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Preparation method of rebaudioside-KA

A glucose and amino acid technology, applied in biochemical equipment and methods, transferases, enzymes, etc., can solve the problems of uncontrollable reaction process, low substrate selectivity, and high cost

Active Publication Date: 2017-09-15
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, since the substrate selectivity of the glycosyltransferase used in the aforementioned patents is not strong, while rebaudioside KA is generated, the glucosyl modification of rebaudioside KA is further catalyzed to generate rebaudioside E, and the reaction process is also difficult. Uncontrollable, so the final product is a mixture of rebaudioside KA and E
At the same time, the structures of the two products are similar, the separation is difficult, the cost is high, and it is not suitable for industrial applications

Method used

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  • Preparation method of rebaudioside-KA

Examples

Experimental program
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Effect test

Embodiment 1

[0030] Embodiment 1 Glycosyltransferase recombinant expression in Escherichia coli

[0031] Design OleD homologous proteins: HXSW-GT-09 and HXSW-GT-10 were obtained by selecting multiple key amino acid residue mutations based on OleD structure and catalytic mechanism; HXSW-GT-11 and HXSW-GT-12 were obtained based on multiple sequences Alignment and OleD structure were obtained by mutation of key amino acid residues and motif recombination of UDP-glucose or substrate binding related parts. HXSW-GT-09 (amino acid sequence shown in SEQ ID NO: 2, nucleotide sequence shown in SEQ ID NO: 7), HXSW-GT-10 (amino acid sequence shown in SEQ ID NO: 3, nucleotide acid sequence as shown in SEQ ID NO:8), HXSW-GT-11 (amino acid sequence as shown in SEQ ID NO:4, nucleotide sequence as shown in SEQ ID NO:9), HXSW-GT-12 (amino acid The sequence is shown in SEQ ID NO:5, and the nucleotide sequence is shown in SEQ ID NO:10).

[0032] The genes of the glycosyltransferase OleD (the amino acid sequ...

Embodiment 2

[0036] Example 2 Recombinant expression of glycosyltransferase in yeast

[0037] The glycosyltransferase OleD and its homologous protein genes were respectively constructed into the Pichia pastoris protein expression vector pPIC9K, and then the recombinant expression plasmid was transformed into Pichia pastoris GS115 by electric shock, and positive transformants were screened according to the Pichia pastoris operation manual of Invitrogen Company (G418 resistance screening). Select a single clone and inoculate it in LB medium, culture it overnight at 30°C to obtain a bacterial solution, connect it to BMGY medium at a 1% inoculum size (v / v), culture it at 30°C and 250 rpm for 48 hours, collect the bacteria by centrifugation, then resuspend the bacteria and mix Transplanted into BMMY, the expression was induced by methanol at 30°C and 250rpm for 5 days. After the induced expression was completed, SDS-PAGE was used to analyze the expression of the target protein in the medium su...

Embodiment 3

[0038] Example 3 Recombinant expression of glycosyltransferase in Bacillus subtilis

[0039] Glycosyltransferase OleD and its homologous protein genes were respectively constructed into Bacillus subtilis secretion expression vector pHT43, the recombinant expression plasmid was transformed into Bacillus subtilis WB800 by chemical method, and positive clones were screened on LB solid medium containing ampicillin. Pick positive colonies and inoculate them into LB liquid medium containing ampicillin, and cultivate to OD at 37°C 600 =0.5, add IPTG to induce overnight, centrifuge to obtain the supernatant of the culture medium, and analyze the expression of the target protein in the supernatant by SDS-PAGE. The results showed that the secreted expression levels of glycosyltransferases OleD, HXSW-GT-09, HXSW-GT-10, HXSW-GT-11 and HXSW-GT-12 in Bacillus subtilis WB800 were higher than those in yeast, respectively 0.33μg / ml, 0.31μg / ml, 0.27μg / ml, 0.25μg / ml, 0.28μg / ml, verified the fea...

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Abstract

Rebaudioside-KA is a nature-derived novel tetranuclear diterpenoids sweetener compound with low calorific value and high sweetness, has very low content in Stevia rebaudiana, and is difficult to obtain in in a large quantity and in a low cost and green manner. The invention establishes a green biosynthetic method for rebaudioside-KA from rubusoside, which can be easily obtained in a large quantity, as a substrate by fixed-site glycosylation by using glycosyltransferase OleD and a homologous protein thereof. The invention realizes green biological preparation of high-purity rebaudioside-KA through optimizing reaction conditions. The sweetness of rebaudioside-KA is equivalent to the sweetness of rubusoside (over 300 times o sucrose sweetness), and the rebaudioside-KA has pure taste and no bitter after-taste, so that the rebaudioside-KA is more suitable for being used as a sweetener or a correctant in food, beverage, medicament, alcohols, nutritional health products, condiment, chemical necessities, oral hygiene products, cosmetics and other products and fields.

Description

technical field [0001] The invention belongs to the technical field of natural product biosynthesis and food chemical industry, and specifically relates to the biosynthesis of natural non-sugar sweetener rebaudioside KA catalyzed by glycosyltransferase rubusoside. Background technique [0002] Obesity, diabetes, hyperlipidemia and related metabolic diseases caused by excessive intake of high-calorie foods and beverages have become major health problems plaguing the international community. The research and development of low (no) calorie and non-sugar sweeteners has attracted more and more attention. At present, the number of non-sugar sweeteners widely used in the world is small, which can be divided into two categories according to the source: the first category is natural sweeteners, which refer to products extracted from natural organisms such as stevioside and mogroside , rubusoside, glycyrrhizin, etc. The second category is chemically synthesized sweeteners, which ref...

Claims

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Application Information

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IPC IPC(8): C12P19/56C12P19/18C12N9/10
CPCC12N9/1051C12P19/18C12P19/56
Inventor 吴旭日陈依军金月
Owner CHINA PHARM UNIV
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