Abstract: The present invention relates to an enzyme combination, genetically engineered bacteria, and application thereof in producing D-psicose. Glucose isomerase and D-psicose 3-epimerase from specific sources are separately co-expressed in Bacillus subtilis, a combination enzyme containing glucose isomerase and D-psicose 3-epimerase is obtained through fermentation, and the combination enzyme is used to catalyze a glucose substrate to perform isomerization to eventually prepare D-psicose. The method provided in the present invention can clearly increase a conversion rate of D-psicose, which greatly reduces the time of converting glucose into D-psicose. In addition, inexpensive glucose is used as a raw material, so that the production costs of D-psicose are greatly reduced. In the present invention, fructose syrup may be synchronously produced, so that the input for a whole set of production lines of fructose syrup is omitted, thereby greatly improving the economic benefits.

Abstract: The present invention relates to an enzyme combination, genetically engineered bacteria, and application thereof in producing D-psicose. In the present invention, glucose isomerase and D-psicose 3-epimerase from specific sources are co-expressed in Bacillus subtilis. A crude enzyme preparation is obtained by fermenting obtained engineered bacteria, and isomerization is performed by using high-concentration glucose as a substrate. Fructose syrup and D-psicose syrup are obtained by performing isolation, purification, and concentration. The enzyme combination provided in the present invention has a high catalytic rate for a substrate, and can clearly increase a conversion rate of D-psicose. In addition, inexpensive glucose is used as a raw material, so that the production costs of D-psicose are greatly reduced.

Abstract: A method for preserving a fermentation broth of an engineered strain of D-psicose-3-epimerase includes: adding a lysozyme into a fermentation broth of an engineered strain of D-psicose-3-epimerase, stirring to make the OD600 of the fermentation broth less than or equal to 1 to obtain an enzyme solution; and adjusting the enzyme solution to be weakly alkaline with an alkali liquor, and storing the weakly alkaline enzyme solution at room temperature. According to the invention, the enzyme solution is obtained by adding the lysozyme after fermentation, and then adjusted to be weakly alkaline. The lysozyme can dissolve the engineered strain to release the enzyme, and can also inhibit the growth of microorganisms. The alkaline environment of the enzyme solution can further inhibit the growth of other microorganisms in the enzyme solution. Therefore, the storage life of the enzyme solution is prolonged while ensuring the enzyme activity.

Abstract: The present invention relates to the technical field of microbial fermentation engineering, and specifically to a method for producing psicose 3-epimerase by high-density fermentation. In view of the low expression level of psicose 3-epimerase and other problems existing in the current fermentation, by controlling the feeding rate in the fermentation process, improving the culture temperature in the middle and late stages of fermentation and other measures in the present invention, the OD value during the fermentation with recombinant Bacillus subtilis and the total enzyme activity of psicose 3-epimerase in the fermentation broth are significantly increased, the expression of psicose 3-epimerase is markedly improved, and the production cost of allulose is reduced. Therefore, the present invention has a very broad prospect of application in industry.

Abstract: The invention provides a D-psicose crystal and a preparation method thereof. The method comprises: adding a seed crystal to a D-psicose solution and stirring uniformly, subjecting the solution to crystallization by gradient cooling and constant-temperature crystallization alternately at 50-30° C., stopping the crystallization until the temperature of the solution is 30° C., separating the crystal by centrifugation, washing and drying, to obtain a D-psicose crystal. Rather than evaporative crystallization, organic solvent-assisted crystallization and ultrasonication, controllable crystallization by gradient cooling, constant-temperature crystallization, and other technical means are employed. Thus, the invention has low requirements for equipment, simple process, low energy consumption, and low preparation cost, and thus particularly suitable for large-scale industrial production and processing. The purity of the D-psicose crystal obtained is ?98.

Abstract: The present invention relates to the technical field of microbial fermentation engineering, and specifically to a method for producing psicose 3-epimerase by high-density fermentation. In view of the low expression level of psicose 3-epimerase and other problems existing in the current fermentation, by controlling the feeding rate in the fermentation process, improving the culture temperature in the middle and late stages of fermentation and other measures in the present invention, the OD value during the fermentation with recombinant Bacillus subtilis and the total enzyme activity of psicose 3-epimerase in the fermentation broth are significantly increased, the expression of psicose 3-epimerase is markedly improved, and the production cost of allulose is reduced. Therefore, the present invention has a very broad prospect of application in industry.

Abstract: The invention provides a D-psicose crystal and a preparation method thereof. The method comprises: adding a seed crystal to a D-psicose solution and stirring uniformly, subjecting the solution to crystallization by gradient cooling and constant-temperature crystallization alternately at 50-30° C., stopping the crystallization until the temperature of the solution is 30° C., separating the crystal by centrifugation, washing and drying, to obtain a D-psicose crystal. Rather than evaporative crystallization, organic solvent-assisted crystallization and ultrasonication, controllable crystallization by gradient cooling, constant-temperature crystallization, and other technical means are employed. Thus, the invention has low requirements for equipment, simple process, low energy consumption, and low preparation cost, and thus particularly suitable for large-scale industrial production and processing. The purity of the D-psicose crystal obtained is ?98.