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KR960034421A - Method for mass production and recovery of heat-resistant D-hydantoinase in soluble state in host cell - Google Patents

Method for mass production and recovery of heat-resistant D-hydantoinase in soluble state in host cell Download PDF

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KR960034421A
KR960034421A KR1019950006592A KR19950006592A KR960034421A KR 960034421 A KR960034421 A KR 960034421A KR 1019950006592 A KR1019950006592 A KR 1019950006592A KR 19950006592 A KR19950006592 A KR 19950006592A KR 960034421 A KR960034421 A KR 960034421A
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hydantoinase
heat
resistant
soluble
cells
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KR1019950006592A
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KR0148468B1 (en
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김학성
성문희
이승구
홍승표
이동철
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조병희
동서석유화학 주식회사
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C12N9/86—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in cyclic amides, e.g. penicillinase (3.5.2)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/30—Extraction; Separation; Purification by precipitation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
    • C12Y305/02—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in cyclic amides (3.5.2)
    • C12Y305/02002—Dihydropyrimidinase (3.5.2.2), i.e. hydantoinase

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  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
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  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Microbiology (AREA)
  • Medicinal Chemistry (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

본 발명은 고온균에서 유래된 내열성 D-히단토이나아제(D-hydantoinase)-코딩 유전자를 포함하는 고발현성 플라스미드, 이에 의해 형질전환된 숙주세포, 이를 배양하여 D-히단토이나아제를 대량 생산하는 방법 및 배양배지로부터 D-히단토이나아제를 간단하고 효과적으로 분리회수하는 방법을 제공한다.The present invention provides a high expression plasmid containing a heat-resistant D-hydantoinase-coding gene derived from a high temperature bacterium, a host cell transformed thereby, and cultivating the same to produce a large amount of D-hydantoinase. And a method for separating and recovering D-hydantoinase from the culture medium simply and effectively.

Description

내열성 D-히단토이나아제를 숙주세포 내에서 가용성인 상태로 대량 생산하는 방법 및 회수방법Method for mass production and recovery of heat-resistant D-hydantoinase in soluble state in host cell

본 내용은 요부공개 건이므로 전문내용을 수록하지 않았음Since this is an open matter, no full text was included.

제1도는 본 발명에 따라 D-히단토이나아제-코딩 유전자를 클로닝하는 방법의 도식도이다, 제2도는 D-히단토이나아제 유전자를 포함하는 10.5kb Sau3A 1 DNA 단편의 제한효소지도이다, 제3도는 D-히단토이나아제 유전자 단편을 포함하는 각종 재조합 벡터를 제작하는 과정의 도식도이다.FIG. 1 is a schematic of a method of cloning a D-hydantoinase-encoding gene according to the present invention. FIG. 2 is a restriction map of a 10.5 kb Sau3A 1 DNA fragment comprising a D-hydantoinase gene. 3 is a schematic of the process of constructing a variety of recombinant vectors comprising the D-hydantoinase gene fragment.

Claims (5)

바실러스 스티아로더모필러스 SD-1(KCTC 8551P)에서 유래되며, 가용성의 내열성 D-히단토이나아제를 코딩하는 3.1kb 크기의 유전자를 함유하는 플라스미드 pHU183.Plasmid pHU183, derived from Bacillus stearothermophilus SD-1 (KCTC 8551P), containing a 3.1 kb gene encoding the soluble heat resistant D-hydantoinase. 제1항의 플라스미드에 의해 형질전환된 대장균 DH5αF′(KCTC 0147BP).E. coli DH5αF '(KCTC 0147BP) transformed with the plasmid of claim 1. 제2항의 대장균을 배양배지에서 배양하여 세포내에 가용성의 내열성 D-히탄토이나아제를 축적시키고 균체로부터 D-히단토이나아제를 회수함을 특징으로 하는 배양법에 의한 가용성의 내열성 D-히단토이나아제의 생산방법.Soluble heat-resistant D-hydantoinia by culturing method characterized by culturing E. coli in a culture medium to accumulate soluble heat-resistant D-hitantinase in cells and recovering D-hydantoinase from cells. Method of producing 제3항에 있어서, 배양배지는 표 1에 기재된 것임을 특징으로 하는 생산방법.The production method according to claim 3, wherein the culture medium is as described in Table 1. 내열성의 D-히단토이나아제-코팅 유전자를 포함하는 벡터에 의해 형질전환된 내열성의 D-히단토이나아제 생산능을 갖는 비(非)내열성 균주를 배양하여 D-히단토이나아제를 생산하고, 배양된 균체로부터 D-히단토이나아제를 분리회수하는 방법에 있어서, 상기 분리회수 방법이 다음 단계 (a)~(c)를 포함함을 특징으로 하는 내열성의 D-히단토이나아제의 분리방법. (a) 내열성 D-히단토이나아제가 가용성 상태로 생산된 균주를 수확후 분쇄용 완충액에 녹이고 호모게나이저로 균체를 파쇄시키는 단계; (b) 균체 파쇄액을 원심분리하여 얻은 무세포 조추출액을 10~20mM의 Mm++화합물 존재하에 50~65℃에서 30분 내지 2시간동안 열처리하여 비(非)내열성 숙주세포의 단백질을 침전시키는 단계; 및 (c) 원심분리하여 침전물을 제거하고 D-히단토이나아제를 포함하는 상등액을 회수하는 단계.D-hydantoinase is produced by culturing a non-heat resistant strain having a heat-resistant D-hydantoinase producing ability transformed by a vector containing a heat-resistant D-hydantoinase-coated gene. In the method for separating and recovering D-hydantoinase from cultured cells, the separation recovery method comprises the following steps (a) to (c): Way. (A) dissolving the strain produced in the heat-soluble D- hydantoinase soluble state in the grinding buffer and crushing the cells with a homogenizer; (b) The cell-free crude extract obtained by centrifugation of the cell lysate was heat-treated at 50-65 ° C. for 30 minutes to 2 hours in the presence of 10-20 mM Mm ++ compound to precipitate protein of non-heat-resistant host cell. Making a step; And (c) centrifuging to remove the precipitate and recovering the supernatant comprising D-hydantoinase. ※ 참고사항 : 최초출원 내용에 의하여 공개하는 것임.※ Note: The disclosure is based on the initial application.
KR1019950006592A 1995-03-27 1995-03-27 Preparation of thermostable d-hydantoinase which is soluble in host cell KR0148468B1 (en)

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