阅读说明：本技术 一种酶法制备鼠李糖基果糖的方法 (Method for preparing rhamnosyl fructose by enzyme method ) 是由 葛林 杨成德 苗向阳 严丹红 于 2021-09-22 设计创作，主要内容包括：本发明属于糖工程技术领域,具体涉及一种α-L-鼠李糖苷酶的制备方法及其在催化果糖制备鼠李糖基果糖的方法应用,所述α-L-鼠李糖具有具有反向水解能力,当以L-鼠李糖为糖基供体,果糖为糖基受体时,该酶能反向水解合成新型二糖鼠李糖基果糖,摩尔转化率为23.4%。通过本发明制备的α-L-鼠李糖苷酶具有反向水解活性,可应用于新型二糖鼠李糖基果糖的酶法制备,并且温度稳定性较好,摩尔转化率较高。(The invention belongs to the technical field of sugar engineering, and particularly relates to a preparation method of alpha-L-rhamnosidase and application of the alpha-L-rhamnosidase in a method for preparing rhamnosyl fructose by catalyzing fructose, wherein the alpha-L-rhamnose has reverse hydrolysis capacity, and when L-rhamnose is taken as a glycosyl donor and fructose is taken as a glycosyl acceptor, the enzyme can be subjected to reverse hydrolysis to synthesize novel disaccharide rhamnosyl fructose, and the molar conversion rate is 23.4%. The alpha-L-rhamnosidase prepared by the invention has reverse hydrolysis activity, can be applied to the enzymatic preparation of novel disaccharide rhamnosyl fructose, and has good temperature stability and high molar conversion rate.)

1. A method for preparing rhamnosyl fructose by an enzyme method is characterized in that alpha-L-rhamnosidase with glycosylation activity is used for preparing rhamnosyl fructose.

2. The method for preparing rhamnosyl fructose by an enzymatic process according to claim 1, wherein the alpha-L-rhamnosidase is derived from Aspergillus oryzaeAspergillus oryzaeThe preparation method comprises the following steps:

a. aspergillus oryzaeAspergillus oryzaeDuring fermentation, appropriate amount of L-rhamnose or rutin or naringin or hesperidin is added for induction to obtain crude enzyme solution;

b. and (3) purifying the crude enzyme solution by ultrafiltration, a DEAE SFF anion column and a Superdex 20010/300 GL molecular sieve to obtain the electrophoretically pure alpha-L-rhamnosidase.

3. The method for preparing rhamnosyl fructose by an enzymatic method according to claim 2, wherein the step a specifically comprises the following steps:

firstly, activating a strain by using a PDA (PDA) culture medium, then resuspending the activated Aspergillus oryzae spores by using sterile physiological saline, inoculating 3 mL of the activated Aspergillus oryzae spores into 250 mL of liquid induction culture medium, and performing constant-temperature shaking culture at 28 ℃ and 180 r/min for 7 days;

the formula of the liquid induction culture medium is as follows: 0.5 g/L KCl, 1.5 g/L KH₂PO₄，4 g/L NH₄Cl, 5 g/L yeast extract, 1 g/L casamino acid, 5 g/L inducer, 1 mL/L Vishinic microelement, adjusting the pH of the culture medium to 6.0.

4. The enzymatic method for preparing rhamnosyl fructose according to claim 3, wherein the inducer is L-rhamnose.

5. The method for preparing rhamnosyl fructose by an enzymatic method according to claim 2, wherein the step b specifically comprises the following steps:

the supernatant was collected by centrifugation at 12000 rpm at 4 ℃ to obtain the crude enzyme, followed by separation and purification:

(1) concentrating with ultrafiltration membrane with molecular weight of 10000 Da to 15 mL, dialyzing with 20 mmol/L pH 7.5 Tris-HCl buffer solution for four times, each for 6 h;

(2) the dialyzed crude enzyme was applied to a packed DEAE SFF column, and the column-bound protein was eluted with a gradient of NaCl at a concentration of 0-1 mol/L;

(3) collecting the eluate step by step, measuring enzyme activity and detecting a target protein band by SDS-PAGE protein electrophoresis;

(4) taking enzyme solution eluted by NaCl with proper concentration, concentrating to 300 mu L, dialyzing with 20 mmol/L Tris-HCl buffer solution with pH 7.5 for four times, and dialyzing for 6 h each time;

(5) the dialyzed crude enzyme was applied to a packed Superdex 20010/300 GL Chromatographic Separation Column, equilibrated with 20 mmol/L pH 7.5 Tris-HCl buffer, at a flow rate of 0.3 mL/min and eluted;

(6) collecting eluate, measuring enzyme activity, detecting target protein band by SDS-PAGE protein electrophoresis, selecting high-purity sample, dialyzing at 4 deg.C in 20 mmol/L citric acid-disodium hydrogen phosphate buffer solution with pH of 4.5 for four times, each time for 6 hr; adding glycerol to 30%, mixing, and storing at-20 deg.C.

6. The method for preparing rhamnosyl fructose by an enzymatic method according to claim 1, characterized by comprising the following steps:

adding 0.05-1.0 mol/L of fructose and 0.05-3.0mol/L of L-rhamnose into a reaction system, controlling the concentration of alpha-L-rhamnosidase to be 1-10U/mL, adding a buffer solution with the pH of 3.0-7.0, controlling the reaction temperature to be 40-80 ℃, and reacting in a water bath for 12-72 hours to obtain the rhamnosyl fructose.

7. The method for preparing rhamnosyl fructose by an enzymatic process according to claim 6, wherein the pH of the synthesized rhamnosyl fructose is 4.5; the temperature for synthesizing the rhamnosyl fructose is 65 ℃; the addition amount of fructose for synthesizing rhamnosyl fructose is 0.1 mol/L, and the addition amount of rhamnose is 0.2 mol/L; the reaction time for synthesizing the rhamnosyl fructose is 36 h, and the concentration of the alpha-L-rhamnosidase is 2U/mL.

8. Rhamnosylfructose obtained by an enzymatic process according to any one of claims 1 to 7.

9. The method for detecting the quality of rhamnosyl fructose according to claim 8, characterized by comprising the following steps: detecting by using high performance liquid chromatography-evaporative light scattering emission, wherein the detection conditions are as follows: a chromatographic column: prevail Carbohydrate ES column (5 μm; 4.6X 250 mm); sample introduction amount: 10 mu L of the solution; mobile phase: acetonitrile: ultrapure water =70:30 (V: V); time: and 15 min.

10. The method for detecting the quality of rhamnosyl fructose according to claim 8, characterized by comprising the following steps: detection was performed using LC-MS, mass spectrometer model: waters LTQ Orbitrap XL LC/MS; n is a radical of₂ Airflow rate: 5L/min, voltage: 3.5 kV, pressure: 25 psi, temperature: at 300 ℃.

Technical Field

The invention belongs to the technical field of sugar engineering, and particularly relates to a preparation method of alpha-L-rhamnosidase and application of the alpha-L-rhamnosidase in catalyzing fructose to prepare rhamnosyl fructose.

Background

Oligosaccharides are widely distributed in nature and play an important role in many processes such as membrane structure regulation, cell-cell recognition, communication, cell adhesion, and viral infection. Conventional chemical synthesis of oligosaccharides requires complex steps to control the stereospecificity and regiospecificity of the reaction. Compared with the prior art, the method for synthesizing the oligosaccharide by enzyme has the advantages of one-step completion, strong specificity, environmental friendliness and the like. Glycosyltransferases and glycoside hydrolases may be used to synthesize specific oligosaccharides. The use of glycosyltransferase to synthesize oligosaccharides requires the use of expensive glycosyl donors, whereas the use of glycoside hydrolases to synthesize oligosaccharides requires the use of inexpensive and readily available glycosyl donors, and thus glycoside hydrolases are more advantageous than glycosyltransferases for large-scale oligosaccharide production.

alpha-L-rhamnosidase is an important glycoside hydrolase and can hydrolyze non-reducing rhamnose residues of a plurality of natural products, and a small part of the alpha-L-rhamnosidase has reverse hydrolysis capacity and can be used for synthesizing a compound containing rhamnosyl by reverse hydrolysis. alpha-L-rhamnosidase, e.g.from Aspergillus terreus CCF 3059, is able to link L-rhamnose to aromatic alcohols by reverse hydrolysis.

Rhamnosyl-rich oligosaccharides and polysaccharides obtained from cultures of Klebsiella pneumoniae and Klebsiella planticola protect normal human dermal fibroblasts from AGE (advanced glycation end products) -induced cytotoxicity. This suggests potential therapeutic applications in combating the effects of hyperglycemia-induced cytotoxicity, such as the treatment of type II diabetes. In addition, two rhamnoses containing long chain fatty alcohol glycosides, derived from the bark of the stem of the Shaosao Dimocarpus fumatus, were cytotoxic to the oral epithelial carcinoma KB cells in vitro. Although rhamnosyl-containing compounds have many specific activities, few rhamnosyl compounds are synthesized enzymatically at present. This may be due to the lack of α -L-rhamnosidase with broad receptor specificity. At present, no relevant patent for synthesizing the rhamnosyl-containing compound by an enzyme method exists.

Disclosure of Invention

Aiming at the problems, the invention discloses a method for preparing rhamnosyl fructose by an enzymatic method.

The invention comprises the following technical scheme:

a method for preparing rhamnosyl fructose by an enzyme method utilizes alpha-L-rhamnosidase with glycosylation activity to prepare rhamnosyl fructose.

Further, in the method for preparing rhamnosyl fructose by using the enzyme method, the alpha-L-rhamnosidase is derived from Aspergillus oryzae and is prepared by the following steps:

a. when Aspergillus oryzae is fermented, adding a proper amount of L-rhamnose or rutin or naringin or hesperidin for induction to obtain a crude enzyme solution;

b. and (3) purifying the crude enzyme solution by ultrafiltration, a DEAE SFF anion column and a Superdex 20010/300 GL molecular sieve to obtain the electrophoretically pure alpha-L-rhamnosidase.

Further, in the method for preparing rhamnosyl fructose by using the enzymatic method, the step a specifically comprises the following steps:

firstly, activating a strain by using a PDA (PDA) culture medium, then resuspending the activated Aspergillus oryzae spores by using sterile physiological saline, inoculating 3 mL of the activated Aspergillus oryzae spores into 250 mL of liquid induction culture medium, and performing constant-temperature shaking culture at 28 ℃ and 180 r/min for 7 days;

the formula of the liquid induction culture medium is as follows: 0.5 g/L KCl, 1.5 g/L KH2PO4, 4 g/L NH4Cl, 5 g/L yeast extract, 1 g/L casamino acid, 5 g/L inducer, 1 mL/L Vishinic microelement, adjusting the pH of the culture medium to 6.0.

Further, in the method for preparing rhamnosyl fructose by using the enzyme method, the inducer is L-rhamnose.

Further, in the method for preparing rhamnosyl fructose by using the enzymatic method, the step b specifically comprises the following steps:

the supernatant was collected by centrifugation at 12000 rpm at 4 ℃ to obtain the crude enzyme, followed by separation and purification:

(1) concentrating with ultrafiltration membrane with molecular weight of 10000 Da to 15 mL, dialyzing with 20 mmol/L pH 7.5 Tris-HCl buffer solution for four times, each for 6 h;

(2) the dialyzed crude enzyme was applied to a packed DEAE SFF column, and the column-bound protein was eluted with a gradient of NaCl at a concentration of 0-1 mol/L;

(3) collecting the eluate step by step, measuring enzyme activity and detecting a target protein band by SDS-PAGE protein electrophoresis;

(4) taking enzyme solution eluted by NaCl with proper concentration, concentrating to 300 mu L, dialyzing with 20 mmol/L Tris-HCl buffer solution with pH 7.5 for four times, and dialyzing for 6 h each time;

(5) the dialyzed crude enzyme was applied to a packed Superdex 20010/300 GL Chromatographic Separation Column, equilibrated with 20 mmol/L pH 7.5 Tris-HCl buffer, and eluted at a flow rate of 0.3 mL/min.

(6) Collecting eluate, measuring enzyme activity, detecting target protein band by SDS-PAGE protein electrophoresis, selecting high purity sample, dialyzing at 4 deg.C in 20 mmol/L citric acid-disodium hydrogen phosphate buffer solution with pH of 4.5 for four times, and dialyzing for 6 hr each time. Adding glycerol to 30%, mixing, and storing at-20 deg.C.

Further, the method for preparing rhamnosyl fructose by using the enzyme method comprises the following steps:

adding 0.05-1.0 mol/L of fructose and 0.05-3.0mol/L of L-rhamnose into a reaction system, controlling the concentration of alpha-L-rhamnosidase to be 1-10U/mL, adding a buffer solution with the pH of 3.0-7.0, controlling the reaction temperature to be 40-80 ℃, and reacting in a water bath for 12-72 hours to obtain the rhamnosyl fructose.

Further, in the method for preparing rhamnosyl fructose by the enzyme method, the pH value of the synthesized rhamnosyl fructose is 4.5; the temperature for synthesizing the rhamnosyl fructose is 65 ℃; the addition amount of fructose for synthesizing rhamnosyl fructose is 0.1 mol/L, and the addition amount of rhamnose is 0.2 mol/L; the reaction time for synthesizing the rhamnosyl fructose is 36 h, and the concentration of the alpha-L-rhamnosidase is 2U/mL.

Further, the rhamnosyl fructose is prepared by the enzyme method.

Further, the quality detection method of rhamnosyl fructose comprises the following steps:

detecting by using high performance liquid chromatography-evaporative light scattering emission, wherein the detection conditions are as follows: a chromatographic column: prevail Carbohydrate ES column (5 μm; 4.6X 250 mm); sample introduction amount: 10 mu L of the solution; mobile phase: acetonitrile: ultrapure water =70:30 (V: V); time: and 15 min.

Further, the quality detection method of rhamnosyl fructose comprises the following steps: detection was performed using LC-MS, mass spectrometer model: waters LTQ Orbitrap XL LC/MS; n2 airflow rate: 5L/min, voltage: 3.5 kV, pressure: 25 psi, temperature: at 300 ℃.

The invention has the following beneficial effects:

1. the invention discloses a method for producing alpha-L-rhamnosidase by inducing wild strain Aspergillus oryzae with an inducer, and the optimal reaction temperature of the alpha-L-rhamnosidase is found to be 65 ℃, and the temperature stability is good.

2. The invention prepares the novel disaccharide rhamnosyl fructose by utilizing the alpha-L-rhamnosidase with reverse hydrolysis capacity for the first time, the highest molar conversion rate can reach 23.4 percent, and the synthesis of the novel disaccharide rhamnosyl fructose is reported for the first time at present.

3. The invention explores and discloses an optimal reaction condition for converting fructose into rhamnosyl fructose by enzyme.

Drawings

FIG. 1 is a schematic diagram of an enzymatic conversion of fructose to rhamnosyl fructose;

FIG. 2 shows the effect of different inducers on the production of alpha-L-rhamnosidase;

FIG. 3 is an SDS-PAGE pattern of the purification process of α -L-rhamnosidase (M: standard protein; 1: crude enzyme after ultrafiltration; 2: enzyme after separation using DEAE SFF anion column; 3: enzyme treated using Superdex 20010/300 GL molecular sieves);

FIG. 4 is a graph of the effect of different reaction conditions on rhamnosyl fructose production;

FIG. 5 is an HPLC-ELSD detection profile of rhamnosylfructose;

FIG. 6 is an LC-MS detection map of rhamnosyl fructose.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. The proper amount of the compound is determined by the ordinary technicians in the field according to the national technical specifications and the actual production conditions. The starting materials described in the present invention are all commercially available unless otherwise specified.

The application of the principles of the present invention will be further described with reference to the accompanying drawings and specific embodiments. FIG. 1 is a schematic diagram of the enzymatic conversion of fructose into rhamnosyl fructose according to the present invention.

Example 1

Screening for inducers inducing Aspergillus oryzae to produce alpha-L-rhamnosidase.

The formula of the Aspergillus oryzae liquid culture medium is as follows: 0.5 g/L KCl, 1.5 g/L KH2PO4, 4 g/L NH4Cl, 5 g/L yeast extract, 1 g/L casamino acid, 5 g/L inducer, 1 mL/L Vishinic microelement, adjusting the pH of the culture medium to 6.0. The strain was activated with PDA medium, then the activated Aspergillus oryzae spores were resuspended in sterile physiological saline, 3 mL were inoculated into 250 mL of liquid induction medium, and cultured for 7 days at 28 ℃ under shaking at 180 r/min at constant temperature. Rutin, L-rhamnose, naringin and hesperidin are respectively selected as inducers for inducing Aspergillus oryzae to produce alpha-L-rhamnosidase. The results showed that the enzyme production of Aspergillus oryzae was highest with L-rhamnose as the inducer, and the enzyme activity was 1.6U/mL (as shown in FIG. 2).

Example 2

Purification of Aspergillus oryzae alpha-L-rhamnosidase.

The directional enzyme production is carried out by taking L-rhamnose as an inducer. The supernatant was centrifuged at 12000 rpm at 4 ℃ to obtain the crude enzyme. Separation and purification of crude enzyme: (1) the mixture was concentrated to 15 mL using an ultrafiltration membrane (Millipore, Billerica, MA, USA) of 10000 Da molecular weight and dialyzed against 20 mmol/L Tris-HCl buffer pH 7.5 in four portions for 6 h each. (2) The dialyzed crude enzyme was applied to a packed DEAE SFF column, and the column-bound protein was eluted with a gradient of NaCl at a concentration of 0-1 mol/L; (3) collecting the eluate step by step, measuring enzyme activity and detecting a target protein band by SDS-PAGE protein electrophoresis; (4) taking enzyme solution eluted by NaCl with proper concentration, concentrating to 300 mu L, and dialyzing with 20 mmol/L Tris-HCl buffer solution with pH 7.5 for four times for 6 h each time. (5) The dialyzed crude enzyme was applied to a packed Superdex 20010/300 GL Chromatographic Separation Column (10X 300 mm, GE Healthcare), equilibrated with 20 mmol/L pH 7.5 Tris-HCl buffer, and eluted at a flow rate of 0.3 mL/min. (6) Collecting eluate, measuring enzyme activity, detecting target protein band by SDS-PAGE protein electrophoresis, selecting high purity sample, dialyzing at 4 deg.C in 20 mmol/L citric acid-disodium hydrogen phosphate buffer solution with pH of 4.5 for four times, and dialyzing for 6 hr each time. Adding glycerol to 30%, mixing, and storing at-20 deg.C. The results of SDS-PAGE are shown in FIG. 3.

Example 3

The process research of preparing rhamnosyl fructose by enzymatic catalytic conversion of fructose.

In order to obtain the optimum pH, the optimum temperature, the optimum L-rhamnose content and the optimum reaction time for synthesizing rhamnosyl fructose by Aspergillus oryzae source alpha-L-rhamnosidase, the following experiments are set:

influence of different pH on the synthesis of rhamnosylated fructose by alpha-L-rhamnosidase: to 50. mu.L of the reaction system, 0.1 mol/L of fructose, 0.3 mol/L of rhamnose, with enzyme concentration controlled to be 2U/mL, 50 mmol/L of citric acid-disodium hydrogenphosphate buffer solution with pH values of 3.5, 4.0, 4.5, 5.0, 5.5 and 6.0, respectively, were added, and the reaction was carried out in a water bath at a temperature controlled to be 60 ℃ for 18 hours, and the residual amount of fructose was measured using HPLC-ELSD after sample treatment.

Influence of different temperatures on the synthesis of rhamnosylated fructose by α -L-rhamnosidase: adding 0.1 mol/L fructose and 0.3 mol/L rhamnose into a 50 mu L reaction system, controlling the enzyme concentration to be 2U/mL, adding 50 mmol/L citric acid-disodium hydrogen phosphate buffer solution with the pH value of 4.5, controlling the temperature to be 50 ℃, 55 ℃, 60 ℃, 65 ℃, 70 ℃ and 75 ℃ respectively, reacting for 18 h in a water bath, and detecting the residual quantity of the fructose by using HPLC-ELSD after sample treatment.

Influence of different addition amounts of L-rhamnose on the synthesis of rhamnosylated fructose by alpha-L-rhamnosidase: 0.1 mol/L of fructose is added into a 50 mu L reaction system, the enzyme concentration is controlled to be 2U/mL, 50 mmol/L of citric acid-disodium hydrogen phosphate buffer solution with pH 4.5 is added, the temperature is controlled to be 65 ℃, rhamnose is respectively added to be 0.05 mol/L, 0.1 mol/L, 0.15 mol/L, 0.2 mol/L, 0.25 mol/L, 0.3 mol/L, 0.35 mol/L, 0.4 mol/L, 0.45 mol/L and 0.5 mol/L, the mixture is reacted in a water bath for 18 h, and the residual quantity of the fructose is detected by using HPLC-ELSD after sample treatment.

Influence of different reaction times on the synthesis of rhamnosylated fructose by alpha-L-rhamnosidase: adding 0.1 mol/L fructose and 0.2 mol/L rhamnose into a 50 mu L reaction system, controlling the enzyme concentration to be 2U/mL, adding 50 mmol/L citric acid-disodium hydrogen phosphate buffer solution with the pH value of 4.5, controlling the temperature to be 65 ℃, reacting for 6 h, 12 h, 18 h, 24 h, 30 h, 36 h, 42 h, 48 h, 54 h and 60 h in a water bath respectively, and detecting the residual amount of the fructose by using HPLC-ELSD after sample treatment.

The result is shown in figure 4, and the optimum pH value of the synthesized rhamnose fructose is 4.5; the optimal temperature for synthesizing the rhamnose fructose is 65 ℃; the optimal rhamnose addition amount for synthesizing the rhamnose fructose is 0.2 mol/L; the optimal reaction time for synthesizing the rhamnose fructose is 36 h, and the highest conversion rate reaches 23.4%.

Example 4

And detecting results of HPLC-ELSD and LC-MS after the fructosyl glycosylation reaction.

Detecting by using high performance liquid chromatography-evaporative light scattering emission (HPLC-ELSD) after the fructosyl glycosylation reaction, wherein the detection conditions are as follows: a chromatographic column: prevail Carbohydrate ES column (5 μm; 4.6X 250 mm); sample introduction amount: 10 mu L of the solution; mobile phase: acetonitrile: ultrapure water =70:30 (V: V); time: and 15 min. As shown in FIG. 5, a new product rhamnosylfructose was produced after the glycosylation reaction.

The rhamnosyl fructose is detected by using LC-MS, and the model of a mass spectrometer is as follows: waters LTQ Orbitrap XL LC/MS, N₂ Airflow rate: 5L/min, voltage: 3.5 kV, pressure: 25 psi, temperature: at 300 ℃. The results are shown in FIG. 6It is shown that the molecular weight of rhamnosylfructose is 326, which corresponds to the theoretical molecular weight.

The above examples 1-4 show that the alpha-L-rhamnosidase prepared by the present invention has reverse hydrolysis activity, can be applied to enzymatic preparation of a novel disaccharide rhamnosyl fructose, and has good temperature stability and high molar conversion rate.

The above are only preferred embodiments of the present invention, and the scope of the present invention should not be limited thereby, and all the equivalent changes and modifications made by the claims and the summary of the invention should be covered by the protection scope of the present patent application.