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Aspergillus niger strain bfa010-7 with high production of L-lactone hydrolase and its application in the preparation of d-pantolactone

The technology of pantolactone and Aspergillus niger strain is applied in the field of bioengineering, and can solve the problems of undisclosed mycelial cell immobilization method, difficulty in separation and recovery of immobilized cells, and high viscosity of reaction liquid, and achieves simple and feasible technology. , shorten the production cycle, the effect of high fermentation yield

Active Publication Date: 2021-04-13
SUZHOU BAIFUAN ENZYME TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, when the immobilized mycelium cells are applied, the reaction solution has high viscosity and poor mass transfer, and after a long period of use, the separation and recovery of the immobilized cells will be difficult due to the breakage of the mycelium
Patent CN 101343627A discloses a method for producing D-pantolactonase by solid fermentation of Fusarium moniliforme, which solves the influence of agitation and shear force on mycelium in the liquid fermentation process, but the patent does not disclose the mycelium cells Immobilization method

Method used

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  • Aspergillus niger strain bfa010-7 with high production of L-lactone hydrolase and its application in the preparation of d-pantolactone
  • Aspergillus niger strain bfa010-7 with high production of L-lactone hydrolase and its application in the preparation of d-pantolactone
  • Aspergillus niger strain bfa010-7 with high production of L-lactone hydrolase and its application in the preparation of d-pantolactone

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 Isolation, screening and identification of Aspergillus niger strain BFA010

[0042] More than 200 soil samples were collected in different locations and environments in Shanghai, Jiangsu, Zhejiang, Shandong, Guangxi and other provinces and cities. When collecting soil samples, the surface soil was shoveled, and deep soil samples were collected. The collected soil samples were used within 10 days. Screening of D-pantolactone-producing strains.

[0043] About 100 mg of soil samples were taken, added to 3 ml of enriched medium, incubated at 30°C, 200 rpm, and shaken for 3 days, and the enriched culture solution was detected by liquid chromatography for the enzymatic hydrolysis product pantothenic acid. The enriched culture solution with significant pantoic acid generation was taken and 100 μl of the bacterial solution was spread on a PDA solid medium plate containing calcium carbonate and pantoic acid lactone, and cultured at 30 °C for 3 days to isolate the Asper...

Embodiment 2

[0050] Example 2 Molecular identification of Aspergillus niger species

[0051] Genomic DNA of Aspergillus niger BFA010 was extracted by secondary precipitation method. The Aspergillus niger BFA010 obtained in Example 1 was inoculated into a liquid rich medium and cultured for 2 days, the mycelium was collected by centrifugation, washed twice with deionized water, and the water was sucked dry. Transfer 1 g of mycelium to a pre-cooled mortar, add liquid nitrogen to freeze, and grind it into a fine powder with a pestle. The mycelium powder was quickly transferred to a 20 ml centrifuge tube, 10 ml of SDS-EB buffer (10 mM Tris-HCl, 0.1 mM EDTA, 4% SDS, pH 8.0) was added, and the mixture was vortexed to mix well. The mixture was incubated in a water bath at 65°C for 20 min, and centrifuged at 10,000 rpm for 15 min. Transfer 8ml of supernatant to a new centrifuge tube, add 0.5ml of potassium acetate buffer (8M, pH 4.2), mix well, ice bath for 45min, centrifuge at 10000rpm for 20mi...

Embodiment 3

[0056] Example 3 Plasma mutation breeding of Aspergillus niger BFA010

[0057] Aspergillus niger BFA010 was inoculated on the slant of solid medium and cultured at 30°C for 48h. Add 1 ml of sterile water to the slanted solid medium, and scrape the spores of Aspergillus niger into the sterile water with an inoculating loop. Dilute the spore suspension to about 10 7 Spores / ml suspension, placed in a plasma box, and mutagenesis was performed. The working gas is high-purity helium, the irradiation power is 40W, the irradiation distance is 4mm, the plasma temperature is less than 30°C, the gas flow rate is 10L / min, the bacterial solution is 10μL, and the treatment time is 40s.

[0058] The mutagenized spore suspension was spread on a solid rich medium plate containing 20 g / L pantolactone and 5 g / L calcium carbonate, and cultured at 30° C. for 48 h, and colonies with obvious clear circles were selected. According to the method described in Example 1, the isolated Aspergillus nige...

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Abstract

The invention discloses a black mold BFA010-7 with high production of D-pantolactonase obtained through soil screening and low-temperature plasma mutagenesis, the preservation number is CGMCC 14631, and the above-mentioned Aspergillus niger strain BFA010-7 is prepared Application of D‑pantotolactone. In the present invention, the Aspergillus niger mycelium is grown and propagated in the sponge block by embedding, and then the immobilized cell catalyst is prepared by a chemical cross-linking method. The whole process is simple, easy, cheap and practical, and easy to scale up. The prepared immobilized cell catalyst has the advantages of high activity, good operational stability, easy separation and recovery, etc., and has a good application prospect in the industrial production process of calcium D-pantothenate.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and more specifically relates to soil screening and mutation breeding of a high-yield D-pantolactonase strain, fermentation culture and cell immobilization of the mutant strain, and biohydrolysis of immobilized cells. Application in the preparation of optically active D-pantolactone. Background technique [0002] D-pantothenolactone is an important chiral intermediate for the synthesis of D-panthenol, D-pantothenic acid and D-calcium pantothenate. D-pantoate lactonase is used to preferentially catalyze the hydrolysis of D-pantoate lactone in the racemic substrate, and the hydrolyzed D-pantoate is collected and converted into D with high optical purity after acidification and heating. - Pantolactone. The D-lactone hydrolase catalyst with high activity, high selectivity and high stability is the core technical element for efficient and low-cost production of D-pantolactone and its derivati...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/14C12N9/18C12N11/093C12P17/04C12R1/685
CPCC12N1/14C12N9/18C12N11/08C12P17/04C12N1/145C12R2001/685
Inventor 潘江许建和郑高伟张志钧钱小龙赵朋戴忆思
Owner SUZHOU BAIFUAN ENZYME TECH
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