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Isopentenyl transferase mutant and method for producing cannabinoid phenol

A technology of isopentenyl and transferase, applied in the biological field, can solve the problem of low purity of catalytic activity and achieve the effect of high production and application prospects

Pending Publication Date: 2022-04-15
嘉兴欣贝莱生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In recent years, the biosynthesis and production of CBG have attracted much attention. At present, the existing technology is catalyzed by prenyltransferase (NphB), and synthesized by olivetol (Olivetol) and geranyl pyrophosphate (GPP) The role of CBG, prenyltransferase is to transfer a prenyl group to another group, but the existing prenyltransferase has problems of catalytic activity and low purity

Method used

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  • Isopentenyl transferase mutant and method for producing cannabinoid phenol
  • Isopentenyl transferase mutant and method for producing cannabinoid phenol
  • Isopentenyl transferase mutant and method for producing cannabinoid phenol

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0157] Example 1: Construction of recombinant strains and expression process of mutants

[0158] (1) Construction of expression plasmids

[0159] The NphB coding sequence derived from Streptomyces sp. species is codon-optimized according to the codon preference of Escherichia coli (E.coli), and then according to figure 1 Connected to the pet-28a plasmid (purchased from Beijing Suo Laibao Biotechnology Co., Ltd.), transformed into competent E. coli (purchased from Nanjing Novizan Biotechnology Co., Ltd.), cultured for 8h-12h, streaked and plated to prepare pET - Monoclonal strain of 28a-NphB.

[0160] (2) Expression and purification of protein:

[0161] Prepare protein purification buffer: 50mM Tris, 150mM NaCl, PH=8

[0162] a. Pick the monoclonal strain of pET-28a-NphB or its strain stored at -80°C and inoculate it into a small test tube containing 5 mL of LB liquid medium (Kan+, 100 μg / mL), and culture it overnight at 37°C and 220 rpm as seeds liquid.

[0163] b. Transf...

Embodiment 2

[0182] Example 2: Olivol and GPP are catalyzed by protease NphB to generate CBG in vitro

[0183] This example aims to utilize the wild-type and M1-M19 mutants produced in Example 1 to catalyze the production of CBG from olivetol and GPP in vitro.

[0184] The way it is synthesized is as follows:

[0185]

[0186] Reaction buffer: 50 mM Tris-HCl, pH=8.0.

[0187] MgCl 2 The final concentration was 5 mM.

[0188] The final concentration of GPP was 2.5 mM.

[0189] The final concentration of Olivetol: 5mM.

[0190] The amount of NphB protease is 50ng (that is, the amount of NphB protease added is 1ng / uL).

[0191] The reaction temperature was 24°C.

[0192] Reaction time: 12h.

[0193] The total reaction system is: 50uL.

[0194] After the reaction was extracted twice with ethyl acetate, the solution was evaporated to dryness by a vacuum rotary evaporator, and finally dissolved with 50 uL of methanol to obtain a product containing CBG.

Embodiment 3

[0195] Embodiment 3: The detection of liquid phase and mass spectrometry is carried out to the CBG that reaction generates

[0196] The purpose of this example is to identify the product generated in Example 2.

[0197] Experimental equipment: ultra-high performance liquid chromatography-mass spectrometry (Shimadzu LC-30A, SCIEX TripleTOF6600).

[0198] Chromatographic conditions: flow rate: 0.4mL / min; column temperature 30°C; chromatographic column: Agilent Eclipse plus C18 (100mm×2.1mm, 3.5μm); mobile phase A: 5mM ammonium bicarbonate aqueous solution; mobile phase B: 0.005% formic acid acetonitrile Solution; Gradient: 0min~2min: 1%B, 2min~2.1min: 1~10%B, 2.1min~12min: 10~80%B, 12min~12.1min: 80~100%B, 12.1min~20min: 100B%.

[0199] Mass spectrometry conditions: ESI ion source; negative ion IDA detection mode; ion source parameters: ion voltage 4500 V, declustering potential 80 V, source temperature 600 ℃, curtain gas 35 psi, nebulizer gas 55 psi, heater gas 55 psi; primar...

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Abstract

According to the isopentenyl transferase mutant and the method for producing cannabinoid phenol, existing isopentenyl transferase is subjected to site-directed mutagenesis, so that olive alcohol and geranyl pyrophosphate can be helped to catalyze to generate cannabinoid phenol with relatively high catalytic activity and purity, and the isopentenyl transferase mutant has relatively high production and application prospects.

Description

technical field [0001] The application relates to the field of biotechnology, in particular to a prenyltransferase mutant and a method for producing cannabigerol. Background technique [0002] Cannabinoids refer to a class of compounds that activate cannabinoid receptors in cells. At present, more than 70 kinds of cannabinoids have been isolated from cannabis plants, mainly including: tetrahydrocannabinol (THC), cannabigerol (CBG), cannabigerolic acid (Cannabigerolic Acid, CBGA), cannabidiol (Cannabidiol, CBD), Cannabinol (CBN), Cannabichromene (CBC), Tetrahydrocannabivarin (THCV), Cannabigerovarin (CBGV) and Cannabigerolic acid (Cannabigerovarinic Acid, CBGVA) and so on. [0003] Cannabigerol (CBG), which has no hallucinogenic side effects, has important application value in the fields of medicine, food, health care and cosmetics. CBG combines with human CB1 and CB2 receptors to perform functions such as anti-tumor, anti-inflammation, anti-oxidation, nerve protection, im...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/54C12N15/70C12N1/21C12P7/22C12R1/19
Inventor 夏文豪陈贤情李珍珠逯晓云王筱刘诗梦杨月黄利辉李子鹤王千江会锋王文
Owner 嘉兴欣贝莱生物科技有限公司
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