<i>Bacillus subtilis </i>strain, culture method and use thereof

Abstract

A Bacillus subtilis strain or a progeny thereof, a method of culturing the same and uses thereof. The invention also relates to a culture and lysate of the Bacillus subtilis strain or its progeny, and a method for producing D-psicose epimerase and producing D-psicose using the Bacillus subtilis strain or its progeny.

Claims

1. A Bacillus subtilis strain BLCY-005 or a progeny thereof, wherein the strain BLCY-005 was deposited on Oct. 26, 2016 in the China General Microbiological Culture Collection Center, with an accession number of CGMCC No. 13152.

2. A method for culturing the Bacillus subtilis strain according to claim 1, comprising a step of inoculating the strain or a progeny thereof into a medium.

3. The method according to claim 2, wherein the method comprises the following steps: (1) inoculating the strain or a progeny thereof into a solid medium and culturing at a temperature of 25-40° C. for 6-12 hours to obtain an activated strain; (2) inoculating the activated strain obtained in the step (1) into a seed medium and culturing at a temperature of 25-40° C. for 6-12 hours to obtain a seed liquid; (3) inoculating the seed liquid obtained in step (2) into a fermentation medium in a volume ratio of 1-10%, and culturing at a temperature of 25-40° C. for 30-48 h to obtain a bacterial fermentation broth.

4. A culture comprising the Bacillus subtilis strain or a progeny thereof according to claim 1 and a culture medium.

5. A lysate of a strain, wherein the strain is the Bacillus subtilis strain or a progeny thereof according to claim 1.

6. A composition comprising fructose and at least one component selected from the group consisting of: (1) the strain or a progeny thereof according to claim 1; (2) a culture comprising the Bacillus subtilis strain or a progeny thereof; and (3) a lysate of the Bacillus subtilis strain or a progeny thereof.

7. A method for preparing a D-psicose epimerase, comprising a step of lysing the Bacillus subtilis strain or a progeny thereof according to claim 1 and isolating the D-psicose epimerase, or a step of isolating the D-psicose epimerase from a culture comprising the Bacillus subtilis strain or a progeny thereof or a lysate of the Bacillus subtilis strain or a progeny thereof.

8. A method for preparing a D-psicose, comprising any one of the following steps: contacting the strain or a progeny thereof according to claim 1 with fructose; contacting a culture comprising the strain or a progeny thereof with fructose; contacting a lysate of the strain or a progeny thereof with fructose; and isolating a D-psicose epimerase from the strain or a progeny thereof according to claim 1, or a culture comprising the strain or a progeny thereof or a lysate of the strain or a progeny thereof, and contacting the D-psicose epimerase with fructose.

9. The method according to claim 2, wherein the method comprises the following steps: (1) inoculating the strain or a progeny thereof into a solid medium, and performing cultivation; (2) inoculating the strain in the solid medium into a first medium, and performing cultivation; (3) optionally, inoculating the strain in the first medium into a second medium, and performing cultivation.

10. The method according to claim 9, wherein the method is characterized by one or more of the following items: (a) the cultivation is performed at a temperature of 25-40° C. in step (1); (b) the cultivation is performed at a temperature of 25-40° C. in step (2); (c) the cultivation is performed at a temperature of 25-40° C. in step (3); (d) the first medium is a seed medium; (f) the second medium is a fermentation medium.

11. The method according to claim 3, wherein the method is characterized by one or more of the following items: (a) the seed medium comprises: 1% peptone, 0.5% yeast extract powder, 1% sodium chloride, 0.01% anhydrous magnesium sulfate, 0.02% potassium dihydrogen phosphate, and the balance of water (all percentages are weight percentages) in step (2); (b) the seed medium has a pH of 6.0-7.0 in step (2); (c) the fermentation medium comprises: 3% yeast extract powder, 2% corn steep powder, 1% glucose, 0.01% anhydrous magnesium sulfate, 0.02% diammonium hydrogen phosphate, 0.02% ammonium sulfate, and the balance of water in step (3); (d) the fermentation medium has a pH of 6.0-7.0 in step (3); (e) the solid medium is LB medium in step (1).

12. The composition according to claim 6, wherein the fructose is obtained or provided in form of an aqueous crystalline fructose solution, liquid fructose, glucose-fructose syrup, dried fructose, or fructose syrup.

13. The method according to claim 7, wherein the method comprises the following steps: (1) culturing the strain under a condition that allows the growth of the strain or a progeny thereof to obtain a fermentation broth of the strain; and (2) isolating the D-psicose epimerase from the fermentation broth obtained in step (1).

14. The method according to claim 13, wherein the D-psicose epimerase is isolated from the fermentation broth by centrifugation, filtration, dialysis, or chromatography in step (2).

15. The method according to claim 13, wherein the D-psicose epimerase is isolated from the fermentation broth in step (2) by the following steps: (2a) centrifuging the fermentation broth obtained in step (1) to obtain a precipitate; (2b) thermally denaturing the precipitate obtained in step (2a); (2c) drying the product of step (2b) to obtain the D-psicose epimerase.

16. The method according to claim 15, wherein the method is characterized by one or more of the following items: (i) the precipitate obtained in step (2a) is thermally denatured at a temperature of 50-60° C. in step (2b); (ii) the product of step (2b) is vacuum freeze-dried in step (2c); (iii) the fermentation broth is centrifuged under a condition of 10000 r/min for 10 min at 4° C. in step (2a).

17. The method according to claim 8, wherein the fructose is obtained or provided in form of an aqueous crystalline fructose solution, liquid fructose, glucose-fructose syrup, dried fructose, or fructose syrup.

18. The method according to claim 8, wherein the method comprises the following steps: (1) isolating a D-psicose epimerase from the strain or a progeny thereof according to claim 1, or a culture comprising the strain or a progeny thereof or a lysate of the strain or a progeny thereof; (2) contacting the D-psicose epimerase obtained in step (1) with fructose; (3) optionally recovering D-psicose from the product of step (2).

19. The method according to claim 18, wherein the D-psicose epimerase is contacted with fructose under a condition suitable for D-psicose epimerase activity in step (2).

20. The method according to claim 18, wherein the D-psicose epimerase is contacted with fructose under a condition of pH 5.5-6.5 in step (2).

Description

EXAMPLE 1

Mutagenesis and Screening of Bacillus subtilis

(1) 1.1 Enrichment and Culture

(2) The soil near the pilot plant in Bailong Chuangyuan, Dezhou, Shandong Province, was chosen. After the topsoil was removed with a spade, about 10 g of soil was collected from the position 5-15 cm below the ground. After being diluted 10 times with sterile water, the soil was added to LB medium for enrichment and culturing at 30-38° C. for 24 h.

(3) 1.2 Purification and Isolation

(4) Streaking method was used, in which 2 ml of the bacterial solution enriched and cultured in step 1.1 was added and diluted in a large test tube containing 5 ml of sterile water, fully shaking for dispersion. One loop of the diluent was picked up by an inoculating loop in a sterile manner, and was streaked parallel 3 to 4 times on one side of a plate medium, to carry out the first steaking; then the culture dish was rotated about 60 degrees, the leftover on the loop was burn down. After cooling, the second steaking was carried out in the same manner as the first steaking; subsequently, the third and the forth steaking were carried out in the same way. After steaking were completed, the dish lid was closed and the dish was inverted. After culturing at 28-38° C. for 24 hours, single colonies were picked and inoculated on 10 slant mediums, which were numbered as 01 to 10.

(5) The slant seeds numbered 01 to 10 were inoculated into shake flask mediums and cultured at 28-38° C. for 24 hours. The enzyme activity of conversion of D-psicose into D-fructose was measured for the shake flask fermentation broths numbered 01 to 10. The shake flask numbered 06 showed the highest enzyme activity of 75 U/ml.

(6) The components of the plate medium were as follows, all by weight percentages:

(7) peptone 1%, yeast extract powder 0.5%, sodium chloride 1%, balance of water, natural pH;

(8) The components of the slant medium were as follows, all by weight percentages:

(9) peptone 1%, yeast extract powder 0.5%, sodium chloride 1%, agar powder 1%, balance of water, natural pH;

(10) The components of the shake flask medium were as follows, all by weight percentages:

(11) yeast extract powder 3%, corn steep powder 2%, glucose 1%, anhydrous magnesium sulfate 0.01%, diammonium hydrogen phosphate 0.02%, ammonium sulfate 0.02%, balance of water, pH 6.0˜7.0.

(12) 1.3 Mutagenesis and Screening Ultraviolet mutagenesis was performed on the strain numbered 06. The ultraviolet mutagenesis was performed using a 15 W ultraviolet lamp at a distance of 20 cm, and the irradiation time was 120 s. The resulting high-yield strain was further subjected to nitrosoguanidine mutagenesis to finally obtain a strain named BLCY-005 that produced D-psicose epimerase with high conversion rate. The strain was deposited at the China General Microbiological Culture Collection Center on Oct. 26, 2016 (address: Institute of Microbiology, Chinese Academy of Sciences, Building 3, #1 West Beichen Road, Chaoyang District, Beijing) under the accession number of CGMCC No. 13152. Under optimal condition, the enzyme activity of the D-psicose 3-epimerase produced by the strain reached 143 U/ml.

(13) Enzyme activity measurement method: In a reaction system of 1 ml, 800 μl of a reaction substrate solution was added, in which the reaction substrate solution was a solution containing 100 g/L D-fructose dissolved in 50 ml of phosphate buffer (pH 7.0), then added with 200 μl of the fermentation broth, kept at 55° C. for 10 min, then boiled for 10 min to stop the enzyme reaction.

(14) The yield of D-psicose was measured by HPLC, and the enzyme activity was calculated. Enzyme activity unit (U): the amount of enzyme required to catalyze and produce 1 μmol D-psicose per minute.

EXAMPLE 2

(15) The culture method for the Bacillus subtilis BLCY-005 described in Example 1 comprised the following steps:

(16) (1) the Bacillus subtilis BLCY-005 was inoculated into LB medium and activated at 35° C. for 12 hours to obtain an activated strain;

(17) (2) the activated strain obtained in step (1) was inoculated into a seed medium and cultured at 35° C. for 12 hours to prepare a seed liquid;

(18) the components of the seed medium were as follows, all by weight percentages:

(19) peptone 1%, yeast extract powder 0.5%, sodium chloride 1%, anhydrous magnesium sulfate 0.01%, potassium dihydrogen phosphate 0.02%, balance water, pH 6.8;

(20) (3) the seed liquid obtained in step (2) was inoculated into the fermentation medium at a volume ratio of 5%, and cultured at 35° C. for 48 hours to obtain a bacterial fermentation broth;

(21) the components of the fermentation medium were as follows, all by weight percentages:

(22) yeast extract powder 3%, corn steep powder 2%, glucose 1%, anhydrous magnesium sulfate 0.01%, diammonium hydrogen phosphate 0.02%, ammonium sulfate 0.02%, balance water, pH 6.8.

(23) The dry enzyme preparation was prepared by centrifugation, washing, sterilization and drying. The optimum enzyme activity was 143 U/ml under the optimum pH of 5.5 to 6.5. The enzyme activity was much higher than 75 U/ml of the original strain.

COMPARATIVE EXAMPLE 1

(24) The culture method for the Bacillus subtilis BLCY-005 described in Example 1 comprised the following steps:

(25) (1) the Bacillus subtilis BLCY-005 was inoculated into LB medium, and cultured at 28° C. for 12 hours to obtain an activated strain;

(26) (2) the activated strain obtained in step (1) was inoculated into a seed medium, and cultured at 28° C. for 12 hours to prepare a seed liquid;

(27) the components of the seed medium were as follows, all by weight percentages:

(28) peptone 1%, yeast extract powder 0.5%, sodium chloride 1%, anhydrous magnesium sulfate 0.01%, potassium dihydrogen phosphate 0.02%, balance water, pH 5.0;

(29) (3) the seed liquid obtained in step (2) was inoculated into a fermentation medium at a volume ratio of 5%, and cultured at 28° C. for 48 h to obtain the bacterial fermentation broth;

(30) the components of the fermentation medium were as follows, all by weight percentages:

(31) yeast extract powder 3%, corn steep powder 2%, glucose 1%, anhydrous magnesium sulfate 0.01%, diammonium hydrogen phosphate 0.02%, ammonium sulfate 0.02%, balance water, pH 5.0.

(32) The dry enzyme preparation obtained after centrifugation, washing, inactivation and drying was assayed for the enzyme activity at optimum pH of 5.5 to 6.5, and the enzyme activity was 107 U/ml.

(33) From the above results, it can be seen that when the culture conditions were not within the scope of the claims of the present invention, the conversion rate of D-psicose was significantly reduced.

(34) Although specific embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details according to all the teachings that have been disclosed, and these changes are all within the protection scope of the present invention. The protection scope of the invention is given by the appended claims and any equivalents thereof.