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Engineering Study on Extracellular Enzyme Production by Escherichia coli Harboring an Excretion Vector

Research Project

Project/Area Number 02650711
Research Category

Grant-in-Aid for General Scientific Research (C)

Allocation TypeSingle-year Grants
Research Field 発酵工学
Research InstitutionNagoya University

Principal Investigator

YAMANE Tsuneo  Nagoya University Dept. of Food Sci. & Technol. Professor, 農学部, 教授 (70026102)

Co-Investigator(Kenkyū-buntansha) HORIKOSHI Koki  Tokyo Inst. Technol Dept. of Bioeng. Professor, 工学部, 教授 (80087551)
Project Period (FY) 1990 – 1991
Project Status Completed (Fiscal Year 1991)
Budget Amount *help
¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 1991: ¥300,000 (Direct Cost: ¥300,000)
Fiscal Year 1990: ¥1,500,000 (Direct Cost: ¥1,500,000)
KeywordsExcretion Vector / Escherichia coli / beta-Penicllinase / Aubomated Fed-batch Culture / Turbidity Sensor / Recombinant Microorganism / tac promoter / kil gene / tacプモロ-タ- / Kil遺伝子 / 分泌ベクタ-
Research Abstract

1. Fed-batch Culture of Recombinant Escherichia coli Carrying a Secretion Vector (Tsuneo YAMANE) Effects of medium composition and culture condition on the secretion of penicillinase of alkalophilic Bacillus sp. by E. coli havoring a secretion vector were studied. LB medium + glycerol enhanced the enzyme secretion. Next, two-stage fed-batch cultures were in a 3L bioreactor where enough LB + glycerol were supplied upto ca. 20 gDC/L of cell concentration in order to suppress cell lysis as much as possible, followed by controlled feeding of LB + glycerol. The limited feeding of glycerol was effective for the extracellular enzyme production and its overfeeding gave abundant cell growth with lesser formation of the enzyme. The highest enzyme activity was obtained when glycerol's specific supply rate was 0.10[g(g DC) ^<-1>h^<-1>], which was 20 times as much as that of a batch culture and the enzyme productivity [(Unit)L ^<-1>h^<-1>] was raised 5 -fold. Secretion in the second growth phase was more than 90%. A little cell lysis was observed even in the best fed-batch culture, implying further improvements in the weak expression of Kil gene are required.
2. Improvement of Secretion Vector (Koki Horikoshi)
A secretion vector, pEAP 85, which carried both promoter of penicillinase gene and tac promoter, was constructed with the aim of increasing ability of secretion vector of kil gene. Cyclodextrine synthetase (CGTase) gene of an alkalophilic Bacillus sp. was introduced to the vector plasmid, and its secretory production was studied. CGTase was highly expressed in the recombinant E. coli and most CGTase was secreted into the culture supernatant.

Report

(3 results)
  • 1991 Annual Research Report   Final Research Report Summary
  • 1990 Annual Research Report
  • Research Products

    (22 results)

All Other

All Publications (22 results)

  • [Publications] Takahiro SUZUKI: "Phenomenological Background and Some Preliminary Trials of Automated Supply in pH-Stat Modal Fed-batch Culture Uisng a Setpoint of High Limit" Journal of Fermentation Technology. 69. 292-297 (1990)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] Edgard E.TULIN: "Characteristics of Batch Culture of Recombinant Bacillus brevis Excreting a Foreign Esterase" Biotechnolgy and Bioengineering. 38. 1249-1252 (1991)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] Yusuke AMAKI: "Purification and Properties of Thermostable Esterase of Bacillus stearothermophilus Produced by Recombinant Bacillus brevis" Bioscience,Biotechnology,and Biochemistry. 56. 238-241 (1992)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] Tsuneo YAMANE: "Fed-batch Culture Automated by Uses of Continuously Measured Cell Concentration and Culture Volume" Biotechnology and Bioengineering.

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] Koki HORIKOSHI: "Genetic Application of Alkaliphilic Microorganisms" FEMS Microbiology Reviews. 75. 279-286 (1990)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] George GEORGANTA: "Expression of CGTase of Alkalophilic Bacillus No.38-2 in Various Hosts" Starke. 43. 361-363 (1991)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] 山根 恒夫: "生物反応工学" 産業図書株式会社, 311 (1991)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] Takahiro SUZUKI, Tsuneo YAMANE and Shoichi SHIMIZU: "Phenomenological background and some preliminary trials of automated substrate supply in pH-stat modal fed-batch culture using a setpoint of high limit" Journal of Fermentation and Bioengineerring. 69(5). 292-297 (1990)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] Edgard E. TULIN, Hiroaki TAKAGI, Shunsaku UEDA, Hideo YAMAGATA, Tsuneo YAMANE and Shigezo UDAKA: "Characteristics of batch culture of a recombinant Bacillus brevis excreting a foreign esterase" Biotechnology and Bioengineering. 38(10). 1247-1252 (1991)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] Yusuke AMAKI, Edgard E. TULIN, Shunsaku UEDA, Kunio OHMIYA and Tsuneo YAMANE: "Purification and properties of a thermostable esterase of Bacillus stearothermophilus produced by recombinant Bacillus brevis" Bioscience, Biotechnology and Biochemistry. 56(2). 238-241 (1992)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] Tsuneo YAMANE, Wataru HIBINO, Kazuto ISHIHARA, Yasuyuki KADOTANI and Minoru KOMINAMI: "Fed-batch culture automated by uses of continuously measured cell concentration and culture volume" Biotechnology and Bioengineering. (1992)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] Koki HORIKOSHI: "genetic application of alkalophilic microorganisms" FEMS Microbiology Reviews. 75(6). 279-286 (1990)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] George GEORGANTA, Toshiaki KUDO and Koki HORIKOSHI: "Expression of CGTase of alkalophilic Bacillus No. 38-2 in various hosts" Starke. 43(7). 361-363 (1991)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] Tsuneo YAMANE: Sangoyo ToshoPub., Co. Ltd.,. Bioreaction Engineering 2nd edition (in Japanese), 311 (1991)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] Koki HORIKOSHI: Kodansha Co., Ltd.Microorganisms in Alkaline Environments (in English), 83-96 (1991)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] Edgard E.TULIN: "Characteristics of Batch Cultureof aRecombinant Bacillus brevis Excreting a Foreign Esterase" Biotechnology and Bioengineering. 38. 1249-1252 (1991)

    • Related Report
      1991 Annual Research Report
  • [Publications] Yusuke AMAKI: "Purification and Properties of Thermostable Esterase of Bacillus stearothermophilus Produced by Recombinant Bacillus brevis" Bioscience,Biotechnology and Biochemistry. 56. 238-241 (1992)

    • Related Report
      1991 Annual Research Report
  • [Publications] Tsuneo YAMANE: "Fedーbatch Culture Automated by Uses Continuously Measured Cell Concentration and Culture Volume" Biotechnology and Bioengineering.

    • Related Report
      1991 Annual Research Report
  • [Publications] George GEORGANTA: "Expression of the CGTase Gene of Alkalophilic Bacillus No.38ー2 in Various Hosts" Starke. 43. 361-363 (1991)

    • Related Report
      1991 Annual Research Report
  • [Publications] Koki HORIKOSHI: "Microorganisms in Alkaline Environments" Kodansha CO.,Ltd., 83-96 (1991)

    • Related Report
      1991 Annual Research Report
  • [Publications] Takahiro SUZUKI: "Phenomenological Background and Some Prelimirary Trials of automated Substrate Supply in pHーstat modal Fedーbatch Culture Using a Setpoint of High Limit" Journal of Fermentation and Biotechnology. 69. 292-297 (1990)

    • Related Report
      1990 Annual Research Report
  • [Publications] 山根 恒夫: "生物反応工学(改訂版)" 産業図書株式会社, 200 (1991)

    • Related Report
      1990 Annual Research Report

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Published: 1990-04-01   Modified: 2016-04-21  

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