Patent Publication Number: US-2022220525-A1

Title: Enzymatic method for preparing rebaudioside c

Description:
FIELD OF INVENTION 
     The present invention relates to a method for preparing Rebaudioside C, in particular to a biological preparation method of Rebaudioside C. 
     BACKGROUND 
     Sweetening agents are a class of food additives that have wide applications in the production of food, beverages, and candies. They may be added in the food production process, or alternatively may be used through appropriate dilution as a substitute for sucrose in household baking. Sweetening agents include natural sweetening agents, for example, sucrose, high fructose corn syrup, honey, etc., and artificial sweetening agents, for example, aspartame, saccharine, etc. Steviosides are a class of natural sweetening agents extracted from the plant  Stevia rebaudiana , and are widely used in food products and beverages at present. The extract of  Stevia rebaudiana  contains a variety of steviosides comprising Rebaudioside. Naturally extracted steviosides vary widely in composition across different batches, and need subsequent purification. 
     In the conventional method of producing Rebaudioside C, Rebaudioside C is extracted from  Stevia rebaudiana  leaves. For example, as disclosed in U.S. Pat. No. 8,501,261, about 111 g of product with 87.6% purity can be obtained by extracting from 10 kg of  Stevia rebaudiana  leaves. Because the percentage of Rebaudioside C found in  Stevia rebaudiana  leaves is relatively lower (about 10% of the total dry weight), the production cost of Rebaudioside C is relatively higher than that of Rebaudioside A (about 60% of the total dry weight). Furthermore, because of the limited yield, the commercial application of Rebaudioside C is hindered. 
     SUMMARY 
     The technical problem to be solved by the present invention is to overcome the defects of the prior art. The present invention achieves so by providing a method for preparing Rebaudioside C using an enzymatic method. With such a method, Rebaudioside C product with high purity can be produced at a lower cost and a shorter production cycle. 
     The following technical solution is employed by the present invention to solve the technical problem described above. 
     A method for preparing Rebaudioside C using an enzymatic method. In the method, dulcoside C is used as a substrate; and in the presence of a glycosyl donor, Rebaudioside C is produced by means of a reaction under the catalysis of recombinant cells containing UDP-glycosyltransferase and/or UDP-glycosyltransferase prepared therefrom. 
     A method for preparing Rebaudioside C using an enzymatic method. In the method, rubusoside is used as a substrate; and in the presence of a glycosyl donor, Rebaudioside C is produced by means of a reaction under the catalysis of recombinant cells containing UDP-glycosyltransferase and/or UDP-glycosyltransferase prepared therefrom. 
     Preferably, the glycosyl donor comprises one or two of glucosyl donor and rhamnosyl donor, the glucosyl donor is UDP-glucose or a UDP-glucose regeneration system (2007,  FEBS Letters,  581, 2562-2566) consisting of sucrose, sucrose synthase and UDP, and the rhamnosyl donor is UDP-rhamnose. Herein, a UDP-glucose regeneration system consisting of sucrose, sucrose synthase and UDP is preferred. The price of UDP-glucose is higher. The cost can be greatly reduced by using the UDP-glucose regeneration system. 
     Preferably, the UDP-glucosyltransferase (i.e., uridine diphosphate glucosyltransferase, UGT for short, which has been known) comprises one or two of UGT-A from  Stevia rebaudiana  and UGT-B from  Oryza sativa.    
     Preferably, the UDP-glucosyltransferase is UGT-A from  Stevia rebaudiana , and the amino acid sequence of the UGT-A is at least 60% consistent with Sequence 2 as shown in the Sequence Listing. 
     More preferably, the amino acid sequence of the UGT-A is at least 70% consistent with Sequence 2 as shown in the Sequence Listing. 
     Further, the amino acid sequence of the UGT-A is at least 80% consistent with Sequence 2 as shown in the Sequence Listing. 
     Further, the amino acid sequence of the UGT-A is at least 90% consistent with Sequence 2 as shown in the Sequence Listing. 
     According to one specific aspect, the amino acid sequence of the UGT-A is fully consistent with Sequence 2 in the Sequence Listing. 
     Preferably, the UDP-glucosyltransferase comprises UGT-A from  Stevia rebaudiana  and UGT-B from  Oryza sativa ; the UDP-glycosyltransferase is added into the reaction system in two steps, the UGT-B is firstly added in the first step and the UGT-A is then added in the second step. 
     Preferably, the amino acid sequence of the UGT-A is at least 60% consistent with Sequence 2 as shown in the Sequence Listing; and/or the amino acid sequence of the UGT-B is at least 60% consistent with Sequence 4 as shown in the Sequence Listing. 
     More preferably, the amino acid sequence of the UGT-A is at least 70% consistent with Sequence 2 as shown in the Sequence Listing; and/or the amino acid sequence of the UGT-B is at least 70% consistent with Sequence 4 as shown in the Sequence Listing. 
     Further, the amino acid sequence of the UGT-A is at least 80% consistent with Sequence 2 as shown in the Sequence Listing; and/or the amino acid sequence of the UGT-B is at least 80% consistent with Sequence 4 as shown in the Sequence Listing. 
     Further, the amino acid sequence of the UGT-A is at least 90% consistent with Sequence 2 as shown in the Sequence Listing; and/or the amino acid sequence of the UGT-B is at least 90% consistent with Sequence 4 as shown in the Sequence Listing. 
     According to the present invention, the reaction may be carried out in an aqueous system with temperature of 4-50° C. and pH of 5.0-9.0. Preferably, the reaction is carried out in an aqueous system with temperature of 34-45° C. and pH of 7.5-8.5. 
     More preferably, the reaction is carried out in a phosphoric acid buffer solution. 
     More preferably, the reaction system comprises the recombinant cells containing UDP-glycosyltransferase and a cell-permeable agent. Further, the cell-permeable agent is toluene, and the volume specific concentration of toluene in the reaction system is 1-3%. 
     More preferably, all raw materials used for reaction are added to a reaction kettle to be uniformly mixed and then placed at a set temperature for reaction while stirring. After reaction is completed, the product Rebaudioside C can be obtained through purification. A specific purification method is a post-treatment including resin separation. According to this purification method, the product Rebaudioside C with purity up to 95% can be obtained. 
     Preferably, the recombinant cells are microorganism cells. More preferably, the microorganism is  Escherichia coli, Saccharomyces cerevisiae  or  Pichia pastoris.    
     According to one specific aspect of the present invention, in the first-step reaction, the substrate is rubusoside, the UDP-glycosyltransferase is UGT-B from  Oryza sativa , and the sequence of the amino acid of UGT-B from  Oryza sativa  is at least 80% consistent with Sequence 4. In the second-step reaction, the substrate is reaction solution containing the product dulcoside A in the first-step reaction, the UDP-glycosyltransferase is UGT-A from  Stevia rebaudiana , and the sequence of the amino acid of UGT-A from  Stevia rebaudiana  is at least 80% consistent with Sequence 2. 
     According to another aspect of the present invention, the substrate is dulcoside A, the UDP-glycosyltransferase is UGT-A from  Stevia rebaudiana , and the sequence of the amino acid of UGT-A from  Stevia rebaudiana  is at least 80% consistent with Sequence 2. 
     Compared with the prior art, the present invention has the following advantages by performing the foregoing technical solution: 
     The method for preparing Rebaudioside C using the enzymatic method provided by the present invention has important application values. Because the growth rate of microorganisms is much faster than that of plants, by adopting the method provided by the present invention, the production cost can be greatly reduced, the production cycle is shortened, and the competitiveness of the product is greatly improved. In addition, since the content of steviosides in plants is low and there are many steviosides with different structures, it is very difficult to extract pure products. Compared with the existing methods for extracting Rebaudioside C from  Stevia rebaudiana  leaves, by adopting the method using the enzymatic method as provided in the present invention, products with higher purity can be extracted. The products can thus be more economically applied in the food industry such as in beverages. Furthermore, the application scope of Rebaudioside C will be further expanded. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     For structural formulas of rubusoside, dulcoside A and Rebaudioside C, respectively refer to formulas I, II and III. 
     
       
         
         
             
             
         
       
     
     The present invention mainly provides two routes for synthesizing Rebaudioside C: 
     
       
         
         
             
             
         
       
     
     The UGT-A or UGT-B used in the present invention may exist in the form of lyophilized enzyme powder or in the recombinant cells. 
     The method for obtaining UGT-A or UGT-B is as follows: 
     A recombinant  Escherichia coli  (or its microorganism) expression strain of UGT-A or UGT-B is obtained by utilizing a molecular cloning technique and genetic engineering technique; then the recombinant  Escherichia coli  is fermented to obtain recombinant cells containing UGT-A or UGT-B, or lyophilized powder of UGT-A or UGT-B is prepared by the recombinant cells. 
     The molecular cloning technique and genetic engineering technique described herein are known ones, unless otherwise specified. For the molecular cloning technique, refer to  Molecular Cloning: A Laboratory Manual  (3rd Edition) (by J. Sambrook, 2005) 
     The expression steps of the recombinant strain herein constructed by employing a genetic engineering technique are as follows: 
     (1) (According to Sequences 1 and 2 as shown in the Sequence Listing, or according to Sequences 3 and 4), needed gene fragments are genetically synthesized, linked into a vector pUC57, and added with restriction enzyme digestion sites NdeI and BamHI at both ends. 
     (2) Each gene fragment is inserted into the corresponding restriction enzyme digestion site of an expression vector pET30a through double enzyme digestion and linking such that each gene is placed under the control of a promoter T7; 
     (3) Recombinant plasmids are transformed into  Escherichia coli  BL21 (DE3) and the expression of the target protein is induced by using IPTG to obtain a recombinant  Escherichia coli  expression strain of UGT-A or UGT-B. 
     Recombinant cells containing UGT-A or UGT-B, or lyophilized powder of UGT-A or UGT-B is prepared by utilizing the recombinant  Escherichia coli  expression strain containing UGT-A or UGT-B. 
     The recombinant  Escherichia coli  expression strain containing UGT-A or UGT-B is inoculated into 4 ml of liquid LB culture medium according to a ratio of 1%, shaking culture is carried out at 37° C. (200 rpm) for one night, the culture stood for one night is transferred into 50 ml of liquid LB culture medium according to an inoculation amount of 1%, shaking culture is carried out at 37° C. (200 rpm) till the OD600 value reaches 0.6-0.8, MIPTG with final concentration of 0.4 mM is added, and shaking culture is carried out at 20° C. for one night. After induction, cells are collected by means of centrifugation (8,000 rpm, 10 min), the cells are resuspended with 5 ml of 2 mmol/L phosphate buffer solution (pH 7.0) to obtain the recombinant cells, the cells are further ultrasonically disrupted in an ice bath, the disruption liquid is centrifuged (8,000 rpm, 10 min), and the supernatant is collected and lyophilized for 24 h to obtain the lyophilized powder. 
     The present invention will be described below in more detail in connection with specific examples. 
     EXAMPLE 1 
     Preparation of Recombinant  Saccharomyces cerevisiae  Cells Containing UGT-A 
     According to Sequences 1 and 2 as shown in the Sequence Listing, gene fragments containing UGT-A were genetically synthesized, added with restriction enzyme digestion sites NdeI and BamHI at both ends, and linked into a vector pUC57 (produced by SUZHOU GENEWIZ BIOTECHNOLOGY CO., LTD.). The UGT gene fragments were digested by restriction enzyme NdeI and BamHI, purified fragments were recovered, and T4 ligase was added to link the fragments to the corresponding restriction enzyme digestion sites pET30a, so as to transform it into a BL21 (DE3) strain. 
     The UGT strain was inoculated into 4 ml of liquid LB culture medium according to a ratio of 1%, shaking culture was carried out at 37° C. (200 rpm) for one night, the culture stood for one night was transferred into 50 ml of liquid LB culture medium according to an inoculation amount of 1%, shaking culture was carried out at 37° C. (200 rpm) till the OD 600  value reached 0.6-0.8, MIPTG with final concentration of 0.4 mM was added, and shaking culture was carried out at 20° C. for one night. After induction, cells were collected by means of centrifugation (8,000 rpm, 10 min), and the cells were resuspended with 5 ml of 2 mmol/L phosphate buffer solution (pH 7.0) to obtain the recombinant cells containing UGT-A for catalysis. 
     EXAMPLE 2 
     Preparation of Lyophilized Powder of UGT-A 
     The recombinant cells containing UGT-A prepared in example 1 were ultrasonically disrupted in an ice bath, the disruption liquid was centrifuged (8,000 rpm, 10 min), and the supernatant was collected and lyophilized for 24 h to obtain lyophilized powder of UGT-A. 
     EXAMPLE 3 
     Preparation of Recombinant  Saccharomyces cerevisiae  Cells Containing UGT-B 
     According to Sequences 3 and 4, gene fragments containing UGT-B were genetically synthesized, added with restriction enzyme digestion sites NdeI and BamHI at both ends, and linked into a vector pUC57 (produced by SUZHOU GENEWIZ BIOTECHNOLOGY CO., LTD.). The UGT gene fragments were digested by restriction enzyme NdeI and BamHI, purified fragments were recovered, and T4 ligase was added to link the fragments to the corresponding restriction enzyme digestion sites pET30a, so as to transform it into a BL21 (DE3) strain. 
     The UGT strain was inoculated into 4 ml of liquid LB culture medium according to a ratio of 1%, shaking culture was carried out at 37° C. (200 rpm) for one night, the culture stood for one night was transferred into 50 ml of liquid LB culture medium according to an inoculation amount of 1%, shaking culture was carried out at 37° C. (200 rpm) till the OD 600  value reached 0.6-0.8, MIPTG with final concentration of 0.4 mM was added, and shaking culture was carried out at 20° C. for one night. After induction, cells were collected by means of centrifugation (8,000 rpm, 10 min), and the cells were resuspended with 5 ml of 2 mmol/L phosphate buffer solution (pH 7.0) to obtain the recombinant cells containing UGT-B for catalysis. 
     EXAMPLE 4 
     Preparation of Lyophilized Powder of UGT-B 
     The recombinant cells containing UGT-B prepared in example 3 were ultrasonically disrupted in an ice bath, the disruption liquid was centrifuged (8,000 rpm, 10 min), and the supernatant was collected and lyophilized for 24 h to obtain lyophilized powder of UGT-B. 
     EXAMPLE 5 
     Synthesis of Rebaudioside C Under Catalysis of UDP-Glycosyltransferase by Using Dulcoside A as the Substrate (Route 1) 
     In this example, UGT-A lyophilized powder prepared according to the method in example 2 was used to catalyze the synthesis of Rebaudioside C. In this example, a UDP-glucose regeneration system consisting of sucrose, sucrose synthase from  Arabidopsis thaliana  (hereinafter referred to as AtSUS1) and UDP was used as a glucosyl donor. 
     In the reaction system, 1 L of 0.05 mol/L phosphate buffer solution (pH 8.0), 2 g of UDP and 8 g of dulcoside A, 50 g of sucrose, 10 g of UGT-A lyophilized powder and 3 g of AtSUS1 lyophilized powder were sequentially added and uniformly mixed, then the mixture was placed in a 40° C. water bath for 16 h, and stirring was carried out at 300 rpm for reaction. After the reaction, 500 μl of reaction solution was taken and uniformly mixed with anhydrous methanol with equal volume, centrifugation at 8,000 rpm was carried out for 10 min, the supernatant was enabled to pass through a filter membrane, and then detection was carried out by using high-performance liquid chromatography (chromatographic conditions: chromatographic column: Aglient eclipse SB-C18 4.6*150 mm; detection wavelength: 210 nm; mobile phase: 0.1% formic acid aqueous solution:acetonitrile=65%:35%; flow rate: 1.0 mL/min; column temperature: 30° C.). The conversion rate of dulcoside A was more than 90%. After the supernatant was purified by post-processing such as separation by silica gel resin and crystallization, 5.6 g of Rebaudioside C was obtained, and the purity was greater than 90%. 
     EXAMPLE 6 
     Synthesis of Rebaudioside C Under Catalysis of Recombinant Cells Containing UDP-Glycosyltransferase by Using Rubusoside as the Substrate (Route 2) 
     In this example, UGT-A lyophilized powder prepared according to the method in example 2 and UGT-B lyophilized powder prepared according to the method in example 4 were used to catalyze the synthesis of Rebaudioside C. 
     First-step reaction: 1 L of 0.05 mol/L phosphate buffer solution (pH 8.0), 4.5 g of UDP rhamnose, 6.5 g of rubusoside and 10 g of UGT-B lyophilized powder were sequentially added into the reaction system, uniformly mixed and then placed in a 40° C. water bath, and stirring was carried out at 300 rpm for reaction for 16 h. Second-step reaction: after the first-step reaction, the reaction solution was boiled for 10 min, the pH value was regulated to 8.0, 2 g of UDP, 50 g of sucrose, 10 g of UGT-A lyophilized powder and 3 g of AtSUS1 lyophilized powder were added, uniformly mixed and then placed in a 40° C. water bath, and stirring was carried out at 300 rpm for reaction for 16 h. After the reaction, 500 μl of reaction solution was taken and uniformly mixed with anhydrous methanol with equal volume, centrifugation at 8,000 rpm was carried out for 10 min, the supernatant was enabled to pass through a filter membrane, and then detection was carried out by using high-performance liquid chromatography (chromatographic conditions: chromatographic column: Aglient eclipse Cl8 4.6*150 mm; detection wavelength: 210 nm; mobile phase: 0.1% formic acid aqueous solution:acetonitrile=65%:35%; flow rate: 1.0 mL/min; column temperature: 30° C.). The conversion rate of rubusoside was more than 90%. After the supernatant was purified by post-processing such as separation by silica gel resin and crystallization, 5.2 g of Rebaudioside C was obtained, and the purity was greater than 90%. 
     EXAMPLE 7 
     Synthesis of Rebaudioside C Under Catalysis of UDP-Glycosyltransferase by Using Duleoside A as the Substrate 
     In this example, recombinant cells containing UGT-A prepared according to the method in example 1 were used to catalyze the synthesis of Rebaudioside C. 
     1 L of 0.05 mol/L phosphate buffer solution (pH 8.0), 2 g of UDP, 50 g of dulcoside A, 50 g of sucrose, 200 ml of toluene, 40 g of UGT-A whole cells and 12 g of AtSUS1 whole cells were sequentially added into the reaction system, uniformly mixed and then placed in a 40° C. water bath, and stirring was carried out at 300 rpm for reaction for 16 h. After the reaction, 500 μl of reaction solution was taken and centrifuged, the supernatant was added and uniformly mixed with anhydrous methanol with equal volume, centrifugation at 8,000 rpm was carried out for 10 min, the supernatant was enabled to pass through a filter membrane, and then detection was carried out by using high-performance liquid chromatography (chromatographic conditions: chromatographic column: Aglient eclipse SB-C18 4.6*150 mm; detection wavelength: 210 nm; mobile phase: 0.1% formic acid aqueous solution:acetonitrile=65%:35%; flow rate: 1.0 mL/min; column temperature: 30° C.). The conversion rate of dulcoside A was more than 90%. After the supernatant was purified by post-processing such as separation by silica gel resin and crystallization, 5.5 g of Rebaudioside C was obtained, and the purity was greater than 90%. 
     EXAMPLE 8 
     Synthesis of Rebaudioside C Under Catalysis of Recombinant Cells Containing UDP-Glycosyltransferase by Using Rubusoside as the Substrate 
     First-step reaction: 1 L of 0.05 mol/L phosphate buffer solution (pH 8.0), 4.5 g of UDP rhamnose, 6.5 g of rubusoside, 20 ml of toluene, and 40 g of UGT-A whole cells were sequentially added into the reaction system, uniformly mixed and then placed in a 40° C. water bath, and stirring was carried out at 300 rpm for reaction for 16 h. Second-step reaction: after the first-step reaction, the reaction solution was boiled for 10 min, the pH value was regulated to 8.0, 2 g of UDP, 50 g of sucrose, 40 g of UGT-A whole cells and 12 g of AtSUS1 whole cells were added, uniformly mixed and then placed in a 40° C. water bath, and stirring was carried out at 300 rpm for reaction for 16 h. After the reaction, 500 μl of reaction solution was taken and centrifuged, the supernatant was added and uniformly mixed with anhydrous methanol with equal volume, centrifugation at 8,000 rpm was carried out for 10 min, the supernatant was enabled to pass through a filter membrane, and then detection was carried out by using high-performance liquid chromatography (chromatographic conditions: chromatographic column: Aglient eclipse SB-C18 4.6*150 mm; detection wavelength: 210 nm; mobile phase: 0.1% formic acid aqueous solution:acetonitrile=65%:35%; flow rate: 1.0 mL/min; column temperature: 30° C.). The conversion rate of rubusoside was more than 90%. After the supernatant was purified by post-processing such as separation by silica gel resin and crystallization, 5.0 g of Rebaudioside C was obtained, and the purity was greater than 90%. 
     The above-described examples are merely for the illustration of the technical concept and features of the present invention; the object is only to allow those skilled in the art to understand the present invention and implement it accordingly, and the scope of the present invention is not limited thereto; any equivalent variations or modifications derived from the essence of the present invention shall fall within the protection scope of the present invention.