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Transaminase and application thereof in preparation of (R)-alpha-methyltryptamine compound

A technology of methyltryptamine and transaminase, applied in microorganism-based methods, applications, transferase and other directions, can solve the problems of complex post-processing and high production cost, and achieve the effects of simple post-processing, high conversion rate and high ee value

Pending Publication Date: 2021-12-31
ABIOCHEM BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The technical problem to be solved by the present invention is to overcome the need to use D-alanine as an amino donor and produce Due to defects such as high cost and relatively complicated post-treatment, the present invention provides a transaminase, a method of using it to catalyze 3-indolylacetone compounds to prepare (R)-α-methyltryptamine compounds, and its application

Method used

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  • Transaminase and application thereof in preparation of (R)-alpha-methyltryptamine compound
  • Transaminase and application thereof in preparation of (R)-alpha-methyltryptamine compound
  • Transaminase and application thereof in preparation of (R)-alpha-methyltryptamine compound

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] According to the enzyme gene sequence of transaminase as shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3 (Table 1), the whole gene synthesis transaminase enzyme gene is connected into pET21a carrier at NdeI, HindIII site, It was verified by sequencing that the gene was successfully connected to the corresponding carrier. The company for gene synthesis and sequencing is Sangon Bioengineering Co., Ltd. (Shanghai) Co., Ltd. (698 Xiangmin Road, Songjiang District, Shanghai).

[0061] The vector containing the transaminase gene is transformed into the host E. coli BL21 (DE3) competent cell to obtain engineering strains respectively containing each transaminase. After the engineering bacteria containing the transaminase gene were activated by streaking on the plate, a single colony was picked and inoculated into 5 mL LB liquid medium containing 100 μg / mL ampicillin antibiotic, and cultured with shaking at 37°C for 12 hours. Transfer to 150mL fresh LB liquid medium also con...

Embodiment 2

[0064] The screening of embodiment 2 transaminases

[0065] The bacteria sludge of the three transaminases obtained in Example 1 and the triethanolamine buffer were homogenized at a mass ratio of 1:4 to obtain a transaminase enzyme liquid for use.

[0066] Add 625 μL of 200 mM PBS (phosphate buffered saline) with a pH of 7.0, 1 mL of transaminase enzyme solution, 100 μL of 50 mM pyridoxal phosphate (PLP), 62.5 μL of 4M isopropylamine hydrochloride, and 212.5 μL of water into the reaction vessel. Substrate (3-indolylacetone) DMSO solution (0.0086g substrate dissolved in 500μL DMSO) was added, and the reaction was carried out at 30°C. Samples were taken regularly, and the conversion rate and ee value were measured by HPLC.

[0067] The experimental results are shown in Table 2 below.

[0068] Table 2

[0069]

[0070]

[0071] \ in the table represents (because the conversion rate is too low) not detected.

[0072] It can be seen from the table that in the reaction sys...

Embodiment 3

[0073] The pH optimization in the transamination reaction of embodiment 3

[0074] Homogenize the Enz.01 bacteria sludge obtained in Example 1 and the triethanolamine buffer solution at a mass ratio of 1:4 to obtain a transaminase enzyme solution for use.

[0075] Add 0.8mL triethanolamine buffer solution with different pH values, 60mg transaminase homogenized enzyme solution, 12μL 16mg / mL pyridoxal phosphate (PLP) and 33mg isopropylamine hydrochloride in the reaction vessel, and adjust the pH to the values ​​shown in the table. After stirring at the pH value indicated at 3, add the substrate (3-indolyl acetone) DMSO solution (20 mg substrate dissolved in 0.4 ml DMSO), react at 35°C while controlling the pH. Samples were taken regularly, and the conversion rate was measured by HPLC.

[0076] The experimental results are shown in Table 3 below.

[0077] table 3

[0078] pH 3h Conversion % 6h conversion rate% 16h Conversion % 7.5 20 31.3 40.4 8.0 2...

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Abstract

The invention provides transaminase. An amino acid sequence of the transaminase is shown as SEQ ID NO. 1. The invention also provides the application of the transaminase in preparation of the (R)-alpha-methyltryptamine compound. The transaminase provided by the invention is high in enzymatic activity, high in stereoselectivity and lower in process cost. When the transaminase provided by the invention is used for catalyzing a 3-indolyl acetone compound to prepare the (R)-alpha-methyltryptamine compound, both the conversion rate and an ee value are relatively high, the transaminase can be applied to a reaction system without D-alanine, for example, isopropylamine hydrochloride is used for replacing D-alanine to serve as an amino donor, and aftertreatment is simpler.

Description

technical field [0001] The invention relates to the technical field of biocatalysis, and relates to a transaminase and a method for preparing (R)-alpha-methyltryptamine compounds by catalyzing the same and its application. Background technique [0002] Monoterpene indole alkaloids are a class of natural products with important medicinal value, but the content of monoterpene indole alkaloids in plants is extremely low (about 0.0002% of fresh weight), and the plant hosts are mostly woody, growing The cycle is long and it is difficult to meet the growing market demand. Therefore, a large amount of chemical or biological synthesis needs to be carried out to meet the needs of the market. [0003] Isosidine is a key skeleton compound in the synthesis of monoterpene indole alkaloids, which is synthesized by the aggregation of tryptamine and ring-opened loganin. Therefore, the preparation method of tryptamine, an important raw material in the synthesis pathway of isosidine, is par...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/54C12N15/70C12N1/21C12P41/00C12P17/10C12R1/19
CPCC12N9/1096C12N15/70C12P41/006C12P17/10C12Y206/01
Inventor 冯庆梅焦琦丁少南程占冰田振华张涛肖宇
Owner ABIOCHEM BIOTECH CO LTD
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