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1. WO2020009168 - NOVEL HYDROXYNITRILE LYASE MUTANT

Publication Number WO/2020/009168
Publication Date 09.01.2020
International Application No. PCT/JP2019/026527
International Filing Date 03.07.2019
IPC
C CHEMISTRY; METALLURGY
12
BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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MICRO-ORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICRO-ORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
15
Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
09
Recombinant DNA-technology
11
DNA or RNA fragments; Modified forms thereof
52
Genes encoding for enzymes or proenzymes
60
Lyases (4)
C CHEMISTRY; METALLURGY
12
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MICRO-ORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICRO-ORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
1
Micro-organisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving micro-organisms or compositions thereof; Processes of preparing or isolating a composition containing a micro-organism; Culture media therefor
14
Fungi; Culture media therefor
15
modified by introduction of foreign genetic material
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MICRO-ORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICRO-ORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
1
Micro-organisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving micro-organisms or compositions thereof; Processes of preparing or isolating a composition containing a micro-organism; Culture media therefor
14
Fungi; Culture media therefor
16
Yeasts; Culture media therefor
19
modified by introduction of foreign genetic material
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BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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MICRO-ORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICRO-ORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
1
Micro-organisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving micro-organisms or compositions thereof; Processes of preparing or isolating a composition containing a micro-organism; Culture media therefor
20
Bacteria; Culture media therefor
21
modified by introduction of foreign genetic material
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MICRO-ORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICRO-ORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
5
Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
10
Cells modified by introduction of foreign genetic material, e.g. virus-transformed cells
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MICRO-ORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICRO-ORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
9
Enzymes, e.g. ligases (6.); Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating, or purifying enzymes
88
Lyases (4.)
C CHEMISTRY; METALLURGY
12
BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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MICRO-ORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICRO-ORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
15
Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
09
Recombinant DNA-technology
63
Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
7
Preparation of oxygen-containing organic compounds
02
containing a hydroxy group
22
aromatic
C CHEMISTRY; METALLURGY
12
BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
13
Preparation of nitrogen-containing organic compounds
C12N 15/60 (2006.01)
C12N 1/15 (2006.01)
C12N 1/19 (2006.01)
C12N 1/21 (2006.01)
C12N 5/10 (2006.01)
C12N 9/88 (2006.01)
CPC
C12N 15/63
C12N 5/10
C12N 9/88
C12P 13/00
C12P 7/22
Applicants
  • 公立大学法人 富山県立大学 PUBLIC UNIVERSITY CORPORATION TOYAMA PREFECTURAL UNIVERSITY [JP/JP]; 富山県射水市黒河5180 5180, Kurokawa, Imizu-shi, Toyama 9390398, JP
Inventors
  • 浅野 泰久 ASANO Yasuhisa; JP
  • チャイケーヨ・シリポーン CHAIKAEW Siriporn; JP
  • ニュイラート・エム NUYLERT Aem; JP
  • 元島 史尋 MOTOJIMA Fumihiro; JP
Agents
  • 中谷 智子 NAKATANI Tomoko; JP
Priority Data
2018-12710503.07.2018JP
Publication Language Japanese (JA)
Filing Language Japanese (JA)
Designated States
Title
(EN) NOVEL HYDROXYNITRILE LYASE MUTANT
(FR) NOUVEAU MUTANT D'HYDROXYNITRILE LYASE
(JA) 新規ヒドロキシニトリルリアーゼ変異体
Abstract
(EN)
The activity of the wild-type hydroxynitrile lyase (HNL) obtained to date is insufficient for industrial use, and HNL having better activity has been demanded. The present inventors introduced various amino acid mutations predicted on the basis of the three-dimensional structures of HNL and (R)-mandelonitrile into wild-type HNL and measured the activities of the resulting HNL. As a result, it was discovered that amino acid modification of specific sites brings about elevation of activity common to millipede-derived HNL. Therefore, the present invention pertains to an (R)-HNL mutant protein having one or more amino acid substitutions in a millipede-derived (R)-HNL protein and having (R)-HNL activity. More specifically, the present invention pertains to HNL having amino acid substitutions in the third β-sheet structure (β3), the fourth β-sheet structure (β4), and the fifth β-sheet structure (β5) of (R)-HNL.
(FR)
L'activité de l'hydroxynitrile lyase (HNL) de type sauvage obtenue jusqu'ici est insuffisante pour une utilisation industrielle, et une HNL ayant une meilleure activité a été demandée. Les présents inventeurs ont introduit diverses mutations d'acide aminé prédites sur la base des structures tridimensionnelles de HNL et de (R)-mandélonitrile dans une HNL de type sauvage et mesuré les activités de la HNL résultante. Ainsi, il a été découvert que la modification d'acides aminés de sites spécifiques entraîne une élévation d'activité commune à une HNL dérivée de mille-pattes. Par conséquent, la présente invention concerne une protéine mutante (R)-HNL ayant une ou plusieurs substitutions d'acides aminés dans une protéine dérivée de mille-pattes (R)-HNL et ayant une activité (R)-HNL. Plus spécifiquement, la présente invention concerne une HNL ayant des substitutions d'acides aminés dans la troisième structure de feuillets β (β3), la quatrième structure de feuillets β (β4) et la cinquième structure de feuillets β (β5) de (R)-HNL.
(JA)
これまで得られた野生型ヒドロキシニトリルリアーゼ(HNL)の活性は産業用としては十分ではなく、更にすぐれた活性を有するHNLが求められていた。本発明者らは、HNLと(R)-マンデロニトリルの立体構造に基づき予測された種々のアミノ酸変異を野生型HNLに導入し、その活性を測定した。その結果、特定の部位のアミノ酸改変がヤスデ由来のHNLに共通した活性上昇をもたらすことを見出した。よって、本発明は、ヤスデ由来の(R)-HNLタンパク質における1種類以上のアミノ酸置換を有し、かつ、(R)-HNL活性を有する、(R)-HNL変異体タンパク質に関する。より具体的には、(R)-HNLの3番目のβシート構造(β3)、4番目のβシート構造(β4)、5番目のβシート構造(β5)におけるアミノ酸置換を有するHNLに関する。
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