FEMS Microbiology Letters69 (lge~0)145-1:52 145 Publishedby Elsevier FEMSLE03998 Production of novel pullulanases at high concentrations by two newly isolated thermophilic clostridia M. Klingeberg 1, H. Hippe 2 and G. Antranikian 3 ) Teefinische Universitiit ttamburg-Harburg, Hamburg, ~lnstitut flit Mikrobialagie der Unwersitiil Giittingen, ti&tingen and ' German Collection of Microorganisms and Cell Cultures (DSM), Braunschweig F.R.G. Receivedit Januar~1990 Revision r'ec¢ived 23 Januar'j 1090 Accepted24 January I990 Key words: CIostridium thermohydrosulfuricum; Thermophilic bacteria; Pullulanase type II; Enzyme secretion 1. SUMMARY Two thermophilic bacteria, which are capable of vowing on starch at 60-70°C under anaerobic conditions, were isolated from a sugar refinery in Uelzen and from Solar lake in Israel. On the basis of their physiological characteristics they were identified as Clostridium thermohydrosulfuricum Uel 1 and C. thermohydrosulfuricum Sol 1, respec- tively. The product pattern of glucose polymer hydrolysis showed that both strains secreted en- zymes that possess amylolytic and pullulytic activ- ities. The major product formed was maltose. In addition, ,t.ghicosidase activity could be detected in the supernatams of Uel 1 strain. Compared to most anaerobes investigated these isolates secreted extremely high concentrations of pulhilanases in batch culture. Up to g57~ of the total enzyme synthesized was detected in the culture fluid. Un- like the pullulanases of type I, which can only Correspondence 1o: Prof. Dr. G. Anlrar~tdan. lastilut fiir Mikrobiologie tier Georg-August Univcrsit/it Gi~ttingen. Grlm- bachstrass¢ 8. 3400Ge, tting~nF.R.G, attack the a-l,6-glycosidic linkages, the pul- lulanases of both dostridial strains were also ca- pable of hydrolyzing a-l,4-1inkages. The enzyme system of both bacteria was found to be highly thermoactive; optimal activity was detected at pH 5,0 and 85°C. Even at 950C and without the addition of metal ions still 15% to 25% of en- zymatic activity was detectable. 2. INTRODUCTION Aerobic as well as anaerobic bacteria have de- veloped various strategies for the utilization of starch 11,2], a highly branched polysaecharide which is composed of glucose units bound by a-lA- and a-l,6-glycosidie linkages. In the last decade a number of thermophilic anaerobic eubacteria were isolated. Most of them belong to the genera Clostridium, Thermoanaerobacter, Thermobacteroides and Dictyogtomus. The major- ity of these organisms synthesize a-amylase as well as puilulanase. The formation of pullulanase, which is capable of hydrolyzing a-l,6-glycosidic linkages in pullulan in addition to a-l,4-1inkages 0378-1097/90/$03.50© 1990Federation of EuropeanMicrobiological Societies