Gene sequence and amino acid constitution of (D)-specific carbonyl reduction enzyme of Morganella morganii proteus

A technology of carbonyl reductase and proteus, applied in the field of bioengineering, can solve the problems of blank genome sequence and few studies

Inactive Publication Date: 2009-07-22
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] At present, some stereoselective oxidoreductase genes have been obtained by genetic engineering, but for the stereoselective oxidoreductase gen...

Method used

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  • Gene sequence and amino acid constitution of (D)-specific carbonyl reduction enzyme of Morganella morganii proteus
  • Gene sequence and amino acid constitution of (D)-specific carbonyl reduction enzyme of Morganella morganii proteus
  • Gene sequence and amino acid constitution of (D)-specific carbonyl reduction enzyme of Morganella morganii proteus

Examples

Experimental program
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Embodiment 1

[0016] Example 1: The (D)-specific carbonyl reductase of M. morganii CMCC (B) 49208 was obtained by conventional protein separation and purification methods. Take a certain amount of enzyme solution and add 0.2mol / L pH 7.5 phosphate buffer, 150mg / L MAK, 100μmol / L glucose, 10mol / L NADH, 15U / mL glucose dehydrogenase, make up to 1mL with double distilled water, and incubate at 30℃ Reaction for 10 hours, HPLC (Agilent 1100 serices chromatograph, ZORBAX SB C18 column, G1314A ultraviolet detector, column temperature: 40°C, detection wavelength: 220nm, injection volume: 10μL, mobile phase: methanol: 0.02mol / L diphosphate Potassium hydrogen buffer: acetic acid: triethylamine=4:96:0.2:0.13 (v:v:v:v)). The detection result shows that more than 89% of the MAK in the reaction solution is converted into d-pseudoephedrine.

[0017] Using the MADLI-TOF-MS method to obtain the peptide fingerprint of the carbonyl asymmetric reductase, such as figure 1 shown. NCBI used MASCOT to identify the...

Embodiment 2

[0028] Embodiment 2: Get the upper and lower two parts ("IAIHDTTL" and "IATYVAAD") nucleotide sequence design and merge primers:

[0029] The upstream primer Z1 is: 5'-ATHGCNATHCAYGAYACNAC-3';

[0030] The downstream primer Z1 is: 5'-GCNGCNACRTANGTNGCDAT-3'

[0031] PCR amplification was performed on the bacterial genome of M. morganii CMCC(B)49208. Reaction conditions: pre-denaturation at 95°C for 5 minutes, denaturation at 95°C for 30 seconds, annealing at 53°C for 45 seconds, extension at 72°C for 1 minute, and extension at 72°C for 10 minutes after 30 cycles to obtain a gene fragment of about 900 bp. Reclaim the specificity fragment about 900bp, determine this gene fragment sequence, after this section sequence is compared with the database published above the NCBI website, obtain the leucine dehydrogenase gene ( leucine dehydrogenase LeuDH gene).

[0032] Several gene sequences with high homology were selected and their conserved sequences were analyzed to design prime...

Embodiment 3

[0039] Example 3: After the extracted plasmid pKK223 was digested with PstI, it was ligated with the same digested target gene fragment and then transformed into E.coli JM109 competent cells, coated with LB plates containing ampicillin, randomly picked transformants, and extracted Recombinant plasmid, PstI enzyme digestion to verify that the recombinant plasmid inserted into the target gene is then digested with EcoRI to verify that the recombinant plasmid with the forward connection of the target gene is obtained, and the recombinant bacteria containing the recombinant plasmid are further verified for enzyme activity.

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Abstract

The invention discloses the gene sequence and the amino acid composition of a D-specific carbonyl reductase of Morganella morganii with the CMCC(B) number of 49208, and belongs to the technical field of biological engineering. The invention determines that the D-specific carbonyl reductase of the Morganella morganii with the CMCC(B) number of 49208 has a gene nucleotide sequence with the SEQ ID NO of 1 and the amino acid composition with the SEQ ID NO of 2, the D-specific carbonyl reductase gene is used for decomposing recemic compounds with biological methods, and by utilizing the gene, a recombinant Morganella morganii bacteria can be used for racemic chiral separation and creating further research conditions of diversity, transforming ways and reaction mechanisms of a stereoselective microbial oxidoreductase that is used in chiral separation.

Description

technical field [0001] Gene sequence and amino acid composition of (D)-carbonyl reductase of Morganella morganii CMCC(B)49208, the (D)-specific carbonyl reductase gene is used for biological resolution of racemic A compound belongs to the technical field of bioengineering. Background technique [0002] Optically pure d-pseudoephedrine is a multifunctional and multipurpose drug in medicine. It is mainly used clinically for the treatment of bronchial asthma, allergic reactions, hypotension and nasal congestion caused by nasal mucosal congestion and swelling. Because the ephedrine molecule contains composite functional groups, it is often used in organic synthesis to develop novel drugs. Therefore, it is very meaningful to carry out the research of ephedrine resolution method. [0003] The preparation of optically pure chiral compounds by biological transformation has the advantages of mild reaction conditions, single product, high stereoselectivity, regioselectivity and che...

Claims

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Application Information

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IPC IPC(8): C12N9/02C12N15/53
Inventor 石贵阳张梁丁重阳张鹏华卢燕王正祥
Owner JIANGNAN UNIV
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