A Plate Culture Method for the Simultaneous Detection of Bacteria Producing Pullulan- and/or Starch-Hydrolyzing Enzymes

Kanno, Mutsuo; Tomimura, Eijiro

doi:10.1080/00021369.1985.10866937

Cited by 4 publications

(6 citation statements)

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Section: Introductionmentioning

confidence: 99%

“…Pullulan is a linear polysaccharide of maltotriose repeating units linked via a-(1,6) glycosidic bonds, produced by the ascomycete fungus Aureobasidium pullulans (Pouliot et al, 2005). Although pullulan is not found in animals, it is commonly used as a substrate to identify pullulanases with a-(1,6) glycosidase activity Morgan et al, 1979;Kanno & Tomimura, 1985).In group A streptococci (GAS), group B streptococci (GBS) and Streptococcus pneumoniae, cell-wall-anchored enzymes that can hydrolyse pullulan have been characterized Abbreviations: Erm, erythromycin; GAS, group A streptococci; GBS, group B streptococci; Spc, spectinomycin. (Bongaerts et al, 2000; Hytönen et al, 2003; Santi et al, 2008).…”

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ApuA, a multifunctional α-glucan-degrading enzyme of Streptococcus suis, mediates adhesion to porcine epithelium and mucus

et al. 2010

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We have identified apuA in Streptococcus suis, which encodes a bifunctional amylopullulanase with conserved a-amylase and pullulanase substrate-binding domains and catalytic motifs. ApuA exhibited properties typical of a Gram-positive surface protein, with a putative signal sequence and LPKTGE cell-wall-anchoring motif. A recombinant protein containing the predicted N-terminal a-amylase domain of ApuA was shown to have a-(1,4) glycosidic activity. Additionally, an apuA mutant of S. suis lacked the pullulanase a-(1,6) glycosidic activity detected in a cell-surface protein extract of wild-type S. suis. ApuA was required for normal growth in complex medium containing pullulan as the major carbon source, suggesting that this enzyme plays a role in nutrient acquisition in vivo via the degradation of glycogen and food-derived starch in the nasopharyngeal and oral cavities. ApuA was shown to promote adhesion to porcine epithelium and mucus in vitro, highlighting a link between carbohydrate utilization and the ability of S. suis to colonize and infect the host. INTRODUCTIONStreptococcus suis is a major porcine pathogen of significant commercial importance worldwide. In suckling and weaning pigs, it is the principal cause of acute meningitis, but can infect other organs leading to arthritis, serositis, endocarditis, otitis media and bronchopneumonia (Beineke et al., 2008;Madsen et al., 2002). Healthy pigs asymptomatically colonized with S. suis form a reservoir for this disease and play a major role in its epidemiology (Arends et al., 1984). To date, 33 different capsule serotypes of S. suis have been identified, but serotype 2 is most commonly associated with disease worldwide Lun et al., 2007). Serotype 2 strains were also associated with recent large outbreaks of severe human infections in China and elsewhere in Asia (Mai et al., 2008;Tang et al., 2006;Wertheim et al., 2009). The recently obtained genome sequences of two virulent Chinese S. suis serotype 2 strains (98HAH12 and 05ZYH33) and P1/7, the European reference strain (http://www.sanger.ac.uk/Projects/S_suis/), led to the identification of a large number of potential surface and secreted proteins that might play a role in virulence, including a number of putative carbohydratedegrading enzymes (Baums & Valentin-Weigand, 2009). Genes that encode carbohydrate-degrading enzymes are common in the genomes of other streptococcal pathogens and play a role in nutrient acquisition for growth and colonization on mucosal surfaces (Rollenhagen & Bumann, 2006;Shelburne et al., 2006Shelburne et al., , 2008a. Dietary sources of highly polymerized a-glycans such as starch and glycogen are abundant in the human colon (Levitt et al., 1987) and oropharynx (Mormann & Muhlemann, 1981;Shelburne et al., 2005 Shelburne et al., , 2007Virtaneva et al., 2005), as well as the epithelium of the vagina and lung (Gourlay et al., 2009;Gregoire et al., 1971; Santi et al., 2008;van Bueren et al., 2007). Degradation of starch and glycogen proceeds in most organisms via the action of amylases a...

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Section: Introductionmentioning

confidence: 99%

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ApuA, a multifunctional α-glucan-degrading enzyme of Streptococcus suis, mediates adhesion to porcine epithelium and mucus

et al. 2010

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“…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. (12). Red-colored pullulan was prepared by dissolving 100 g of pullulan PFlO (Hayashibara Co., Ltd., Okayama, Japan) in 2 liters of distilled water.…”

Section: Methodsmentioning

confidence: 99%

Description of Bacillus naganoensis sp. nov.

Tomimura

Zeman

Frankiewicz

et al. 1990

International Journal of Systematic Bacteriology

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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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“…8 ) This bacterial strain was identified as Bacillus acidocaldarius, based mainly on its 'aerobic, rod-shaped, spore-forming, thermophilic, and acidophilic characteristics and the G+C content of its DNA (62mol%). The extracellular amylase production of the strain started at the onset of the post-logarithmic phase of growth, but ceased when the growth reached the stationary phase (a cycloserineresistant mutant of the strain continued amylase production, even during the stationary phase of growth).…”

Section: Microbiological Properties and Amylase Productionmentioning

confidence: 99%

“…4 ) We have isolated many aerobic and anaerobic thermophiles under neutral and acidic conditions, and checked for their productivities of starch-and pullulan-hydrolyzing enzymes. 8 ) Among them, one B. acidocaldarius strain (named A-2) produced an amylase which showed very high stability to heat under acidic conditions compared to the enzymes of the B. acidocaldalius strains so far reported. This report describes the purification procedures and some properties of the A-2 amylase.…”

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confidence: 99%

A Plate Culture Method for the Simultaneous Detection of Bacteria Producing Pullulan- and/or Starch-Hydrolyzing Enzymes

Cited by 4 publications

References 1 publication

ApuA, a multifunctional α-glucan-degrading enzyme of Streptococcus suis, mediates adhesion to porcine epithelium and mucus

ApuA, a multifunctional α-glucan-degrading enzyme of Streptococcus suis, mediates adhesion to porcine epithelium and mucus

Description of Bacillus naganoensis sp. nov.

ABacillus acidocaldariusα-Amylase That is Highly Stable to Heat under Acidic Conditions

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