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Esterase with racemic phenyl glycidyl ester split activity, encoding gene and application thereof

A technology for encoding glycidyl ester and esterase, which is applied in the field of enzyme engineering and can solve the problems of loss of target optical product and reduction of product optical activity.

Inactive Publication Date: 2012-04-18
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This non-absolutely selective enzyme can easily result in reduced optical activity of the product or loss of the desired optical product

Method used

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  • Esterase with racemic phenyl glycidyl ester split activity, encoding gene and application thereof
  • Esterase with racemic phenyl glycidyl ester split activity, encoding gene and application thereof
  • Esterase with racemic phenyl glycidyl ester split activity, encoding gene and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] The acquisition of the esterase MHest gene of embodiment 1 of the present invention

[0035] (1) Establishment and screening of genomic plasmid library of Microbacterium liquefaciens

[0036] Purchase the strain amp tube of Microbacterium liquefaciens 1.1934 at the China Common Microorganism Culture Collection and Management Center, break the mouth of the tube and add 1mL liquid nutrient gravy agar medium (peptone 10g / L, beef extract 3g / L L, NaCl 5g / L, distilled water 1L, pH7.0), and then the suspension of the bacterial strain was inserted into 100mL of the same medium for culture, the shaker condition was 220 rpm, 30°C, 48 hours. After the cultivation was completed, the bacteria were collected by centrifugation at 12000 rpm.

[0037] Genomic DNA from Microbacteria liquefaction was purified and extracted using a bacterial genome extraction kit (Shanghai Sangong), and the operation method was carried out according to the instructions provided by the kit. The genomic DN...

Embodiment 2

[0049] Embodiment 2 recombinant protein expression and purification

[0050] The constructed plasmid pETMHest was introduced into Escherichia coli E.coil BL21(DE3) by electrotransformation method to obtain transformant E.coil BL21(pETMHest). Inoculate the transformant into a test tube of LB liquid medium (containing kanamycin), culture overnight at 37°C, and transfer to 400 mL of LB liquid medium (containing kanamycin) at a transfer rate of 1%. , cultured at 37°C, and when the OD value was 0.6-0.8, IPTG with a final concentration of 1 mM was added for induction culture for 3 hours, and the cells were collected by centrifugation. Suspend the bacteria in the binding buffer (50mM TrisHCl, 20mM imidazole, 50mMNaCl, pH8.0,), perform ultrasonic disruption (300W, ultrasonic 3 seconds, 1 second interval, 90 cycles in total), centrifuge at 14000RPM, and collect the supernatant solution, the supernatant was added to 1mL nickel affinity column (Novogen), combined with 5mL wash buffer (5...

Embodiment 3

[0052] The immobilization of embodiment 3 esterase

[0053] The purified protein was immobilized by sodium alginate method. The immobilization method was as follows. Add 0.3 g of sodium alginate (purchased from Bailingwei Company) to 10 mL of water and heat it to 121 ° C to completely dissolve it. Cool to 30°C, add esterase dissolved in pH 7.0 phosphate buffer solution, the concentration of the esterase in the sodium alginate solution is 4g / L, stir the mixture slightly to mix, carefully inject the mixture with a syringe Drop into ice-cold 0.1mol / L CaCl 2 1.5mol / L boric acid solution, the sodium alginate and CaCl 2 The molar ratio of the immobilized enzyme was controlled at 23:10, and the immobilized enzyme was suspended in the above boric acid solution for 3 hours, keeping the temperature at 4°C. After immobilization, the immobilized enzyme was collected by filtration, washed with distilled water and stored in a refrigerator at 4°C.

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Abstract

The invention provides an esterase MHest derived from microbacterium liquefaciens and an encoding gene thereof and also provides the application of the esterase in hydrolyzing and splitting racemic phenyl glycidyl ester. The esterase of the invention is a protein of the (a) or the (b): (a) the protein consisting of amino acid residue sequences of SEQ ID NO:1 in a sequence table; and (b) the protein which is formed by substituting and / or deleting and / or adding one or more amino acid residues of the SEQ ID NO:1 in the sequence table, has racemic phenyl glycidyl ester split activity and is derived from the SEQ ID NO:1. (2R, 3S)-phenyl glycidyl ester with the optical purity of 99.6 percent can be obtained by splitting the racemic phenyl glycidyl ester by using the esterase, and the yield is over 42 percent.

Description

technical field [0001] The invention belongs to the field of enzyme engineering, and specifically relates to an esterase with the activity of splitting racemate phenyl glycidyl ester, its encoding gene, and the application of the esterase. Background technique [0002] At present, resource, energy and environmental crises have threatened the survival and development of human beings. Biotransformation uses microorganisms or enzymes as catalysts to replace non-renewable resources with renewable resources, and is an effective means for large-scale production of chemicals, medicines, energy, materials, etc. needed by humans. [0003] (2R,3S)-Phenyl glycidyl ester is an important intermediate in the synthesis of anticancer drug paclitaxel and cardiovascular drug diltiazem. Currently, methods for synthesizing chiral (2R, 3S)-phenylglycidyl esters are mainly divided into chemical synthesis, chiral auxiliary co-crystallization and biological enzymatic conversion. The chemical meth...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/16C12N15/55C12N15/63C12N5/10C12N1/15C12N1/19C12N1/21C12P41/00
Inventor 王建军吴胜
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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