본문 바로가기
KERD
논문KCI 등재

Bacillus subtilis에서 발현된 재조합 Endoxylanase농축과 안정화 공정의 최적화

Process Optimization for Concentration and Stabilization of Recombinant Endoxylanase Expressed in Bacillus subtilis

권현주|김병우|박정하|김영만|남수완|최영록
한국미생물·생명공학회 (The Korean Society For Microbiology And Biotechnology)|2004.12|한국미생물·생명공학회지 (Microbiology and Biotechnology Letters)|제32권 제4호|pp. 322-327

0

조회

0

다운로드

0

인용

0

참고문헌

주제분류생물학

초록

구성적 promoter인 PJH promoter 하류에 연결된 Bacillus sp. 유래의 endoxylanase를 B. subtilis에서 과발현 하였다. 발현된 plasmid, pJHKJ4는 자체 promoter(PB)와 Bacillus의 강력한 promoter(PJH)가 tandem으로 위치하여 transcription 되었다. B. subtilis DB431에 형질전환된 균주를 glucose가 함유된 LB 배지에서 배양 후 총활성 및 분비효율은 140 U/ml으로 나타났다. 배양상등액은 ultrafiltration(MW cut-off 10 kDa and 30 kDa)과 염석법으로 농축하였다. 효소의 농축에서 ultrafiltration이 70% ammonium sulfate precipitation보다 효과적이었다. 50oC에서 농축된 효소액의 안정화는 NaCl, glycerol, sorbitol, and CaCl2 등의 다양한 안정제를 농도별로 첨가하여 안정제의 효과를 검토한 결과, 가장 효과적인 안정제는 NaCl과 CaCl2이었다.

Abstract

A strong constitutive PJH promoter from Bacillus sp. was applied to overexpress the endoxylanase gene (639 bp) in Bacillus subtilis. The expression plasmid, pJHKJ4, was designed to contain the PJH promoter and open reading frame of endoxylanase including its own promoter. The plasmid was introduced into B. subtilis DB431 and the resulting transformant was grown on LB glucose medium. At the end of cultivation, the endoxylanase activity in the culture supernatant reached about 140 U/ml. The enzyme in the supernatant was concentrated by ultrafiltration (MW cut-off 10 kDa and 30 kDa) and ammonium sulfate precipitation. For the concentration of the enzyme, ultrafiltration was more efficient than 70% ammonium sulfate precipitation. The stabilization of concentrated enzyme solution at 50oC was examined with various stabilizers such as NaCl, glycerol, polyethylene glycol, sorbitol, and CaCl2. The most effective stabilizers were found to be NaCl and CaCl2.

참고문헌 (24)

  1. "Effect of various additives and solvents on thermostability of cyclodextrin glucanotransferase from Bacillus stearothermopilus", pp.368~371 (1991)
  2. "Application of enzymes in the pulp and paper industy.", 15, pp.147~157 (1999)
  3. "Purification and characterization of extracellular xylanase of Bacillus stearothermophilus", pp.592~597 (1991)
  4. "Molecular cloning of a Bacillus subtilis xylanase gene in Escherichia coli", pp.59~65 (1983)
  5. "Molecular cloning and the nucleotide sequence of a Bacillus sp", 8:, pp.28~33 (1998)
  6. "Conformational adaptability in enzymes", pp.397~648 (1973)
  7. "A novel thermostable xylanase from Thermomonospora sp influence of additives on thermostability", 78:, pp.221~224 (2001)
  8. "Constitutive overexpression of endoxylanase gene in Bacillus subtilis", 10:, pp.551~553 (2000)
  9. "A fibrinolytic enzyme from Bacillus amyloliquefaciens D4-7 isolated from Chungkook-Jang; it s characterization and influence of additives on thermostability", 31, pp.271~276 (2003)
  10. "Cleavage of structural proteins during the assembly of the head of bacteriophage T4", pp.680~227 685 (1970)
  11. "Purification and characterization of a new xylanase", pp.3212~3218 (1993)
  12. "Use of dinitrosalicylic acid reagent for determination of reducing sugar", pp.426~428 (1959)
  13. "The release of enzymes from E coli by osmotic shock and during the formation of spheroplast", pp.3685~3692 (1965)
  14. "Extremophiles as a source of novel enzymes for industrial application", pp.711~729 (1999)
  15. "Molecular cloning of the genes for xylan degradation of Bacillus pumilus and their expression in Escherichia coli", pp.335~341 (1983)
  16. "Effects of xylanase pretreatment procedures on nonchlorine bleaching", cesterni, pp.492~495 (1994)
  17. "Molecular cloning and expression of xylanases from an alkalophilic thermophilic Bacillus", pp.343~59 347 (1993)
  18. "Measurement of protein using bicinchoninic acid", pp.76~85 (1985)
  19. "Microbial xylanases for paper industry", 77, pp.137~142 (1999)
  20. "Purification and characterization of xylanase II from Aspergillus niger SFN-416", 24, pp.687~692 (1996)
  21. "Xylanases in bleaching from an idea to the industry", pp.338~350 (1994), Linco
  22. "Xylanase treatment for the peroxide bleaching of oxygen delignified kraft pulps derived from three softwood species", 48:, pp.137~145 (1996)
  23. "Multiplicity of b-1 4-xylanase in microorganism", pp.305~317 (1988)
  24. "High-level secretory production of intact biologically active staphylokinase from Bacillus subtilis", 62, pp.87~96 (1998)

관련 논문AI추천

두 종류 Bacillus속 균주의 Xylanases 특성 비교윤기홍, 진현경한국미생물·생명공학회 · 한국미생물·생명공학회지2016 · 제44권 제3호Xylanase, Cellulase의 생산성이 높은 Bacillus sp.의 분리 및 효소생산을 위한 배지조건의 최적화김창원, 송민동, 양시용, 정형원, 하종규한국미생물·생명공학회 · 한국미생물·생명공학회지2003 · 제31권 제4호Purification and Characterization of Xylanase from Bacillus sp. A-6최석호한국미생물·생명공학회 · 한국미생물·생명공학회지2009 · 제37권 제2호Paenibacillus woosongensis로부터 대장균에 Xylanase 10A의 유전자 클로닝과 정제 및 특성분석윤기홍한국미생물·생명공학회 · 한국미생물·생명공학회지2020 · 제48권 제2호대장균에서 Bacillus subtilis의Mannanase 유전자 과잉발현권민아, 손지영, 윤기홍한국미생물·생명공학회 · 한국미생물·생명공학회지2004 · 제32권 제3호Xylanase를 생산하는 Bacillus sp. AMX-4 균주의 분리와 효소 생산성설숙자, 윤기홍, 이미성, 조효찬, 최준호한국미생물·생명공학회 · 한국미생물·생명공학회지2002 · 제30권 제2호

이 간행물의 다른 논문 (14건)

청국장으로부터 면역증강활성이 우수한Bacillus pumilus JB-1의 뷴리 및 분리균의청국장 발효특성김영숙, 권기석, 권정숙, 손호용, 권하영pp. 291-296넙치(Paralichthys olivaceus) HSP90β 유전자의 분자생물학적 연구이재형, 김영태pp. 297-306미역 포자엽에서 분리한 항산화물질의 특성양지영, 김상권, 유미영pp. 307-311고추역병과 시들음병을 방제하는 토착길항세균Pseudomonas fluorescens 4059 의 선발과길항기작김상달, 정희경pp. 312-316반응표면분석법에 의한 미역 포자엽의 항산화물질추출 조건 최적화김경환, 김상권, 유미영, 이재우, 양지영pp. 317-321
원문 보기