CN116004417A

CN116004417A - Bacillus subtilis and application thereof

Info

Publication number: CN116004417A
Application number: CN202210834070.1A
Authority: CN
Inventors: 柏文琴; 郑宏臣; 谭明; 郑雯; 付晓平; 赵兴亚
Original assignee: Tianjin Institute of Industrial Biotechnology of CAS
Current assignee: Tianjin Institute of Industrial Biotechnology of CAS
Priority date: 2022-07-14
Filing date: 2022-07-14
Publication date: 2023-04-25

Abstract

The invention discloses an application of bacillus subtilis in glycosylation modification. Specifically, the invention screens out a bacillus subtilis strain from the environment and uses the bacillus subtilis strain for glycosylation modification. The strain is preserved in the China general microbiological culture Collection center with the preservation number of CGMCC No.19322, stevioside and rebaudioside A are taken as substrates, and the bacillus subtilis fermentation supernatant is added for a conversion reaction of 12h to generate the rebaudioside E, wherein the yield is up to 78.9%; after 30h conversion reaction, the rebaudioside D is produced, and the yield is up to 39.7%.

Description

Bacillus subtilis and application thereof

Technical Field

The invention relates to screening and application of a bacillus subtilis glycosyltransferase production strain, and belongs to the fields of microorganisms and bioengineering.

Background

Glycosylation is an important post-modification reaction in the biosynthesis of natural products, most active natural products being present in the form of glycosides. In recent years, many studies have been made on the synthesis of glycoside compounds using glycosyltransferases derived from different sources, such as improving the patentability of natural products using glycosyltransferases, and modifying some natural products to produce sugar substitute products such as sweeteners. Along with the global high incidence of diabetes and the like, various sugar substitute products are also emerging on the market, wherein steviol glycoside compounds are distinguished on the market due to the high-power sweetness of the steviol glycoside compounds, the steviol glycoside compounds are various, and the most current application comprises: stevioside, rebaudioside a, rebaudioside D, rebaudioside M, rebaudioside E, rebaudioside C, wherein the sweetness of rebaudioside D and rebaudioside E is about 200-250 times that of sucrose. The sweetener which can produce different tastes by compounding different rebaudioside compounds on the market is added into foods and beverages, and secondly, the production cost of a plurality of stevioside compounds is high in consideration of the production cost, so that the compounding of different stevioside compounds is a necessary way for reducing the cost.

At present, the method for producing stevioside compounds is diversified, mainly comprises the steps of producing glycosyltransferase by using escherichia coli and a yeast expression system to synthesize stevioside in an in-vitro reaction system, and then catalyzing and synthesizing stevioside compounds by using escherichia coli or yeast cells. The third method is to modify stevioside compound, and cyclodextrin glucosyl oxidase is commonly used.

However, the yield and productivity of stevioside compounds produced by in vitro reactions of glycosyl transfer expressed by E.coli are relatively high by several existing strategies, but E.coli has potential safety hazards as a food production strain and the endotoxin removal cost of the produced E.coli is high. Second, the modification of steviol glycosides is often not specific to the glycosylation product and is inefficient.

The bacillus is widely distributed in soil environment, the variety of strains provides abundant strain resources for the industrial fields of foods, enzyme preparations, biological medicines and the like, most bacillus grows fast, enzyme is easy to secrete out of cells and be separated, has low requirements on nutrition, does not generate toxin, and is approved as a food safety strain by the United states food and drug administration.

Therefore, the bacillus is used as chassis cell to produce stevioside derivative.

Disclosure of Invention

The invention solves the technical problem of providing a glycosyltransferase production strain and application thereof.

The invention firstly provides a glycosyltransferase producing strain which is classified and named as bacillus subtilis SH-12 (Bacillus sp.) and is preserved in China general microbiological culture collection center CGMCC (China general microbiological culture collection center) at 1 month and 9 days in 2020, wherein the preservation address is North Chen Xiyu No.1 (China academy of sciences microbiological culture Collection center) in the Korean region of Beijing city, and the preservation number is CGMCC No. 19322. The glycosylase producing strain is obtained by performing primary screening on the characteristics of a plate colony in silt soil at the downstream of a hot spring of a Trojan horse in Yunnan, performing shake flask culture one by adopting primary fermentation, and comparing the enzyme activities of glycosyltransferases through enzyme activity measurement. The strain is used as an initial strain, glycosyltransferase is produced through seed culture and liquid submerged fermentation, and the crude enzyme liquid obtained by fermentation is used as a catalyst to produce the rebaudioside D and the rebaudioside E.

The invention thus provides a process for preparing glycosyltransferases, which is characterized by the use of the said Bacillus subtilis preparation. In a specific embodiment, fermentation liquor is obtained through fermentation, and supernatant fluid is obtained through centrifugation. Preferably, the more purified glycosyltransferase is further obtained by purification.

Furthermore, the present invention provides a method for producing rebaudioside E, comprising reacting stevioside and a rebaudioside a mixture (e.g. produced by extraction of stevia rebaudiana leaves) as substrates, and UDP, sucrose synthase, sucrose, and glycosyltransferase derived from the strain of the present invention to form a reaction system, thereby producing rebaudioside E; or a mixture of stevioside and rebaudioside A is used as a substrate, and UDPG and glycosyltransferase derived from the strain form a reaction system to react to generate rebaudioside E.

Preferably, the reaction pH is from 6 to 10, more preferably from 7 to 9.

Preferably, the reaction temperature is 25-45 ℃, more preferably 30-40 ℃.

Preferably, UDP is used in an amount of 1mM-10mM, preferably 2mM-8mM, more preferably 3mM-6mM.

Preferably, the optimal substrate concentration (mg/ml) and enzyme activity (U/ml) ratio for preparing rebaudioside E by catalyzing stevioside glycosylation is 10 according to the result and combining energy consumption and other factors: 1.

preferably, the reaction time is from 5 to 20 hours, more preferably from 10 to 15 hours.

The present invention also provides a process for preparing a mixture of rebaudioside D and rebaudioside E, characterized in that the reaction time is prolonged to produce rebaudioside E and rebaudioside D on the basis of the above-mentioned process for preparing rebaudioside E, the reaction time is 25-50 hours, preferably 30-40 hours.

Preferably, the reaction pH is from 6 to 10, more preferably from 9 to 10.

Preferably, the reaction temperature is 25-45 ℃, more preferably 30-40 ℃.

According to the result and combining factors such as energy loss, the optimal substrate concentration (mg/ml) and enzyme activity (U/ml) ratio of catalyzing stevioside glycosylation to prepare rebaudioside D are 2:1 to 30:1, preferably 2:1 to 10:1, while combining the yield of rebaudioside E, preferably substrate concentration with enzyme activity 5: the yields of both 1-rebaudioside D and rebaudioside E were higher.

After the reaction was completed for a certain period of time, methanol was added to terminate the reaction.

In one embodiment, the reaction system employs Tris-HCl buffer.

According to the invention, bacillus subtilis SH-12 capable of expressing glycosyltransferase is screened, the mixture of stevioside and rebaudioside A can be converted into the mixture of rebaudioside D or the mixture of rebaudioside E and rebaudioside D by using the supernatant produced by fermenting the bacillus subtilis, and the glycosyltransferase produced by using the bacillus subtilis as an expression system is used for synthesizing stevioside compounds, so that the food safety is higher.

The glycosyltransferase produced by the bacillus subtilis has high-efficiency and specific transglycosylation activity, and can specifically identify the site of the C19 site and carry out 1-2 glycosidic bond transglycosylation reaction on the site. Converting stevioside to Cheng Laibao di-glycoside E; the conversion of rebaudioside A to rebaudioside D. And the bacillus expression system is extracellular expression, and the extracellular expression is simple to operate in the post-processing treatment process, so that the cost is saved.

The glycosyl conversion system constructed by experiments of the invention obtains the wild bacillus for producing glycosyltransferase, has a substrate conversion rate of 40-80%, also has strains capable of producing rebaudioside E and rebaudioside D, and has good application prospect.

In addition, the mixture of the rebaudioside E and the rebaudioside D and the stevioside and the rebaudioside A which are not completely converted by the reaction system form a primary sweetener with a compound effect, and four different stevioside compounds are subjected to subsequent purification together, so that the purification cost can be saved compared with the independent purification, and the cost is saved for further market application.

Drawings

Fig. 1: bacillus subtilis SH-12.

Fig. 2: HPLC detection diagrams of mixtures of rebaudioside E and rebaudioside D were generated.

Biological material preservation information:

the bacterial strain SH-12 is classified and named as bacillus subtilis @Bacillu subtilis) The strain is preserved in China general microbiological culture collection center (CGMCC) in 1 and 9 days of 2020, wherein the preservation unit address is Beijing Chaoyang area North Xiylu No.1 (China academy of sciences microbiological institute), and the preservation number is CGMCC No. 19322.

Detailed Description

The methods in the following examples are conventional methods unless otherwise specified.

Enrichment medium: peptone 10g/L, yeast extract 3g/L, naCl 10g/L, pH7.2.

Screening the culture medium: 10g/L peptone, 3g/L yeast extract, 10g/L NaCl, 20g/L agar, pH7.2.

Fermentation medium: 10g/L peptone, 5g/L yeast extract, 5g/L NaCl, 20g/L glucose, pH7.2.

EXAMPLE 1 screening of wild type Bacillus

The inventor collects environmental samples for genomics analysis and strain screening in Yunnan round Ma Requan, inner Mongolia environmental soil, tianjin great harbor sea area and the like.

Enrichment and screening of strains: weighing 5g of an environmental sample, adding 50ml of sterile water, and culturing for 2 hours at 220rpm in an oscillating way; 10ml of supernatant is taken after standing for 30min and inoculated into a liquid screening culture medium filled with 90ml, the culture is carried out for 2d in a shaking table with constant temperature of 37 ℃, bacterial suspension with proper dilution is taken to be coated and separated on the screening culture medium, and the culture is carried out for 24h at 37 ℃. The single clone cultured by the flat plate is subjected to high-flux screening to select about 600 strains of bacteria for further glycosyltransferase screening.

EXAMPLE 2 production of glycosyltransferase by fermentation with wild type Bacillus

(1) Fermentation culture

The wild bacillus preserved from-80 ℃ glycerol pipe is inoculated on a solid LB plate by a three-area lineation method, cultured in a 37 ℃ incubator for 12h hours, inoculated on an activated single colony on the LB solid plate to LB liquid culture medium for 12 hours, inoculated in 20mL fermentation culture medium at 37 ℃ and 220rpm culture medium for shaking culture for 24 hours according to an inoculum size, the fermentation broth is centrifugally taken out, the fermentation supernatant is added into an enzyme reaction system for screening glycosyltransferase, 50% of each mixture of stevioside and rebaudioside A is used as a substrate, and a control group is arranged for terminating the reaction in advance. Strains producing glycosyltransferases are selected by the methods described above.

The enzyme activity determination method comprises the following steps: in a 1ml reaction system, the mixture of substrate stevioside and rebaudioside A is 0.5mM,UDPG 1.5mM,0.05M Tris-HCl buffer (pH 7.0), and glycosyltransferase crude enzyme solution (supernatant obtained by bacillus fermentation) is 500ul, and the mixture is subjected to shaking reaction at 35 ℃ for 12h. After the reaction, boiling, centrifuging to remove protein, treating with twice the volume of methanol, centrifuging, collecting filtrate by filtration, and performing HPLC analysis. The amount of stevioside consumption of 50uM substrate per ml of enzyme solution per hour was defined as 1U enzyme activity. The liquid phase detection method comprises the following steps: chromatographic conditions: chromatographic column: waters C18,4.6 mm. Times.250 mm,5 μm) with water as the mobile phase: acetonitrile (ph 7.0) =30:70, flow rate 1mL/min, column temperature 40 ℃. The detection is carried out by an ultraviolet detector, the wavelength is 210nm, the sample injection amount is 10 mu L, and the analysis time is 25min.

The bacillus subtilis capable of glycosylating stevioside and rebaudioside A is obtained by screening 600 strains of bacteria, and is subjected to biological material preservation, the preservation number is CGMCC NO.19322, and the screening result is shown in figure 1.

(2) 16S rDNA sequence homology analysis

Extracting genome DNA of the bacterium and taking the genome DNA as a template, and adopting a primer F: 5'-AGAGTTTGATCCTGGCTCAG-3' and primer R: and 5'-GGTTACCTTGTTACGACTT-3', performing PCR amplification to obtain a PCR amplification product. The PCR amplification product was sequenced.

Sequencing results show that the PCR amplification product contains SEQ ID NO:1, and a DNA molecule shown in the specification.

Setting SEQ ID NO:1, and performing a BLAST alignment on Genbank. The comparison result shows that the homology between the strain obtained by screening and bacillus subtilis (Bacillus subtilis) is highest. Thus, the bacterium was identified as bacillus subtilis (Bacillus subtilis). The bacillus subtilis colony is round or elliptical, has smooth edges, can secrete viscous liquid after being cultured for a period of time, and can drip down.

Example 3: glycosyltransferase catalyzed production of rebaudioside D and rebaudioside E mixtures

In 1ml of the reaction system, substrate stevioside and rebaudioside A were each subjected to shaking reaction at 35℃for 30 hours in 0.5mM,UDPG 1.5mM,0.05M Tris-HCl buffer (pH 7.0) with 3U of crude glycosyltransferase (supernatant obtained by fermentation of Bacillus in example 2). After the reaction, boiling, centrifuging to remove protein, treating with twice the volume of methanol, centrifuging, collecting filtrate by filtration, and performing HPLC analysis. The amount of stevioside consumption of 50uM substrate per ml of enzyme solution per hour was defined as 1U enzyme activity. The detection conditions are described in example 2. Under the conditions of the HPLC analysis, the retention time of stevioside was 13.2min, the retention time of rebaudioside A was 12.2, the retention time of rebaudioside E was 11.8min, and the retention time of rebaudioside D was 7.6min, and the results are shown in FIG. 2.

Example 4: catalytic glycosylation to rebaudioside E using UDPG regenerative circulatory system

A UDPG regeneration circulation system consisting of sucrose, sucrose synthase and UDP is adopted for preparing the rebaudioside E. The optimization conditions are as follows:

(1) Optimal reaction pH

The optimum reaction pH (5 ml) was investigated: the substrate stevioside and rebaudioside a mixtures were 10mm each, udp 10mm, sucrose synthase (source:Arabidopsis thaliana) Pure enzyme solution 500ul, sucrose 0.1M, crude glycosyltransferase (supernatant obtained by fermentation of Bacillus in example 2) 3U, and shaking reaction at 35℃for 12h. After the reaction, boiling, centrifuging to remove protein, treating with twice the volume of methanol, centrifuging, filtering, and collecting filtrate for HPLC analysis, wherein the analysis method is shown in example 2. Under the above HPLC analysis conditions, the yield of the glycosyltransferase-catalyzed synthesis of rebaudioside E was calculated according to the area normalization method, and the results are shown in Table 1.

According to the results, the optimal pH for preparing rebaudioside E by bacillus glycosylation of this glycosyltransferase is 7.0.

TABLE 1 yields of rebaudioside E at different pH conditions

Reaction pH	5.0	6.0	7.0	8.0	9.0	10.0
							Yield rate	4.6%	25.7%	33.2%	30.3%	28.3%	16.9%

(2) Optimum reaction temperature

The optimum reaction pH (5 ml) was investigated: the substrate stevioside and rebaudioside A mixtures were 10mM each, 10mM UDP, 500ul of sucrose synthase pure enzyme solution, 0.1M sucrose, 3U of glycosyltransferase crude enzyme solution, and shaken at 35℃for 12h. After the reaction, boiling, centrifuging to remove protein, treating with twice the volume of methanol, centrifuging, filtering, and collecting filtrate for HPLC analysis, wherein the analysis method is shown in example 2. The rebaudioside E yields at different pH conditions are shown in table 2. According to the results, the optimal reaction temperature for preparing rebaudioside E by bacillus glycosylation of this glycosyltransferase is 35 ℃.

TABLE 2 rebaudioside E yields at different reaction temperatures

Reaction temperature	15℃	25℃	30℃	35℃	40℃	45℃
							Yield rate	10.4%	26.7%	30.9%	32.1%	30.6%	29.7%

(3) Optimal reaction time

The optimal reaction time (5 ml) was examined: the substrates stevioside and rebaudioside A were 10mM each, UDP 10mM, sucrose synthase pure enzyme solution 500ul, sucrose 0.1M, glycosyltransferase crude enzyme solution 3U, shake reacted at 35℃5-15 h. After the reaction, boiling, centrifuging to remove protein, treating with twice the volume of methanol, centrifuging, filtering, and collecting filtrate for HPLC analysis, wherein the analysis method is shown in example 3. The rebaudioside D yields at different reaction times are shown in table 3. According to the results, the optimal reaction time for preparing rebaudioside E by bacillus glycosylation of this glycosyltransferase is 12h.

TABLE 3 rebaudioside E yields at different reaction times

Reaction time h	5 h	7h	10h	12h	15h
						Rebaudioside D yield	10.3%	14.5%	26.2%	32.4%	32.3%

(4) Optimum UDP usage

The optimum UDP usage (5 ml) was investigated: the substrate stevioside and rebaudioside A mixture 10mM, UDP 1-10mM, pH7.0, glycosyltransferase crude enzyme solution 3U, shake reaction at 35 deg.C for 12 hr respectively. After the reaction, the protein was removed by heating, and twice the volume of methanol was added for treatment, and the filtrate obtained by centrifugal filtration was analyzed by HPLC, and the analysis method was as in example 2. The rebaudioside E yields for different UDP amounts are shown in table 4. According to the results, the best UDP usage for preparing rebaudioside E by the bacillus-fermented glycosyltransferase-catalyzed stevioside glycosylation is 5mM, and the best molar concentration ratio of the bacillus-fermented glycosyltransferase to the substrate is 1:2.

TABLE 4 rebaudioside E yields with different UDP usage

UDP concentration	Substrate concentration: UDP (user Datagram protocol)	Yield of rebaudioside E
			1mM	1：0.1	32.8%
2 mM	1：0.2	35.9%
			3 mM	1：0.3	38.7%
4 mM	1：0.4	40.6%
			5 mM	1：0.5	44.8%
6 mM	1：0.6	39.7%
			8mM	1：0.8	35.6%
10mM	1：1	32.1%

(5) Optimal substrate concentration and enzyme dosage

Optimum substrate concentration (5 ml) was investigated: the substrate stevioside and rebaudioside A are 1mg/ml-25mg/ml, UDP is 5 times of the molar concentration of the substrate, the pH value is 7.0,3U glycosyltransferase crude enzyme solution, and the substrate stevioside and rebaudioside A are respectively subjected to shaking reaction at 35 ℃ for 12 hours. After the reaction, heating to remove protein, adding 2 times of methanol for treatment, centrifuging and filtering to obtain filtrate, and performing HPLC analysis, wherein the analysis method is shown in example 2. The rebaudioside E yields for the different steviol concentration conditions are shown in table 5. According to the result and combining factors such as energy loss, the optimal substrate concentration (mg/ml) and enzyme activity (U/ml) ratio of catalyzing stevioside glycosylation to prepare the rebaudioside E are 10:1.

TABLE 5 yield of rebaudioside E at different substrate concentrations

Stevioside and rebaudioside a concentration	Substrate concentration, enzyme Activity	Yield of rebaudioside E
			1.2 mg/ml	2:1	81.2%
3 mg/ml	5:1	78.7%
			6 mg/ml	10:1	49.3%
12 mg/ml	20:1	33.8%
			18 mg/ml	30:1	20.5%
24 mg/ml	40:1	8.7%

Example 6: catalytic glycosylation to produce rebaudioside D and rebaudioside E mixtures using UDPG regeneration circulatory system

According to the results of embodiments 3 and 4, in the presence of both stevioside and rebaudioside a, the extended reaction time produces rebaudioside D along with rebaudioside E. A UDPG regeneration cycle system consisting of sucrose, sucrose synthase and UDP was used to prepare the rebaudioside E and rebaudioside D mixtures. The optimization conditions are as follows:

(1) Optimal reaction pH

The optimum reaction pH (5 ml) was investigated: the substrates stevioside and rebaudioside a were 10mm each, udp 10mm, sucrose synthase (source:Arabidopsis thaliana) 500ul of pure enzyme solution, 0.1M of sucrose, 3U of glycosyltransferase crude enzyme solution, and the reaction pH is 5-10, and the reaction is carried out by shaking at 35 ℃ for 24 hours. After the reaction, boiling, centrifuging to remove protein, treating with twice the volume of methanol, centrifuging, filtering, and collecting filtrate for HPLC analysis, wherein the analysis method is shown in example 2. The rebaudioside E yields at different pH conditions are shown in table 6. According to the results, the optimal pH for the preparation of rebaudioside D and E mixtures by bacillus glycosylation of this glycosyltransferase is 9.0.

TABLE 6 yield of rebaudioside D at different pH conditions

Reaction pH	5.0	6.0	7.0	8.0	9.0	10.0
							Rebaudioside D yield	1.8%	6.7%	10.2%	14.9%	18.6%	17.8%

(2) Optimum reaction temperature

The optimum reaction temperature (5 ml) was examined: the substrates stevioside and rebaudioside A are 10mM, UDP is 10mM, sucrose synthase pure enzyme solution is 500ul, sucrose is 0.1M, glycosyltransferase crude enzyme solution is 3U, pH value is 8.0, and shaking reaction is 24h at 15-45 ℃. After the reaction, boiling, centrifuging to remove protein, treating with twice the volume of methanol, centrifuging, filtering, and collecting filtrate for HPLC analysis, wherein the analysis method is shown in example 2. The rebaudioside D yields at different temperature conditions are shown in table 7. According to the results, the optimal reaction temperature for preparing rebaudioside E by bacillus glycosylation of this glycosyltransferase is 35 ℃.

TABLE 7 rebaudioside D yields at different reaction temperatures

Reaction temperature (DEG C)	15	25	30	35	40	45
							Rebaudioside D yield	4.3%	8.9%	13.2%	18.1%	15.4%	10.9%

(3) Optimal reaction time

The optimal reaction time (5 ml) was examined: the substrates stevioside and rebaudioside A are 10mM, UDP 10mM, sucrose synthase pure enzyme solution 500ul, sucrose 0.1M, glycosyltransferase crude enzyme solution 3U, pH value 8.0, shake reaction 15-40 h at 35 ℃. After the reaction, boiling, centrifuging to remove protein, treating with twice the volume of methanol, centrifuging, filtering, and collecting filtrate for HPLC analysis, wherein the analysis method is shown in example 2. The rebaudioside D yields at different reaction times are shown in table 8. According to the results, the optimal reaction time for the preparation of rebaudioside E by bacillus glycosylation of this glycosyltransferase is 30h.

TABLE 8 yield of rebaudioside D at different reaction times

Reaction time h	15h	20h	25h	30h	40h
						Rebaudioside D yield	7.3%	14.5%	17.2%	19.4%	19.1%

(4) Optimum UDP usage

The optimal UDP-glucose dose (5 ml) was investigated: the substrate stevioside and rebaudioside A mixture 10mM, UDP 1-10mM, pH 8.0, glycosyltransferase crude enzyme solution 3U, shake reaction at 35 deg.C for 30 hr respectively. After the reaction, the protein was removed by heating, and twice the volume of methanol was added for treatment, and the filtrate obtained by centrifugal filtration was analyzed by HPLC, and the analysis method was as in example 2. The rebaudioside D yields for different UDP amounts are shown in table 9. According to the results, the best UDP amount used in the preparation of rebaudioside D by the Bacillus-fermented glycosyltransferase-catalyzed stevioside glycosylation was 5mM, and the best molar concentration ratio of the UDP to the substrate was 1:2.

TABLE 9 rebaudioside D and rebaudioside E yields with different UDP usage

UDP concentration	Substrate concentration: UDP (user Datagram protocol)	Yield of rebaudioside D	Rebaudioside E yield
				1mM	1：0.1	15.1%	33.8%
2 mM	1：0.2	16.6%	37.9%
				3 mM	1：0.3	18.2%	38.9%
4 mM	1：0.4	25.1%	41.6%
				5 mM	1：0.5	35.3%	45.9%
6 mM	1：0.6	22.7%	40.7%
				8mM	1：0.8	19.6%	36.5%

(5) Optimal substrate concentration and enzyme dosage

Optimum substrate concentration (5 ml) was investigated: the substrate stevioside and rebaudioside A are 1mg/ml-25mg/ml, UDP is 5 times of the molar concentration of the substrate, the pH value is 9.0,3U crude enzyme solution, the reaction pH value is 8.0, and the reaction is respectively carried out by shaking at 35 ℃ for 30 hours. After the reaction, heating to remove protein, adding 2 times of methanol for treatment, centrifuging and filtering to obtain filtrate, and performing HPLC analysis, wherein the analysis method is shown in example 2. The rebaudioside D yields for the different steviol concentration conditions are shown in table 10.

TABLE 10 yields of rebaudioside D and rebaudioside E at different substrate concentrations

Stevioside and rebaudioside a concentration	Substrate concentration, enzyme Activity	Yield of rebaudioside D	Rebaudioside E yield
				1.2 mg/ml	2:1	45.2%	80.2%
3 mg/ml	5:1	39.7%	78.9%
				6 mg/ml	10:1	39.3%	50.3%
12 mg/ml	20:1	36.8%	34.6%
				18 mg/ml	30:1	17.1%	21.5%
24 mg/ml	40:1	4.7%	8.9%

Based on the results and in combination with factors such as energy loss, the optimal substrate concentration (mg/ml) and enzyme activity (U/ml) ratio for preparing rebaudioside D by catalyzing stevioside glycosylation is 20:1, and the yield of the comprehensive rebaudioside E is preferably 5: the yields of both 1-rebaudioside D and rebaudioside E were higher.

Claims

1. A method for preparing glycosyltransferase, which is characterized in that bacillus subtilis with the preservation number of CGMCC No.19322 is utilized for preparation.

2. The method according to claim 1, wherein the fermentation broth is obtained by fermenting the bacillus subtilis, and the supernatant is obtained by centrifugation; preferably, the more purified glycosyltransferase is further obtained by purification.

3. A method for preparing rebaudioside E, wherein a mixture of stevioside and rebaudioside a is used as a substrate, and UDP, sucrose synthase, sucrose and glycosyltransferase according to claim 1 or 2 form a reaction system, and the reaction system is used to produce rebaudioside E; or a mixture of stevioside and rebaudioside A is used as a substrate, and UDPG and glycosyltransferase as set forth in claim 1 or 2 form a reaction system to react to produce rebaudioside E;

preferably, the reaction pH is from 5 to 10, more preferably from 7 to 9;

preferably, the reaction temperature is 15-45 ℃, more preferably 30-40 ℃;

preferably, UDP is used in an amount of 1mM-10mM, preferably 2mM-8mM, more preferably 3mM-6mM;

preferably, the ratio of substrate concentration (mg/ml) to enzyme activity (U/ml) is 5:1;

4. A process for preparing a mixture of rebaudioside D and rebaudioside E by extending the reaction time based on the reaction system in the process of claim 3 to produce a mixture of rebaudioside E and rebaudioside D, preferably for 15-50 hours, more preferably 30-40 hours;

preferably, the reaction pH is from 6 to 10, more preferably from 9 to 10;

preferably, the reaction temperature is 25-45 ℃, more preferably 30-40 ℃;

the ratio of substrate concentration (mg/ml) to enzyme activity (U/ml) was 2:1 to 30:1, preferably 2:1 to 10:1, and the yield of the comprehensive rebaudioside E is preferably 5:1.

5. the method of claim 3 or 4, wherein the reaction is terminated by adding methanol after the reaction is completed for a certain period of time; the reaction system adopts Tris-HCl buffer solution.

6. The method of claim 3 or 4, wherein the stevioside and rebaudioside a mixture is produced by stevia rebaudiana leaf extraction.

7. A process for preparing a sweetener, wherein the sweetener is a mixture of the compounds of claim 5.

8. The method of making according to claim 7, further purifying to obtain a mixture of rebaudioside E, rebaudioside D and a mixture of not fully converted stevioside and rebaudioside a.

9. A sweetener made by the method of claim 7 or 8.