Gene for coding nitrilase of alcaligenes and method for preparing single enantiomorph of mandelic acid using same

A technology for nitrilase and alcaligenes, which is applied in the field of base sequences encoding nitrilase, can solve the problems of poor stability, low catalytic efficiency, low enzyme yield and the like, and achieves excellent effects.

Inactive Publication Date: 2010-05-05
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the main problems of the methods reported above are: the yield of enzymes in wild strains is very low, the catalytic activity is not high, the stability of enzymes is poor, and they cannot tolerate high concentrations of substrates, so their overall catalytic efficiency is not good. High, difficult to apply to mass production of mandelic acid

Method used

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  • Gene for coding nitrilase of alcaligenes and method for preparing single enantiomorph of mandelic acid using same
  • Gene for coding nitrilase of alcaligenes and method for preparing single enantiomorph of mandelic acid using same
  • Gene for coding nitrilase of alcaligenes and method for preparing single enantiomorph of mandelic acid using same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Cultivation of Alcaligenes sp.ECU0401

[0034] Alcaligenes sp.ECU0401 was cultured in a liquid medium with the following composition at 30° C. and 180 rpm for 48 hours with shaking to prepare cells expressing nitrilase activity.

[0035] Culture Medium Composition

[0036] Glycerin: 10g / L; peptone: 15g / L; yeast extract: 8g / L; KH 2 PO 4 : 4g / L; NaCl: 2g / L; MgSO 4 : 0.2g / L; FeSO 4 : 0.05g / L; pH 7.0.

[0037] The fermentation broth was centrifuged at 12,000×g for 10 min to recover the cells, washed twice with saline, and stored in a refrigerator at 4°C for future use.

Embodiment 2

[0038] Example 2 Preparation of Alcaligenes Genomic DNA

[0039] After activation, the bacteria cells Alcaligenes sp.ECU0401 were inoculated in 1.5ml fermentation medium, cultured at 30°C for 12 hours, and the cells were collected by centrifugation (12000rpm, 3min). The cells were washed twice with TE solution (10000 rpm, centrifuged for 3 min), and resuspended in 300 μl TE solution. Add 200 μl of 10% SDS and 5 μl of 20 mg / ml proteinase K, mix well, and incubate at 30° C. for 2 hours. After the bacterial cells burst, a large amount of polysaccharides are released, add a mixture of 500μl 5mol / L sodium chloride and 500μl CTAB / NaCl solution, invert the centrifuge tube up and down, mix well, incubate at 55°C for 10min, then take it out and mix again , and incubated at 55°C for another 10min. Cool the mixture in an ice bath, add an equal volume of phenol chloroform (phenol: chloroform: isoamyl alcohol = 25: 24: 1) to extract twice, centrifuge at 12000 rpm for 10 min. Take the su...

Embodiment 3

[0040] Example 3 Preparation of Genomic DNA Library of Alcaligenes sp.ECU0401

[0041] The genomic DNA obtained in Example 2 was completely digested with the restriction endonuclease Sau I, separated by agarose gel electrophoresis, segmented according to the length of the DNA, and the DNA fragments above 2 kb were collected, and the 5' end was restricted. The endonuclease Sau I digested and dephosphorylated vector pUC18 was ligated, and the resulting plasmid library was transformed into Escherichia coli DH5α, and the transformants were spread on LB agar plates containing ampicillin (100 μg / ml) and kept at 37°C. cultured to form single colonies.

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Abstract

The invention relates to a base sequence for coding nitrilase of alcaligenes, relating to a carrier comprising the base sequence and a transformant comprising the base sequence or the carrier, wherein the carrier comprises the base sequence, or a composite unit connecting the base sequence with a corresponding signal sequence. The invention also relates to an amino acid sequence coded by the base sequence, and further relates to a technical method for preparing single enantiomorph of mandelic acid and analogue thereof from corresponding mandelonitrile by utilizing the recombinant expression nitrilase as catalyst.

Description

technical field [0001] The present invention relates to a base sequence encoding nitrilase, a plasmid containing the base sequence, and a transformant transformed from the plasmid. The present invention also relates to a genetically engineered recombinant nitrilase that can catalyze the efficient and enantioselective hydrolysis of mandelonitrile into (R)-(-)-mandelic acid, and use this enzyme to prepare (R) from the corresponding mandelonitrile -Technical methods of (-)-mandelic acid and its analogues. The present invention also relates to a method for preparing (R)-(-)-mandelic acid and its analogs using the transformant. The present invention also relates to the preparation of corresponding (R)-(-)-mandelic acid single enantiomers and their analogs by utilizing nitrilase transformants, cultures thereof or processed products thereof to catalyze the asymmetric hydrolysis of mandelonitrile Methods. technical background [0002] Mandelic acid, or α-hydroxyphenylacetic acid,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/55C12N9/78C12N15/63C12N1/00C12P7/42C12R1/05
Inventor 许建和张志钧刘俊峰潘江
Owner EAST CHINA UNIV OF SCI & TECH
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