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Technical method for preparing phenylalanine hydroxylase

A technology of phenylalanine carboxylase and phenylalanine dehydrogenase, which is applied in the field of high-yield L-phenylalanine dehydrogenase, can solve the problems such as tedious and time-consuming experimental purposes of the purification method, and achieves high yield, The effect of simple production method

Inactive Publication Date: 2010-12-01
北京协和洛克生物技术有限责任公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Phenylalanine dehydrogenase is also usually purified by multi-stage column chromatography, but these purification methods are somewhat tedious and time-consuming for experimental purposes

Method used

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  • Technical method for preparing phenylalanine hydroxylase
  • Technical method for preparing phenylalanine hydroxylase
  • Technical method for preparing phenylalanine hydroxylase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Preparation of chromosomal DNA containing L-phenylalanine dehydrogenase gene

[0028] Inoculate the following media with Bacillus sphaericus: 2g / l L-phenylalanine, 5g / l K 2 HPO4, 10g / l digested protein, 1g / l NaCl and 0.5g / l MgSO 4 ·7H 2 O, pH7.0. Shake culture at 30°C for about 10 hours until OD616=1.1.

[0029] Bacteria solution was centrifuged to obtain 10g wet bacteria, and chromosomal DNA was extracted by known methods, that is, bacteria were suspended in 40ml buffer solution TEN (10mM Tris-HCl, pH 7.6, 1mM EDTA and 10mM NaCl), centrifuged, collected cell. Then the collected cells were suspended in 20ml buffer SET (20% sucrose, 50mM Tris-HCl, pH 7.6 and 50mM EDTA), 10mg lysozyme was added, and incubated at 37°C for 30min. After confirming the formation of bacterial cells under a microscope, 10 ml of buffer solution SET and 0.25 g of SDS were added to the suspension to lyse the bacterial cells. Lysates were extracted with phenol / chloroform, and DNA wa...

Embodiment 2

[0031] Embodiment 2 Chromosomal DNA is inserted in the vector

[0032] Plasmid pUC9 was used as a vector, and 10 μg of pUC9 was digested with Hind III at 37°C. Then, it was treated with calf thymophosphatase at 37°C for 2 hours, and then with ethanol for 1 hour.

[0033] The treated solution was subjected to agarose electrophoresis (1%), and the 2.7kb DNA fragment was isolated from pUC9 by electroelution method, extracted with phenol / chloroform, and then the carrier DNA was precipitated with ethanol, and dissolved in 20 μl sterile in the water.

[0034] On the other hand, as described in Example 1, 50 μg of chromosomal DNA was digested with 50u Hind III at 37°C, extracted with phenol / chloroform, and then precipitated with ethanol for 16 hours, and the obtained DNA was dissolved in 50 μl sterile water . The 0.5 μg vector DNA and 2 μg chromosomal DNA were mixed, and treated with T4 DNA ligase at 12.5° C. for 16 hours in the presence of ATP and dithiothreitol to form a recombi...

Embodiment 3

[0036] The screening of embodiment 3L-phenylalanine dehydrogenase positive bacteria

[0037] Transfect the above transformation mixture onto a nitrocellulose membrane. When each piece of nitrocellulose membrane reaches 500-1000 cloned recombinants, stick the nitrocellulose membrane on an LB plate containing 50 μg / ml ampicillin and incubate at 37°C 16 hr, followed by counterstaining with fresh nitrocellulose membranes incubated at 37°C for 3 hr.

[0038] The nitrocellulose membrane was immersed in lysate (5 mg / ml lyase, 10 mM Tris-HCl, pH 8.5, 1.5 mM NAD+, 0.8 mMINT, and 0.32 mM phenazine dimethyl sulfate), and dark red transformants were selected. One L-phenylalanine dehydrogenase-positive colony provides about 2000 ampicillin-resistant transformants. Plasmids were extracted from L-phenylalanine dehydrogenase-positive colonies, and the recombinant plasmid from pUC9 was used as a vector, which was called pBPDH1.

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Abstract

The invention belongs to the technical field of bioengineering and relates to a method for preparing recombinant L-phenylalanine hydroxylase, in particular to a method for obtaining high-yield L-phenylalanine hydroxylase by embedding a programmable microorganism source L-phenylalanine hydroxylase gene into an appropriate expression vector to establish recombinant plasmid and a transformed cell and using a simple and effective affinity method. The L-phenylalanine hydroxylase obtained by the method has high yield, high activity and simple and convenient production method. The invention also comprises the new recombinant plasmid and transformed cell established in the method. The L-phenylalanine hydroxylase prepared by the method can be used for developing a screening reagent of newborn phenylketonuria and producing L-phenylalanine.

Description

technical field [0001] The present invention relates to a method for preparing L-phenylalanine dehydrogenase, specifically, the present invention provides a method for cloning L-phenylalanine dehydrogenase gene by genetic engineering technology, constructing its expression plasmid, A method for transforming cells to obtain high-yield L-phenylalanine dehydrogenase. technical background [0002] Phenylalanine dehydrogenase was first discovered in microorganisms by Hummel et al. in 1984. After more than 20 years, many scholars have isolated phenylalanine dehydrogenase from some bacteria and actinomycetes, and produced Most of the microorganisms with phenylalanine dehydrogenase are aerobic Gram-positive bacteria, such as Brevibacterium sp., Thermoactinomyces, Rhodocaccus sp., Bacillus sp. .)Wait. [0003] Although phenylpyruvate is an ideal substrate, PheDH and phenylpyruvate analogs limit its substrate activity. Enzymatic reductive amination of phenylpyruvate is very useful ...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N15/70C12N9/06C12P13/22C12Q1/32C12R1/19C12R1/07
Inventor 王晓华
Owner 北京协和洛克生物技术有限责任公司
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