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Gene for coding penicillium chrysogenum phenylacetic acid hydroxylase and its use

A technology of phenylacetic acid hydroxylase activity and Penicillium chrysogenum, applied in the direction of enzymes, biochemical equipment and methods, using vectors to introduce foreign genetic material, etc., can solve problems such as the decline of phenylacetic acid oxidation ability

Inactive Publication Date: 2007-06-13
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

Examples

Experimental program
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Effect test

Embodiment 1

[0031] [Example 1]: pahB gene cloning

[0032] Culture the Penicillium chrysogenum Wis54-1255 (ATCC28089) in YPD (1% yeast extract, 2% peptone, 2% glucose) medium for 24 hours, filter and collect the hyphae, and take 0.1 g of wet hyphae N 2 Grind, add 1ml Trizol reagent (Invitrogen, 15596-026) to extract the total RNA of Penicillium chrysogenum, dissolve the total RNA of Penicillium chrysogenum in DEPC water, and store it at -70°C for use.

[0033] In order to remove possible DNA contamination, take 5μg of total RNA from Penicillium chrysogenum, add 5μl RQ1 RNase-Free DNase (Promega, M6101) and 10μl RQ1 RNase-FreeDNase reaction buffer (10×) into the 100μl reaction system. Incubate at 37°C for 30 minutes, extract with phenol:chloroform, precipitate with ethanol, and dissolve in 10μl DEPC water. Take 5 μl of RNA sample from which DNA has been removed, and perform reverse transcription with random primers (RandomHexamers, Promega, C1181) to obtain cDNA. The reverse transcriptase used...

Embodiment 2

[0037] [Example 2]: The expression of pahB gene is induced by phenylacetic acid

[0038] Press 2×10 Penicillium chrysogenum Wis54-1255 (ATCC28089) 6 Conidia / ml were inoculated into the seed medium and cultured at 26°C for 24 hours. The seed medium formula 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; pH 5.8. Then, the seed culture was inoculated into the fermentation medium with an inoculum of 5%, and cultivated 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; pH6.5.

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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 a new isolated polynucleotide, in particular to a new gene encoding phenylacetate hydroxylase (pahB) from Penicillium chrysogenum. [0002] The invention also relates to the polypeptide encoded by the gene. [0003] The 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 penicillin and cephalosporins are the most important antibacterial drugs, occupying 2 / 3 of the world's anti-infective drug market, and are currently known antibiotics with the lowest toxicity, the most varieties, and the most clinical applications. A broad class of antibiotics. [0006] Penicillin is produced by the filamentous fungus Penicillium chrysogenum. Due to the broad market demand, people have put a lot of energy into the improvement ...

Claims

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Application Information

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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