Dephosphorylation enzyme of new psicose-6-phosphoric acid, composition for producing psicose comprising same, and method for preparing psicose using same
A technology of phosphatase and psicose, applied in the field of enzymes, can solve the problems of unsuitable enzymes and poor heat resistance, etc.
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[0064] The psicose-6-phosphate phosphatase, α-glucan phosphorylase, phosphoglucomutase (or phosphomannose mutase), glucose-6-phosphate phosphatase used in the method for preparing psicose of the present invention, 6-phosphate isomerase, psicose-6-phosphate-3-epimerase (or ribulose-5-phosphate-3-epimerase), pullulanase (or isoamylase enzyme), 4-α-glucanotransferase and polyphosphoglucokinase, with little or no side reaction with the final product psicose.
[0065] In the invented method for producing psicose, optimal / maximum amount of psicose can be produced by decomposing high-concentration starch in a complex composition of phosphate sugar-inverting enzymes, and up to 8 enzymes can be used , to ensure the maximum production of allulose.
[0066] First, the enzyme that degrades starch and produces glucose-1-phosphate, namely glucan phosphorylase (glycogen phosphorylase, EC 2.4.1.1), uses α-1,4-starch specific to produce glucose-1-phosphate. Secondly, in the intermediate com...
Embodiment 1
[0079] Embodiment 1: the preparation of the recombinant expression vector of the microorganism of each enzyme and transformation
[0080] In order to provide the necessary enzymes for the pathway to produce psicose, a heat resistance gene was selected as shown in Table 2 below.
[0081] Table 2
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[0089] Using gene synthesis or chromosomal DNA (genomic DNA) of each cultured strain, the gene for the selected amino acid was amplified by polymerase chain reaction (PCR), and the amplified DNA was amplified using restriction enzymes NdeI and XhoI or SalI The plasmid vector pET2la (Novagen) was inserted to construct the recombinant expression vector, which was used for E. coli expression. Transformed microorganisms were prepared by transforming the expression vector into E. coli BL21(DE3) strain using general transformation techniques (see Sambrook et al. 1989).
[0090]In particular, the allulose o...
Embodiment 2
[0092] Embodiment 2: the preparation of recombinant enzyme
[0093] To prepare the recombinant enzyme, each transformed microorganism prepared in Example 1 was inoculated into a culture tube containing 5 mL of LB liquid medium. Seed culture was carried out in a shaking incubator at 37°C until the absorbance at 600 nm reached 2.0. The culture solution obtained by the seed culture was inoculated into a culture bottle containing an LB liquid medium, followed by main culture. When the absorbance of the culture at 600 nm reached 2.0, 1 mM IPTG was added to induce the expression and production of the recombinant enzyme. The culture was carried out at a stirring speed of 180 rpm and a temperature of 37°C. The culture solution was centrifuged at 8000×g and 4° C. for 20 minutes to recover cells. The recovered cells were washed twice with 50 mM Tris-HCl (pH 8.0) buffer and resuspended in the same buffer, and the cells were disrupted using an ultrasonic homogenizer. The cell lysate w...
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