Sucrose phosphorylase-producing preristeria megatherium and application of preristeria megatherium

Abstract

The present invention provides a sucrose phosphorylase producing P.megatherium SLJTM1 strain and an application thereof, the P.megatherium SLJTM1 strain is separated from soil of a sugarcane field of Guangxi guest, the preservation number of the P.megatherium SLJTM1 strain is GDMCC NO: 65139, the preservation date is September 14, 2024, the preservation address is Microbial Culture Collection Center in China, Guangzhou and Guangdong Province, the preservation number is CGMCC NO: 65139, the preservation number is CGMCC NO: 65139, and the preservation number is CGMCC NO: 65139. The strain is preserved in Guangdong Microbial Culture Collection Center, the strain has relatively high enzyme yield activity and good sucrose phosphorylase production stability, the enzyme activity of the enzyme produced by the strain reaches 84.37 U, and the yield of 2-O-D-glucopyranosyl-L-ascorbic acid (AA-2G) prepared by conversion of the enzyme produced by the strain reaches 19.35 g / L.

Description

[Technical field]

[0001] The invention relates to the technical field of microorganisms, and in particular to Priesteria gigantea producing sucrose phosphorylase and application thereof. [Background technology]

[0002] Vitamin C, also known as L-ascorbic acid, is a common water-soluble vitamin widely distributed in fresh vegetables, fruits, and green plants. 2-O-α-D-pyranose glucopyranosyl-L-ascorbic acid (AA-2G), as a derivative of vitamin C, has the advantages of being non-reducing, not easily oxidized, and having a particularly stable aqueous solution. AA-2G can be hydrolyzed by α-glucosidase to generate vitamin C and glucose, so it can exert the efficacy of vitamin C; it has the effects of scavenging free radicals and promoting collagen synthesis. AA-2G is considered to be the best substitute for vitamin C and is widely used in food, medical, and cosmetics.

[0003] Research on AA-2G in my country is constantly advancing. At present, as a synthetic vitamin C derivative, enzymatic synthesis is the only way to synthesize AA-2G. The glucoside on the donor is transferred to C2 of vitamin C by glycosyltransferase. The enzymes reportedly used to synthesize AA-2G include α-glucosidase, α-amylase, sucrose phosphorylase, cyclodextrin glucosyltransferase and dextran sucrase. Some scholars have also explored its chemical synthesis method, but the results show that the synthesis process of this scheme is complicated and the cost is too high, which is far from industrial production. On the one hand, the low enzyme activity of the industrial enzyme itself leads to increased production costs; on the other hand, low substrate conversion rate and imperfect separation and purification methods are also another important reason hindering its industrial production.

[0004] The present invention utilizes the self-screened sucrose phosphorylase enzymatic synthesis, that is, the sucrose phosphorylase enzyme solution catalyzes sucrose and L-ascorbic acid to synthesize 2-OD-pyranosyl-L-ascorbic acid, and the reaction has the advantages of being green, efficient and mild. [Summary of the invention]

[0005] In view of this, the object of the present invention is to provide a Priesteria gigantea producing sucrose phosphorylase and application thereof.

[0006] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0007] A Priestia megaterium strain SLJTM1, whose preservation number is GDMCCNO: 65139, preservation date: September 14, 2024, preservation address: Guangdong Microbiological Culture Collection Center, Guangzhou, China, preservation unit: Guangdong Microbiological Culture Collection Center.

[0008] It is further explained that the Priestia megaterium strain SLJTM1 is isolated from the soil of a sugarcane field in Laibin, Guangxi.

[0009] The present application also provides a microbial preparation comprising the Priestia megaterium strain SLJTM1 as described above.

[0010] Further, the microbial preparation contains a viable count of not less than 1.0×10 8 cells / mL of Priesteria megaterium strain SLJTM1 cells.

[0011] The present application also provides a method for producing sucrose phosphorylase, and the specific method is: under sterile operation, a sterile inoculation loop is used to inoculate the Priesteria gigantea strain SLJTM1 into a high-temperature sterilized fermentation medium (pH = 6.0), and after constant temperature fermentation and cultivation at 37°C and 180 r / min in a shaking incubator for 24 hours, the fermentation liquid is inoculated into a new fermentation medium according to 10% of the inoculation amount, and the culture is carried out at a constant temperature of 37°C for 84 hours in a shaking incubator, and the supernatant is obtained by centrifugation at 10000 r / min and 15 min, which is the crude sucrose phosphorylase enzyme solution.

[0012] It is further described that the fermentation medium is: glucose 90g / L, KH2PO4 10g / L, Na2HPO4 15g / L, NH4Cl 10g / L, yeast powder 100g / L, MgSO4·7H2O 5g / L, CaCl2 2g / L, initial pH is 6.0, and wet heat sterilization is performed at 121°C;

[0013] The present application also provides a use of the Priesteria gigantea strain SLJTM1 in producing 2-O-α-D-glucopyranosyl-L-ascorbic acid products.

[0014] The present application also provides a method for preparing the above-mentioned 2-O-α-D-pyranosyl-L-ascorbic acid, the method comprising the following steps:

[0015] (1) inoculating the preserved Priesteria gigantea strain SLJTM1 on a slant culture medium, placing the culture in a 37°C constant temperature incubator for 2 days to obtain activated strains, eluting the strains with sterile water to obtain a suspension of Priesteria gigantea strain SLJTM1, inoculating the suspension of strains into a shake flask fermentation medium; culturing the culture in a shaker at 37°C and 180 r / min for 84 h, and centrifuging the culture in a centrifuge at 10,000 r / min for 15 min to obtain a supernatant, which is a crude sucrose phosphorylase enzyme solution;

[0016] The slant culture medium is PS culture medium; PS culture medium: 0.5g sucrose, 0.02g KH2PO4, 0.61g Na2HPO4, 0.1g NH4Cl, 0.01g yeast extract, 0.05g MgSO4·7H2O, 0.5mg CaCl2, 1g agar powder, 100mL distilled water, natural pH, 121°C wet heat sterilization;

[0017] The shake flask fermentation medium is: glucose 90g / L, KH2PO4 10g / L, Na2HPO4 15g / L, NH4Cl 10g / L, yeast powder 100g / L, MgSO4·7H2O 5g / L, CaCl2 2g / L, initial pH is 6.0, and wet heat sterilization is performed at 121°C;

[0018] (2) Take 10 g of sucrose and 7 g of ascorbic acid as substrates, add 100 mL of 50 mmol / L, pH 6.0 phosphate buffer, and use 30 mL of enzyme solution as a catalyst. React in a shaker at 42°C for 12 h to obtain 2-O-α-D-pyranosyl-L-ascorbic acid.

[0019] It is further described that the concentration of the suspension of Priesteria gigantea strain SLJTM1 is 1.0×10 6 Pieces / ml.

[0020] The present application also discloses the application of the Priesteria gigantea strain SLJTM1 in the fields of food, cosmetics or pharmaceuticals.

[0021] The phosphate buffer of the present invention can be purchased on the market and is produced by Hefei Bomei Biotechnology Co., Ltd.

[0022] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0023] The Priestia megaterium strain SLJTM1 of the present invention is a strain isolated and screened from the soil of a sugarcane field in Laibin, Guangxi. The study found that the strain is the Priestia megaterium strain SLJTM1 that produces sucrose phosphorylase. In order to study the enzyme production ability of the strain, a single factor study was conducted. The applicant determined through the study that the optimal conditions for the fermentation enzyme production of the Priestia megaterium strain SLJTM1 are: fermentation time 84h, inoculation amount 10%, fermentation temperature 37°C, glucose 90g / L, KH2PO4 10g / L, Na2HPO4 15g / L, NH4Cl 10g / L, yeast powder 100g / L, MgSO4·7H2O 5g / L, CaCl2 2g / L, the initial pH was 6.0; the enzyme activity of the bacteria reached 84.37U / mL, which showed that the bacteria had high enzyme activity and good stability; and the yield of 2-O-α-D-pyranoglucopyranosyl-L-ascorbic acid prepared by the bacteria's enzyme production conversion reached 19.35g / L.

Brief Description of the Drawings

[0024] Figure 1 This is a morphological picture of the Priesteria gigantea strain SLJTM1 on a plate according to an embodiment of the present application;

[0025] Figure 2 This is a graph showing the effect of different fermentation times on the enzyme production capacity of Priesteria gigantea SLJTM1;

[0026] Figure 3 This is a graph showing the effect of different carbon sources on the enzyme production capacity of Priesteria gigantea SLJTM1;

[0027] Figure 4 This is a graph showing the effect of different glucose concentrations on the enzyme production capacity of Priesteria gigantea SLJTM1;

[0028] Figure 5 This is a diagram showing the effect of different nitrogen sources on the enzyme production capacity of Priesteria gigantea SLJTM1;

[0029] Figure 6 This is a graph showing the effect of different yeast powder concentrations on the enzyme production capacity of Priesteria gigantea SLJTM1;

[0030] Figure 7 This is a graph showing the effect of different fermentation medium pH on the enzyme production capacity of Priesteria gigantea SLJTM1;

[0031] Figure 8 This is a graph showing the effect of different inoculation amounts of bacteria on the enzyme production capacity of Priesteria gigantea SLJTM1;

[0032] Fig. 9 This graph shows the effect of different fermentation temperatures on the enzyme production capacity of Priesteria gigantea SLJTM1. [Specific implementation method]

[0033] All features disclosed in this specification, or steps in all methods or processes disclosed, except mutually exclusive features and / or steps, can be combined in any manner.

[0034] Any feature disclosed in this specification (including any appended claims and abstract), unless otherwise stated, is merely an example of a series of equivalent or similar features.

[0035] 1. Screening of bacterial strains

[0036] Soil samples were taken from the sugarcane field in Laibin, Guangxi. PS medium was used to form a screening medium to screen strains producing sucrose phosphorylase. The process was as follows: Initial screening of strains: 1 g of soil was added to 9 mL of sterile saline to make 10 -1 The sample solution of concentration was diluted to 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 Concentration, set aside for use. Take the last three dilutions, separate the strains on PS medium plates by spreading method, separate by streaking on the plates, purify repeatedly, and obtain pure culture of isolated strains. Store the plates in a 4℃ refrigerator.

[0037] PS medium is PS agar slant solid medium;

[0038] The formula of PS medium is: 0.5g sucrose, 0.02g KH2PO4, 0.61g Na2HPO4, 0.1g NH4Cl, 0.01g yeast extract, 0.05g MgSO4·7H2O, 0.5mg CaCl2, 1g agar powder, 100mL distilled water, natural pH, and moist heat sterilization at 121℃.

[0039] 2. Identification of bacterial strains and optimization of fermentation conditions

[0040] 1. Identification of bacterial species

[0041] (1) Observation of colony morphology: Inoculate the slant bacteria into a plate with PS medium, incubate at 37°C for 48 hours, and observe the colony morphology. Single colonies are large, light yellow, round or oval, with rough and shiny edges, and can be transparent or opaque.

[0042] (2) Identification of strains: The purified strains were sent to Guangdong Microbial Culture Collection Center for identification. Nucleotide BLAST analysis showed that the 16S r DNA sequence homology with the Priestia megaterium strain was 100%, and it was preliminarily determined that the Priestia megaterium SLJTM1 strain was Priestia megaterium.

[0043] The preservation information of the screened Priestia megaterium strain SLJTM1 is as follows: the name is Priestia megaterium strain SLJTM1, its classification name is Priestia megaterium SLJTM1, the preservation number is GDMCC NO: 65139, the preservation date is September 14, 2024, the preservation address is: 5th Floor, Laboratory Building, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province, and the preservation unit is Guangdong Provincial Microbiological Culture Collection Center.

[0044] The morphological characteristics of Priestia megaterium SLJTM1 on a plate according to the present invention are as follows: Figure 1 As shown, the colonies of this strain are milky white in color, round in shape with ridges, and have a smooth surface texture.

[0045] 2. Study on optimization of enzyme production conditions of Priestia megaterium strain SLJTM1

[0046] (1) Effect of fermentation time on enzyme production

[0047] The initial fermentation medium was inoculated with Priestia megaterium SLJTM1, and the fermentation was carried out for 60, 72, 84, 96, and 108 hours at a temperature of 37°C, pH 6, a liquid volume of 100 mL / 250 mL, an inoculum of 10%, and a rotation speed of 180 r / min, and the supernatant was taken to measure the enzyme activity. The results are shown in Table 1 and Figure 2 .

[0048] The initial fermentation medium is: lactose 50g / L, KH2PO4 10g / L, Na2HPO4 15g / L, NH4Cl 10g / L, yeast powder 100g / L, MgSO4·7H2O 5g / L, CaCl2 2g / L.

[0049] Table 1 Effect of different fermentation times on the enzyme production capacity of Priesteria gigantea SLJTM1

[0050] project 60h 72h 84h 96h 108h Enzyme activity (U / mL) 20.12 36.48 54.15 48.76 37.52

[0051] (2) Optimization of carbon source and its concentration

[0052] Priestia megaterium SLJTM1 was inoculated based on the initial fermentation medium. The carbon source variables were sucrose, starch, lactose, glucose, and maltose. The liquid medium was prepared and placed on a shaker at 37°C, with a speed of 180 r / min, and fermented for 84 hours. The medium was centrifuged at 10000 r / min for 10 minutes. On this basis, the effect of glucose concentration (3, 5, 7, 9, 11%) on the enzyme production of the strain was further explored, and the supernatant was taken to measure the enzyme activity. The results are shown in Table 2 and Figure 3 , 4 .

[0053] Table 2 Effect of different carbon sources on the enzyme production capacity of Priesteria gigantea SLJTM1

[0054]

[0055] (3) Optimization of nitrogen source

[0056] Priestia megaterium SLJTM1 was inoculated on the basis of the initial fermentation medium, and beef extract, peptone, yeast powder, corn flour, and soybean powder were selected as the only organic nitrogen source in the fermentation medium. The liquid culture medium was prepared and placed on a shaker at 37°C, with a speed of 180r / min, for 84h of fermentation culture, and centrifuged at 10000r / min for 10min. On this basis, the effect of yeast powder concentration (4, 6, 8, 10, 12%) on the enzyme production of the strain was further explored, and the supernatant was taken to measure the enzyme activity. The results are shown in Table 3 and Figure 5 , Figure 6 .

[0057] Table 3 Effect of different nitrogen sources on the enzyme production capacity of Priesteria gigantea SLJTM1

[0058]

[0059] (4) Effect of pH on enzyme activity

[0060] The initial fermentation medium was inoculated with Priestia megaterium SLJTM1, and the initial pH of the fermentation medium was adjusted to 3, 4, 5, 6, and 7 by hydrochloric acid. The supernatant was taken to measure the enzyme activity. The liquid medium was prepared and placed on a shaker at 37°C and a speed of 180 r / min for 84 hours of fermentation. The fermentation liquid was taken out and centrifuged at 10000 rpm for 10 minutes, and the supernatant was taken to measure the enzyme activity. The results are shown in Table 4 and Figure 7 .

[0061] Table 4 Effect of different fermentation medium pH on enzyme production capacity of Priesteria gigantea SLJTM1

[0062]

[0063]

[0064] (5) Effect of inoculation amount on enzyme production

[0065] The initial fermentation medium was inoculated with Priestia megaterium SLJTM1 at 6%, 8%, 10%, 12%, and 14% of the inoculation volume. The liquid medium was prepared and placed on a shaker at 37°C, with a rotation speed of 180 r / min, and the fermentation culture was carried out for 84 hours. The fermentation liquid was taken out and centrifuged at 10000 rpm for 10 minutes, and the supernatant was taken to measure the enzyme activity. The results are shown in Table 5 and Figure 8 .

[0066] Table 5 Effect of different inoculation amounts of bacterial species on the enzyme production capacity of Priesteria gigantea SLJTM1

[0067] project 6％ 8％ 10％ 12％ 14％ Enzyme activity (U / mL) 41.47 56.62 68.75 51.34 40.33

[0068] (6) Effect of fermentation temperature on enzyme production

[0069] The initial fermentation medium was inoculated with Priestia megaterium SLJTM1, and fermented at 31, 33, 35, 37, and 40°C for 84 hours under the conditions of pH 6, liquid volume 100mL / 250mL, inoculation volume 10%, and rotation speed 180r / min, and the supernatant was taken to measure the enzyme activity. The results are shown in Table 6 and Fig. 9 .

[0070] Table 6 Effect of different fermentation temperatures on the enzyme production capacity of Priesteria gigantea SLJTM1

[0071] project 31℃ 33℃ 35℃ 37℃ 39℃ Enzyme activity (U / mL) 44.29 55.68 64.34 69.86 47.15

[0072] The enzyme activity data of the single-factor experimental optimization test in Tables 1 to 6 can determine that the optimal conditions for enzyme production by fermentation of Priestia megaterium strain SLJTM1 are: fermentation time 84h, inoculation amount 10%, fermentation temperature 37°C, glucose 90g / L, KH2PO4 10g / L, Na2HPO4 15g / L, NH4Cl 10g / L, yeast powder 100g / L, MgSO4·7H2O 5g / L, CaCl2 2g / L, and initial pH 6.0.

[0073] The above-obtained optimal conditions for fermentation and enzyme production of Priestia megaterium SLJTM1 were used to ferment and culture Priestia megaterium SLJTM1, and the fermentation broth was centrifuged at 10000 rpm for 10 min, and the supernatant was taken to measure the enzyme activity. After detection, the enzyme activity reached 98.4 U / mL.

[0074] 3. Application of Priestia megaterium strain SLJTM1: Production of 2-O-α-D-pyranosyl-L-ascorbic acid

[0075] The method for preparing AA-2G by transformation using Priestia megaterium strain SLJTM1 is as follows:

[0076] (1) Preparation of crude sucrose phosphorylase solution

[0077] Under aseptic operation, Priestia megaterium strain SLJTM1 was inoculated with a sterilized inoculation loop into a high-temperature sterilized fermentation medium (glucose 90 g / L, KH2PO4 10 g / L, Na2HPO4 15 g / L, NH4Cl 10 g / L, yeast powder 100 g / L, MgSO4·7H2O 5 g / L, CaCl2 2 g / L, initial pH 6.0). After constant temperature fermentation and cultivation at 37°C and 180 r / min shaking for 24 h, the fermentation broth was inoculated into a new fermentation medium (glucose 90 g / L, KH2PO4 10 g / L, Na2HPO4 15 g / L, NH4Cl 10 g / L, yeast powder 100 g / L, MgSO4·7H2O 5 g / L, CaCl2 2g / L, initial pH 6.0), incubate in a shaking incubator at 37°C and 180r / min for 24h, and then centrifuge at 10000r / min for 15min to obtain the supernatant, which is the crude sucrose phosphorylase solution.

[0078] (2) Catalytic preparation of 2-O-α-D-pyranosyl-L-ascorbic acid (AA-2G)

[0079] Take 10 g of sucrose and 7 g of ascorbic acid as substrates, add 100 mL of 50 mmol / L, pH 6.0 phosphate buffer, use 30 mL of enzyme solution as catalyst, react in a shaker at 42 ° C for 12 hours, and you can get 2-O-α-D-pyranosyl-L-ascorbic acid.

[0080] After measurement, the AA-2G yield reached 19.35g / L.

[0081] The formula of phosphate buffer is: produced by Hefei Bomei Biotechnology Co., Ltd.

[0082] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A Priestia megaterium strain SLJTM1, whose deposit number is GDMCCNO: 65139, deposit date: September 14, 2024, deposit address: Guangdong Microbiological Culture Collection Center, Guangzhou, China, deposit unit: Guangdong Microbiological Culture Collection Center.

2. The Priestia megaterium strain SLJTM1 according to claim 1, characterized in that The strain was isolated from the soil of a sugarcane field in Laibin, Guangxi.

3. A microbial preparation comprising the Priestia megaterium strain SLJTM1 as claimed in claim 1.

4. The microbial preparation according to claim 3, characterized in that The number of viable bacteria in the microbial preparation is not less than 1.0×10 8 / mL of Priesteria megaterium SLJTM1 cells.

5. A method for producing sucrose phosphorylase, characterized in that: The specific method is as follows: under aseptic operation, Priesteria gigantea SLJTM1 is inoculated into a high-temperature sterilized fermentation medium (pH = 6.0) using a sterilized inoculation loop, and cultured in a shaking incubator at 37°C and 180 r / min for 24 hours, then the fermentation liquid is inoculated into a new fermentation medium at a constant temperature of 37°C for 84 hours in a shaking incubator according to 10% of the inoculation amount, and centrifuged in a centrifuge at 10000 r / min for 15 minutes to obtain a supernatant, which is a crude sucrose phosphorylase enzyme solution.

6. A method for producing sucrose phosphorylase according to claim 5, characterized in that: The fermentation medium comprises: 90 g / L glucose, 10 g / L KH2PO4, 15 g / L Na2HPO4, 10 g / L NH4Cl, 100 g / L yeast powder, 5 g / L MgSO4·7H2O, and 2 g / L CaCl2, and the initial pH is 6.

0.

7. Use of Priesteria gigantea SLJTM1 as claimed in claim 1 in producing 2-O-α-D-glucopyranosyl-L-ascorbic acid products.

8. A method for preparing 2-O-α-D-glucopyranosyl-L-ascorbic acid as claimed in claim 7, characterized in that: The method comprises the following steps: (1) inoculating the preserved Priesteria gigantea SLJTM1 on a slant culture medium, placing the culture in a 37°C constant temperature incubator for 2 days to obtain activated strains, eluting the strains with sterile water to obtain a suspension of Priesteria gigantea SLJTM1, inoculating the strain suspension into a shake flask fermentation medium; culturing the culture in a shaker at 37°C and 180 r / min for 84 h, and centrifuging the culture in a centrifuge at 10,000 r / min for 15 min to obtain a supernatant, which is a crude sucrose phosphorylase enzyme solution; The slant culture medium is PS culture medium; PS culture medium: 0.5g sucrose, 0.02g KH2PO4, 0.61g Na2HPO4, 0.1g NH4Cl, 0.01g yeast extract, 0.05g MgSO4·7H2O, 0.5mg CaCl2, 1g agar powder, 100mL distilled water, natural pH, 121°C wet heat sterilization; The shake flask fermentation medium is: glucose 90g / L, KH2PO4 10g / L, Na2HPO4 15g / L, NH4Cl 10g / L, yeast powder 100g / L, MgSO4·7H2O 5g / L, CaCl2 2g / L, initial pH is 6.0, and wet heat sterilization is performed at 121°C; (2) Take 10 g of sucrose and 7 g of ascorbic acid as substrates, add 100 mL of 50 mmol / L, pH 6.0 phosphate buffer, and use 30 mL of enzyme solution as a catalyst. React in a shaker at 42°C for 12 h to obtain 2-O-α-D-pyranosyl-L-ascorbic acid.

9. The method according to claim 8, characterized in that The concentration of the Priesteria gigantea SLJTM1 bacterial suspension is 1.0×10 6 Pieces / ml.

10. Use of Priesteria gigantea SLJTM1 according to claim 1 in the fields of food, cosmetics or pharmaceuticals.

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