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A kind of biological preparation method of p-nitrobenzyl alcohol malonate monoester

A technology for microorganisms and amino acids, applied in the field of preparing p-nitrobenzyl alcohol malonate monoester, can solve the problems of uneconomical, low yield, etc., and achieves reducing the generation of amide by-product III, improving the yield, realizing effects of industrialization

Active Publication Date: 2020-08-28
TAIZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the low yield of the target product, compound I, this method is not economical

Method used

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  • A kind of biological preparation method of p-nitrobenzyl alcohol malonate monoester
  • A kind of biological preparation method of p-nitrobenzyl alcohol malonate monoester
  • A kind of biological preparation method of p-nitrobenzyl alcohol malonate monoester

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] The construction of embodiment 1 nitrilase SEQ ID NO:1 expression strain

[0053] The gene sequence encoding nitrilase SEQ ID NO:1 was synthesized from the whole gene, and connected with the vector plasmid pET21a (purchased from Novagen) through Nde I and Xho I double restriction sites to obtain a recombinant plasmid. The Escherichia coli DH5α competent was transformed by calcium chloride method, and the genetic engineering plasmid with correct DNA sequence and capable of expressing nitrilase was obtained after culturing and extracting the plasmid. The genetic engineering plasmid constructed above was transformed into competent Escherichia coli BL21(DE3) by the calcium chloride method, coated on an LA plate, placed in a constant temperature incubator at 37°C, and cultured upside down overnight. On the next day, pick 3-5 positive clones from the LA plate, and perform IPTG-induced enzyme production test on the positive clones. For the positive identification above, it is...

Embodiment 2

[0054] The construction of embodiment 2 amidase SEQ ID NO:2 expression strain

[0055] The gene sequence encoding amidase SEQ ID NO: 2 was synthesized from the whole gene, and connected with the vector plasmid pET21a (purchased from Novagen) through Nde I and Xho I double restriction sites to obtain a recombinant plasmid. The Escherichia coli DH5α competent was transformed by calcium chloride method, and the genetic engineering plasmid with correct DNA sequence and amidase expression was obtained after culturing and extracting the plasmid. The genetic engineering plasmid constructed above was transformed into competent Escherichia coli BL21(DE3) by the calcium chloride method, coated on an LA plate, placed in a constant temperature incubator at 37°C, and cultured upside down overnight. On the next day, pick 3-5 positive clones from the LA plate, and perform IPTG-induced enzyme production test on the positive clones. For the positive identification above, it is the engineering...

Embodiment 3

[0056] Embodiment 3 Escherichia coli engineering strain shake flask fermentation

[0057] 3.1 The nitrilase-producing Escherichia coli engineered strain obtained in Example 1 was inoculated into LB medium, and placed on a constant temperature shaker at 37° C. at 200 rpm for shaking flask culture overnight. The next day, add IPTG to a final concentration of 0.5mmol / L, and induce at 30°C and 200rpm for 6-8 hours. Subsequently, the cells containing nitrilase were collected by centrifugation at 4° C. (6000 rpm, 5 min). Wash the cells containing nitrilase twice with 0.9% sodium chloride solution. Discard the sodium chloride solution for the last wash, and store the cells containing nitrilase in a -80°C refrigerator.

[0058] 3.2 Inoculate the amidase-producing Escherichia coli engineered strain obtained in Example 2 into LB medium, and place it on a constant temperature shaker at 37° C. at 200 rpm for shaking flask culture overnight. The next day, add IPTG to a final concentrati...

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Abstract

The invention provides a method for preparing p-nitrobenzyl alcohol malonate monoester by double-enzyme catalysis, comprising the steps of: using 2-cyanoacetic acid-(4-nitrophenyl) methyl ester as a substrate, using It is obtained through combined catalysis of nitrilase derived from Alcaligenes faecalis and amidase derived from Alcaligenes faecalis. The method of the invention has high product yield, few by-products, mild reaction conditions and little environmental pollution, and is suitable for industrialized production.

Description

technical field [0001] The invention belongs to the technical field of biocatalysis, and in particular relates to a method for preparing p-nitrobenzyl malonate monoester through combined catalysis of nitrilase and amidase. Background technique [0002] The compound shown in formula I p-nitrobenzyl alcohol malonate (or called p-nitrobenzyl alcohol malonate) is a multipurpose pharmaceutical intermediate, such as an important raw material for the preparation of meropenem. The English name is Mono-4-nitrobenzyl Malonate, and the CAS number is 77359-11-6. It has a high market demand and a wide range of uses. [0003] [0004] The preparation method of p-nitrobenzyl malonate is mainly chemical synthesis. The chemical synthesis method, for example, uses malonic acid and p-nitrobenzyl alcohol as raw materials, and is prepared through steps such as esterification, alkali-acid adjustment and refining (see invention patent US5087734). However, the chemical synthesis method consume...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P13/00
CPCC12P13/008
Inventor 杨仲毅钟永军何昕蔚罗希蒋晶晶余达勇
Owner TAIZHOU UNIV
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