A process for the identification and preparation of a (r)-specific omega-transaminase

A technology for transaminase and amino, which is applied in the field of identification and preparation of (R)-specific ω-transaminase, which can solve the problems of unobtainable and economically feasible methods

Inactive Publication Date: 2015-04-01
LONZA LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, there is still a need to provide other (R)-selective ω-transaminases and means and methods for obtaining them, thereby producing R-enantiomers, especially optically active amines, such as for pharmaceutical or agrochemical applications, but these have so far Still not available at all or in an economically viable manner, preferably on an industrial scale

Method used

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  • A process for the identification and preparation of a (r)-specific omega-transaminase
  • A process for the identification and preparation of a (r)-specific omega-transaminase
  • A process for the identification and preparation of a (r)-specific omega-transaminase

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Embodiment 1

[0154] Embodiment 1-identification of (R)-selective ω-transaminase

[0155] The amino acid sequence (SEQ ID no.5) of the (R)-selective ω-TA of Mycobacterium aureus given in EP 1038953A1 and the (R)-selective ω-TA of Arthrobacter given in EP 0987332A1 The amino acid sequence (SEQ ID no. 7) was used as the query biomolecule sequence. The biomolecule library used in this embodiment is the pubmed protein database of NCBI ( http: / / www.ncbi.nlm.nih.gov / pubmed (July 13, 2009).

[0156] The amino acid sequence (SEQ ID no.5 or 7) from Mycobacterium aureus or Arthrobacter ω-TA has been used as query sequence, using standard parameters (BLOSUM62 scoring matrix, word length: 3, gap penalty: presence-11, Extension-1) A BLAST search is performed to identify a first set of 100 different amino acid sequences from different organisms, all of which have a minimum of 30% sequence identity to the query sequence.

[0157] BLAST was performed on July 13, 2009 using "non-redundant protein sequen...

Embodiment 2-

[0162] Example 2- Preparation and analysis of transaminases from Aspergillus terreus, Mycobacterium vanbaalenii and Rhizobium japonicus

[0163] 2.1 ω-TA from Mycobacterium vanbaalenii (entry 9 in Table 1, SEQ ID no.49 and 50), ω-TA from Aspergillus terreus (entry 1 in Table 1, SEQ ID no.3 and 4) have been obtained , omega-TA from Rhizobium japonicum (entry 11 in Table 1, SEQ ID nos.1 and 2), and used (for Escherichia coli) in the form of codon usage (codon usage) suitable for use in Expression of suoshu enzyme in Escherichia coli. Hereinafter the Mycobacterium va nbaalenii transaminase is referred to as Mva-TA, the Aspergillus terreus transaminase as Ate-TA and the Rhizobium japonicum transaminase as Mlo-TA.

[0164] 2.2 Acetophenone test

[0165] Mva-TA and Ate-TA convert (R)-α-MBA faster than the (S)-enantiomer in the acetophenone assay using 2.5 mM amine and 2.5 mM pyruvate at pH 7.5 and 30°C At least 100 times.

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Abstract

The present invention relates to a process for the identification and preparation of a (r)-specific omega-transaminase, in particular a processes for the screening, preparation and characterization of (R)-selective omega-transaminases, to transaminases obtained thereby and their uses in various transamination processes.

Description

[0001] This application is a divisional application of the Chinese patent application with the application number 201080039297.2, the application date is August 13, 2010, and the invention title is "Method for Identifying and Preparing (R)-Specific ω-Transaminase". technical field [0002] The present invention relates to methods for screening, characterizing and preparing (R)-specific ω-transaminases, the obtained ω-transaminases and their applications in various transamination processes. Background technique [0003] Chiral amines play important roles in the pharmaceutical, agrochemical, and chemical industries. They are often used as intermediates or synthones to prepare various physiologically, eg pharmaceutically active substances, such as cephalosporin or pyrrolidine derivatives. Among the many applications of chiral amines, only one specific optically active form (the (R) or (S) enantiomer) is physiologically active. Therefore, there is a need to provide a method for...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P13/00C40B50/06G16B30/10
CPCC12N9/1096C12P13/001G16B30/00G16B30/10C12N9/10C12N9/88
Inventor 马蒂亚斯·赫内乌韦·博恩朔伊尔卡伦·罗宾斯塞巴斯蒂安·舍茨勒
Owner LONZA LTD
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