Febs Letters Volume 57, Number 1-3./Volume 58, Number 1. [antikvár]

Volume 57, number 1 l-EBS LILTTERS September 1975 IDENTIFICATION OF ISOAMYLASE, A GLYCOGEN-DEBRANCHING ENZYME, FROM BA CILL US AMY L OLJQ UEFA CI ENS Herbert URLAUB and Günter WÖBER Biochemie (Fachbereich Chemie}, der Philipps-Universität, D-355Ö Marburg (Lahnj, Lahnberge, West Germany Received 28 May 1975 1. Introduction Bacillus amyloliquefaciens is well Icnown for its production of an extracellular a-amylase. Tlie enzyme is secreted into tlie culture fluid during the exponential phase of growth when the bacteria are grown with an amylaceous carbon source, e.g. maltose, maltodex-trins, soluble starch, or glycogen, a-Amylase has been purified, crystallized and investigated in depth with regard to its physical properties, amino acids composition, mode of action and product specificity [1]. a-Amylase will only iiydrolize 1,4-a-glucosidic linkages in appropriate substrates and does not attack 1,6-a-bonds of branched a-glucans. The enzyme is thought to function in the degradation of ci-glucans making these macromolecules available to the cell as a meta-bolizable carbon source. In the course of an investigation of a-glucan metabolism in bacteria [2,3,4], we found it worthwhile to re-examine, in B. amyloliquefaciens, the enzymes possibly involved in the utilization of branched a-glucans. This report communicates the identification of a hydrolase which will selectively cleave 1,6-a:-glucosidic bonds in branched Q-glucans. According to its substrate specificity, this enzyme may be named iso-amylase or glycogen 6-glucanohydrolase (EC 3.2.1.68). 2. Materials and methods 2.1. Chemicals and accessory enzymes Carbohydrates used as a carbon source in growth experiments and as a substrate in enzyme digests were obtained from the following suppliers: Amylose No. 4561, maltose No. 5910 and 5912, D-glucose No. 8346, soluble starch No. 1253 from E. Merck, Darmstadt, GFR; pullulan from Serva, Heidelberg, GFR; amylopectin from Koch-Light, Colnbrook, U.K.; oyster glycogen, type II, from Sigma, St. Louis, USA. Phyto-glycogen was isolated from sweet corn [5], commercial amylopectin was freed from small amounts of amylose [6], a maltodextrin mixture (average chain length 7 glucosyl units) was a gift of Corn Products International, USA. D-Glucose oxidase (grade 111) and horse radish peroxidase (grade II) were purchased from Boehringer Mannheim, GFR, glucoamylase was a partly purified [7] commercial preparation ("Diazyme", Miles Labs., Elkhart, USA). In the standard purification of ^-amylase from sweet potatoes [8], a procedure removing traces of a-glucosidase was included [9]. All other chemicals were of the highest grade commercially available. 2.2. Culture conditions o/B. amyloliquefaciens B. amyloliquefaciens ATCC 23350 was grown aerobically at 32°C in liquid media of the following composition per litre: Maltose or another carbon source as indicated in Results and discussion, 10 g; NH4 CI, 7 g; Naa SO4 ¦ 10 Hj 0, 1 g; KHj PO4, 3.25 g; K2HPO4, 12.50 g; aspartic acid, 1 g; sodium glutamate, 1 g; stock solution of trace elements according to Chen and Segel [10], 2 ml. Cells harvested in their mid-exponential phase of growth on maltose were used as the source of isoamylase. Roughly the same amount of activity of isoamylase, in addition to an a-amylase, was also present in the culture fluid. A cell sonicate was purified by removal of nucleic acids with streptomycin sulphate and by fractional precipitation with North-Holland Publishing Company - Amsterdam 1