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Cyclodextrin-Glucanotransferase von Klebsiella pneumoniae

1. Synthese, Reinigung und eigenschaften des Enzyms von K. pneumoniae M 5 al

Cyclodextrin glucanotransferase from Klebsiella pneumoniae

1. Formation, purification and properties of the enzyme from Klebsiella pneumoniae M 5 al

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Abstract

  1. 1.

    The strain M 5 al of Klebsiella pneumoniae grows excellently with starches. We were able to show that besides the pullulanase associated with the external membrane of the cells the bacterium produces an inducible, extracellular cyclodextrin glucanotrans-ferase [1,4-α-d-glucan-4-α-(1,4-α-glucano)-transferase (cyclising) (EC 2.4.1.19)]. Potato starch and cyclohexaamylose or cycloheptaamylose were found to be the best “inducing” carbon sources for the synthesis of the enzyme. When the bacteria are grown batchwise, maltose is a poorly “inducing” carbon source; larger quantities of the enzyme are synthesized by continuous cultivation with maltose as growth limiting factor.

  2. 2.

    For the determination of the cyclodextrin glucanotransferase-activity an assay method was worked out.

  3. 3.

    The enzyme could be separated from the culture filtrate and purified to more than 90% in few steps. At a total yield of 61.2% related to the activity of the culture filtrate employed we received an enzyme solution with the specific activity of 26.6 units/mg protein. Some properties of the enzyme are described.

  4. 4.

    The products formed from amylopectin by the enzyme were analyzed. Somewhat more than half the amylopectin was found as cyclodextrins. 29.3% of the cyclodextrin fraction were cycloheptaamylose, 47.2% cyclohexaamylose and 10.7% exo-branched cyclohexaamylose. 12.8% of cyclohexaamylose were obtained from a cyclodextrin glucanotransferase-limit dextrin after debranching by pullulanase and exposing the product to the action of the glucanotransferase again.

  5. 5.

    The importance of the cyclodextrin glucanotransferase for the utilization of starches by this strain of Klebsiella pneumoniae is discussed. After a first characterization the enzyme is compared to the amylase of Bacillus macerans.

Zusammenfassung

  1. 1.

    Der Stamm M 5 al von Klebsiella pneumoniae wächst ausgezeichnet mit Stärken. Wir konnten zeigen, daß das Bacterium neben der mit der externen Membran der Zellen assoziierten Pullulanase eine induzierbare, extracelluläre Cyclodextrin-Glucanotransferase [1,4-α-d-Glucan-4-α-(1,4-α-glucano)-transferase (cyclising) (EC 2.4.1.19)] produziert. Als beste “induzierende” Kohlenstoffquellen erwiesen sich Kartoffelstärke und Cyclohexaamylose bzw. Cycloheptaamylose. Bei statischer Kultivierung ist Maltose eine nur schlecht “induzierende” Kohlenstoffquelle, größere Mengen des Enzyms werden bei kontinuierlicher Züchtung mit diesem Zucker als wachstumslimitierendem Faktor synthetisiert.

  2. 2.

    Für die Bestimmung der Cyclodextrin-Glucanotransferase-Aktivität wurde eine Testmethode ausgearbeitet.

  3. 3.

    Das Enzym konnte in wenigen Schritten aus dem Kulturfiltrat abgetrennt und in guten Ausbeuten zu über 90% rein erhalten werden. Bei einer Gesamtausbeute von 61,2%, bezogen auf die mit dem Kulturfiltrat eingesetzte Aktivität, erhielten wir eine Enzymlösung mit der spezifischen Aktivität von 26,6 E/mg Protein. Einige Eigenschaften des Enzyms werden beschrieben.

  4. 4.

    Die durch das Enzyme aus Amylopectin gebildeten Produkte wurden analysiert. Etwas mehr als die Hälfte des Amylopectins fand sich als Cyclodextrine. 29,3% der Cyclodextrinfraktion waren Cycloheptaamylose, 47,2% Cyclohexaamylose und 10,7% exo-verzweigte Cyclohexaamylose. 12,8% Cyclohexaamylose konnten aus einem Cyclodextrin-Glucanotransferase-Grenzdextrin nach Entzweigung durch Pullulanase und erneuter Einwirkung der Glucanotransferase erhalten werden.

  5. 5.

    Die Bedeutung der Cyclodextrin-Glucanotransferase für die Verwertung von Stärken durch diesen Stamm von Klebsiella pneumoniae wird diskutiert. Nach einer ersten Charakterisierung wird das Enzym mit der amylase von Bacillus macerans verglichen.

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  1. Chemisches Laboratorium der Universität, Albertstr. 21, D-7800, Freiburg i.Br., Bundesrepublik Deutschland

    Hans Bender

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Bender, H. Cyclodextrin-Glucanotransferase von Klebsiella pneumoniae . Arch. Microbiol. 111, 271–282 (1977). https://doi.org/10.1007/BF00549366

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