Production, Purification, and Characterization ofThermoanaerobacterium thermosaccharolyticum Glucoamylase

Abstract

Glucoamylase from Thermoanaerobacterium thermosaccharolyticum ATCC 7956 (DSM 571) was produced in extracellular form. It was purified to homogeneity by two separate methods, one with two chromatographic steps and the other with three. This glucoamylase is closely related to glucoamylases from Clostridium sp. G0005 and T. thermosaccharolyticum DSM 572. Activities and KM values with maltose substrate are less than one‐tenth and about fourfold, respectively, those of Aspergillus niger glucoamylase. T. thermosaccharolyticum glucoamylase is about twenty times as thermostable as A. niger glucoamylase and its optimal pH is somewhat higher at 4.9; however, it is produced in much lower activities. Sorbitol strongly stabilizes A. niger glucoamylase.

Purification and biochemical characterization of extracellular glucoamylase from Paenibacillus amylolyticus strain

“…Upon Periodic acid-Schiff (PAS) staining, P. amylolyticus glucoamylase demonstrated negative results (data shown in supplementary data). The enzyme was found to be non-glycosylated which was in accordance to the previously reported bacterial glucoamylases [12,40]. The thin layer chromatogram of glucoamylase showed the presence of sole D -glucose units, indicating that the purified enzyme was a true glucoamylase.…”

“…The electrophoretic experiments conducted on P. amylolyticus glucoamylase confirmed that the enzyme is a single polypeptide chain. A bacterial glucoamylase producer S. solfataricus showed molecular size of 65 kDa [12], whereas other bacterial strains depicted a slightly lower M r as reported in L. amylovorus of 47 kDa, and some showed slightly higher M r of 77 kDa in T. thermosaccharolyticum ATCC 7956 [40]. On the other hand, the reported fungal glucoamylase producers also showed similar molecular size of 66 kDa in T. lanuginosus [41], C. lunata [42], and T. reesei [43].…”

Isomaltose formation by free and immobilized dextransucrase

“…The most applicable methods for stabilizing enzymes are modifications of the protein structure by direct mutagenesis (Matthews 1993a,b;Park 2000), immobilization of the enzyme (Buchholz & Klein 1987;Cao 1999) or the addition of additives like substrates, products, inhibitors, cofactors, metal ions, proteins, sugars, polyols and natural or synthetic polymers (Bryjak & Noworyta 1994;Bryjak 1995;Athès et al 1999;Feng et al 2002;Kü hlmeyer & Klein 2002), which suppress protein unfolding while retaining catalytic activity. The decrease of enzyme activity E as a function of time may be described by a first-order reaction in many cases:…”

Purification, Characterization, and Subsite Affinities ofThermoactinomyces vulgarisR-47 Maltooligosaccharide-metabolizing Enzyme Homologous to Glucoamylases

“…According to this equation, the subsite affinities A 3 , A 4 , A 5 , A 6 , and A 7 were determined to be 0.11, À0:67, 7) and maltoheptaose (lanes [8][9][10][11][12][13][14]. Lanes 1 and 8, substrate only.…”

“…Most fungal GAs have a starch-binding domain in addition to a catalytic domain, and prefer starch over maltooligosaccharides, indicating that the primary substrate of these enzymes is starch. 2,3) Among the bacterial and archaeal enzymes, GAs from thermophilic clostridia have been studied the most, [4][5][6][7][8] and the crystal structure of a clostridial species, Thermoanaerobacterium thermosaccharolyticum, GA has been recently determined. 9) However, except for the GAs from thermophilic clostridia, relatively little is known about the bacterial and archaeal GAs.…”