Method for catalytically synthesizing vitamin K2 through NovQ aromatic isopentenyl transferase

A technology for prenyl and vitamin, which is applied in the field of aromatic prenyl transferase catalyzed synthesis of fine chemicals, can solve the problems of difficulty in meeting large-scale production needs, complex purification process, low synthesis efficiency, etc., and achieves the production process. The effect of green environmental protection, simple production process and low production cost

Active Publication Date: 2016-08-17
HEFEI INSTITUTES OF PHYSICAL SCIENCE - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, microbial fermentation produces vitamin K 2 The metabolic process is very lengthy, with many branch nodes, inhibited by various metabolites, the overall synthesis efficiency is low, and the follow-up purification process is complicated, so it is currently difficult to meet the needs of large-scale production
At present, vitamin K is synthesized by single-enzyme in vitro catalysis at home and abroad. 2 Haven't seen any reports yet

Method used

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  • Method for catalytically synthesizing vitamin K2 through NovQ aromatic isopentenyl transferase
  • Method for catalytically synthesizing vitamin K2 through NovQ aromatic isopentenyl transferase
  • Method for catalytically synthesizing vitamin K2 through NovQ aromatic isopentenyl transferase

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Experimental program
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Effect test

Embodiment 1

[0029] 5.17g (30mmol) vitamin K 3 Dissolve in 40mL aqueous ethanol (1:1, v / v), add 1.13g (30mmol) NaBH in portions 4 , at N 2 Under protection, control the temperature not to exceed 50°C and stir the reaction for 12 hours until the system becomes a light green transparent liquid. After neutralizing to pH 4.0 with 100mmol / L dilute hydrochloric acid, extract the product three times with 120mL ethyl acetate, concentrate the extract to about 8mL under nitrogen protection, freeze and crystallize at -80°C, filter to retain the crystals, and concentrate the supernatant to 1.6mL, freeze and crystallize at -80°C, filter, combine the filtered crystals twice to obtain 4.73g vitamin K 3 Hydroquinone, yield 89.5%. After being oxidized in the air, it is detected by HPLC, and its peak characteristics are as follows: figure 1 Shown (peak time 3.19min, detector A248nm).

Embodiment 2

[0031] 107.44g (780mmol) vitamin K 3 Dissolve in 800mL aqueous ethanol (3:1, v / v), add 45.40g (1200mmol) NaBH in portions 4 , at N 2 Under protection, the temperature was controlled not to exceed 50°C, and the reaction was stirred for 6 hours until the system became a light green transparent liquid. After neutralizing to pH 4.0 with 100mmol / L dilute hydrochloric acid, the product was extracted three times with 2.4L ethyl acetate, and the extract was placed in N 2 Concentrate to about 0.177L under protection, freeze and crystallize at -20°C, filter to retain the crystals, concentrate the supernatant to about 0.039L, freeze and crystallize at -20°C, filter, combine the crystals filtered twice to obtain 96.07g of vitamin K 3 Hydroquinone, yield 90.9%.

Embodiment 3

[0033] 68.87g (400mmol) vitamin K 3 Dissolve in 500mL ethanol aqueous solution (2:1, v / v), add 11.35g (300mmol) NaBH 4 , at N 2 Under protection, the temperature was controlled not to exceed 50°C, and the reaction was stirred for 18 hours until the system became a light green transparent liquid. After being neutralized to pH 4.0 with 100mmol / L dilute hydrochloric acid, the product was extracted three times with 1500mL ethyl acetate, and the extract was placed in N 2Concentrate to about 91mL under protection, freeze and crystallize at -50°C, filter to retain the crystals, concentrate the supernatant to about 16.5mL, freeze and crystallize at -50°C, filter, combine the crystals filtered twice to obtain 57.85g of vitamin K 3 Hydroquinone, yield 82.1%.

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Abstract

The invention discloses a method for catalytically synthesizing vitamin K2 through NovQ aromatic isopentenyl transferase. The method comprises the steps that 1, vitamin K3 is dissolved in an ethanol aqueous solution, NaBH4 is added, under the protection of N2, the temperature is controlled to be not more than 50 DEG C, stirring reaction is conducted till the system becomes light green transparent liquid, after neutralization is conducted till the pH is 4.0, extraction is conducted on a product with ethyl acetate, after concentration is conducted, freezing crystallization is conducted, clean liquid is filtered, and vitamin K3 hydroquinone is obtained; 2, vitamin K3 hydroquinone and DMAPP are added into an enzymatic reaction system, reaction is conducted under the closed and N2 protection conditions, after the reaction is completed, Fe<3+> is added, N2 protection is removed, after adequate oxidation is conducted, extraction is conducted with ethyl acetate, and the product is obtained. According to the method for catalytically synthesizing the vitamin K2 through the NovQ aromatic isopentenyl transferase, the core link adopts a biological enzyme catalytic engineering technology, and the cheap vitamin K3 is utilized for catalytically synthesizing high-value vitamin K2; the production technology is simple, the production cost is low, and the production process is green and environmentally friendly.

Description

technical field [0001] The invention belongs to the field of fine chemicals catalyzed by aromatic prenyl transferase, and specifically relates to a NovQ aromatic prenyl transferase catalyzed synthesis of vitamin K 2 Methods. Background technique [0002] Vitamin K 2 It is a member of the vitamin K family, and its molecular structure is characterized by 3-methyl-1,4-naphthoquinone whose 2-position is substituted by a polyisoprene side chain. According to the number of polymerized units of its polyisoprene side chain, it can be divided into MK 1 、MK 2 、MK 4 And so on 14 kinds. In nature, vitamin K 2 Mainly present in prokaryotes, it is a component of the electron transport chain on the cell membrane, and it is also the final active form of various vitamin K used in the human body. [0003] Vitamin K was first recognized by people because of its coagulation activity, it is the coenzyme of coagulation factor γ-carboxylase, and it is also a necessary condition for the synt...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P7/66
CPCC12P7/66
Inventor 郑之明李哲敏赵根海刘会王鹏王丽吴荷芳尉鸿飞孙孝娟王晗孙小雯
Owner HEFEI INSTITUTES OF PHYSICAL SCIENCE - CHINESE ACAD OF SCI
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