CN118048428A - Preparation method of rebaudioside B and application of glycoside hydrolase in preparation of rebaudioside B - Google Patents

Abstract

The invention discloses a preparation method of rebaudioside B and application of glycoside hydrolase in preparation of rebaudioside B. The preparation method comprises the step of contacting beta-glucosidase with a substrate to perform a reaction; the substrate is rebaudioside A or rebaudioside D. The glycoside hydrolase provided by the invention has strong catalytic capability and high yield of the rebaudioside B when preparing the rebaudioside B, thereby reducing the production cost and being beneficial to industrial production.

Description

Preparation method of rebaudioside B and application of glycoside hydrolase in preparation of rebaudioside B

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a preparation method of rebaudioside B and application of glycoside hydrolase in preparation of rebaudioside B.

Background

Because of the problems of weight gain, induced diabetes, hypertension, etc. caused by excessive intake of sugar, more and more consumers choose low calorie sugar substitutes. Stevioside (Steviol glycosides, also called steviol glycoside) produced by stevia rebaudiana (Stevia rebaudiana Bertoni) has the characteristics of high sweetness, low heat energy, no participation in metabolism in human bodies, health care function and the like, is praised as a new sugar source with the most development prospect, and has wide application prospect and high economic value. However, it is not enough that some steviol glycosides have, in addition to sweetness, afterbitter and licorice tastes, which have to be faced with difficulties in many applications.

Steviol glycosides (steviol glycoside compounds) have the following structural formula:

The steviol glycosides described above have a common aglycone: steviol (Steviol), differing in the number and type of glycosyl groups attached at the C-13 and C-19 positions, mainly includes eight glycosides, stevioside (Stevioside), rebaudioside A (rebaudiosid A, reba A, RA), rebaudioside B (rebaudiosid B, reba B, RB), rebaudioside C, rebaudioside D (rebaudiosid D, reba D, RD), rebaudioside E, dulcoside, steviolbioside, etc. Stevia leaves are capable of accumulating up to 10-20% (on a dry weight basis) steviol glycosides. The main glycosides found in stevia leaves are rebaudioside a (2-10%), stevioside (2-10%) and rebaudioside C (1-2%). Other glycosides, such as rebaudiosides B, D, E and F, steviolbioside and rubusoside, are found at much lower levels (about 0-0.2%).

Rebaudioside a has a sweetness of 150 to 320 times that of sucrose, however, even in a highly purified state, rebaudioside a still has undesirable taste attributes such as bitter, sweet aftertaste, licorice taste, etc. Although the sweetness of the rebaudioside B is weaker than that of the rebaudioside A, the rebaudioside B has no peculiar smell, can be independently used as a sweetener, has the effect of reducing blood sugar, and is a stevioside compound with great application potential.

The current preparation method of rebaudioside B is a chemical hydrolysis method, for example, patent CN104725437A discloses a method for preparing rebaudioside B and laminariae disaccharide by alkaline hydrolysis of rebaudioside I. The chemical hydrolysis method has extreme reaction conditions, can generate a large amount of hazardous waste reagents and is not friendly to the environment.

CN102796790a discloses a method for converting steviolbioside into rebaudioside B by an enzymatic method, which uses steviolbioside as a raw material, sucrose as a sugar donor, and converts the steviolbioside into rebaudioside B under the catalysis of glycosyltransferase (from aspergillus niger cic 40417), the method requires adding sugar source sucrose, the cost is high, and the conversion rate of the obtained rebaudioside B is between 70 and 75%, and the conversion rate needs to be further improved.

CN112322686a discloses a method for producing rebaudioside B by enzymatic method, which takes rebaudioside a, rebaudioside D, rebaudioside I and rebaudioside M as substrates, beta-galactosidase (derived from sulfolobus, aspergillus niger or kluyveromyces) as catalyst, and catalyzes the substrate reaction in aqueous solution to obtain rebaudioside B, the method takes 80g/L RA as substrate, 100000U/L glycoside hydrolase is added, glycerol and mercaptoethanol are not added, the reaction is carried out for 6 hours at 37 ℃, the conversion rate of rb is only 22.5%, the required enzyme amount is large, and the conversion rate is low; when 30% of glycerol and 5% of beta-mercaptoethanol are added as a protective agent and an activator of the enzyme, respectively, the conversion rate is improved, but the corresponding reaction cost is increased and the environment is not friendly.

With the development of technology, it is a great trend to replace chemical methods with biological methods. However, the currently disclosed glycoside hydrolase has fewer varieties, lower enzyme activity and lower product yield, and is not suitable for industrial production, so that the novel glycoside hydrolase with high expression and high enzyme activity can be mined and identified, the preparation efficiency of stevioside can be further improved, and the stevioside has great market prospect and research value.

Disclosure of Invention

The technical problem to be solved by the invention is the defect of low yield of the existing stevioside production enzyme when catalyzing stevioside to produce rebaudioside B, so that a preparation method of the rebaudioside B and application of the glycoside hydrolase in the preparation method are provided. The glycoside hydrolase is beta-glucosidase (also known as beta-glucosidase). The beta-glucosidase used in the invention has strong catalytic activity, and the prepared rebaudioside B has high yield, reduces the cost of reaction and is beneficial to industrial production.

The invention solves the technical problems through the following technical proposal.

In a first aspect, the invention provides a method of preparing rebaudioside B, the method comprising the step of contacting beta-glucosidase with a substrate to react; the substrate is rebaudioside A or rebaudioside D.

In some embodiments of the invention, the β -glucosidase is a β -glucosidase derived from pichia pastoris (Komagataella pastoris) or pichia intramedullary (Kuraishia capsulata).

In some embodiments of the invention, the β -glucosidase is an enzyme corresponding to NCBI accession number ANZ76010.1 or xp_ 022460765.1.

In some embodiments of the invention, the beta-glucosidase is used in the form of a wet cell, a bacterial powder, a liquid enzyme, a solid enzyme powder, or an immobilized enzyme.

In the present invention, wet cells refer to a precipitate obtained by centrifuging a culture solution of cells expressing β -glucosidase and discarding the supernatant. The fungus powder is a powder obtained by drying the wet fungus body to remove solvent water and pulverizing the wet fungus body. The liquid enzyme (e.g., crude enzyme liquid) is an enzyme liquid obtained by resuspending the wet cells in a predetermined ratio with a solvent (e.g., water), and preferably is a supernatant obtained by homogenizing the enzyme liquid after resuspending at a predetermined pressure and centrifuging at a predetermined speed. The solid enzyme powder is powder obtained by drying and pulverizing the liquid enzyme. The immobilized enzyme is obtained by immobilizing the enzyme component in the liquid enzyme on an immobilization carrier. Immobilization carriers include resins such as epoxy resins, amino resins, adsorption resins. More specifically, the epoxy resin may beECHFA, reliZymeTMHFA403, reliZymeTMEP, 113, reliZymeTMEP403 and/or/>ECEP。

In some embodiments of the invention, the beta-glucosidase is used in the form of wet bacterial cells, and the ratio of the added mass of the wet bacterial cells to the added mass of the substrate in the reaction system is (0.3-30): 1, preferably (0.48-20): 1, more preferably (0.8-20): 1.

In the present invention, the reaction system refers to a solution system in which each reaction component participating in the reaction is present when the reaction occurs. The reaction system in this embodiment includes components such as wet cells containing beta-glucosidase, substrate, product, and solvent. Other embodiments are the same.

In the present invention, the added mass means the initial mass of the wet cell, substrate and other components when added into the reaction system.

In some preferred embodiments of the present invention, the amount of the wet cell added in the reaction system is 16 to 100g/L, preferably 40 to 80g/L; the addition amount of the wet cell means a ratio of the addition mass of the wet cell to the total volume of the entire reaction system.

In other embodiments of the invention, the beta-glucosidase is used in the form of a liquid enzyme, which is a crude enzyme solution prepared from a bacterium expressing the beta-glucosidase.

In the invention, one preparation example of the crude enzyme solution is to re-suspend wet thalli expressing the beta-glucosidase by using buffer solution according to the ratio of 1:5 (g/mL), then homogenize the heavy suspension for 10-15 min (so as to break the thalli) by 600bar, and centrifuge for 10-15 min at 12000-13000 rpm, and then take the supernatant to obtain the crude enzyme solution.

In the present invention, a phosphate buffer having a pH of 7.0 to 7.5 may be used as the buffer.

In some preferred embodiments of the present invention, the ratio of the addition volume of the crude enzyme solution to the addition mass of the substrate in the reaction system is (1-100): 1, preferably (4-100): 1 in mL/g. The addition volume of the crude enzyme solution refers to the initial volume of the crude enzyme solution at the time of addition to the reaction system.

In the present invention, the mass of the substrate to be added refers to the initial mass of the substrate when it is added to the reaction system.

In some embodiments of the invention, the substrate is added to the reaction system in an amount of 20 to 120g/L, preferably 20 to 60g/L, more preferably 20 to 40g/L; the addition amount of the substrate refers to the ratio of the addition mass of the substrate to the total volume of the whole reaction system.

In some embodiments of the invention, the pH of the reaction system is from 6.0 to 8.0, preferably from 7.2 to 7.5.

In some embodiments of the invention, the temperature of the reaction is 28 to 37 ℃, preferably 30 to 35 ℃;

In some embodiments of the invention, the reaction time of the reaction is from 2 to 24 hours, for example, 2 hours, 4 hours, or 24 hours.

In a second aspect, the invention provides a composition comprising rebaudioside B, the composition prepared by a method as described in the first aspect.

In a third aspect the invention provides an isolated nucleic acid molecule encoding a beta-glucosidase enzyme, which is a beta-glucosidase enzyme used in the method according to the first aspect.

In some embodiments of the invention, the nucleic acid molecule has a nucleotide sequence as set forth in SEQ ID NO. 1 or SEQ ID NO. 2.

In a fourth aspect, the invention provides a use of a nucleic acid molecule according to the third aspect for preparing rebaudioside B.

In a fifth aspect, the invention provides the use of a glycoside hydrolase in the preparation of rebaudioside B, the glycoside hydrolase being a β -glucosidase, the β -glucosidase being a β -glucosidase derived from pichia pastoris (Komagataella pastoris) or pichia intramedullary (Kuraishia capsulata).

In some embodiments of the invention, the β -glucosidase is an enzyme corresponding to NCBI accession number ANZ76010.1 or xp_ 022460765.1.

In some embodiments of the invention, the beta-glucosidase is in the form of a liquid enzyme, a solid enzyme powder, or an immobilized enzyme.

Some embodiments of the present invention also provide for the use of a liquid enzyme in the preparation of rebaudioside B, the liquid enzyme being an enzyme solution of beta-glucosidase.

Some specific embodiments of the present invention also provide an application of the solid-state enzyme powder in preparing rebaudioside B, wherein the solid-state enzyme powder is an enzyme powder of beta-glucosidase.

Some embodiments of the invention also provide an application of the immobilized enzyme in preparing rebaudioside B, wherein the immobilized enzyme is obtained by immobilizing beta-glucosidase on an immobilized carrier.

On the basis of conforming to the common knowledge in the field, the above preferred conditions can be arbitrarily combined to obtain the preferred examples of the invention.

The reagents and materials used in the present invention are commercially available.

The invention has the positive progress effects that: the beta-glucosidase used in the invention has high yield (for example, 99.35 percent) when the rebaudioside A or the rebaudioside D is used as a substrate to prepare the rebaudioside B, and has good application prospect.

Detailed Description

The invention is further illustrated by means of the following examples, which are not intended to limit the scope of the invention. The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications.

E.coli BL21 (DE 3) competent cells were prepared according to the method described in the molecular cloning laboratory Manual written by J.Sam Broker et al. RA, RD, RM and RB standards were purchased from Shanghai Seiyaku Biotechnology Inc.

HPLC detection method:

Chromatographic column: diamonsil C18 (2) (4.6 mm. Times.250 mm,5 μm); mobile phase a: 1.5g of sodium dihydrogen phosphate (NaH ₂PO₄) was weighed, dissolved in 1000mL of water, and the pH was adjusted to 2.6 with phosphoric acid; mobile phase B: acetonitrile; gradient elution was performed under the gradient elution conditions shown in Table 1. Detection wavelength: 210nm; flow rate: 0.8mL/min; sample injection volume: 20. Mu.L; column temperature: 40 ℃. RA retention time was 21.905min; RB retention time is 33.730min; RD retention time 12.614min; the RM retention time is 16.458min.

TABLE 1 gradient elution conditions

Time (minutes)	Mobile phase A%	Mobile phase B%
			0.00	75	25
13.0	75	25
			15.0	68	32
27.0	68	32
			40.0	30	70
45.0	30	70
			45.5	25	25
60.0	75	25

Example 1: beta-glucosidase (beta-Glucosidase, BGL) screening

1.1 Obtaining crude enzyme solution

LB basal medium: 10g/L tryptone, 5g/L Yeast extract (Yeast extract), 10g/L sodium chloride.

LB resistance plates: 10g/L tryptone, 5g/L yeast extract, 10g/L sodium chloride, 50. Mu.g/mL kanamycin, 20g/L agar.

TB basal medium: 2% tryptone, 2.4% yeast extract, 72mM K ₂HPO₄、17mM KH₂PO₄, 0.4% glycerol (glycidol).

Plasmid preparation: the BGL genes which are expressed as shown in Table 2 and have different sources and are subjected to codon optimization are respectively inserted into pET28a plasmids, the enzyme cutting sites are NdeI and HindIII, different pET28a-BGL plasmids are respectively obtained, and each pET28a-BGL plasmid only contains one BGL gene. These plasmids were synthesized by the division of biological engineering (Shanghai).

Plasmid transformation: the different types of pET28a-BGL plasmids were transformed separately as follows. mu.L of pET28a-BGL plasmid with the concentration of 40 ng/. Mu.L is added into 100 mu.L of E.coli BL21 (DE 3) competent cells, placed on ice for 30min, and then subjected to heat shock at 42 ℃ for 45s, and immediately placed on ice for 2-5 min. After 800. Mu.L of LB basal medium was added, the mixture was placed on a shaker at 37℃and incubated for 45min. The incubated bacterial liquid was spread on LB resistant plates (containing kanamycin at a working concentration of 50. Mu.g/mL), inverted in a 37℃incubator, and cultured overnight. And (3) selecting a single colony, inoculating the single colony into an LB basal medium containing 50 mug/mL kanamycin, and obtaining the genetically engineered strain after culturing. Different types of pET28a-BGL plasmids are respectively transformed into competent cells of the escherichia coli to obtain a plurality of genetic engineering strains, and each genetic engineering strain can express one beta-D-glucosidase in a one-to-one correspondence manner. These genetically engineered strains were cryopreserved using glycerol as cryoprotectant (30% final glycerol concentration).

The above-mentioned multiple genetic engineering strains are respectively inoculated into LB resistance plate to make streak activation, then the single colony is picked up and inoculated into 5mL of LB basic culture medium containing 50 mug/mL kanamycin, and vibration-cultured for 12h at 37 deg.C. The inoculated amount was 2% (v/v) and transferred to 150mL of TB basal medium containing kanamycin at a final concentration of 50. Mu.g/mL, when the culture medium was shaken at 37℃until the OD600 reached about 0.8, isopropyl-. Beta. -D-thiogalactopyranoside (IPTG) was added to a final concentration of 0.1mM, and the culture was induced at 25℃for 16 hours. After the culture is finished, the culture solution is centrifuged at 10000rpm for 10min, the supernatant is discarded, wet thalli are collected and stored in a refrigerator at the temperature of minus 20 ℃ for standby.

The wet cells collected after the completion of the culture or the frozen wet cells were washed twice with 50mM Phosphate Buffer (PBS) having a pH of 7.2 to obtain wet cells. After each wash, centrifugation was carried out at 10000rpm for 10min, the supernatant was discarded and the pellet was collected. Then, the wet cells were resuspended in 50mL of PBS having a pH of 7.2 at a ratio of 1:5 (g/mL), homogenized and broken by using a high-pressure homogenizer at 600bar for 10min, and the obtained broken bacterial liquid was centrifuged at 13000rpm for 10min to remove the precipitate, and the obtained supernatant was a crude enzyme liquid containing beta-glucosidase.

The different BGL sources are shown in table 2 below:

TABLE 2 sources of different beta-glucosidase enzymes and NCBI sequence numbers

Enzyme numbering	Source(s)	NCBI sequence number
			BGL1	Komagataella pastoris	ANZ76010.1
BGL2	Kuraishia capsulata	XP_022460765.1
			BGL3	Babjeviella inositovora	XP_018985210.1
BGL4	Komagataella phaffii	XP_002493453.1
			BGL5	Trichoderma reesei	BAA74959.1
BGL6	Talaromyces amestolkiae	XP_040728882.1
			BGL7	Aspergillus niger	ABW87793.1

1.2 Screening of beta-glucosidase

The screening procedure is as follows:

(1) mu.L of substrate, 200. Mu.L of crude enzyme solution and 700. Mu.L of PBS buffer were mixed in a one-to-one correspondence to obtain a mixed solution (total volume: 1 mL). Wherein the substrate is selected from any one of Rebaudioside A (RA) with a final concentration of 2g/L, rebaudioside D (RD) with a final concentration of 3g/L, and Rebaudioside M (RM) with a final concentration of 2 g/L. The final concentration refers to the initial concentration of the above substrate in the mixed solution after addition (i.e., the concentration at which no reaction has occurred or the concentration at which the reaction time is 0 h), and the same applies to the other examples. The crude enzyme solution was selected from any one of the crude enzyme solutions containing BGL1, BGL2, BGL3, BGL4, BGL5, BGL6, and BGL7 obtained in section 1.1 of this example. The buffer was 100mM PBS pH 7.2.

(2) The respective mixed solutions were reacted at 30℃for 4 hours, and after the completion of the reaction, a sample to be measured (volume: 1 mL) was obtained. From each sample to be tested, 500. Mu.L of each sample was taken, 20. Mu.L of 20% hydrochloric acid (to denature the enzyme protein) and 480. Mu.L of deionized water were added, and the mixture was centrifuged at 13000rpm for 5 minutes (to remove the denatured enzyme protein), and 20. Mu.L of each sample was subjected to HPLC analysis after passing through a 0.22 μm filter. The concentration and yield of RB in the sample to be tested were obtained from the HPLC detection results, see table 3 below.

TABLE 3 concentration and yield of RB

Wherein, when RA is used as a substrate to generate RB, the concentration (C _RB) of RB is calculated according to an external standard method.

The calculation method of RB yield is as follows:

In this example, when the RB yield was calculated according to the formula above, C _RA was 2g/L, M _RA was 967.01, and M _RB was 804.87.

The method for calculating the yields of C _RB and RB was the same when RB was generated using RD and RM as substrates.

From the data in Table 3, the concentration of RB produced by BGL2 catalytic substrate was highest, as was the RB yield. Specifically:

1) When RA is taken as a substrate, the concentration of RB generated by catalyzing RA by BGL2 is highest, the concentration of RB generated by catalyzing RA by the rest of enzymes is lower after BGL is performed 1 time. In addition, BGL2 catalyzes RA to produce RB in the highest yield, while BGL1 yields to RB are about 30% lower than the rest of the enzyme yields to RB are low. The data indicate that BGL2 catalyzes RA to produce RB with the highest enzyme activity, and that BGL1 has the next lowest enzyme activity, BGL3-BGL 7.

2) When RD is used as a substrate, the concentration of RB generated by catalyzing RD with BGL2 is highest, BGL is carried out 5 times, and the activity of catalyzing RD to generate RB by the rest enzymes is low. In addition, BGL2 catalyzes RD to RB in the highest yield, while BGL5 catalyzes RD to RB in about 80% lower yield than the rest of the enzyme has low yield to RB. The results show that BGL2 catalyzes RD to generate RB with the highest enzyme activity, BGL5 with the next highest enzyme activity and the rest enzymes with low enzyme activities.

3) When RM is taken as a substrate, the concentration of the RB produced by catalyzing RM is very low, the highest yield is only 4.01%, and the data indicate that the catalytic ability of the BGL1-BGL7 is poor when the RB is produced by taking RM as a substrate, and the corresponding enzyme activity is poor.

Example 2: optimization experiment of reaction pH value of RB (RB-reactive oxygen species) produced by BGL2 (beta-cyclodextrin) catalysis RA (RA)

According to the results of example 1, BGL2 was selected to produce RB using RA as a substrate in this example 2.

The steps of this embodiment are as follows:

2.1. 500. Mu.L of substrate RA at a concentration of 20g/L, 80. Mu.L of crude enzyme solution containing BGL2 prepared in section 1.1 of example 1, 420. Mu.L of PBS buffer solutions with pH values of 100mM of 6.0, 6.5, 7.0, 7.5 and 8.0 were mixed in a one-to-one correspondence so that the final concentration of RA in the obtained mixture (total volume: 1000. Mu.L) became 10g/L. Then reacting for 2 hours at 30 ℃, and obtaining a sample to be detected after the reaction is finished.

2.2. From each sample to be tested, 500. Mu.L of each sample was taken, 20. Mu.L of 20% hydrochloric acid and 480. Mu.L of deionized water were added, respectively, and the mixture was centrifuged at 13000rpm for 5 minutes to remove the enzyme, and 20. Mu.L of each sample was taken after passing through a 0.22 μm filter membrane, and HPLC analysis was performed, and the reaction results are shown in Table 4.

TABLE 4 concentration and yield of RB at different pH values

PH value of	CRB(g/L)	Yield of RB
			6.0	5.45	65.48％
6.5	5.40	64.88％
			7.0	5.50	66.08％
7.5	5.88	70.65％
			8.0	5.35	64.28％

Reaction results: when the pH of the reaction system is 7.5, the concentration of RB generated by catalyzing RA through BGL2 is highest, and 5.88g/L, which shows that the enzyme activity of RB generated by catalyzing RA through BGL2 is highest when the pH value is 7.5, and the pH value is 7.5 and can be used as the optimal pH value when catalyzing BGL 2.

Example 3: optimization experiment of reaction temperature for producing RB by BGL2 catalysis RA

The steps of this embodiment are as follows:

3.1. 500. Mu.L of substrate RA at a concentration of 20g/L, 80. Mu.L of crude enzyme solution containing BGL2 prepared in section 1.1 of example 1, 420. Mu.L of 100mM PBS buffer at pH 7.5 were mixed in one-to-one correspondence, and the final concentration of RA in the resulting mixture (total volume: 1000. Mu.L) was brought to 10g/L. Then respectively reacting for 2 hours at 30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃ and 55 ℃ to obtain a sample to be detected after the reaction is finished.

3.2. From each sample to be tested, 500. Mu.L of each sample was taken, 20. Mu.L of 20% hydrochloric acid and 480. Mu.L of deionized water were added, respectively, and the mixture was centrifuged at 13000rpm for 5 minutes to remove the enzyme protein, and 20. Mu.L of each sample was taken after passing through a 0.22 μm filter membrane, and HPLC analysis was performed, and the reaction results are shown in Table 5.

TABLE 5 concentration and yield of RB at different temperature conditions

Experimental data in table 5 illustrates: at a shorter reaction time (2 h), when the temperature of the reaction system is 35 ℃, the concentration and yield of RB generated by BGL2 catalysis are highest, and when the temperature is 30 ℃, the optimum temperature condition of BGL2 at 35 ℃ is shown under the conditions of shorter reaction time (such as 2h to 4h and the like) and pH value of 7.5. However, if the reaction time is longer, the enzyme activity of BGL2 at 35 ℃ is gradually reduced, so when the reaction time is longer (such as 10h to 24h, etc.), the temperature of the catalytic reaction of BGL2 can be 30 ℃ at this time in order to keep the stable enzyme activity of BGL2 during the longer reaction time.

Example 4: enzyme catalysis experiments of different RA concentrations during production of RB by BGL2 catalysis RA

The steps of this embodiment are as follows:

4.1. 500. Mu.L of substrate RA at a concentration of 40, 80, 120, 160, 200g/L, 160. Mu.L of crude enzyme solution containing BGL2 prepared in section 1.1 of example 1, 340. Mu.L of 100mM PBS buffer at a pH of 7.5 were mixed in one-to-one correspondence, so that the final concentrations of RA in the obtained mixed solution (total volume: 1000. Mu.L) were 20, 40, 60, 80, 100g/L, respectively. Then reacting for 24 hours at 30 ℃, and obtaining a sample to be detected after the reaction is finished.

4.2. From each sample to be tested, 500. Mu.L of each sample was taken, 20. Mu.L of 20% hydrochloric acid and 480. Mu.L of deionized water were added, respectively, and the mixture was centrifuged at 13000rpm for 5 minutes to remove the enzyme protein, and 20. Mu.L of each sample was taken after passing through a 0.22 μm filter membrane, and HPLC analysis was performed, and the reaction results are shown in Table 6.

TABLE 6 concentration and yield of RB at different RA substrate concentrations

Final concentration of RA (g/L)	CRB(g/L)	Yield of RB
			20	16.54	99.35％
40	32.75	98.37％
			60	42.20	84.50％
80	46.62	70.01％
			100	52.56	63.15％

When 160. Mu.L of crude enzyme solution (homogeneous ratio 1:5 (g/mL)) was added to the reaction system, and the final concentration of RA was 20g/L, RA was able to be substantially completely converted to RB, indicating that a substrate concentration of 20g/L was suitable for the enzyme. When 160. Mu.L of crude enzyme solution (homogeneous ratio 1:5 (g/mL)) was added to the reaction system, and the final concentration of RA was 40g/L, RA was also substantially completely converted into RB. When the final concentration of RA is higher, the yield of RB decreases, but still is higher.

Example 5: enzyme addition experiment under high RA concentration when BGL2 catalyzes RA to produce RB

The steps of this embodiment are as follows:

5.1. 500. Mu.L of 200g/L substrate RA, 160. Mu.L, 240. Mu.L, 320. Mu.L and 400. Mu.L of the crude enzyme solution containing BGL2 prepared in section 1.1 of example 1, and 340. Mu.L, 260. Mu.L, 180. Mu.L and 100. Mu.L of 100mM PBS buffer solution having pH 7.5 were mixed in one-to-one correspondence so that the final concentration of RA in the resultant mixture (total volume: 1000. Mu.L) became 100g/L. Then reacting for 24 hours at 30 ℃, and obtaining a sample to be detected after the reaction is finished.

5.2. From each sample to be tested, 500. Mu.L of each sample was taken, 20. Mu.L of 20% hydrochloric acid and 480. Mu.L of deionized water were added, respectively, and the mixture was centrifuged at 13000rpm for 5 minutes to remove the enzyme protein, and 20. Mu.L of each sample was taken after passing through a 0.22 μm filter membrane, and HPLC analysis was performed, and the reaction results are shown in Table 7.

TABLE 7 concentration and yield of RB at different enzyme levels

BGL crude enzyme liquid (mu L)	CRB(g/L)	Yield of RB
			160	52.81	63.45％
240	62.72	75.36％
			320	69.95	84.05％
400	76.01	91.32％

Reaction results: when the final concentration of substrate RA in the reaction system is 100g/L, 400 mu L of crude enzyme solution is added for reaction for 24 hours, RA can be almost completely converted into RB, which means that 400 mu L of BGL2 crude enzyme solution can catalyze 100g/L RA to be almost completely converted into RB after 24 hours of reaction. However, for a high substrate concentration of 100g/L, if the amount of crude enzyme solution is reduced, the productivity for industrial application cannot be achieved within a catalytic time of 24 hours.

From the experimental results, the BGL2 enzyme can produce RB with RA as a substrate under proper conditions and has higher yield.

The reaction parameters in the various examples described above are shown in table 8 below.

TABLE 8 reaction parameters Table in various examples

In Table 8, the mass (g) of the wet cells added and the volume (mL) of the crude enzyme solution added were calculated at a ratio of 1:5.

Claims (10)

1. A method of preparing rebaudioside B, the method comprising the step of contacting beta-glucosidase with a substrate to effect a reaction;

the substrate is rebaudioside A or rebaudioside D.

2. The method of claim 1, wherein the β -glucosidase is a β -glucosidase derived from pichia pastoris (Komagataella pastoris) or pichia intramedullary (Kuraishia capsulata);

Preferably, the beta-glucosidase is an enzyme corresponding to NCBI accession No. ANZ76010.1 or XP_ 022460765.1.

3. The method of claim 1 or 2, wherein the beta-glucosidase is used in the form of a wet cell, a bacterial powder, a liquid enzyme, a solid enzyme powder, or an immobilized enzyme.

4. The method according to claim 3, wherein the beta-glucosidase is used in the form of wet bacterial cells, and the ratio of the mass of the wet bacterial cells to the mass of the substrate added in the reaction system is (0.3-30): 1, preferably (0.48-20): 1, more preferably (0.8-20): 1, a step of;

Preferably, the amount of the wet cell added in the reaction system is 16 to 100g/L, preferably 40 to 80g/L.

5. A method according to claim 3, wherein the β -glucosidase is used in the form of a liquid enzyme; the liquid enzyme is crude enzyme liquid; the crude enzyme solution is prepared from bacteria expressing the beta-glucosidase;

the ratio of the addition volume of the crude enzyme liquid to the addition mass of the substrate in the reaction system is (1-100): 1, preferably (4-100): 1 in mL/g.

6. The method according to any one of claims 3 to 5, wherein the substrate is added in an amount of 20 to 120g/L, preferably 20 to 60g/L, more preferably 20 to 40g/L;

and/or the pH of the reaction system is 6.0 to 8.0, preferably 7.2 to 7.5;

And/or the temperature of the reaction is 28-37 ℃, preferably 30-35 ℃;

And/or the reaction time of the reaction is 2-24h.

7. A composition comprising rebaudioside B, wherein the composition is prepared by the method of any one of claims 1-6.

8. An isolated nucleic acid molecule encoding a β -glucosidase enzyme employed in the method of any of claims 1-6;

preferably, the nucleic acid molecule has a nucleotide sequence as shown in SEQ ID NO. 1 or SEQ ID NO. 2.

9. Use of the nucleic acid molecule of claim 8 for preparing rebaudioside B.

10. Use of a glycoside hydrolase in the preparation of rebaudioside B, the glycoside hydrolase being a β -glucosidase, the β -glucosidase being a β -glucosidase derived from pichia pastoris (Komagataella pastoris) or pichia intramedullary (Kuraishia capsulata);

Preferably, the beta-glucosidase is an enzyme corresponding to NCBI accession No. ANZ76010.1 or XP_ 022460765.1.