A kind of improved transaminase, its encoding gene and genetic engineering bacteria expressing the enzyme

A technology of genetically engineered bacteria and transaminase, applied in genetic engineering, transferase, plant gene improvement, etc., can solve problems such as poor stereoselectivity, low yield, and expensive catalyst, and achieve good industrial application prospects and catalytic activity High, product chiral purity and high yield effect

Active Publication Date: 2020-01-31
盐城仁越生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] The technical problem to be solved in the present invention is: based on the reported asymmetric synthesis of (R)-(+)-1-p-methylphenethylamine, the reaction yield is low, the stereoselectivity is not good, the catalyst is expensive, etc. , the invention provides an improved transaminase, and also discloses the coding gene of the transaminase and the genetically engineered bacteria expressing the enzyme

Method used

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  • A kind of improved transaminase, its encoding gene and genetic engineering bacteria expressing the enzyme
  • A kind of improved transaminase, its encoding gene and genetic engineering bacteria expressing the enzyme
  • A kind of improved transaminase, its encoding gene and genetic engineering bacteria expressing the enzyme

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Embodiment 1

[0025] The construction of embodiment 1 transaminase mutant coding gene

[0026] The synthetic sequence of the whole gene is shown in SEQ ID No.1, and two restriction sites, Nde I and Hind III, were selected and inserted into the pET-28a expression vector, and the obtained recombinant expression vector was named pET28a-AfAT. To construct the mutant library, we designed the following 8 primers, see Table 1 for details:

[0027] Table 1 PCR primer list

[0028]

[0029] Use pET28a-AfAT as a template and use the above primers for PCR amplification. The PCR system is: 10L of 5×PCR buffer, 4L of 2.5mmol / L dNTPs, 0.5L of DNA polymerase, 0.5L of pET28a-AfAT template (containing 0.1g of DNA template), ddH 2O is 31L, the AfAT-up upstream primer (SEQ ID No.5) and H53-down downstream primer (SEQ ID No.8), H53-up upstream primer (SEQ ID No.7) and E115- down downstream primer (SEQ ID No.10), E115-up upstream primer (SEQ ID No.9) and S214-down downstream primer (SEQ ID No.12), S214-u...

Embodiment 2

[0030] The construction of embodiment 2 transaminase mutant library

[0031] The above gene fragments and the vector pET-28a plasmid were subjected to enzyme digestion reactions (Nde I and Hind III, 37°C for 1 h), and the enzyme digested products were gel-cut and recovered, followed by ligation reaction (reaction overnight at 16°C), and transformed into Escherichia coli BL21(DE3) competent cells were screened with kanamycin to obtain positive single clones. Add 0.6 mL of LB medium (containing 50 g / L kanamycin) to each well of a 96-well plate, pick 93 positive clones and 3 BL21(DE3) / pET28a-AfAT clones from each 96-well plate as controls, and pick Twelve 96-well plates were taken and cultured in a shaker at 37°C for 16 hours, and this was the mutant library.

Embodiment 3

[0032] Expression, screening and identification of embodiment 3 transaminase mutants

[0033] Transplant 200 L of the overnight mutant bacterial solution to a new 96-well plate, each well of which contains 1 mL of fresh LB medium, wherein the concentration of kanamycin is 50 g / L, and the concentration of IPTG is 1.0 mmol / L. Induce the culture at 25°C for about 20 hours, centrifuge to discard the supernatant, and collect the bacteria. Add 600L reaction solution to each well, including: 10g / L substrate p-methylacetophenone, 60g / L isopropylamine, 1mmol / L coenzyme pyridoxal phosphate (PLP), 100mmol / L potassium phosphate buffer (pH=8.0), after 24 hours of shaking reaction on a shaking table at 35° C., the reaction solution was analyzed by HPLC to detect the conversion rate of the mutant. Conversion rate detection conditions: Shimadzu2010AHT high performance liquid chromatography; Athena C18-WP (150 × 4.6mm, 5m) chromatographic column; wavelength 214nm; mobile phase A (H 2 O+0.1%T...

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Abstract

The invention relates to improved transaminase and a coding gene thereof. By constructing Escherichia coli genetically engineered bacteria, heterologous expression of the transaminase is realized. Corresponding to amino acid sequence of wild-type transaminase, the 53rd of improved transaminase is serine, the 115th of improved transaminase is alanine, and the 214th of improved transaminase is proline. In comparison with catalytic efficiency of wild-type transaminase derived from aspergillus fumigates Af293, catalytic efficiency of the improved transaminase is at least two times higher than catalytic efficiency of the wild-type transaminase in the aspect of catalyzing p-methylacetophenone asymmetric transamination for generation of (R)-(+)-1-p-methylphenylethylamine.

Description

technical field [0001] The invention belongs to the field of biopharmaceuticals, and in particular relates to an improved transaminase, its coding gene and a genetically engineered bacterium expressing the enzyme. Background technique [0002] (R)-(+)-1-p-methylphenethylamine, as a drug intermediate, has been widely used in organic synthesis and drug synthesis. Under normal temperature and pressure, (R)-(+)-1-p-methylphenethylamine is a colorless liquid, soluble in organic solvents such as ethanol and ethyl acetate, and its structural formula is as follows: [0003] [0004] The currently announced synthetic method of (R)-(+)-1-p-methylphenethylamine is mainly chemical synthesis, and one is to utilize a chiral resolution reagent for racemic 1-(4-tolyl) Ethylamine was resolved, and then the chirally pure target compound was obtained by separation and purification (Tetrahedron: Asymmetry, 2003, 14, 2683–2685). The final yield of this method was low (49.25%), and the chiral...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/10C12N15/54C12N1/21C12R1/19
CPCC12N9/1096
Inventor 韦平和高新星彭加平张鑫
Owner 盐城仁越生物科技有限公司
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