金属イオン制御培養法におけるRhizopus sp. MKU 40の生澱粉分解性グルコアミラーゼの生産 Raw Starch-Digesting Glucoamylase Production of Rhizopus sp. MKU 40 Using a Metal-Ion Regulated Liquid Medium

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Author(s)

    • 森田 洋 MORITA Hiroshi
    • 九州大学農学部食糧化学工学科 Department of Food Science and Technology, Faculty of Agriculture, Kyushu University
    • 水野 康平 MIZUNO Kouhei
    • 九州大学農学部食糧化学工学科 Department of Food Science and Technology, Faculty of Agriculture, Kyushu University
    • 藤尾 雄策 FUJIO Yusaku
    • 九州大学農学部食糧化学工学科 Department of Food Science and Technology, Faculty of Agriculture, Kyushu University

Abstract

液体培地中に添加する金属イオンの種類と濃度を制御することにより,特定の代謝産物を大量に生産させる金属イオン制御培養法の確立を目的として,金属イオンストレス下での微生物の生理的変化を調べた.微生物としてRhizopus sp. MKU 40を用い,菌の生育とグルコアミラーゼ生産を微生物生理の指標とした.その結果,亜鉛,マグネシウム,鉄イオンは本菌の生育およびグルコアミラーゼ生産に必須であった.また,カルシウムイオンの添加により,グルコアミラーゼの生産性はさらに増強した.これらの金属を至適濃度添加させることにより,グルコアミラーゼ活性を3.94U/mLにまで増強させることができた.さらに本酵素の精製は容易で,硫安沈澱,CM-Sephadex C-50イオン交換クロマトグラフィーの2段階で完了した.精製後の比活性値は培養液の4.8倍に達し,活性回収率も86.0%と高かった.またSDS-PAGEにより本酵素の分子量は約80.4kDaと推定された.本菌が生産するグルコアミラーゼの生澱粉分解能について調べたところ,粗酵素では可溶性澱粉分解活性基準で26%,精製酵素では16%程度の高い生澱粉分解活性が得られた.さらに培養液中の蛋白質量あたりの比活性値も21.9U/mg proteinと高かったことから,本培養法は生澱粉分解性グルコアミラーゼの生産に有望であった.

In this study we attempted to control some specified metal-ion concentrations in a liquid medium and found a highly raw starch-digesting glucoamylase (RSDG) producing culture with protease-less activity, which we have called a metal-ion regulated liquid medium . Rhizopus sp. MKU 40 could not grow and produce glucoamylase (GA) in the liquid medium without metal ions (SLS medium). The addition of iron, magnesium, and zinc ions in the liquid medium was essential to growth and GA production of Rhizopus sp. MKU 40. Calcium ions also stimulated its growth and GA production. To summarize, the addition of 75 ppm calcium ions, 2 ppm iron ions, 49 ppm magnesium ions, and 0.7 ppm zinc ion to the SLS liquid medium was the best condition for producing GA and resulted in a high specific activity of GA (21.9 U/mg protein). Specific RSDG activity of the crude enzyme was 5.7 U/mg protein, and the ratio of RSDG activity to GA activity was 0.26. Since the purification was easy, the purification of GA was done in only two steps (ammonium sulfate precipitation and column chromatography on CM-Sephadex C-50) . The enzyme was purified about 4 .8-fold based on specific GA activity with an 86.0% yield based on GA activity from culture supernatant . The enzyme was proved to be homogeneous as judged by SDS-PAGE, and it has a molecular mass of about 80.4 kDa by comparison of its relative mobility on SDS-PAGE with those of standard proteins . For purified enzymes, the ratio of RSDG activity to GA activity was 0.16.

Journal

  • Journal of Applied Glycoscience

    Journal of Applied Glycoscience 46(1), 15-21, 1999-03-31

    The Japanese Society of Applied Glycoscience

References:  15

Cited by:  1

Codes

  • NII Article ID (NAID)
    10008258550
  • NII NACSIS-CAT ID (NCID)
    AN10453916
  • Text Lang
    ENG
  • Article Type
    Journal Article
  • ISSN
    13403494
  • NDL Article ID
    4785614
  • NDL Source Classification
    ZP24(科学技術--化学・化学工業--糖・澱粉)
  • NDL Call No.
    Z17-15
  • Data Source
    CJP  CJPref  NDL  J-STAGE  JASI 
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