Breeding of Yeast Strain Producing Nitrilase and Its Application in Biotransformation of Nitrile Compounds

A nitrilase and yeast strain technology, applied in the field of industrial biology, can solve problems such as yeast strains that have not yet been seen, and achieve the effects of outstanding nitrile transformation ability, low production cost, and increased accumulation

Active Publication Date: 2019-09-20
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, no Saccharomyces strains, especially Pichia pastoris Meyerozyma guilliermondii, have been reported to produce nitrilase for the synthesis of 3-hydroxypropionic acid

Method used

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  • Breeding of Yeast Strain Producing Nitrilase and Its Application in Biotransformation of Nitrile Compounds
  • Breeding of Yeast Strain Producing Nitrilase and Its Application in Biotransformation of Nitrile Compounds

Examples

Experimental program
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Effect test

Embodiment 1

[0034] (1) Enrichment and screening process of bacterial strains

[0035] Collect soil samples around the nitrile compound factory building. When collecting soil samples, first use a small shovel to shovel off the surface soil, and collect soil samples at a depth of 5-15cm. Take 1g of the collected soil and sludge samples, dissolve them in 10mL of 0.9% normal saline, shake fully at 30°C for 30min, and let stand; take 1mL of the soil suspension and put it into the Erlenmeyer flask containing screening medium (50mL / 250mL) was cultured in a reciprocating shaker at 120 rpm at 30°C; after 48 hours, 100 μL of turbid culture solution was spread on a solid screening medium plate, and the plate was placed in a constant temperature incubator for upside-down culture, and cultured at 30°C 48h; Pick a large and strong single colony grown on the screening medium plate, transfer to a fresh screening medium, streak and separate, and transfer for 3 to 5 generations.

[0036] Carry out re-scre...

Embodiment 2

[0049] This example illustrates the method of culturing CGMCC No.12935 inserted into the fermentation medium to obtain bacteria with high activity and high biomass, and biotransforming 3-hydroxypropionitrile to produce 3-hydroxypropionic acid.

[0050] The composition of the fermentation medium (g / L) is: yeast powder 7.5g, peptone 15g, NaCl 1g, KH 2 PO 4 2g, K 2 HPO 4 2g, glycerol 15g, 3-hydroxypropionitrile 1g, pH 7.2;

[0051] Insert CGMCC No.12935 into the medium with 3% inoculum amount, culture at 30°C, 120rpm reciprocating shaker for 36h, centrifuge at 12000rpm for 5min to collect the cultured bacteria, wash the bacteria with 100mM, pH7.2 sodium phosphate buffer cells 2 to 3 times, and resuspend the bacteria in the buffer.

[0052] Mix the substrate solution with the bacterial suspension, and make the final concentration of the substrate 300mM, control the culture conditions at 30°C, 120rpm in a reciprocating shaker, and carry out the transformation reaction. After 3...

Embodiment 3

[0054] Collect the bacteria from the culture medium by centrifugation, wash the bacteria with 100mM, pH7.2 sodium phosphate buffer 2 to 3 times, resuspend to make the bacteria suspension, mix the substrate solution and the bacteria suspension, and make The final concentration of the substrate was 400mM, and the culture conditions were controlled. The transformation reaction was carried out in a reciprocating shaker at 30°C and 120rpm. After 41 hours, the substrate was completely transformed, and the cumulative concentration of 3-hydroxypropionic acid detected at 21.5 hours was the highest, which was 52.90mM .

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Abstract

The invention belongs to the technical field of industrial biology, and in particular relates to a screening method for nitrilase-producing yeast and its application in the biotransformation of nitrile compounds to prepare 3-hydroxypropionic acid. The present invention provides a method for screening yeast producing nitrilase. For the first time, a yeast strain 3H2-2 capable of transforming 3-hydroxypropionitrile into 3-hydroxypropionic acid has been screened, combined with morphology, physiological and biochemical characteristics and 18S and ITS sequencing analysis, it was identified as Pichia guilliermondii (Meyerozyma guilliermondii). The strain has been deposited in the General Microorganism Center of China Committee for Culture Collection of Microorganisms (preservation number CGMCC No. 12935). Adding 3% glucose as an energy source, using the free cells of the strain to carry out transformation experiments on 3-hydroxypropionitrile, can completely convert 500mM 3-hydroxypropionitrile, and the cumulative concentration of 3-hydroxypropionate can reach 216.3mM. Pichia mongolica CGMCC 12935 has the characteristics of strong vitality and outstanding nitrile conversion ability, and a new process has been established for the synthesis of 3-hydroxypropionic acid. This process has the characteristics of mild reaction conditions, low energy consumption, and environmental friendliness.

Description

technical field [0001] The invention belongs to the technical field of industrial biology, and in particular relates to a screening method for nitrilase-producing yeast and its application in the biotransformation of nitrile compounds to prepare 3-hydroxypropionic acid. Background technique [0002] Nitrilase can convert nitrile groups in nitrile substrates into carboxyl groups to prepare organic acids, amino acids and other compounds. It has important applications in the fields of bulk chemicals, food additives and pharmaceutical intermediates. Since Robinson et al. first isolated a ricinine nitrilase-producing Pseudomonas strain from soil in 1964, nitrilase from different sources has been extensively studied. Japanese scholars used Rhodococcus rhodochrous J1 Produces nitrilase, which can hydrolyze a variety of nitrile compounds to prepare organic acids. Since then, nitrilase-producing bacteria from different sources have gradually been reported, including Rhodococcus genu...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/16C12N1/02C12P7/42C12R1/645
CPCC12N1/02C12N1/16C12P7/42C12N1/145C12R2001/645
Inventor 许正宏史劲松张强龚劲松李恒窦文芳
Owner JIANGNAN UNIV
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