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Amidase variant with improved specific activity and application thereof

An amidase and amidase-describing technology, applied in the field of molecular biology, can solve the problems of large amount of enzyme added and high production cost

Pending Publication Date: 2022-03-04
HEC PHARM
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, in the process of catalyzing amidase ketoprofen to (S)-(+)-ketoprofen, the enzyme activity is only 1.1U / mg {Hirrlinger B, Stolz A, Knackmuss HJ.Purification and properties of an amidase from Rhodococcus erythropolis MP50 whichenantioselectively hydrolyzes 2-arylpropionamides.J Bacteriol.1996; 178(12):3501-3507.}, which makes the amount of enzyme added in the catalytic reaction larger and the production cost high

Method used

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  • Amidase variant with improved specific activity and application thereof
  • Amidase variant with improved specific activity and application thereof
  • Amidase variant with improved specific activity and application thereof

Examples

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preparation example Construction

[0084]The present invention provides a method of preparing an amide enzyme variant, and an amide enzyme variant is expressed in E. coli, an enzyme-catalytic reaction is performed using E. coli, wherein the pathogenic pathway is used to catalyze the ketone Loven synthesis:

[0085]

Embodiment 1

[0087] Example 1 Construction of Expression strains of amidase AMD03 E. coli

[0088] According to Rhodococcus Erythropolis MP50 amide AMD03 amino acid sequence, the sequence is shown in SEQ ID NO: 1. Optimization is optimized in accordance with E. coli codons, and sequences are shown in SEQ ID NO: 2. An RBS site is added in front of the amide enzyme AMD03 nucleotide sequence, and the selected RBS sequence is submitted as aaagaggagaaa, and the SUMCE is submitted for synthesis.

[0089] Then, by gene recombination technique, the above-described synthetic nucleotide sequence was cloned to the XBAI of the PET28A vector, and PET28A-AMD03 plasmid, plasmid spectrum figure 1 Indicated. PET28A-AMD03 plasmid transforms BL21 (DE3) induced state cells, conversion of LB + KAN plate (10 g / L protein, 5g / L yeast powder, 10 g / l sodium chloride, 50 μg / ml kazza) Mythromycin, 15g / L agar powder), 37 ° C overnight culture. The resulting transformation was named BL21-AMD03 strain after the PC...

Embodiment 2

[0090] Example 2 induced expression of amidase AMD03

[0091] The BL21-AMD03 strain freezed -80 ° C-80 ° C was separated from the LB + KAN flat scribe line. Picking the single bacteria on the conversion plate to 5 mL liquid LB + KAN (10 g / L protein, 5g / L yeast powder, 10 g / l sodium chloride, 50 μg / ml kanamycin) medium, 37 ° C, 250g / Overnight culture under min, about 12 h.

[0092] Take overnightly activated bacterial liquid, 2ml transfer to fresh 200ml liquid LB + KAN, cultured under 37 ° C, 250 g / min until OD 600 The value reached 0.5-0.8.

[0093] The cultured bacterial solution was taken, and the final concentration of 1 mM IPTG, 30 ° C, 250 g / min, and cultured under the conditions of 5 h.

[0094] After the end of the induction, the total bacterial liquid was collected at 4000 g / min, 4 ° C. Take appropriate bacteria to perform ultrasonic breakage treatment, centrifugation of 10 min at 4 ° C, and take the supernatant to detect protein expression by SDS-PAGE, suc...

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Abstract

The invention provides an amidase variant with improved specific activity. The amidase variant is obtained by amidase from Rhodococcus erythropolis MP50 through point mutation, and the amidase variant has the advantages that the specific activity of the amidase variant is improved, and the specific activity of the amidase variant is improved. The invention also relates to application of the enzyme in preparation of dexketoprofen by a biological enzyme catalysis technology. When the amidase variant disclosed by the invention is used for catalytically producing dexketoprofen, the substrate conversion rate is high, and the e.e. Value of a product is gt; and the enzyme input amount is small, so that the cost of producing dexketoprofen by a biological enzyme synthesis method is effectively reduced.

Description

Technical field [0001] The present invention relates to the field of molecular biology, and more particularly to an amide enzyme variant of enzyme activity and its application in the preparation of right spin lofffen in biomease catalytic techniques. Background technique [0002] Ketofen is 2-aryl propamine non-steroidal anti-inflammatory drug (NSAIDS), and such drugs have a chiral center. Only the right rotor has anti-inflammatory anti-rheumatoid and analgesic effect, and the left-rotor is almost no Pharmacological activity and poisonous side effects {He Fengci, etc. Research progress in ketofen and its chiral enantiomers [J]. Chinese Pharmacy, 2004,15 (4): 244-246; Mei Zhi South, etc. Pharmacokinetics and pharmacodynamics in Fen [J]. Chinese Journal of New Drugs, 1998,7 (005): 339-341}. [0003] Currently, commercially available (S) - (+)-ketone Loven main synthetic methods have traditional chemical synthesis, chiral splitting and biological enzyme complex. [0004] The chemica...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/80C12N15/55C12N15/70C12N1/21C12P7/40C12R1/19C12R1/125C12R1/10C12R1/07C12R1/84C12R1/865C12R1/885C12R1/685C12R1/69
CPCC12N9/80C12N15/70C12P7/40
Inventor 王小龙杨燕花胡盛本鲍素敏屈代鑫封海生
Owner HEC PHARM
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