Method for preparing 2-hydroxy 4-methylthio butyric acid using nitrilase

A technology of methylthiobutyric acid and methylthiobutyric acid ammonium salt is applied in the field of preparing 2-hydroxy-4-methylthiobutyric acid with nitrilase, which can solve the problem of insufficient microbial activity of nitrilase and the unusable issues such as industrial scale

Inactive Publication Date: 2004-12-22
ADISSEO IRELAND LIMITED IE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the method described in this document cannot be used on an industrial scale because of insufficient microbial activity to synthesize these nitrilases

Method used

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  • Method for preparing 2-hydroxy 4-methylthio butyric acid using nitrilase
  • Method for preparing 2-hydroxy 4-methylthio butyric acid using nitrilase
  • Method for preparing 2-hydroxy 4-methylthio butyric acid using nitrilase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0120] Example 1: Purification and N-terminal sequencing of nitrilase

[0121] The nitrilase was purified in four steps. A summary of the purification is shown in Table 1 below.

[0122] Alcaligenes faecalis cells were grown for 24 hours at 30°C in minimal medium containing benzonitrile (0.5 g / l). After the culture was centrifuged, the pellet was resuspended in TG buffer (25 mM Tris-HCl, 10% (w / v) glycerol, pH 7.5). The cell suspension was sonicated and then centrifuged to obtain a crude extract. The crude extract was then treated with ammonium sulfate until 30% saturation. The resulting pellet was resuspended in TG buffer and then dialyzed overnight against 2 liters of the same buffer. The resulting solution was then loaded onto a Q Sepharose Fast Flow HR 26 / 10 anion exchange column pre-equilibrated with TG buffer. The active ingredient was then eluted with a gradient of 0-1M NaCl. The active fraction was loaded onto a Mono Q HR5 / 5 anion exchange column pre-equili...

Embodiment 2

[0126] Embodiment 2: Cloning of Alcaligenes faecalis ATCC8750 nitrilase

[0127] The N-terminal sequence given in Example 1 and the N-terminal sequence of Alcaligenes faecalis JM3 nitrilase (Kobayashi et al., 1993, Proc. Natl. Acad. Sci. USA 90: 247-251 ) were completely identical, while the N-terminus of bacterial nitrilases had 35-57% identity over 14 residues. The inventors hypothesized that the nitrilase of the present invention purified from the ATCC8750 strain was identical to the enzyme described by Kobayashi et al. (cited above). Therefore, the cloning strategy is to carry out PCR reaction on the DNA genome of the ATCC8750 strain with two nucleotide probes determined according to the sequence given by Kobayashi et al. (cited above) to amplify the nitrilase gene.

[0128] Two probes were synthesized, one hybridizing to the 5' portion and the other hybridizing to the 3' portion of the sequence given by Kobayashi et al. (cited above):

[0129] 5' portion (PCRAF1...

Embodiment 3

[0132] Embodiment 3: the sequencing of the 1130pb fragment containing nitrilase activity polypeptide coding DNA

[0133] The insert cloned in plasmid pRPA-BCAT3 has been sequenced by Genome Express S.A. (Grenoble, France) using laboratory prepared DNA preparations (kit Wizzard Midi-prep, Promega). The sequencing strategy for this fragment is shown in Figure 4 , and implemented by conventional methods known to those skilled in the art. Ten internal nucleotide primers (in Figure 4 Winning designations 623-629 and 681-682). The full-length sequence was completed with universal primers "Reverse" and "M13 Forward". Each region is read at least once on each DNA.

[0134] The resulting sequence differs from the published sequence in two places: one in the putative structure serving as a transcription terminator, and the other in the gene for the nitrilase enzyme (called nitB), leading to Asn 279 → Replacement of Asp. Thus on the plasmid pRPA-BCAT1,2,4,5 with two spec...

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Abstract

The invention concerns a method for preparing 2-hydroxy 4-methylthio butyric acid and / or the ammonium salt thereof by enzymatic hydrolysis of 2-hydroxy 4-methythio butyronitrile characterised in that: a) in a first step a biological material having nitrilase activity is prepared, b) in a second step it is immobilised, c) in a third step, 2-hydroxy 4-methylthio butyronitrile is brought in the presence of the immobilised biological material, for obtaining the ammonium salt of 2-hydroxy 4-methylthio butyric acid, d) in a fourth step, optionally, the salt obtained in step c) is transformed into the corresponding acid, and e) in a fifth step, the product resulting from step c) or d) is concentrated.

Description

technical field [0001] The present invention relates to a new preparation method of 2-hydroxy-4-(methylthio)butyric acid (HMTBA) and / or its ammonium salt (HMTBS). Background technique [0002] For a long time, 2-hydroxy-4-(methylthio)butyric acid and its salts have been used in animal feed instead of methionine. Compared with methionine, its advantage is that it is in a liquid state, which is convenient for food manufacturers to use. [0003] The preparation of 2-hydroxy-4-(methylthio)butyric acid by chemical routes has been known for a long time. For example, patents EP142488, EP-143000 may be cited, which describe a two-step process for the hydrolysis of 2-hydroxy-4-methylthiobutyronitrile (HMTBN). The first step is to contact 2-hydroxy-4-methylthiobutyronitrile with a strong mineral acid such as hydrochloric or sulfuric acid. In the second step, after diluting with water, the temperature is raised to completely hydrolyze. 2-Hydroxy-4-(methylthio)butanoic acid is then ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/09C12N1/15C12N1/21C12N9/14C12N9/78C12N11/08C12N15/55C12P11/00C12R1/01C12R1/07C12R1/66
CPCC12N9/78C12P11/00C12N11/08C12N11/089C12N11/096C12N15/52C12N9/14
Inventor O·发沃-布勒J·彼尔阿德C·戴维P·默里尔D·霍伯咨
Owner ADISSEO IRELAND LIMITED IE
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