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New biosynthesis method for preparing deoxy-ribonucleoside triphosphate

A technology for deoxyribonucleoside triphosphate and biosynthesis, which can be applied to fermentation and other directions, can solve problems such as high price, and achieve the effects of easily available and cheap substrates, low process cost, and environmentally friendly process reactions.

Pending Publication Date: 2020-06-30
南京曼蕊生物科技有限公司
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AI Technical Summary

Problems solved by technology

dNTP reagents require high purity and no nuclease pollution. At this stage, most of the domestic dNTP reagent raw materials come from abroad and are expensive.

Method used

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Examples

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Comparison scheme
Effect test

Embodiment Construction

[0011] Application of Penicillium citrinum fermentation to produce phosphodiesterase technology.

[0012] Using the purchased Penicillium citrinum ST817 as the starting strain, a 5'-phosphodiester strain was obtained by ultraviolet (UV), nitrosoguanidine (NTG), high temperature treatment and diethyl sulfate (DES) mutagenesis P.citrinum YB-1, a high-producing strain of enzyme, has a shake flask fermentation enzyme activity of 693U / mL, which is 2.5 times higher than that of the original strain. The genetic stability experiment confirmed that the enzyme production performance of strain YB-1 was stable. The single factor experiment was used to optimize the fermentation medium and culture conditions of the strain P.citrinum YB-1, and the optimal fermentation medium composition was determined as (g / L): glucose 60; peptone 6; KH_2PO_4 0.4; K_2HP_O40.6 MgSO_4·7H_2O 0.3; CaCl_2 0.2; MnSO_4 0.15; The determined optimal culture conditions are: initial pH 5.5, culture temperature 30℃, shake ...

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PUM

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Abstract

The invention relates to a preparation method for artificially synthesizing a necessary DNA raw material, and belongs to the technical field of biosynthesis and biocatalysis of important chemicals. Brand-new enzyme hydrolysis and enzyme catalysis processes are adopted, mackerel sperm DNA is selected and used as a raw material, the mackerel sperm DNA is hydrolyzed by phosphodiesterase to generate adNMP mixture, ion-exchange chromatographic separation and purification are performed, a dNTP mixture is synthesized by whole-cell catalysis of active yeasts, then four high-purity monomeric compoundsare prepared by preparative chromatographic separation and purification, and compounding is performed to form an equimolar dNTP biochemical reagent. The process is environment-friendly in reaction, efficient, low in cost, suitable for industrial production, and extremely competitive, and the product purity reaches 99.9% or above.

Description

Technical field [0001] The invention relates to a brand-new biosynthesis method using enzymatic hydrolysis and yeast catalysis. The substrate is easy to obtain and cheap, the process reaction is green and environmentally friendly and efficient, the product purity is over 99.9%, the overall process cost is low, and it can be industrialized and is highly competitive. force. Background technique [0002] dNTP is the abbreviation of deoxyribonucleoside triphosphate, including dATP, dGTP, dTTP and dCTP. It is an indispensable biochemical reagent for PCR amplification, DNA sequencing, molecular labeling, and cDNA synthesis. The demand is increasing. dNTP reagents require high purity and no nuclease contamination. At this stage, most of the domestic dNTP reagents are sourced from abroad and are expensive. The process route of this project is to use mackerel sperm DNA as the raw material, hydrolyze with phosphodiesterase to generate dNMP (dAMP, dGMP, dTMP, dCMP) mixture, after separati...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/30C12P19/32
CPCC12P19/305C12P19/32
Inventor 张天时张先文
Owner 南京曼蕊生物科技有限公司
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