Extracellular Maltase of Bacillus brevis

Abstract

Bacillus brevis NRRL B-4389 produced extracellular maltase (a-glucosidase; EC 3.2.1.20) only in the presence of short a-1,4-glucosidic polymers, such as maltose and maltotriose. An optimum medium was developed; it contained 2.5% maltose, 0.5% nonfat dry milk, 0.4% yeast extract, and 0.01% CaCl2. The enzyme was produced extracellularly during the logarithmic phase of growth; no cellbound activity was detected at any time. Partial purification of the maltase was accomplished by using diethylaminoethyl cellulose batch adsorption, ammonium sulfate precipitation, and Sephadex G-200 gel filtration. Maltase, isomaltase (oligo-1,6-glucosidase), and glucosyltransferase activities were purified 20.0-, 19.1-, and 11.5-fold, respectively. Some properties of the partially purified maltase were determined: optimum pH, 6.5; optimum temperature, 48 to 50°C; pH stability range, 5.0 to 7.0; temperature stability range, 0 to 50°C; isoelectric point, pH 5.2; and molecular weight, 52,000. The relative rates of hydrolysis of maltose (G2), maltotriose (G3), G4, methyl-aD -maltoside, G40, dextrin, and isomaltose were 100, 22, 12, 10, 10, 8, and 5%, respectively; the Km on maltose was 5.8 mM; Dglucose, p-nitrophenyl-aD -glucoside, and tris(hydroxymethyl) aminomethane were competitive inhibitors; transglucosylase activity of the enzyme on maltose resulted in the synthesis of isomaltose, isomaltotroise, and larger oligosaccharides. Maltases (a-glucosidases; EC 3.2.1.20) presently play an important role in the industrial production of glucose syrups used by the food industry. To the present, only two extracellular maltases from bacteria have been characterized in detail (9, 29). It was the purpose of this study to isolate a bacterium that produced extracellular maltase, to maximize enzyme production, and to purify and characterize the maltase. MATERIALS AND METHODS Isolation and mutagenesis. Over 100 isolates capable of producing extracellular maltase were obtained from soil samples by using the screening procedure of Wang et al. (30). An isolate that produced the most maltase in shake culture was identified as Bacillus brevis (S. J. McWethy, Ph.D. thesis, Iowa State University, Ames, 1977). The isolate was subjected repeatedly to N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis (1). Conditions for mutagenesis were: 100 ,ug of N-methyl-N'-nitro-N-nitrosoguanidine per ml, pH 6.0 (0.05 M tris(hydroxymethyl)aminomethane [Tris]-maleate buffer), and 20 min of exposure at 35°C. Mutants were screened for extracellular maltase production (30), and a three-step mutant that produced elevated levels of maltase was selected for further study.

Characterization of a Maltase from an Early-Diverged Non-Conventional Yeast Blastobotrys adeninivorans

“…As thermostability of S. cerevisiae isomaltases was also elevated in the presence of Tris [55], it may bind to the active site of these enzymes. Literature mining revealed Tris as a competitive inhibitor of Bacillus brevis maltase with the K i of 14.5 mM [56]. In a yeast Brettanomyces bruxellensis (former name Br.…”

Extracellular α-glucosidase of an alkalophilic microorganism, Bacillus sp. ATCC 21591

“…In contrast, enzymes from other alkalophilic organisms have optima between pH 9.5 and 11.5 and are inactive at pH 7.0 [5][6][7][8]13]. Most neutrophilic bacterial a-glucosidases have pH optima in the range 5.0-7.0 [1,2, [14][15][16] and temperature optima at or about Table 3 Substrate specificity of a-glucosidase of Bacillus sp. ATCC [15,16].…”

“…The level of c~-ghicosidase detected in the CFS was approximately 48-times greater than that detected intracellularly -210 units as against 4.4 units after 15 h growth, a-Glucosidases are produced extracellularly by Bacillus subtilis P-I 1 [ 17], Bacillus thermoglucosidius KP 1006 [18][19][20], Bacillus sp. KP 1035 [21], Bacillus brevis [14] and Bacillus arnylolyticus [22] although intracellular a-glucosidases are also well distributed in the genus Bacillus [2,15,16].…”

A cell-associated oligo-1,6-α-glucosidase from an extremely thermophilic anaerobic bacterium, Thermoanaerobium Tok6-B1

“…Firstly, a-D-glucosidase (EC 3.2.1.20) hydrolyses terminal, non-reducing, 1-4-linked (and to a lesser extent, 1-6-linked) aD-glucose residues of disaccharides, oligosaccharides and aryl-glucosides. Enzymes showing preferential specificity towards maltose and maltooligosaccharides, but little activity towards aryl glucosides have been termed 'maltases' and have been reported in mesophilic [3,4,5] and thermophilic bacteria [6].…”