Gene for highly expressing nuclease P1 and its application

A high-efficiency expression and nuclease technology, applied in applications, genetic engineering, plant gene improvement, etc., can solve the problems of four single nucleotide separation difficulties, affect hydrolysis efficiency, increase production costs, etc., and reduce downstream separation processes , Reduce production costs, reduce the effect of by-products

Inactive Publication Date: 2010-02-24
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The nuclease P1 derived from Penicillium citrinum prepared by the fermentation method has high fermentation cost and produces more miscellaneous enzymes, which affects the hydrolysis efficiency and makes it difficult to separate the four single nucleotides in the later stage, which increases the production cost

Method used

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  • Gene for highly expressing nuclease P1 and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1: Construction of recombinant yeast.

[0023] 1. Cloning of nucP gene nucP of Penicillium citrinum nuclease P1.

[0024] (1) Extraction of total RNA from Penicillium citrinum.

[0025] Take 50ml Penicillium citrinum liquid, 4°C, 8000°C r·min -1 Centrifuge for 15 minutes to collect the bacteria, pre-cool the mortar with liquid nitrogen, grind it into powder while adding liquid nitrogen, add 3 to 5 times Trizol reagent; put it at room temperature for 15 minutes, add 1 / 5V chloroform, mix with Votex for 10 seconds, and room temperature for 10 minutes , 4°C, centrifuge at 1200rpm, 15min; remove the supernatant and aliquot 500-600μL, add an equal volume of isopropanol, mix up and down 5 times, let stand for 10min, 12000g, centrifuge at 4°C, 15min; pour the supernatant, absorb water Blot dry with paper, add 1mL of 75% ethanol to wash the precipitate, let stand for 1min, 4°C, centrifuge at 12000g for 10min; discard the supernatant, invert it on a foam plate, blow dry...

Embodiment 2

[0040] Example 2: Induced expression of recombinant Pichia strains in shake flasks.

[0041] Pick a single colony and place it in a 250mL shake flask filled with 25mL BMGY medium, at 30°C, 250-300r min -1 Grow to OD 600 to 2-6. Centrifuge at 1500-3000g for 5min at room temperature, collect the cells, and resuspend the cells with BMMY to make the OD 600 Up to about 1.0 (about 100-200mL). Put the obtained bacterial solution in a 1L shaker flask, seal it with double gauze or cheesecloth, and place it at 28-30°C, 250-300r·min -1 Continue to grow on the shaker. Add 1-5% (v / v) of 100% methanol to the medium every 24 hours. After testing, the enzyme activity reaches 625U / mL, which is 52.43% higher than that of the starting wild strain.

Embodiment 3

[0042] Example 3: High-density fermentation of recombinant Pichia strains in a 5L fermenter.

[0043] The fermentation medium contains 16mL of phosphoric acid per liter, 0.3g of calcium phosphate, 12g of potassium sulfate, 15g of magnesium sulfate, 3.0g of potassium hydroxide, and 27g of glycerin. The fermentation temperature is 28°C, pH 6.0, and dissolved oxygen is controlled at 5LPM. Fermentation is divided into two stages. The first stage is the cell growth stage. It is inoculated and grown in the above-mentioned medium at 10% (v / v). The first stage is cultivated for 16 hours. When the glycerin is exhausted, the dissolved oxygen rises rapidly At this time, turn to the proliferation stage, feed the inducer methanol, the adding volume of the inducer is 3% of the medium volume, and the feeding time is 16h, so that dissolved oxygen is maintained above 5LPM, the total culture time of the proliferation stage is 48h, and the filtration time is 48h. Yeast cells were removed to obta...

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Abstract

The invention discloses a gene highly expressing a nuclease P1 and an expression vector and a host cell comprising the gene and an application for the gene. The invention discloses a recombinant pichia yeast CGMCC No.3188 expressing the gene of nuclease P1 produced from Penicillium citrinum and its construction method. The inventive recombinant engineering strain pichia yeast CGMCC No.3188 can highly express Penicillium citrinum nucP gene. The secretion-expressed nuclease P1 has high phosphodiesterase activity, is used for hydrolyzing RNA, has high product yield and little generated impurity and reduces working procedure of downstream separation as well as decreases production cost. The invention uses the recombinant engineering strain pichia yeast CGMCC No.3188 to perform high density fermentation. The enzyme activity is 1.5 times by the wild strains. The acquired recombinase is applied to RNA solution enzymatic hydrolysis, thus, reaching the hydrolysis rate of more than 40% and the acquired hydrolysate by-product is substantially reduced.

Description

technical field [0001] The invention belongs to the field of bioengineering, in particular to a gene expressing nuclease P1 and its application. Background technique [0002] 5′-nucleotides play an important role in cell structure, metabolism, energy and regulatory functions [Wang Yuqin, Cheng Wufeng, Li Xuanhai. Nucleotides and Nutrition. Foreign Medicine and Health Science Volume, 1999, 26(5): 257- 260], which are indispensable active substances in living organisms. In the food industry, it has expanded from the initial food freshness aid to a functional food additive that can improve the immune function of organisms. It can be added to foods such as bread and biscuits, especially in infant food. It can effectively enhance the ability of infants and young children to resist bacillary dysentery and reduce the occurrence of diarrhea; in the field of medicine, 5'-nucleotides can not only be used as drugs, but also the raw materials for the production of many antiviral and an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/55C12N15/63C12N15/81C12N1/19C12N9/22C12R1/84C12R1/80
Inventor 应汉杰贺沁婷许琳熊健柏建新栗西木
Owner NANJING UNIV OF TECH
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