Analysis of the Amylolytic Enzyme System of Clostridium thermosulfurogenes EM1: Purification and Synergistic Action of Pullulanases and Maltohexaose Forming α‐Amylase

Abstract

During growth of C. thermosulfurogenes EM1 on starch. seven forms of pullulanase and one form of a-amylase were detected after gel electrophoretic separation of proteins. By determining the isoelectric points and N-terminal sequences of various pullulanase species it was evident that no structural differences exist between these proteins. These pullulanases hydrolyzed a-1,4-and a-16-linkages in various soluble sugar polymers causing their breakdown to maltose and maltotriose. Unlikc these enzymes. the a-amylase p r o d u d by C. thermosulficrogenes E M preferentially attackcd non-soluble starch. The main product of hydrolysis was maltohexaose. The combined action Analyse des amylolytlschen Enrymsystems vOn CbsWdhm tfwmosuhlmgmes EMl: Reinigung und synergigtlsohe Wrkung von Pultulana~e uml lueltohexaose Mldender a-Amylase. Bei Wachstum auf Stilrke w d e n bei Clostridium thermosulfurogenes EM1 sieben Pullulanase-Formen sowie eine a-Amylase mit Hilfe von Polyacrylamid-Gelelektrophorese nachgewiesen. Der isoelektrische Punkt der Pullulanase-Formen war identisch wie auch die Nterminalen Aminostiure-Sequenzen der daraufhin untersuchten Pullulanase-Formen. Die Pullulanasen hydrolysierten a-1.6-und Q-1,4glykosidische Bindungen verschiedencr lbslischer Kohlenhydrate, starch/stiirke 44 (1992) Nr. 8. S. 305-312 0 VCH Verlagsgeseilschaft mbH, D-6940 Weinheim, 1992 0038-~6/9uM1o8-03~o3o5so3.H)+.2sN)

Pullulanase of Thermoanaerobacterium thermosulfurigenes EM1 (Clostridium thermosulfurogenes): molecular analysis of the gene, composite structure of the enzyme, and a common model for its attachment to the cell surface

“…(47). Since the different forms of the pullulanase have identical N-terminal amino acid sequences (48), which are in agreement with the deduced amino acid sequence reported here from residue 36 onwards, and since two pullulanase forms from one gene fragment were also observed in E. coli (8), we conclude that the seven major pullulanase forms of T. thermosulfurigenes EM1 are the product of one gene. The discrepancy between the calculated molecular mass and the apparent molecular mass of the largest forms, i.e., 290 kDa, is probably due to glycosylation of the enzyme.…”

“…Strain EM1, which will be renamed accordingly, is known as a potent producer of starch-degrading enzymes (2,19). The thermostable enzymes a-amylase and pullulanase have been purified and characterized (48). Pullulanase (pullulan-6-glucanohydrolase [EC 3.2.1.41]) cleaves a-1,6-glycosidic linkages in pullulan, a polysaccharide produced by the fungus Aureobasidium pullulans that consists of ax-1,6-linked maltotriose units.…”

“…Seven major forms of pullulanase from T. thernosulfurigenes EM1, with molecular masses between 93 and 490 kDa, have been described (48). Interestingly, these various pullulanases have identical isoelectric points and N-terminal amino acid sequences.…”

Isolation of a new Thermoanaerobacterium thermosaccharolyticum strain (FH1) producing a thermostable dextranase.

“…The phenotypic characteristics of isolate FH1 were also consistent with those of the Thermoanaerobacterium species, though none of the latter has been reported to produce dextranase. Members of this genus, however, are known for their ability to produce exoenzymes such as amylases, xylanases, and cellulases, which are active at temperatures above 65°C (Ganghofer et al, 1998;Lee et al, 1993;Liu et al, 1996;Shao and Wiegel, 1995;Spreinat and Antranikian, 1992). The T. thermosaccharolyticum type strain DSM571 showed no growth with dextran as the sole carbon and energy source under the employed conditions (Table 1).…”

Molecular analysis of the amy gene locus of Thermoanaerobacterium thermosulfurigenes EM1 encoding starch-degrading enzymes and a binding protein-dependent maltose transport system

“…Several enzymes have been purified and characterized (21,26,37), and in many cases the corresponding genes have been cloned, analyzed, and expressed in Escherichia coli or Bacillus subtilis (2,7,24,27). On the other hand, regulation of gene expression and enzyme synthesis as well as transport of the soluble products of the depolymerases into the cell have received surprisingly little attention.…”