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Method for preparing nucleases P1 by using fossilized penicillium citrinum

A Penicillium citrinum, nuclease technology, applied in biochemical equipment and methods, microorganism-based methods, immobilized on or in inorganic carriers, etc. Complex, carrier recovery loss and other problems, to shorten the fermentation cycle, improve production intensity and equipment utilization, improve the effect of transmission capacity

Active Publication Date: 2019-06-07
江苏省生产力促进中心 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The technical problem to be solved by the present invention is to provide a novel immobilized carrier of Penicillium citrinum, and apply it to the fermentation of immobilized Penicillium citrinum to prepare nuclease P1, so as to solve the immobilization process of Penicillium citrinum in the prior art It is more complicated, and the loss of carrier recovery is large, which limits the number of times the particles can be reused and is difficult to separate and purify.

Method used

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  • Method for preparing nucleases P1 by using fossilized penicillium citrinum
  • Method for preparing nucleases P1 by using fossilized penicillium citrinum
  • Method for preparing nucleases P1 by using fossilized penicillium citrinum

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Preparation of Example 1 Porous Network Carrier Material

[0059] Carry out corresponding chemical modification to the selected porous mesh material, said chemical modification comprises the following steps:

[0060] 1. Chemical modification of polyurethane foam sponge immobilization material: get polyurethane foam sponge 20g, drop in the sodium hydroxide aqueous solution of 2L epichlorohydrin (the mass fraction of epichlorohydrin and sodium hydroxide is respectively 10%, 5% ), stirred at room temperature for 12h, then washed to neutrality with deionized water to obtain a hydroxylated polyurethane foam sponge; the obtained hydroxylated polyurethane foam sponge was dropped into 1.5L mass fraction of 12% polyethyleneimine aqueous solution, After stirring and soaking for 8 hours, wash with deionized water until no polyethyleneimine is adsorbed on the surface to obtain a polyurethane foam sponge immobilized material with primary amine grafted arms.

[0061] 2. Chemical mod...

Embodiment 2

[0065] Example 2 The immobilized fermentation production of nuclease P1 in a high-throughput shaker

[0066] The soft fiber filler was chemically modified according to the method described in Example 1, and the carrier before and after the modification was used for immobilized fermentation of Penicillium citrinum in a high-throughput shaker at a filling rate of 4-6 g / L.

[0067] Cultivate the Penicillium citrinum strain on the wort slope at 28-30°C for 5-7 days for activation, wash the Penicillium citrinum slope fully with sterile water to obtain a spore suspension, and transfer it to a liquid fermentation medium ( Components: glucose 50g / L, peptone 5g / L, potassium dihydrogen phosphate 0.04g / L, dipotassium hydrogen phosphate 0.04g / L, magnesium sulfate 0.05g / L, calcium chloride 0.05g / L, zinc sulfate 0.05g / L, pH5-7) and immobilized carrier in a high-flux shaker reactor at 28-30°C, 200rpm, 0.05-0.5m 3 Repeated batches of fermentation culture of Penicillium citrinum were carried...

Embodiment 3

[0068] The immobilized fermentation production of nuclease P1 in the mechanical stirring tank of embodiment 3

[0069] Chemically modify the biologically active filler according to the method described in Example 1, fill it in an NBS tank with a filling capacity of 6 to 10 g / L, and carry out the immobilized fermentation test of Penicillium citrinum. The fixation method is as follows: figure 1 shown.

[0070] The preparation process of Penicillium citrinum seeds and the fermentation medium were the same as those in the high-throughput shaker fermentation experiment. During the fermentation process of the NBS tank, the rotation speed is 200rpm, and the ventilation volume is 0.2-1.0m 3 / h, pH 5-7, culture temperature 28-32°C, liquid volume 50%. Monitor the residual sugar and enzyme activity of the fermentation broth. When the activity of nuclease P1 in the fermentation broth no longer increases or decreases, remove all the fermentation broth and add the same volume of fresh med...

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Abstract

The invention discloses a method for preparing nucleases P1 by using fossilized penicillium citrinum. A porous reticular material after chemical modification is used as a fossilizing medium, a fossilizing method suitable for different fermentation vessels is provided, and the method is applied to fossilized fermentation and production of the nucleases P1. A carrier material used for immobilized cells is subjected to the corresponding process of chemical modification, when being used as the fossilizing carrier, the carrier material has the characteristics of being stable, good in biocompatibility, high in mass transfer efficiency and repeatable in use, production and application of repeated batch fermentation of the penicillium citrinum in various fermentation vessels can be realized, the fermentation period is greatly shortened, besides, sugar consumption and the production cost are reduced, and the production rate of the nucleases P1 and the utilization rate of the equipment are greatly improved.

Description

technical field [0001] The invention belongs to the technical field of industrial biology, and in particular relates to a method for preparing nuclease P1 by fermenting immobilized Penicillium citrinum. Background technique [0002] Nuclease P1 is an enzyme preparation with important application value. Its main catalytic properties are to cleave 3',5'-phosphodiester bonds in RNA and DNA, generate 5'-nucleotides and decompose single nucleotides in low temperature environment And the 3'-phosphomonoester bond in oligonucleotides is an essential raw material in the industrial production of nucleotides. The production of nuclease P1 usually adopts solid or liquid fermentation. Solid fermentation is simple and low cost, but it occupies a large area and has low production efficiency, and the conidia produced are easy to pollute the environment. Submerged fermentation avoids the above disadvantages, but the cost is higher. [0003] With the rapid development of immobilization tec...

Claims

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Application Information

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IPC IPC(8): C12N9/22C12N11/14C12N11/08C08J9/40C08J9/42C08J7/12D06M14/00C12R1/80C08L75/04C08L23/06
Inventor 杨艳红陈勇赵南刘庆国曹治邹亚男刘桂文朱杰
Owner 江苏省生产力促进中心
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