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Method for preparing R-o-chloromandelic acid by using nitrilase engineering bacteria

A technology of o-chloromandelic acid and nitrilase, applied in the field of preparing R-o-chloromandelic acid, can solve problems such as low activity, and achieve the effects of lowering price, lowering production cost and high reaction conversion rate

Active Publication Date: 2017-06-16
枣庄市杰诺生物酶有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
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Problems solved by technology

Zhang Chensheng screened the enantioselective nitrilase LaN from Stappiaaggregata from Stappiaaggregata through traditional genome prospecting technology, and the ee value of the product (R)-(-)-o-chloromandelic acid was 96.5%. but low energy

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  • Method for preparing R-o-chloromandelic acid by using nitrilase engineering bacteria
  • Method for preparing R-o-chloromandelic acid by using nitrilase engineering bacteria
  • Method for preparing R-o-chloromandelic acid by using nitrilase engineering bacteria

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Embodiment Construction

[0033] Embodiments of the present invention will be described in detail below in conjunction with examples, but those skilled in the art will understand that the following examples are only for illustrating the present invention, and should not be considered as limiting the scope of the present invention. Those who do not indicate the specific conditions in the examples are carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used were not indicated by the manufacturer, and they were all conventional products that could be purchased from the market.

[0034] 1.1 Gene mining of new nitrilases

[0035] 1.1.1 Sequence screening

[0036] Using the amino acid sequence of nitrilase (bll6402, NP_773042) from Bradyrhizobium japonicum USDA110 as a template, the potential hydrolysis of mandelonitrile was searched by BLASTP (http: / / www.ncbi.nlm.nih.gov / blast / Blast.cgi) program enzyme. We set up three criteria...

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Abstract

The invention relates to the field of biotechnology and especially relates to a method for preparing R-o-chloromandelic acid by using nitrilase engineering bacteria. The method comprises carrying out centrifugation on a culture liquid obtained by fermental cultivation of engineering bacteria containing recombinant nitrilase genes, carrying out a conversion reaction process on o-chloromandelonitrile as a substrate and a buffer solution as a reaction medium in the presence of wet bacteria as catalysts, and after the reaction, carrying out separation purification on the reaction product mixed liquid to obtain R-o-chloromandelic acid, wherein the engineering bacteria containing recombinant nitrilase genes are constructed by nucleotide sequence coding genes shown in the formula SEQ ID NO. 2. The method has a fast reaction rate and a high reaction conversion rate.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for preparing R-o-chloromandelic acid by utilizing nitrilase engineering bacteria. Background technique [0002] (R)-(-)-o-chloromandelic acid is a widely used pharmaceutical intermediate and fine chemical, mainly used in the synthesis of the anti-platelet aggregation drug clopidogrel. As a global blockbuster drug, clopidogrel had global sales of nearly US$10 billion in 2010, and sales of more than US$9 billion in 2011 and 2012. With the expiry of the patent of clopidogrel on May 17, 2012, there was a wave of imitation in the world. And (R)-(-)-o-chloromandelic acid as its important intermediate also rises accordingly, and the demand at home and abroad continues to grow, so how to obtain high-concentration, high-purity (R)-(-)-o-chloromandelic acid Mandelic acid will have important market significance. [0003] The preparation of (R)-(-)-o-chloromandelic acid by nitrilase...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P41/00C12P7/42C12N15/55
CPCC12N9/78C12P7/42C12P41/00C12Y305/05001
Inventor 许向阳何秋霞楚杰刘可春宋在伟韩利文
Owner 枣庄市杰诺生物酶有限公司
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