min, transfer the extraction solution to another new EP tube, repeat the extraction once, combine the extraction solutions, and vacuum centrifuge and dry them. 3) Desalting: After desalting using a self-filled desalting column, vacuum dry it at 45℃ for use.

[0040] 2) Protein sample HPLC-MS analysis: The five enzyme-cleaved peptide samples were analyzed by HPLC-MS, and five original mass spectrometry data were obtained. The LC-MS conditions were as follows:

[0041] 3) Liquid chromatography conditions: A binary gradient system consisting of 0.1% (v / v) formic acid (A) and 0.1% (v / v) formic acid, 80% (v / v) ACN was used for elution analysis. The experiment used a 150 μm id×150 mm column filled with Acclaim PepMap RPLC C 18 , 3μm, Column temperature, injection volume, detection wavelength. Gradient conditions: 0-5min, 5-15% (B); 5-65min, 15-40% (B); 65-75min, 40-100% (B); 75-90min, 100-100% (B); flow rate was 600nL / min.

[0042] Mass spectrometry conditions: 1) Primary mass spectrometry parameters: resolution: 120,000; AGCtarget: 400,000; MaximumIT: 50ms; scanning range: 150 to 2000 m / z. 2) Secondary mass spectrometry parameters: resolution: 60,000; AGCtarget: 50,000; MaximumIT: 118ms; NCE / steppedNCE: 30.

[0043] The results of protein sample HPLC-MS were analyzed. The primary and secondary mass spectra were obtained by setting parameters and screening the software to obtain spectra that can detect protein sequences and merge them to obtain labeled secondary mass spectra, such as the labeled secondary mass spectra of peptides obtained by chymotrypsin digestion. Figure 1 The obtained sequences were spliced ​​according to the splicing algorithm to finally obtain the complete protein sequence of glycosidase 1, and the molecular weight was calculated to be 44445.07Da.

[0044] Example 3

[0045] The HPLC detection of ginsenosides was carried out according to the high performance liquid chromatography method (Appendix ⅥD of Part I of the Chinese Pharmacopoeia 2015 edition).

[0046] 1) Instruments, reagents and drugs:

[0047] High performance liquid chromatograph (Wanyi LC3200), SHIMADZU high performance liquid chromatograph (LC-20A).

[0048] 2) Chromatographic conditions

[0049] Chromatographic conditions C 18 Column (Venusil XBP C 18 Column, 25cm*4.6mm, filler particle size 5μm), acetonitrile as mobile phase A, 0.1% phosphoric acid as mobile phase B, gradient elution, 0-25min, 19-20% A; 25-60min, 20-40% A; 60-80min, 40-100% A, detection wavelength 203nm.

[0050] 3) Sample determination

[0051] Take appropriate amounts of ginsenoside Rb1 and ginsenoside Rd, weigh them accurately, place them in 2 ml volumetric flasks respectively, and add methanol to make a reference solution containing 1.0 mg of ginsenoside Rb1 and 1.0 mg of ginsenoside Rd per 1 ml.

[0052] Take another 1.0 mg of the product, weigh it accurately, put it in a 2 ml volumetric bottle, add methanol to dissolve it to volume as the test solution. Filter it through a microporous membrane, inject it for determination, and calculate the content and conversion rate by the external standard one-point method.

[0053] Conversion rate % = amount of Rd substance generated in the product (mol) / amount of Rb1 substance in the feed solution (mol) × 100%

[0054] Example 4

[0055] Take 10 ml of liquid containing 0.36 g of ginseng extract (ginsenoside Rb1 content 6.1%), add 0.72 g of glycosidase according to the ratio of 1:2 of feed enzyme (mass ratio of ginseng extract to enzyme), keep at 70°C for 1.5 hours, cool, add to the pretreated D101 macroporous resin column (φ15mm×30cm), wash with water, recover the enzyme solution, elute with 200 ml of 70% aqueous ethanol, flow rate 2.0 ml / min, evaporate the eluate to dryness, and obtain the ginsenoside Rd sample. According to the HPLC method of Example 3, the conversion rate of ginsenoside Rd was 37.75%.

[0056] Example 5

[0057] Take 10 ml of liquid containing 0.36 g of ginseng extract (ginsenoside Rb1 content 6.1%), add 0.72 g of glycosidase according to the ratio of feed to enzyme (mass ratio of ginseng extract to enzyme) 1:2, keep at 52.5°C for 1.5 hours, let cool, add to the pretreated D101 macroporous resin column (φ15mm×30cm), wash with water, recover the enzyme solution, elute with 200 ml of 70% aqueous ethanol, flow rate 2.0 ml / min, evaporate the eluate to dryness, and obtain a ginsenoside Rd sample. According to the HPLC method of Example 3, the conversion rate of ginsenoside Rd was 71.2%.

[0058] Example 6

[0059] Take 10 ml of liquid containing 0.36 g of ginseng extract (ginsenoside Rb1 content 6.1%), add 0.91 g of enzyme powder according to the ratio of 1:2.5 of feed to enzyme (mass ratio of ginseng extract to enzyme), keep at 45°C for 2.0 h, add to the pretreated D101 macroporous resin column (φ15 mm×30 cm), wash with water, recover the enzyme solution, elute with 200 ml of 70% aqueous ethanol, elute with 200 ml of 70% aqueous ethanol, evaporate the eluate to dryness, and obtain a ginsenoside Rd sample. According to the HPLC method of Example 3, the conversion rate of ginsenoside Rd is 61.33%.

[0060] Example 7

[0061] Take 10 ml of a liquid containing 10 mg of ginseng extract (ginsenoside Rb1 content 6.1%), add 10 mg of enzyme powder according to a feed-enzyme ratio (mass ratio of ginseng extract to enzyme) of 1:1, maintain at 52.5°C for 6 hours, add to a pretreated D101 macroporous resin column (φ15 mm×30 cm), wash with water, recover the enzyme solution, elute with 200 ml of 70% aqueous ethanol, flow rate 2.0 ml / min, evaporate the eluate to dryness, and obtain a ginsenoside Rd sample. According to the HPLC method of Example 3, the conversion rate of ginsenoside Rd was 65.46%.

[0062] Example 8

[0063] Take 10 ml of liquid containing 0.36 g of ginseng extract (ginsenoside Rb1 content 6.1%), add 0.72 g of glycosidase according to the ratio of feed to enzyme (mass ratio of ginseng extract to enzyme) 1:2, keep at 52.5°C for 2.0 h, let cool, add to the pretreated D101 macroporous resin column (φ15 mm×30 cm), wash with water, recover the enzyme solution, elute with 200 ml of 70% aqueous ethanol, flow rate 2.0 ml / min, evaporate the eluate to dryness, and obtain a ginsenoside Rd sample. According to the HPLC method of Example 3, the conversion rate of ginsenoside Rd was 69.73%.

Claims

1. A glycosidase, characterized in that The amino acid sequence of the glycosidase is shown in SEQ ID NO.

1.

2. A method for preparing ginsenoside Rd, characterized in that: Ginsenoside Rb1 is used as a substrate and the product ginsenoside Rd is prepared under the catalysis of glycosidase; wherein the amino acid sequence of the glycosidase is shown in SEQ ID NO.

1.

3. The method for preparing ginsenoside Rd according to claim 2, characterized in that: The method comprises the following steps: a. adding a ginseng extract aqueous solution of a certain concentration into a reaction container, adding a glycosidase solution according to a feed-enzyme ratio, and converting for a certain time under certain temperature conditions; b. after the conversion is completed, adding the solution to a pretreated D101 macroporous resin column, washing with water, recovering the enzyme solution, eluting with 70% aqueous ethanol, concentrating the eluate, and drying the column.

4. The method for preparing ginsenoside Rd according to claim 2, characterized in that: The concentration of the ginseng extract is 1-36.36 mg / ml.

5. The method for preparing ginsenoside Rd according to claim 2, characterized in that: The feed-enzyme ratio is 1:1 to 1:2.

5.

6. The method for preparing ginsenoside Rd according to claim 2, characterized in that: The temperature is 45-60°C.

7. The method for preparing ginsenoside Rd according to claim 2, characterized in that: The conversion time is 1.5 to 6 hours.

8. The method for preparing ginsenoside Rd according to claim 2, characterized in that: The conversion rate of the product ginsenoside Rd is 37.75% to 71.2%.

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