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D-aminoacylase mutants

A kind of technology of amino acid and acetyl, applied in the field of D-aminoacylase mutant

Inactive Publication Date: 2011-11-23
DAICEL CHEM IND LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no report about the production of D-tryptophan from a higher concentration of the above-mentioned substrates. In the industrial production of D-tryptophan, it is expected that the high-concentration substrate N-acetyl-DL-tryptophan can be hydrolyzed to produce D-tryptophan

Method used

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Examples

Experimental program
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Effect test

Embodiment 1

[0274] Embodiment 1: Preparation of chromosomal DNA of denitrifying Alcaligenes xylose subspecies MI-4 strain

[0275] Chromosomal DNA was prepared from Alcaligenes denitrificans subsp. xylose MI-4 strain (FERM P-9413) according to the method described in Nuclei acids Res. 8, 4321 (1980).

Embodiment 2

[0276] Example 2: Cloning of D-aminoacylase gene by PCR from Alcaligenes denitrificans subsp. xylose MI-4 strain

[0277] According to the description in the reference (Biosci.Biotech.Biochem, 59,2115 (1995)), a sense primer ADD-ATG1 (SEQ ID NO: 4) and an antisense primer ADD-TGA1 (SEQ NOID: 5) were synthesized, they Corresponds to the 5'- and 3'-untranslated regions of the D-aminoacylase gene of Alcaligenes xylosoxydans subsp. xylosoxydans A-6. Perform a PCR reaction using a 50-μl reaction solution containing 70 ng of chromosomal DNA of Alcaligenes denitrificans subsp. xylose MI-4 strain, 1.0 U ExTaq DNA polymerase, Taq polymerase buffer, 0.2 mM dNTP, 5% DMSO, and primers ADD-ATG1 and ADD-TGA1 (10 pmol each), were denatured at 94°C for 30 seconds and extended at 72°C for 2 minutes for 30 cycles. PCR obtained a highly specific PCR product of about 1.5kbp.

Embodiment 3

[0278] Example 3: Sequencing of PCR products.

[0279] The DNA fragment obtained in Example 2 was purified with GFX kit (Pharmacia). The nucleotide sequence of the purified DNA fragments was analyzed. Nucleotide sequence analysis of DNA was performed by PCR in a PRISM310 Genetic Analyzer (Applied Bio Systems) using the BigDyeTerminator Cycle Sequencing ready Reaction Kit (Applied Bio Systems). The primers used were ADD-189R (SEQ ID NO: 6), ADD-524R (SEQ ID NO: 7), ADD-466F (SEQ ID NO: 8), ADD-1032R (SEQ ID NO: 9), ADD- 987F (SEQ ID NO: 10), and ADD-TGA1.

[0280] The amino acid sequence encoded by the obtained sequence is shown in SEQ ID NO:1. The nucleotide sequence of this D-aminoacylase was compared with the known D-aminoacylase clone of Alcaligenes xylosoxidans subspecies xyloseoxidans A-6 strain. Taking the first letter A of the start codon ATG as 1, only two nucleotide substitutions, 360-T->C and 435-C->T, were found between these two clones. The amino acid sequen...

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Abstract

The present invention provides mutant D-aminoacylases and use thereof. The mutant D-aminoacylases are hard to be inhibited by the substrate and, comprise the amino acid sequences of the D-aminoacylase derived from Alcaligenes denitrificans subsp. xylosoxydans MI-4 strain, wherein amino acid residues at specific sites have been modified. The mutants of the present invention have high reaction specificity as well as resistance to inhibition by the substrate. The present invention enables high-yield production of D-amino acids using higher concentrations of N-acyl-DL-amino acid as the substrate.The mutants of the present invention are useful in producing D-tryptophan in particular.

Description

technical field [0001] The present invention relates to D-aminoacylase mutants, genes encoding mutants, methods for producing them, and methods for producing D-amino acids, especially D-tryptophan, using D-aminoacylase mutants. Background technique [0002] Enzymes not only exhibit high catalytic activity but also specificity. The specificity includes stereospecificity as well as substrate specificity and reaction specificity. Although there are some exceptions, the stereospecificity of enzymes is almost absolute. [0003] Recent research has increasingly relied on high-precision techniques. Against such a background, the use of optically active bodies in the fields of pharmaceuticals, insecticides, foods, seasonings, etc. is becoming more and more important. Techniques for separating specific optical isomers are important because the physiological activities of optical isomers are sometimes quite different. Therefore, how to separate (synthesize or decompose) pure optic...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/00C07K14/225C12N9/14C12N15/55C12P13/04C12P21/00C12N1/21C12N9/80C12P13/22
CPCC12P13/227C12N9/80
Inventor 中島贤则山本浩明
Owner DAICEL CHEM IND LTD
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