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Engineering bacterium, construction thereof and method for preparing D-valine by using the same

A construction method and technology for engineering bacteria, applied in the field of protein engineering, can solve the problems of production scale limitation, easy to be contaminated by bacteriophage, etc., and achieve the effects of low production cost, avoiding oxidation, and improving transformation efficiency.

Inactive Publication Date: 2008-09-10
SHANXI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, natural strains such as Agrobacterium are easily contaminated by phages during the fermentation process and need to add methylthioethylhydantoin inducers that cause environmental pollution, so their production scale is greatly limited

Method used

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  • Engineering bacterium, construction thereof and method for preparing D-valine by using the same
  • Engineering bacterium, construction thereof and method for preparing D-valine by using the same
  • Engineering bacterium, construction thereof and method for preparing D-valine by using the same

Examples

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Embodiment 1

[0021] Example 1: Construction of recombinant plasmids and engineered bacteria

[0022] Using the genomic DNA of the strain Pseudomonas putia YZ26 preserved in our laboratory as a template, the primers 5'-CCCGAA TTC CAT ATG TCC CTG TTG ATC CGT-3' and 5'-CCC GGA TCC TCA GCG CTG AACTGG-3' were used to carry out PCR amplification. The PCR product was recovered by 1% agarose gel electrophoresis, digested with Nde I / BamH I, and then connected to the similarly digested vector pET-28a to obtain a recombinant plasmid pET28a-hyd (Figure 2), which was transformed into E. coli DH5α competent cells, single colony PCR screening positive recombinants, Nde I / BamH I double digestion to identify the size of the insert, and finally confirmed by DNA sequence analysis.

[0023] In order to preserve the genome DNA template of Sinorhizobium sp.SS-ori in our laboratory, primers 5'-GGGGA TCC ATG ACA CGT CAG AAG ATA C-3' and 5'-GGG AAG CTT TCA GAA TTC CGC GATCA-3 'Proceed to PCR amplification. The ...

Embodiment 2

[0025] Example 2: Activity detection of engineering bacteria

[0026] According to the catalytic reactivity of the enzyme and the structural properties of the product D-amino acid, OD can be determined by color using the ninhydrin method 570nm The light absorption values ​​were used to calculate the amount of product formed and the enzymatic activity. It can also be directly measured by HPLC method.

[0027] 1. Ninhydrin chromogenic method

[0028] After centrifuging 200 μL of bacterial liquid, discard the supernatant, wash the obtained bacterial cells once with 100 mmol / L PBS (pH 8.0), and resuspend the bacterial cells with 1 mL of 25 mmol / L isopropyl hydantoin dissolved in the same buffer. Suspend with 100 mmol / L PBS, shake at 37 °C for 1 h, centrifuge at 13000 r / min for 10 min, take 400 μL of the reaction solution and add an equal volume of 2 mol / L NaAc buffer (pH 5.5), incubate at 60 °C for 5 min, and then add 400 μL ninhydrin (58mg reduced ninhydrin, 58mg hydrated ninh...

Embodiment 3

[0036] Example 3: Using engineering bacteria E.coli BL21 (DE3) / (pET28a-hyd, pQE30-cab) to transform 5'-isopropyl hydantoin to produce D-valine

[0037] According to Example 1, a single colony was picked from the plate and inoculated in 5 mL LB (containing 50 μg / mL Amp and 40 μg / mL Kan) liquid medium at 37° C. for 10-12 h. The pre-cultured bacteria were transferred to fresh 1L LB liquid culture solution containing antibiotics Amp (50 μg / mL) and Kan (40 μg / mL) at 2% inoculum, and shaken at 37°C until the bacterial concentration reached OD. 600nm When ≈ 1.2, IPTG was added to the final concentration of 0.2 mmol / L, cultured at 20°C for 10-12 h, and the cells were collected by centrifugation.

[0038] Prepare 400mL 50mmol / L 5'-isopropyl hydantoin solution with deionized water, adjust the pH to 9.0 with saturated NaOH, add the collected thalline and put it into a triangular flask, and replace the oxygen in the triangular flask with nitrogen. After sealing, the reaction was shaken a...

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Abstract

The invention provides an engineering strain, which can simultaneously express D-hydantoin enzyme and N-carbamoyl-D-amino amidohydrolase. The construction method of the invention is as follows: a D-hydantoin enzyme gene is cloned to a plasmid ET-28a and an N-carbamoyl-D-amino amidohydrolase gene is cloned to a plasmid pQE-30, and then two recombinant plasmids pET28a-hyd and pQE30-cab are shifted into the same Escherichia coli cell by utilization of a co-conversion technology. The invention also provides a method for producing D-valines by utilization of the engineering strain to convert 5'-opropyl hydantoin. The method is characterized by high optical purity of products, simple manufacturing technique, and environmental protection and so on; moreover, compared with the prior market products, the method is low in production cost.

Description

technical field [0001] The invention belongs to the technical field of protein engineering. It involves artificial construction of engineering bacteria and separation and purification of enzyme-catalyzed products. Specifically, it is the construction of an engineering bacterium that can express recombinant D-hydantoinase and recombinant N-carbamoyl-D-amino acid amidohydrolase at the same time, and the transformation of the bacterium to produce D-valine. Background technique [0002] D-valine is an important organic chirality source, which is mainly used in the fields of chiral drugs and chiral additives. The insecticidal activity of pyrethroids synthesized by replacing L-valine with D-valine is increased by 5000 times, while the toxicity to animals is reduced by 10 times; D-valine is also an important raw material for the synthesis of antibacterial drugs. Such as the synthesis of intermediates and diiodophthaloyl derivatives of the antibacterial active drug dactinomycin, t...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/55C12N15/70C12P13/08C12R1/19
Inventor 钮利喜石亚伟袁静明
Owner SHANXI UNIV
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