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Screening method for Fe<2+> and alpha-ketoglutaric acid-dependent hydroxylase and isoleucine hydroxylase

A technology of ketoglutarate dehydrogenase and ketoglutarate, which can be used in botany equipment and methods, biochemical equipment and methods, and other methods for inserting foreign genetic materials, and can solve the problem of high-activity hydroxylase screening methods Problems such as missing, hydroxyl compound development and application of industrial production constraints, to achieve the effect of improving catalytic efficiency, enzyme activity and thermal stability

Inactive Publication Date: 2018-09-11
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the limitation of insufficient activity of such hydroxylases, the development, application and industrial production of hydroxyl compounds are severely restricted
The lack of screening methods for highly active hydroxylases is the main bottleneck causing the above problems

Method used

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  • Screening method for Fe&lt;2+&gt; and alpha-ketoglutaric acid-dependent hydroxylase and isoleucine hydroxylase
  • Screening method for Fe&lt;2+&gt; and alpha-ketoglutaric acid-dependent hydroxylase and isoleucine hydroxylase
  • Screening method for Fe&lt;2+&gt; and alpha-ketoglutaric acid-dependent hydroxylase and isoleucine hydroxylase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Construction of α-ketoglutarate dehydrogenase coding gene deletion and isocitrate lyase gene deletion strains

[0036] (1) Construction of homology arm overlapping gene fragments for knockout

[0037]According to the nucleotide sequence of sucA shown in Sequence Listing 6, its upstream and downstream homologous sequences were designed. The upstream and downstream gene fragments were obtained by DNA polymerase chain reaction. The upstream homology arm gene fragment and the downstream homology arm gene were used as templates, and sucA-up-1 and sucA-down-2 were used as upstream and downstream primers to perform polymerase chain reaction to obtain overlapping gene fragments of the upstream and downstream homology arms. After purification by 1.0% agarose electrophoresis, it was purified with a gel cutting recovery kit. in,

[0038] sucA upstream homology arm sequence primer:

[0039] sucA-up-1: 5'TCTGGTGGAATCCCGATAAGTT 3'

[0040] sucA-up-2: 5'TTACGACGCTGTTCCTGTTTCTATCC...

Embodiment 2

[0058] Recombinant expression vector containing mutant and construction of strain containing the vector

[0059] Using the isoleucine hydroxylase coding gene ido as a template, the sequence is shown in Sequence Listing 4, and pWSK29-1 and pWSK29-2 were used as primers to perform error-prone PCR, and the product was purified after 1.0% agarose electrophoresis Afterwards, the ido gene and its mutant fragments are obtained by purifying with a gel-cutting recovery kit. Primer sequences are as follows

[0060] pWSK29-1:5'

[0061] AGGGAACAAAAGCTGGAGCTCGAAGGAGATATACAAATGAAAATGAGCGGTTTTTAGCAT 3'

[0062] pWSK29-2:5'

[0063] GGGCCCCCCCTCGAGGTCGACTTATTTGGTTTCTTTATAGCTAAAGGTCA 3'

[0064] The obtained above ido gene fragments were recombined and ligated with the pWSK29 vector after digestion with Sac I and Sal I restriction endonucleases in a ligation system containing Exnase II, and the ligated reaction product was transformed into host cell E.coli From W3110△sucA△aceA competent ...

Embodiment 3

[0066] hyperactive ido mutant ido M screening

[0067] The bacterial strain containing the mutant recombinant expression vector obtained in Example 2 was applied to solid selection medium (glucose 10g / L, MgSO 4 0.24g / L, Na 2 PO 4 ·7H 2 O 13g / L, KH 2 PO 4 2.5g / L, NH 4 SO 4 5g / L, Isoleucine 2g / L, FeSO 4 2g / L, Vc 0.2g / L, agar 20g / L, tap water 1000mL, pH 6.5-7.5) plate, cultured upside down at 37°C for 12 hours, pick 480 larger single colonies on the plate and transfer to 5 blocks containing ampicillin (100μg / mL) liquid screening medium (glucose 10g / L, MgSO 4 0.24g / L, Na 2 PO 4 ·7H 2 O 13g / L, KH 2 PO 4 2.5g / L, NH 4 SO 4 5g / L, Isoleucine 2g / L, FeSO 4 2g / L, Vc 0.2g / L, tap water 1000mL, pH 6.5-7.5) in a 96-well plate, cultured with shaking at 37°C for 24h. Determination of OD by microplate reader 600 . Select the bacterial strain with the highest OD600 and determine the growth curve, such as figure 2 shown. Extract the plasmid and its ido mutant ido M P...

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Abstract

The invention relates to a screening method of high-activity Fe<2+> and alpha-ketoglutaric acid-dependent hydroxylase. The method includes the steps of: constructing a bacterial strain with deletion of alpha-ketoglutaric acid dehydrogenase encoding gene and isocitric acid lyase encoding gene; amplifying Fe<2+> and alpha-ketoglutaric acid-dependent hydroxylase encoding gene through error-prone PCRto obtain a mutant thereof, and connecting the mutant to an expression vector; and converting the expression vector to the bacterial strain with deletion of alpha-ketoglutaric acid dehydrogenase encoding gene and isocitric acid lyase encoding gene and screening a bacterial strain, having high growth speed, on a culture medium containing alpha-ketoglutaric acid and corresponding substrates; extracting a plasmid therefrom, and amplifying a hydroxylase encoding gene mutant. The method achieves high-throughput screening on the high-activity Fe<2+> and alpha-ketoglutaric acid-dependent hydroxylaseand accelerates application process of the enzyme.

Description

technical field [0001] The invention relates to the field of biochemical industry, especially a kind of high activity Fe 2+ And α-ketoglutarate-dependent hydroxylase screening method and isoleucine hydroxylase. Background technique [0002] Hydroxyl compounds are an important class of compounds, especially amino acid hydroxyl compounds, which have attracted much attention due to their unique active functions, and are widely used in food, medicine, chemical industry, and pesticides. For example, 4-hydroxyisoleucine has the activity of promoting insulin secretion dependent on glucose concentration, enhances the absorption of blood sugar by muscle cells, promotes fat metabolism, lowers blood fat and protects liver function; 5-hydroxytryptophan has the effect of improving mild insomnia And the function of chronic stress headache; hydroxyproline has the functions of treating chronic hepatitis and preventing liver cirrhosis. [0003] A considerable part of hydroxyl compounds is ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/90C12N15/53C12N15/60C12N15/10C12N9/02
CPCC12N9/0071C12N9/22C12N9/88C12N15/1031C12N15/70C12N15/902C12N2810/10C12Y114/11C12Y401/03001C12Q2531/113
Inventor 张成林战俊杰王道安何继龙陈宁孟静李英滋朱福周李智祥赵磊徐庆阳谢希贤李燕军范晓光马倩
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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