A new species, BaciZZus naganoensis, is proposed for an obligately aerobic, moderately acidophilic, endospore-forming bacterium that produces a thermostable, aciduric pullulanase (EC 3.2.1.41). The organism was isolated from soil by selection on solid, pullulan-containing medium at pH 4.0 and 30°C. The isolate required a medium pH of less than 6.5 for growth initiation. Fatty acid composition studies revealed that the major fatty acid of cells grown in nutrient broth supplemented with 1 % starch was 14-methylpentadecanoic acid (iso-C,,) at 45 mol%. The guanine-plus-cytosine content of the DNA of this organism was 45 f 2 mol%. A type culture has been deposited with the American Type Culture Collection, Rockville, Md., as strain ATCC 53909.Pullulanases (EC 3.2.1.41) are enzymes which specifically cleave the alpha-1,6-glycosidic linkages of pullulan, starch, glycogen, and amylopectin. Recently, these enzymes have found commercial utility in glucose-and maltose-manufacturing processes (10). Yeasts, higher plants, and microorganisms are known to be producers of pullulanase (17). Within the genus Bacillus, several species and strains have been reported to make pullulanase, including Bacillus sp. strain 202-1 (17), "Bacillus acidopullulyticus" (lo), Bacillus cereus subsp. mycoides (23), Bacillus macerans (l), Bacillus p l ymyxa (9), and Bacillus stearothermopkilus (13,22). Of these, only the "B. acidopullulyticus' ' pullulanase has properties that allow its use in industrial saccharification processes.In our search for a pullulanase that could operate under conventional glucose-manufacturing conditions (i .e., pH 4.3 and 60"C), we assumed that bacteria capable of growth at low pH values or high temperatures or both would produce aciduric or thermostable enzymes. In this paper, we describe the isolation and characterization of a mesophilic, moderately acidophilic bacterium, designated Bacillus naganoensis, that makes a pullulanase with desirable acid and temperature stability characteristics. (20), and 7.5 g of red-colored pullulan. The pH was adjusted with 0.2 N sulfuric acid to pH 4.0. The color of the plates was dark violet. Incorporation of the two colored substrates into the plate medium allowed for preliminary selection of organisms which (i) produced aamylases (enzymes that hydrolyzed the blue-colored starch but not the red-colored pullulan, leaving a red background around a colony); (ii) produced pullulanases (enzymes that hydrolyzed the red-colored pullulan but not the blue-colored starch, leaving a blue background around a colony); or (iii) produced both a-amylases and pullulanases (enzymes that hydrolyzed both the blue-colored starch and the red-colored pullulan, leaving a clear zone of hydrolysis around a colony) * Corresponding author. MATERIALS AND METHODS(12). Red-colored pullulan was prepared by dissolving 100 g of pullulan PFlO (Hayashibara Co., Ltd., Okayama, Japan) in 2 liters of distilled water. The solution temperature was increased to 50"C, and 10 g of Mikacion Brilliant Red 5BS (Mitsubishi Kasei ...

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