Method for generating rebaudioside M by catalyzing rebaudioside A via immobilized glycosyltransferases
Pending Publication Date: 2020-02-28
SINOCHEM HEALTH IND DEV CO LTD +1
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The plant leaf extraction method requires a large amount of stevia raw materials, and the enrichment process is cumbersome. After extraction, it needs to pass through the column many times, desalinat...
Method used
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Embodiment 1
[0024] The source of the UGT2 gene involved in this experiment is stevia, and the source of the UGT1 gene is rice. The sequence identification numbers are shown in Table 1.
[0025] Table 1 Sequence Identification Number
[0026] name serial identification number illustrate Glycosyltransferase gene UGT2 SEQ ID NO.1 nucleic acid Glycosyltransferase UGT2 SEQ ID NO.2 amino acid Glycosyltransferase gene UGT1 SEQ ID NO.3 nucleic acid Glycosyltransferase UGT1 SEQ ID NO.4 amino acid
[0027] 1. Preparation of engineered bacteria containing target gene
[0028] Utilize the plasmid pET-28a(+) to construct the recombinant plasmid pET-28a(+)-UGT2; use pET-28a(+) as the carrier, and the UGT2 gene base sequence (as shown in SEQ ID NO.1) as the target fragment, after Optimizing digestion, recovery, purification and ligation, constructing expression plasmids, transforming the ligation reaction product into E.coli BL21, and obtaining engi...
Embodiment 2
[0048] The chitosan bead after activation is made with following method:
[0049] (1) dissolve chitosan powder with the acetic acid aqueous solution of volume concentration 2%, be heated to 50 ℃, ultrasonic until the solution is uniform and transparent, obtain the chitosan solution of 40g / L;
[0050] (2) said chitosan solution is dripped into concentration and is in 4mM NaOH aqueous solution, leaves standstill, washes, and obtains the chitosan bead that diameter is 0.1-3.5mm;
[0051] (3) According to the ratio of 1g:30mL, the chitosan pellets were mixed with the glutaraldehyde aqueous solution with a volume concentration of 0.1%, and activated by cross-linking to obtain activated chitosan pellets.
Embodiment 3
[0053] The chitosan bead after activation is made with following method:
[0054] (1) dissolving the chitosan powder with an aqueous acetic acid solution with a volume concentration of 1%, heating to 40° C., and ultrasonically until the solution is uniform and transparent to obtain a 20 g / L chitosan solution;
[0055] (2) the chitosan solution is dripped into a 3mM NaOH aqueous solution, left to stand, and washed to obtain chitosan pellets with a diameter of 0.1-3.5mm;
[0056] (3) According to the ratio of 1g:20mL, the chitosan pellets were mixed with the glutaraldehyde aqueous solution with a volume concentration of 0.01%, and activated by cross-linking to obtain activated chitosan pellets.
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Abstract
The invention discloses a method for generating rebaudioside M by catalyzing rebaudioside A via immobilized glycosyltransferases, which comprises the steps of: (1) crosslinking glycosyltransferase UGT1 and glycosyltransferase UGT2 on activated chitosan beads to obtain immobilized glycosyltransferases; (2) adding rebaudioside A into a container, adding uridine diphosphate glucose, and adding the immobilized glycosyltransferases, and catalyzing obtain rebaudioside M. The immobilized glycosyltransferases according to the invention are easy to separate, can be repeatedly used, have high thermal stability and high operation stability, and can, when compared with free enzymes, directly yield a higher amount of rebaudioside M by catalyzing rebaudioside A via the immobilized glycosyltransferases;the free enzymes can only be used once, while the immobilized glycosyltransferases can be reused; after the immobilized glycosyltransferases are reused 10 times, the activity of the immobilized glycosyltransferases is still more than 50% of that of the immobilized glycosyltransferase used for the first time. The method is simple and easy to implement. The process steps are reduced, the cost is reduced, and the production efficiency is improved.
Description
technical field [0001] The invention belongs to the field of bioengineering, and in particular relates to a method for catalyzing rebaudioside A to generate rebaudioside M by an immobilized glycosyltransferase. Background technique [0002] Steviol glycoside is a new type of natural low-calorie sweetener steviol glycoside compound extracted from stevia leaves of Compositae herb, because of its high sweetness, low calorie, non-toxicity, high temperature resistance, acid and alkali resistance and good water solubility It has been certified as safe by the US Food and Drug Administration and can be used in the food industry. Rebaudioside M is a newly discovered glycoside. Compared with other glycosides, its sweetness is high, about 400 times that of sucrose, and its sweetness is pure, and its taste is closer to sucrose. It has good stability and is an ideal natural sweetener product. In 2013, the US FDA approved the use of stevia products containing 50% rebaudioside M extracte...
Claims
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