CN113564080B - Bacillus subtilis for producing sucrose phosphorylase and application thereof - Google Patents
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Abstract
The invention provides a bacillus subtilis for producing sucrose phosphorylase and application thereof, the bacillus subtilis subspecies SLLSM1 strain is separated from soil of Guangxi guest sugarcane field, and the preservation number is CCTCC NO:2021697, date of deposit: the preservation address is as follows at 2021, 6 and 9 months: china, wuhan and Wuhan university, the preservation unit: the strain has high enzyme production activity and good stability of sucrose phosphorylase, the enzyme production activity of the strain reaches 195.3U, the yield of the prepared alpha-arbutin reaches 58.84g/L, and the molar yield of the alpha-arbutin is 59.48 percent.
Description
[ technical field ] A method for producing a semiconductor device
The invention relates to the technical field of microorganisms, and particularly relates to bacillus subtilis for producing sucrose phosphorylase and application thereof.
[ background of the invention ]
Alpha-arbutin (alpha-Ar) is a high-value glucoside, is widely applied to the cosmetic and pharmaceutical industries, and is a safe and reliable whitening agent. The enzyme method is a main method for producing alpha-arbutin, is widely used due to simple raw materials and short production period, but is greatly limited in the yield of the alpha-arbutin at present due to the lack of enzymes with high catalytic efficiency.
The method utilizes the self-screened sucrose phosphorylase enzyme method for catalytic synthesis, namely the sucrose phosphorylase enzyme liquid catalyzes sucrose and hydroquinone to synthesize the alpha-arbutin, and the reaction has the advantages of greenness, high efficiency and mild reaction.
[ summary of the invention ]
In view of this, the present invention aims to provide a bacillus subtilis for producing sucrose phosphorylase and application thereof.
In order to solve the technical problem, the invention adopts the following technical scheme:
a Bacillus subtilis subsp. Spizinii strain SLLSM1 with a preservation number of CCTCC NO:2021697, date of deposit: the preservation address is as follows at 2021, 6 and 9 months: china, wuhan and Wuhan university, the preservation unit: china center for type culture Collection.
Further, the strain SLLSM1 of Bacillus subtilis subsp.Spizinii, which is isolated from the soil of the Guangxi Guest sugarcane field.
The present application also provides a microbial preparation comprising the Bacillus subtilis subsp.
Further, the microbial preparation contains viable bacteria with the number not less than 1.0 x 10 8 one/mL of SLLSM1 cells of subspecies Spanish of Bacillus subtilis.
The application also provides a method for producing sucrose phosphorylase, which comprises the following steps: under aseptic operation, inoculating Bacillus subtilis subspecies SLLSM1 into a fermentation medium (pH = 4.0) which is sterilized at high temperature, performing constant-temperature fermentation culture at 37 ℃ and 180r/min for 24h by using a shaking table, inoculating the fermentation broth into a new fermentation medium according to 8% of the inoculation amount, performing constant-temperature culture at 37 ℃ for 72h by using the shaking table, and centrifuging by using a centrifuge of 10000r/min and 15min to obtain a supernatant, namely the crude sucrose phosphorylase liquid.
Further, the formula of the fermentation medium is as follows: 70g/L potassium nitrate, 70g/L yeast powder, 90g/L glucose and 5g/L, H dipotassium hydrogen phosphate 2 O 1000mL。
The application also provides an application of the bacillus subtilis subspecies SLLSM1 in the production of alpha-arbutin products.
The present application also provides a method for preparing the above-mentioned alpha-arbutin, comprising the steps of:
(1) Inoculating the preserved subspecies SLLSM1 of the Bacillus subtilis to a slant culture medium, placing the culture medium in a constant-temperature incubator at 37 ℃ for 2 days to obtain activated strains, eluting the strains by using sterile water to obtain a suspension of the subspecies SLLSM1 of the Bacillus subtilis, inoculating the suspension of the strains to a shaking fermentation culture medium, carrying out constant-temperature culture on the suspension for 72 hours in a shaking table at 37 ℃ and 180r/min, and centrifuging the suspension by a centrifuge of 10000r/min and 15min to obtain a supernatant which is a sucrose phosphorylase crude enzyme solution;
the slant culture medium is a PS culture medium; the PS culture medium comprises the following components: 0.5g of sucrose and 0.02g of KH 2 PO 4 、0.61g Na 2 HPO 4 、0.1g NH 4 Cl, yeast extract 0.01g, mgSO 0.05g 4 ·7H 2 O、0.5mgCaCl 2 1g of agar powder, 100mL of distilled water, natural pH, and moist heat sterilization at 121 ℃;
the formula of the shake flask fermentation medium is as follows: 70g/L potassium nitrate, 70g/L yeast powder, 90g/L glucose and 5g/L H dipotassium hydrogen phosphate 2 O 1000mL；
(2) Adding 10mL of 30mmol/L, pH which is 6.0 phosphate buffer solution, 5g of sucrose and 50g/L of hydroquinone at the temperature of 50 ℃, adding 3mL of crude enzyme solution, and carrying out shaking table reaction at 180r/min for 5 hours in a dark place to obtain the alpha-arbutin.
Further, the suspension concentration of the Bacillus subtilis subspecies SLLSM1 strain is 1.0 × 10 8 One per ml.
The application also discloses application of the bacillus subtilis subspecies SLLSM1 in the fields of food, cosmetics or pharmacy.
The phosphate buffer solution can be purchased in the market and produced to Heifeibomei biotechnology Limited liability company.
In summary, due to the adoption of the technical scheme, the invention has the beneficial effects that:
the bacillus subtilis subspecies SLLSM1 is a strain separated and screened from soil of Guangxi guest sugarcane field, and is researched and found to be the bacillus subtilis subspecies SLLSM1 for producing sucrose phosphorylase, and in order to research the enzyme production capacity of the strain, the research of single factor and orthogonal experiments is carried out, and the best conditions for producing the enzyme by fermenting the bacillus subtilis subspecies SLLSM1 are determined by the research of the applicant: the fermentation time is 72h, the inoculum size is 8%, the fermentation temperature is 37 ℃, the potassium nitrate is 70g/L, the yeast powder is 70g/L, the glucose is 90g/L, the dipotassium phosphate is 5g/L, and the initial pH is 4.0; the enzyme activity of the bacterium for producing the enzyme reaches 195.3U/mL, so that the bacterium has high enzyme production activity and good stability; the yield of the alpha-arbutin prepared by the enzyme-producing transformation of the strain reaches 58.84g/L, and the molar yield of the alpha-arbutin is 59.48 percent.
[ description of the drawings ]
FIG. 1 is a morphological diagram of the Bacillus subtilis subspecies SLLSM1 strain on a plate according to the examples of the present application;
FIG. 2 is a graph showing the effect of different fermentation times on the enzyme productivity of Bacillus subtilis subspecies Spanish;
FIG. 3 is a graph showing the effect of different carbon sources on the ability of Bacillus subtilis subspecies Spanish to produce enzyme;
FIG. 4 is a graph showing the effect of different glucose concentrations on the enzyme-producing ability of Bacillus subtilis subspecies Spanish;
FIG. 5 is a graph showing the effect of different inorganic nitrogen sources on the ability of Bacillus subtilis subspecies Spanish to produce enzyme;
FIG. 6 shows different KNO 3 A graph showing the effect of concentration on the enzyme productivity of Bacillus subtilis subspecies stevensis;
FIG. 7 is a graph showing the effect of different organic nitrogen sources on the enzyme-producing ability of Bacillus subtilis subspecies Spanish;
FIG. 8 is a graph showing the effect of different yeast powder concentrations on the enzyme productivity of Bacillus subtilis subspecies Spanish;
FIG. 9 is a graph showing the effect of different inorganic salt ions on the enzyme productivity of Bacillus subtilis subspecies Spanish;
FIG. 10 is a graph showing the effect of different fermentation medium pH on the enzyme productivity of Bacillus subtilis subspecies Spanish;
FIG. 11 is a graph showing the effect of different inoculum sizes of the strains on the enzyme productivity of subspecies Spanish of Bacillus subtilis;
FIG. 12 is a graph showing the effect of different fermentation temperatures on the enzyme productivity of Bacillus subtilis subspecies Spanish.
[ detailed description ] A
All of the features disclosed in this specification, or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations of features and/or steps that are mutually exclusive.
Any feature disclosed in this specification (including any accompanying claims, abstract) is merely an example of a generic series of equivalent or similar features, unless explicitly described as such.
1. Screening of strains
The soil sample is taken from soil of Guangxi guest sugarcane field, a PS culture medium is used for forming a screening culture medium to screen strains producing sucrose phosphorylase, and the process comprises the following steps: primary screening of strains: primary screening of strains: adding 1g of soil into 9mL of sterile physiological saline to prepare 10 -1 Shaking and mixing the sample solution with the concentration uniformly, and diluting the sample solution to 10 degrees by degrees -2 、10 -3 、10 -4 、10 -5 、10 -6 And (5) concentration and standing for later use. Taking the last 3 dilutions, performing strain separation on PS culture medium plate by coating method, streaking and separating on the plate, and repeatedly purifying to obtain pure culture of isolated strain. The plates were stored in a refrigerator at 4 ℃.
The PS culture medium is a PS agar slant solid culture medium;
the formula of the PS culture medium is as follows: 0.5g sucrose, 0.02gKH 2 PO 4 、0.61gNa 2 HPO 4 、0.1gNH 4 Cl, yeast extract 0.01g, mgSO 0.05g 4 ·7H 2 O、0.5mgCaCl 2 And 1g of agar powder, 100mL of distilled water, natural pH and 121 ℃ damp-heat sterilization.
2. Identification of strains and optimization of fermentation conditions
1. Identification of the species
(1) And (3) observing colony morphology: the slant strain was spotted on a plate containing PS medium, and cultured at 37 ℃ for 48 hours to observe colony morphology.
(2) 16S rDNA identification of the strain: the purified strain is sent to Meiji biological medicine science and technology Limited company in Shanghai for identification. The SLLSM1 strain was initially determined to be a Bacillus subtilis subspecies by a Nucleotide BLAST analysis with 100% homology to the 16S R DNA sequence of the Bacillus subtilis subspecies.
The selected Bacillus subtilis subspecies SLLSM1 has the following preservation information: the name is Bacillus subtilis subspecies SLLSM1, which is classified and named as Bacillus subtilis subspecies SLLSM1, and the preservation number is CCTCC NO:2021697, date of deposit: the preservation address is as follows at 2021, 6 and 9 months: wuchang Lodojia mountain in Wuhan city, hubei province, preservation unit: china center for type culture Collection.
The morphological characteristics of the bacillus subtilis subspecies SLLSM1 on the plate are shown in fig. 1, the bacterial colony of the strain is yellowish, and the surface of the bacterial colony is rough and opaque.
2. Optimization research of enzyme production conditions of bacillus subtilis subspecies SLLSM1
The PS culture medium formula is as follows: 0.5g of sucrose and 0.02g of KH 2 PO 4 、0.61g Na 2 HPO 4 、0.1g NH 4 Cl, yeast extract 0.01g, mgSO 0.05g 4 ·7H 2 O、0.5mgCaCl 2 And 1g of agar powder, 100mL of distilled water, natural pH and 121 ℃ damp-heat sterilization.
(1) Influence of fermentation time on enzyme production
Inoculating Bacillus subtilis subspecies SLLSM1 to PS culture medium, fermenting at 37 deg.C, pH natural, liquid loading amount of 100ml/250ml, inoculation amount of 1ml, and rotation speed of 180r/min for 36, 48, 60, 72, and 84h respectively, and taking supernatant to measure enzyme activity. The results are shown in Table 1 and FIG. 2.
TABLE 1 ability to produce enzyme by different fermentation times for subspecies Spanish of Bacillus subtilis
| Item | 36h | 48h | 60h | 72h | 84h |
| Enzyme activity (U/mL) | 57.33 | 92.71 | 164.63 | 186.96 | 173.73 |
(2) Optimization of carbon source and its concentration
Variables of carbon source are sucrose, starch, lactose, glucose, maltose based on PS medium. Preparing a liquid culture medium, performing fermentation culture for 72h, centrifuging at 10000rpm for 10min at 37 ℃ on a shaking table at the rotating speed of 180r/min, further researching the influence of the glucose concentration (5, 7, 9, 11, 13%) on the enzyme production of the strain, and taking the supernatant to measure the enzyme activity. The results are shown in Table 2 and FIGS. 3 and 4.
TABLE 2 ability of different carbon sources to produce enzymes from subspecies Spanish of Bacillus subtilis
(3) Optimization of nitrogen sources
The nitrogen source variable is NaNO based on PS medium 3 、NH 4 Cl、KNO 3 、(NH 4 )SO 4 And urea. Preparing a liquid culture medium, centrifuging for 10min at 37 ℃ and 180r/min on a shaking table at 72h and 10000rpm, discussing the influence of potassium nitrate (5, 7, 9, 11 and 13%) with different concentrations on the enzyme production of the strain on the basis, and taking supernatant to measure the enzyme activity. The results are shown in Table 3 and FIGS. 5 and 6.
TABLE 3 ability of different inorganic nitrogen sources to produce enzyme from subspecies Spiladelphia subtilis
11 percent of glucose is used as a carbon source, 7 percent of potassium nitrate is used as an inorganic nitrogen source, and urea, peptone, yeast powder, corn flour and soybean flour are respectively selected as the only organic nitrogen source in the fermentation medium. Preparing a liquid culture medium, centrifuging for 10min at 37 ℃ and 180r/min on a shaking table at 72h and 10000rpm, further exploring the influence of the concentration (5, 7, 9, 11 and 13%) of the yeast powder on the enzyme production of the strain on the basis, and taking the supernatant to test the enzyme activity. The results are shown in Table 4 and FIGS. 7 and 8.
TABLE 4 ability of different organic nitrogen sources to produce enzyme from subspecies stressful bacillus subtilis
(4) Effect of inorganic salt ions on enzyme Activity
Respectively selecting 11 percent of glucose as a carbon source, 7 percent of potassium nitrate as an inorganic nitrogen source and 9 percent of yeast powder as an organic nitrogen source, and respectively selecting MgSO (MgSO) 4 、CuSO 4 、CaCl 2 、K 2 HPO 4 、ZnSO 4 As the only inorganic saltAnd (4) adding the active ingredients. Preparing a liquid culture medium, performing fermentation culture for 72h at 37 ℃ on a shaking table and a rotating speed of 180r/min, taking out fermentation liquor, centrifuging for 10min at 10000rpm, and taking supernatant to measure enzyme activity. The results are shown in Table 5 and FIG. 9.
TABLE 5 ability of various inorganic salt ions to produce enzyme from subspecies Spanish of Bacillus subtilis
| Item | MgSO 4 | CuSO 4 | CaCl 2 | K 2 HPO 4 | ZnSO 4 |
| Enzyme activity (U/mL) | 115.70 | 125.68 | 84.61 | 193.72 | 124.23 |
(6) Effect of pH on enzyme Activity
Adjusting the initial pH of the culture medium to 3, 4, 5, 6, 7 with hydrochloric acid, and collecting supernatant to determine enzyme activity. Preparing a liquid culture medium, performing fermentation culture for 72h at 37 ℃ on a shaking table and a rotating speed of 180r/min, taking out fermentation liquor, centrifuging for 10min at 10000rpm, and taking supernatant to measure enzyme activity. The results are shown in Table 6 and FIG. 10.
TABLE 6 ability of different fermentation media pH to produce enzyme from Bacillus subtilis subspecies Stevensis
| Item | pH(3) | pH(4) | pH(5) | pH(6) | pH(7) |
| Enzyme activity (U/mL) | 165.58 | 192.07 | 164.87 | 134.39 | 92.52 |
(7) Effect of inoculum size on enzyme production
Inoculating Bacillus subtilis subspecies SLLSM1 to the fermentation medium, and inoculating 6%, 8%, 10%, 12%, and 14% of the strains respectively. Preparing a liquid culture medium, performing fermentation culture for 72h at 37 ℃ on a shaking table and a rotating speed of 180r/min, taking out fermentation liquor, centrifuging for 10min at 10000rpm, and taking supernatant to measure enzyme activity. The results are shown in Table 7 and FIG. 11.
TABLE 7 ability of different inoculum sizes of strains to produce enzyme from subspecies of Bacillus subtilis
| |
6％ | 8％ | 10％ | 12％ | 14％ |
| Enzyme activity (U/mL) | 107.28 | 189.51 | 116.86 | 84.57 | 52.08 |
(8) Influence of fermentation temperature on enzyme production
Inoculating Bacillus subtilis subspecies SLLSM1 to the fermentation culture medium, performing shake culture at 28 deg.C, 30 deg.C, 35 deg.C, 37 deg.C and 40 deg.C respectively at rotation speed of 180r/min for 72h, centrifuging the fermentation liquid at 10000r/min for 10min, and collecting the supernatant to measure enzyme activity. The results are shown in Table 8 and FIG. 12.
TABLE 8 enzyme-producing ability of different fermentation temperatures of strains on subspecies schwersonii of Bacillus subtilis
| |
28 |
30℃ | 35℃ | 37 |
40℃ |
| Enzyme activity (U/mL) | 81.90 | 131.86 | 163.07 | 185.40 | 111.32 |
Through the enzyme activity data of the single-factor experiment optimization detection in tables 1 to 8, the optimal conditions for producing the enzyme by fermenting the subspecies SLLSM1 of the bacillus subtilis can be determined as follows: the fermentation time is 72h, the inoculation amount is 8%, the fermentation temperature is 37 ℃, the potassium nitrate is 70g/L, the yeast powder is 70g/L, the glucose is 90g/L, the dipotassium phosphate is 5g/L, and the initial pH is 4.0.
The optimal conditions for producing the enzyme by fermenting the bacillus subtilis subspecies SLLSM1 are adopted, the bacillus subtilis subspecies SLLSM1 is subjected to fermentation culture, the fermentation liquid is centrifuged for 10min at 10000rpm, and the supernatant is taken to measure the enzyme activity. The enzyme activity reaches 195.3U/mL through detection.
3. The application of the Bacillus subtilis subspecies SLLSM1 comprises the following steps: product for producing alpha-arbutin
The method for preparing the alpha-arbutin by utilizing the transformation of the subspecies SLLSM1 of bacillus subtilis comprises the following steps:
(1) Preparation of crude enzyme solution of sucrose phosphorylase
In a sterile procedure, the Bacillus subtilis subspecies SLLSM1 was inoculated with sterile inoculations into a sterile, optimized medium (potassium nitrate)70g/L of yeast powder, 70g/L of glucose, 90g/L of glucose and 5g/L, H of dipotassium hydrogen phosphate 2 O1000 mL and pH = 4.0), fermenting and culturing for 24h at 37 ℃ and 180r/min by a shaking table at constant temperature, taking the fermentation liquor, inoculating the fermentation liquor into a new optimized enzyme-producing culture medium (70 g/L of potassium nitrate, 70g/L of yeast powder, 90g/L of glucose and 5g/L, H of dipotassium hydrogen phosphate 2 O1000 mL and pH = 4.0), culturing for 72h at 37 ℃ and 180r/min by a shaking table at constant temperature, and centrifuging by a centrifuge of 10000r/min and 15min to obtain supernatant, namely the crude sucrose phosphorylase liquid.
(2) Catalytic preparation of alpha-arbutin
Adding 10mL of 30mmol/L, pH which is 6.0 phosphate buffer solution, 5g of sucrose and 50g/L of hydroquinone at the temperature of 50 ℃, adding 3mL of sucrose phosphorylase crude enzyme solution, and carrying out shaking table light-shielding reaction for 5h at the speed of 180r/min to obtain the alpha-arbutin.
Measuring the content of alpha-arbutin in the reaction solution according to a spectrophotometry method, and calculating the conversion rate.
Through determination, the yield of the alpha-arbutin reaches 58.84g/L, and the molar yield of the alpha-arbutin is 59.48 percent.
Although the invention has been described in detail with respect to the general description and the specific embodiments thereof, it will be apparent to those skilled in the art that modifications and improvements can be made based on the invention. Accordingly, it is intended that all such modifications and alterations be included within the scope of this invention as defined in the appended claims.
Claims (8)
1. Bacillus subtilis subspecies stevensisBacillus subtilis subsp. SpizizeniiThe strain SLLSM1 has a preservation number of CCTCC NO:2021697, date of deposit: the preservation address is as follows at 2021, 6 and 9 months: china, wuhan and Wuhan university, the preservation unit: china center for type culture Collection.
2. Comprising the Bacillus subtilis subspecies Spanish of claim 1Bacillus subtilis subsp. SpizizeniiMicrobial preparation of strain SLLSM 1.
3.The microbial preparation of claim 2, wherein the microbial preparation contains viable count of not less than 1.0 x 10 8 one/mL of SLLSM1 cells of subspecies Spanish of Bacillus subtilis.
4. A method for producing sucrose phosphorylase is characterized in that under aseptic operation, bacillus subtilis subspecies SLLSM1 is inoculated to a fermentation medium which is sterilized at high temperature through sterilization, the pH of the fermentation medium is =4.0, after fermentation culture is carried out for 24 hours at constant temperature of 37 ℃ and 180r/min by a shaking table, fermentation liquor is taken according to 8% of inoculum size and is inoculated to a new fermentation medium, the fermentation medium is cultured for 72 hours at constant temperature of 37 ℃ by the shaking table, and the fermentation liquor is centrifuged by a 10000r/min and 15min centrifuge to obtain supernatant which is crude sucrose phosphorylase liquid; the preservation number of the bacillus subtilis subspecies SLLSM1 is CCTCC NO:2021697.
5. the method of claim 4, wherein the fermentation medium is formulated as: potassium nitrate 70g/L, yeast powder 70g/L, glucose 90g/L, dipotassium hydrogen phosphate 5g/L, H 2 O 1000 mL。
6. Use of the Bacillus subtilis subspecies Stevensis of claim 1Bacillus subtilis subsp. SpizizeniiThe method for preparing alpha arbutin by using the strain SLLSM1 is characterized by comprising the following steps:
(1) Inoculating the preserved subspecies SLLSM1 of the Bacillus subtilis to a slant culture medium, placing the culture medium in a constant-temperature incubator at 37 ℃ for 2 days to obtain activated strains, eluting the strains by using sterile water to obtain a suspension of the subspecies SLLSM1 of the Bacillus subtilis, inoculating the suspension of the strains to a shaking fermentation culture medium, carrying out constant-temperature culture on the suspension for 72 hours in a shaking table at 37 ℃ and 180r/min, and centrifuging the suspension by a centrifuge of 10000r/min and 15min to obtain a supernatant which is a sucrose phosphorylase crude enzyme solution;
the slant culture medium is a PS culture medium; the PS culture medium comprises the following components: 0.5 Sucrose (g) and KH (0.02 g) 2 PO 4 、0.61 g Na 2 HPO 4 、0.1 g NH 4 Cl, 0.01g yeast extract, 0.05g MgSO 4 ・7H 2 O、0.5 mg CaCl 2 1g of agar powder, 100mL of distilled water, natural pH and damp-heat sterilization at 121 ℃;
the formula of the shake flask fermentation medium is as follows: potassium nitrate 70g/L, yeast powder 70g/L, glucose 90g/L, dipotassium hydrogen phosphate 5g/L, H 2 O 1000 mL；
(2) Adding 10mL of 30mmol/L, pH which is 6.0 phosphate buffer solution, 5g of sucrose and 50g/L of hydroquinone into the mixture at the temperature of 50 ℃, adding 3mL of crude enzyme solution into the mixture, and carrying out shaking table reaction on the mixture at 180r/min in a dark place for 5h to obtain the alpha arbutin.
7. The method of claim 6, wherein the suspension of Bacillus subtilis subspecies SLLSM1 is at a concentration of 1.0 x 10 8 one/mL.
8. The Bacillus subtilis subspecies Stevensis of claim 1Bacillus subtilis subsp. SpizizeniiThe application of the strain SLLSM1 in the field of preparing cosmetics or medicines.
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| Title |
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| Cloning and expression of the sucrose phosphorylase gene in Bacillus subtilis and synthesis of kojibiose using the recombinant enzyme;Miaomiao Wang et al.;《Microb Cell Fact》;20180215;第17卷(第1期);第1-9页 * |
| 蔗糖磷酸化酶的半理性设计及生产α-熊果苷的条件优化;沈洋等;《食品与发酵工业》;20200731;第46卷(第13期);第1-9页 * |
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