Preparation method for archaea thermophilic esterase and (S)-ketoprofen

An archaea and esterase technology, applied in the field of bioengineering, to achieve the effect of high protein purity and good acid resistance

Inactive Publication Date: 2013-07-10
ZHENGZHOU UNIVERSITY OF LIGHT INDUSTRY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Most commercial lipases are enzymes at medium temperature, and the reaction pH is also around neutral. There is ...

Method used

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  • Preparation method for archaea thermophilic esterase and (S)-ketoprofen
  • Preparation method for archaea thermophilic esterase and (S)-ketoprofen
  • Preparation method for archaea thermophilic esterase and (S)-ketoprofen

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Embodiment Construction

[0018] An archaeal thermophilic esterase with the following DNA sequence (ORF: TM1160, gene sequence number NC_000853.1):

[0019] atgttaatca aaagctttgc aatcgagagt aatatattaa aagataatcc tttaaaagat 60

[0020] cccaatgtta gaaaagtcta ttctatatagag gttggagatg taactcaagc tccaatagta 120

[0021] atttatttga gtggattttt ctcttcctct attacactct taaattatga tcctttatca 180

[0022] gaatctataa atgaaaaact agatagatta cataaagaag ggaaaatcaa gaatttaata 240

[0023] tttatacttc cagatctttt tacaaaatta ggtgggaatc aatacataaa ttcaacagct 300

[0024] gtaggaatgt acgaggattt tattataaaa gaattaatcc catatctttt tgatatatat 360

[0025] ggaaagaagg atgtaatatt aatgggaaag tcttctggtg gttacggttc aatagtttta 420

[0026]gctatgaaat atccagaaat aatacgcggc ctaataaatc attctggaga ttcttatttt 480

[0027] gaatatgttt acttacctta ttttcctcat gctattagac atataaggag agaaggtgga 540

[0028] cttaataagt ggttggaaaa gtactggagt aaggaaaata agaaggataa agatgatctt 600

[0029] aatacattaa atattattgc catggccgca ttttatccc cagaagggac agaaattgt...

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Abstract

The invention discloses a preparation method for archaea thermophilic esterase and (S)-ketoprofen. The method comprises preparation of a strain, a plasmid, enzymes and a culture medium, PCR amplification and construction of recombinant plasmid, and inducible expression of a gene and purification of protein. Specifically, the method comprises the following steps: (1) designing a pair of primers, carrying out amplification with a whole genome sequence of Thermotogamaritima as a template, carrying out enzyme digestion with endonuclease, connecting the primers to pET15b which has undergone enzyme digestion by same endonuclease, transforming ligation productsinto escherichia coli DH5 alpha, carrying out screening to obtain a recombinant plasmid and carrying out double digestion and sequencing confirmation; and (2) carrying out inducible expression of the gene and purification of the protein so as to obtain the archaea thermophilic esterase. According to the invention, through usage of a genetic engineering method, the gene of the esterase is cloned and expressed in escherichia coli, the protein of the esterase is prepared, and the protein has a high purity (more than 95%). The optimal reaction temperature and the reaction pH value of the esterase are 70 DEG C and 5.0 to 5.5, respectively; the esterase has good acid resistance and can maintain about 50% of its activity when treated for 60 min in a reaction system where the temperature is 70 DEG C and the pH value is 4.5.

Description

technical field [0001] The invention designs an archaeal thermophilic esterase and a method for preparing (S)-ketoprofen, belonging to the technical field of bioengineering. Background technique [0002] 2-Arylpropionic acid drugs (2-APA) belong to non-steroidal anti-inflammatory analgesics (NSAIDS). a class of drugs. There are 3 commonly used ibuprofen, naproxen and ketoprofen, and there is a chiral carbon atom in its α position, and there is a pair of optical enantiomers in (S)-configuration and (R)-configuration. Supplied in the form of racemate. Among them, the anti-inflammatory and analgesic effect of ketoprofen is 150 times that of aspirin, and the antipyretic effect is 100 times that of aspirin. It is clinically used to treat chronic rheumatoid arthritis, degenerative arthritis, trauma and postoperative pain. Pharmacological experiments show that the anti-inflammatory and analgesic effect of its (S)-isomer is 2 times that of racemic ketoprofen, and the activity of ...

Claims

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Application Information

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IPC IPC(8): C12N15/55C12N15/70C12N9/16C12P7/40
Inventor 魏涛张飞冯昕孙浩姜春鹏
Owner ZHENGZHOU UNIVERSITY OF LIGHT INDUSTRY
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