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Omega-transaminase from bacillus pumilus and application in biological amination

A transaminase, amino technology, applied in the field of bioengineering

Active Publication Date: 2019-03-01
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, there are no reports on the coding sequence and preparation method of Bacillus pumilus ω-transaminase at home and abroad

Method used

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  • Omega-transaminase from bacillus pumilus and application in biological amination
  • Omega-transaminase from bacillus pumilus and application in biological amination
  • Omega-transaminase from bacillus pumilus and application in biological amination

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1 Obtaining of Bacillus pumilus ω-transaminase gene

[0052] Firstly, the whole gene DNA of Bacillus pumilus W3 was extracted, and specific primers were designed according to the ω-transaminase gene sequence.

[0053] Using the whole gene DNA of Bacillus pumilus W3 as a template, and using FWD-ota and REW-ota as primers for PCR, a 915bp product fragment was obtained. After the product was sequenced and compared with NCBI, it was found that it was related to the Bacillus pumilus W3ω-transaminase gene The sequence of (Sequence ID: MH196528) is consistent, and the sequence ID is expected to be published on NCBI in May 2019.

[0054] Wherein, design primer FWD-ota (sequence as shown in SEQ ID NO.4) and REW-ota (sequence as shown in SEQ ID NO.5), utilize PCR to the DNA coding frame 5' and 3' of Bacillus pumilus W3ω-transaminase XhoI and PstI restriction enzyme sites (without signal peptide) are respectively introduced on both sides, the underline is the restriction ...

Embodiment 2

[0070] The predicted crystal structure of Bacillus pumilus W3ω-transaminase and its amino acid similarity with Arthrobacter sp.KNK 168ω-transaminase were obtained by "homology modeling".

[0071] The full length of the Bacillus pumilus ω-transaminase gene ota3 is 915bp, and the predicted open reading frame of the new protein is located at 30-915 nucleotides, encoding 304 amino acid residues, and the molecular weight is 33.4kDa.

[0072] Submit the amino acid sequence of Bacillus pumilus ω-transaminase to the SWISS-MODEL protein online modeling server (http: / / swissmodel.expasy.org / ) for homology modeling, and then use Discovery studio software to analyze the homology of Bacillus pumilus ω-transaminase protein Modeling structural analysis (see figure 2 ): The template for homology modeling of this protein is 5e25.1.A, and the sequence homology similarity with the template is 51.21%.

[0073] The ω-transaminase gene sequence of Bacillus pumilus obtained from the screened Bacill...

Embodiment 3

[0074] Example 3 Construction of Bacillus pumilus ω-transaminase prokaryotic expression vector, recombinant expression and protein expression thereof

[0075] 1. Construction of prokaryotic expression vector

[0076] (1) Primer design: design primers from the mature peptide sequence after the signal peptide (as shown in SEQ ID NO: 4 and SEQ ID NO: 5)

[0077] (2) PCR reaction, using the cloning vector pMD-19T-ota3 as a template, annealing at 62°C, 35 cycles.

[0078] (3) The PCR product of ota3 and the plasmid pColdⅡ were double-digested with XhoI and PstI.

[0079] Table 3 Double Enzyme Digestion System

[0080] Element

Usage amount

Purification of PCR products / plasmids

30μl

10*quitcut buffer

5μl

QuitCut SnaBI

1μl

Quit Cut Not I

1μl

wxya 2 o

13μl

total capacity

50μl

[0081] Digest at 37℃ for 2hr

[0082] (4) Connect, transform, and carry out double-enzyme digestion identification (see ...

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Abstract

The invention discloses Omega-transaminase from bacillus pumilus and application in biological amination, and belongs to the technical field of bioengineering. In the invention, an Omega-transaminasegene is separated and identified from bacillus pumilus for the first time, and by adopting an escherichia coli recombination expression system to produce codon optimized Omega-transaminase, the yieldcan be remarkably increased, and the requirements of industrial application are met more easily. The invention discovers that the most proper action pH of the Omega-transaminase is 7.0 and the most proper action temperature is 45 DEG C when (R)-Alpha-phenethylamine serves as a substrate; meanwhile, the R-Omega-transaminase has excellent pH stability and temperature stability, and the catalytic activity on the experimental group (taking (R)-Alpha-phenethylamine as a substrate) is higher than that on the control group (taking (S)-Alpha-phenethylamine as a substrate), indicating that the recombinant Omega-transaminase has a function of synthesizing R-chiral amine with perfect selectivity and has relatively great application potential.

Description

technical field [0001] The invention relates to an omega-transaminase from Bacillus pumilus and its application in biological amination, belonging to the technical field of bioengineering. Background technique [0002] Aminotransferase (transaminase) belongs to the class of transferases and is usually used to catalyze the transfer of an amino group from an amino donor compound to an amino acceptor compound. It is ubiquitous in animals, plant tissues and many microorganisms. Among them, animal tissues such as myocardium, brain, liver, and kidney, as well as mung bean sprouts have relatively high levels of transaminases. The increase of transaminase content in the human body often leads to obesity and metabolic syndrome, eventually leading to the formation of fatty liver. Transaminase participates in the decomposition and synthesis of amino acids in biocatalytic reactions. It catalyzes the transfer of the α-amino group of an amino acid to the ketone group of another α-keto a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P13/00C12P13/04C12N9/10C12N15/54C12R1/19
CPCC12N9/1096C12P13/001C12P13/04
Inventor 廖祥儒翟李欣赖英杰杨邵岚蔡宇杰管政兵
Owner JIANGNAN UNIV
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