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A kind of nitrilase and application thereof

A nitrilase and expression vector technology, applied in the field of nitrilase, can solve problems such as low activity, and achieve the effects of lowering prices, lowering energy consumption and three-waste emissions, and having broad application prospects

Active Publication Date: 2020-10-09
枣庄市杰诺生物酶有限公司
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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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  • A kind of nitrilase and application thereof
  • A kind of nitrilase and application thereof
  • A kind of nitrilase and application thereof

Examples

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Embodiment Construction

[0027] 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.

[0028] 1.1 Gene mining of new nitrilases

[0029] 1.1.1 Sequence screening

[0030] 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 present invention relates to the technical field of biology, particularly to a nitrilase and applications thereof, wherein the amino acid sequence of the nitrilase is represented by SEQ ID NO:1. According to the present invention, the nitrilase can completely hydrolyze 600 mM substrate within 12 h, and the 112 g / L product o-chloromandelic acid is produced due to the substrate flowing addition and the dilution effect of the pH value control, wherein the reaction conversion rate in the whole reaction process is remained at above 90%, the flowing addition is stopped after the reaction is performed for 10 h, and the substrate is completely converted after the reaction is continuously performed for 2 h; and the reaction rate is rapid, and the reaction conversion rate is high.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a nitrilase and its application. 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 enzymatic method has been reported at home and abroad, but...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/78C12P41/00C12P7/42C12N15/55C12N15/70C12N1/21C12R1/19
CPCC12N9/78C12N15/70C12N2800/101C12P7/42C12P41/00C12Y305/05001
Inventor 许向阳刘可春楚杰何秋霞宋在伟栗建中
Owner 枣庄市杰诺生物酶有限公司
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