Immobilized whole-cell catalyst and its preparation method and application

A whole-cell catalyst and fixative technology, applied in biochemical equipment and methods, botany equipment and methods, applications, etc., can solve problems such as high cost and large pollution

Inactive Publication Date: 2011-11-30
SYNCOZYMES SHANGHAI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The traditional synthesis method is to use the synthetic route of organic ch...

Method used

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  • Immobilized whole-cell catalyst and its preparation method and application
  • Immobilized whole-cell catalyst and its preparation method and application
  • Immobilized whole-cell catalyst and its preparation method and application

Examples

Experimental program
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Effect test

Embodiment 1

[0026] Preparation of whole-cell catalysts comprising D-lactate dehydrogenase from Staphylococcus epidermidis ATCC 12228 and formate dehydrogenase from Lodderomyces elongisporus NRRL YB-4239:

[0027] (1) Preparation of recombinant plasmid pACYCDuet-1-SeLDH:

[0028] Genomic DNA of Staphylococcus epidermidis was extracted according to the general method in the field ("Molecular Cloning Experiment Guide", Science Press, 3rd edition), and the D-type lactate dehydrogenase gene fragment was amplified using specific primers, and this fragment was used Restriction endonucleases NcoI and HindIII were used for double digestion, and the gene fragment was purified by agarose gel electrophoresis, ligated with pACYCDuet-1 digested by the same restriction enzyme, and transformed into E. coli host strain Top10 cells. The transformant was picked and inoculated into LB liquid medium, cultured at 37°C for 20 hours, and the recombinant plasmid was extracted with a plasmid extraction kit (Broadt...

Embodiment 2

[0041] Immobilized Whole Cell Preparation:

[0042] Dissolve 3.0 g of polyethylene glycol in 35 ml of water, add 5.0 g of polyvinyl alcohol (PVA) and raise the temperature to 90-95°C to dissolve. After the polyvinyl alcohol is completely dissolved, lower the temperature to 25°C-30°C, add 4 grams of recombinant cells (wet weight), mix well, use a disposable straw to absorb the mixture, and pour it on the plane to form a disc, and Incubate at 30°C for 1 to 1.5 hours. Transfer to 0.1M sodium sulfate solution to stabilize for 2 hours, filter dry and wash twice with water, and place in 10mM sodium phosphate buffer for use.

[0043] The cell immobilization reagent polyvinyl alcohol can also be replaced by polyacrylamide or calcium alginate with the same effect.

[0044]

Embodiment 3

[0046] Synthesis of (R)-2-hydroxy-4-phenyl-butyrate using a whole-cell catalyst comprising D-type lactate dehydrogenase and formate dehydrogenase:

[0047]Add 100 mM phosphate buffer (pH adjusted to 6.5), 40 g / L cell concentration (wet weight) whole-cell catalyst BL21(DE3) (pET21a-LeFDH, pACYCDuet-1-SeLDH) (containing Lactate dehydrogenase type D from Staphylococcus epidermidis and formate dehydrogenase from Lutheran yeast), (R)-2-hydroxy-4-phenyl-butyric acid sodium salt and 75 mM ammonium formate at a final concentration of 30 mM. The reaction mixture was stirred at 30°C for 1.5 hours. Samples were taken at regular intervals, and the amount of product formed was determined by HPLC. The conversion was greater than 99% after 1.5 hours of reaction. After the reaction was completed, the cells were recovered by centrifugation, the supernatant of the centrifuged liquid was acidified to pH 3 with 30% hydrochloric acid, extracted once with 120 ml of ethyl acetate, and then extract...

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Abstract

The invention relates to the field of biotechnology in the pharmaceutical industry, and discloses the preparation of an immobilized whole-cell catalyst and its application in the synthesis of a Puley intermediate (R)-2-hydroxy-4-phenyl-butyric acid. The recombinant cells containing the D-type lactate dehydrogenase gene and the formate dehydrogenase gene are established and fixed to prepare the immobilized whole cell catalyst. Using this catalyst to synthesize the intermediate (R)-2-hydroxy-4-phenyl-butyric acid of lisinopril, the reaction conditions are mild, the action is specific, and there are no by-products. It is used in medicine and food industries. value.

Description

technical field [0001] The invention belongs to the field of biotechnology in the pharmaceutical industry, and relates to the preparation of an immobilized whole-cell catalyst and its application in the synthesis of Pulley intermediate (R)-2-hydroxy-4-phenyl-butyric acid. Background technique [0002] Lisinopril (Lisinopril) is an antihypertensive drug with a strong angiotensin-converting enzyme inhibitory effect. The traditional synthesis method is to use the organic chemical synthesis route (US 2007 / 0093664 A1, CN 1053437 C), which causes great pollution and high cost. [0003] Coenzyme NAD used in biocatalysis + It can be used for substrate conversion, but it cannot be regenerated and needs to be supplemented with new coenzymes. Therefore, if the coenzyme can be regenerated during the reaction, the cost can be greatly reduced. Contents of the invention [0004] The invention aims to provide an immobilized whole cell catalyst and a preparation method. [0005] The pr...

Claims

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Application Information

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IPC IPC(8): C12N11/10C12N11/08C12N15/53C12N15/63C12N1/21C12P7/42C12R1/19
Inventor 梁晓亮王娟王波曾聪明孙勇文军
Owner SYNCOZYMES SHANGHAI
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