Gene for coding penicillium chrysogenum phenylacetic acid hydroxylase and its use

A technology for the activity and encoding of phenylacetic acid hydroxylase, which is applied in the fields of enzymes, biochemical equipment and methods, and the introduction of foreign genetic material using vectors, etc.

Inactive Publication Date: 2010-11-17
NORTH CHINA PHARMA COMPANY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Traditional mutagenesis and isolation and screening resulted in a significant decrease in the phenylacetic acid oxidation ability of the enzyme in high-yield Penicillium chrysogenum, and an increase in the penicillin production capacity

Method used

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  • Gene for coding penicillium chrysogenum phenylacetic acid hydroxylase and its use
  • Gene for coding penicillium chrysogenum phenylacetic acid hydroxylase and its use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] [Example 1]: pahB gene cloning

[0033] Cultivate Penicillium chrysogenum Wis54-1255 (ATCC28089) in YPD (1% yeast extract, 2% peptone, 2% glucose) medium for 24h, filter and collect mycelium, take 0.1g of wet mycelium liquid N 2 After grinding, add 1ml Trizol reagent (Invitrogen, 15596-026) to extract the total RNA of Penicillium chrysogenum, dissolve the obtained total RNA of Penicillium chrysogenum in DEPC water, and store it at -70°C for future use.

[0034] To remove possible DNA contamination, take 5 μg of total RNA from Penicillium chrysogenum, add 5 μl RQ1 RNase-Free DNase (Promega, M6101), 10 μl RQ1 RNase-FreeDNase reaction buffer (10×), and add 5 μl RQ1 RNase-Free DNase reaction buffer (10×) to the 100 μl reaction system. Incubate at ℃ for 30 minutes, extract with phenol: chloroform, precipitate with ethanol, dissolve in 10 μl DEPC water. Take 5 μl of the DNA-depleted RNA sample and perform reverse transcription with random primers (RandomHexamers, Promega, C1...

Embodiment 2

[0038] [Example 2]: The expression of pahB gene is induced by phenylacetic acid

[0039] Penicillium chrysogenum Wis54-1255 (ATCC28089) was pressed 2×10 6 Conidia / ml was inoculated into the seed medium and cultured at 26°C for 24 hours. The formulation of the seed medium was (g / l): glucose, 30; lactose, 10; cottonseed meal, 10; yeast powder, 10; corn pulp, 10; (NH 4 ) 2 SO 4 , 2; KH 2 PO 4 , 1; CaCO 3 , 5; pH5.8. The seed culture was then inoculated into the fermentation medium at a 5% inoculum size and cultured at 26°C. The fermentation medium is (g / l): lactose, 120; cottonseed meal, 30; corn steep liquor, 20; (NH 4 ) 2 SO 4 , 5; KH 2 PO 4 ,1;K 2 SO 4 , 5; CaCO 3 , 10; pH 6.5.

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Abstract

This invention involves the new encoding hydroxylase gene (pahB) from Penicillium chrysogenum, the gene encoding the peptide and containing the gene expression vector. It also demonstrated that the phenyl acetic acid can significantly induce the expression of pahB. The invention also provides a direction and target for increasing the production of penicillin from genitic modified Penicillium Chrysogenum, and is of great importance to the production of penicillin.

Description

technical field [0001] The present invention relates to novel isolated polynucleotides, in particular to a novel gene (pahB) encoding phenylacetate hydroxylase from Penicillium chrysogenum. [0002] The present invention also relates to the polypeptide encoded by the gene. [0003] The present invention also relates to an expression vector containing the gene. [0004] The invention also provides the application of the new gene in increasing the penicillin production of Penicillium chrysogenum. Background technique [0005] β-lactam antibiotics represented by penicillins and cephalosporins, as the most important antibacterial drugs, occupy 2 / 3 of the world's anti-infective drug market. A broad class of antibiotics. [0006] Penicillin is produced by the filamentous fungus Penicillium chrysogenum. Due to the large market demand, people have invested a lot of energy in the improvement of penicillin-producing bacteria. Based on traditional mutagenesis screening techniques,...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/52C12N9/00C12N15/63C12P37/00
Inventor 王富强任志红刘静戴梦郑桂珍王泽建赵颖王红怡贾茜贺建功
Owner NORTH CHINA PHARMA COMPANY
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