Cyclodextrin glycosyltransferase mutant, coding gene and application of cyclodextrin glycosyltransferase mutant

A technology of glycosyltransferase and encoding gene is applied in the application field of biocatalytic preparation of α-glucosyl hesperidin, which can solve the problem that the yield and purity of the product are difficult to balance, and the cyclodextrin glycosyltransferase cannot realize hesperidin. Efficient conversion, affecting the water solubility of products, etc., to achieve the effect of high yield and high purity

Pending Publication Date: 2022-03-01
浙江渚隆生物科技有限公司
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Problems solved by technology

However, cyclodextrin glycosyltransferase cannot achieve efficient conversion of hesperidin. The inventor's research group submitted an application number 201911420161.5 in the early stage, and the name is "An alkaline cyclodextrin glucosyltransferase in the production of α-glucose Application of Hesperidin in Hesperidin", the application provides a cyclodextrin glycosyltransferase derived from Bacillus alkalophilus, the amino acid sequence is shown in SEQ ID NO.2, the enzyme in 5% orange peel 88% of the substrate conversion rate can be achieved when the glycoside dosage is 15%, and the conversion rate is reduced to 58% when the 15% hesperidin dosage is used. The remaining hesperidin is difficult to separate, which seriously affects the water solubility of the final product, which leads to Product yield and purity are difficult to balance

Method used

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  • Cyclodextrin glycosyltransferase mutant, coding gene and application of cyclodextrin glycosyltransferase mutant
  • Cyclodextrin glycosyltransferase mutant, coding gene and application of cyclodextrin glycosyltransferase mutant
  • Cyclodextrin glycosyltransferase mutant, coding gene and application of cyclodextrin glycosyltransferase mutant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Example 1: Construction of recombinant engineering bacteria expressing novel cyclodextrin glycosyltransferase mutants

[0018] Refer to the alkalophilic bacillus reported in the NCBI database ( Bacillus sp. ) source of cyclodextrin glucosyltransferase (NCBI accession number NO.AAV38118), add His-tag purification tag, and submit it to Gene Synthesis Company (Hua Da Gene Qinglan Biotechnology Co., Ltd.), after codon optimization , artificially synthesized cyclodextrin glucosyltransferase coding gene (nucleotide sequence shown in SEQ ID NO.1, amino acid sequence shown in SEQ ID NO.2), and cloned into the expression vector pET22b between NcoI / BamHI , to obtain the recombinant plasmid pET22b-CGTase-WT. The primers shown in Table 1 were used to amplify the recombinant plasmid pET22b-CGTase-WT by PCR to obtain recombinant plasmids pET22b-CGTase-Y217F, pET22b-CGTase-G283V, pET22b-CGTase-G283T and pET22b-CGTase-G283L. The PCR reaction system is: 20 μLddH 2 O, 25 μL 2 × Phant...

Embodiment 2

[0026] Example 2: Preparation of novel cyclodextrin glycosyltransferase mutants

[0027] The positive clones described in Example 1 were inoculated on LB liquid medium containing 50 μg / mL ampicillin for resuscitation, 37° C., and after 10 hours of overnight culture in a shaker at 200 rpm, transfected with an inoculum volume concentration of 5%. Inoculated into fresh LB liquid medium containing 50 μg / mL ampicillin, cultured in a shaker at 37°C and 200 rpm for 3 hours; inoculated into the fermentation medium containing 50 mg / L ampicillin with an inoculum of 5% volume concentration , cultured at 35°C for 5h, then lowered the fermentation temperature to 22°C, added glycerol at a rate of 3g / h / L initial fermentation medium, and then added lactose at a rate of 5.4g / h / L initial fermentation medium , where glycerol was added for 15 hours, lactose was added for 5 hours, and the fermentation was continued at 22° C. for 16 hours to obtain a cyclodextrin glycosyltransferase-containing ferm...

Embodiment 3

[0032] Example 3: Application of a fermentation broth containing a novel cyclodextrin glycosyltransferase mutant in the synthesis of α-glucosyl hesperidin

[0033] (1) Catalytic conversion with 10% dextrin dosage and different hesperidin dosage

[0034] According to the method described in Example 2, the fermentation broth containing CGTase-Y217F / G283T cyclodextrin glycosyltransferase mutant, the catalytic process is as follows: take 50mL water and add 10%, 20%, 30% hesperidin respectively, and then add 20% % dextrin to obtain the substrate solution, and the pH was adjusted to 10.0 by adding 2 mol / L NaOH solution dropwise. Dilute the fermentation broth to 2% wet cell concentration and then add 2mol / L NaOH solution dropwise to adjust the pH to 10.0. The substrate solution and fermentation broth were mixed at a volume ratio of 1:1, and catalyzed in a constant temperature water bath magnetic stirrer at 40 °C for 16 h. During the catalytic process, the pH was adjusted by titratin...

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Abstract

The invention relates to a cyclodextrin glycosyl transferase mutant, a coding gene and application thereof. The novel cyclodextrin glycosyl transferase mutant is obtained by mutating wild type cyclodextrin glycosyl transferase from bacillus alcalophilus (Bacillus sp.), the amino acid sequence of the wild type cyclodextrin glycosyl transferase is as shown in SEQ ID NO.2, the mutant is obtained by performing single-point or two-point mutation on the 217th site and the 283th site of the wild type cyclodextrin glycosyl transferase, and the mutant has a nucleotide sequence as shown in SEQ ID NO.1. The novel cyclodextrin glycosyl transferase mutant has a nucleotide sequence as shown in SEQ ID NO.2. The novel cyclodextrin glycosyl transferase mutant has a nucleotide sequence as shown in SEQ ID NO.2. The amino acid sequence of the mutant is preferably as shown in SEQ ID NO. 4. The novel cyclodextrin glycosyl transferase mutant is obtained through a site-directed mutagenesis technology, the mutant with the amino acid sequence shown as SEQ ID NO.4 has the highest enzyme activity, the highest conversion rate of a substrate can reach 94.8%, and the novel cyclodextrin glycosyl transferase mutant has the advantages that the novel cyclodextrin glycosyl transferase mutant is a novel cyclodextrin glycosyl transferase mutant for improving a biological catalytic synthesis technology of alpha-glucosyl hesperidin; the method has important significance in realizing high-yield and high-purity alpha-glucosyl hesperidin.

Description

technical field [0001] The invention relates to a cyclodextrin glycosyltransferase mutant and its coding gene, and its application in the biocatalytic preparation of α-glucosyl hesperidin. Background technique [0002] Among the main functional components of citrus peel, hesperidin is one of the functional components with relatively high added value. It is a dihydroflavone with a chemical structure shown in formula (I). Dihydroflavonoids are one of the 60 kinds of flavonoids in citrus. Hesperidin mainly exists in the peel, juice and seeds of Rutaceae citrus plants, especially in the peel, with a content of about 3%. Hesperidin is weakly acidic, insoluble in water, natural and non-toxic. It has the functions of maintaining normal osmotic pressure of blood vessels, enhancing capillary toughness, shortening bleeding time, and reducing vascular fragility. It has also been found recently that it can lower blood pressure. , anti-allergic, lowering low-density cholesterol so as t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/54C12N15/70C12N1/21C12P19/60C12P19/18C12R1/19
CPCC12N9/1074C12Y204/01019C12N15/70C12P19/60C12P19/18
Inventor 陈小龙陈翰驰陆跃乐朱林江刘怡
Owner 浙江渚隆生物科技有限公司
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