Pullulan Degrading Enzymes of Bacterial Origin

Pytka, Monika; Bardowski, Jacek

doi:10.1080/10408410490435115

Cited by 117 publications

(65 citation statements)

References 108 publications

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“…Upstream of each functional domain lie two tandem repeats belonging to carbohydrate-binding motif family 41 (CBM41), which bind tightly to a-glucan polysaccharides containing a-(1,4) glycosidic and a-(1,6) glycosidic linkages (van Bueren et al, 2007). Within the protein, two pairs of four regions highly conserved in aamylase-like proteins were identified (I, II, III and IV) which form the catalytic triad Asp-Glu-Asp (Doman- Pytka & Bardowski, 2004;Kuriki & Imanaka, 1999).…”

Section: Resultsmentioning

confidence: 99%

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: Resultsmentioning

confidence: 99%

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

et al. 2010

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“…Amylopullulanases, or type II pullulanases, exhibit both ␣-amylase and pullulanase activities and can therefore cleave both ␣-1,4-and ␣-1,6-glucosidic bonds (20). There are a number of other homologs on the genome (24), some with putative signal sequences for secretion, expected to harbor the ability to degrade ␣-linked polysaccharides.…”

Section: Resultsmentioning

confidence: 99%

Disruption of a Sugar Transporter Gene Cluster in a Hyperthermophilic Archaeon Using a Host-Marker System Based on Antibiotic Resistance

et al. 2007

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We have developed a gene disruption system in the hyperthermophilic archaeon Thermococcus kodakaraensis using the antibiotic simvastatin and a fusion gene designed to overexpress the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase gene (hmg Tk ) with the glutamate dehydrogenase promoter. With this system, we disrupted the T. kodakaraensis amylopullulanase gene (apu Tk ) or a gene cluster which includes apu Tk and genes encoding components of a putative sugar transporter. Disruption plasmids were introduced into wild-type T. kodakaraensis KOD1 cells, and transformants exhibiting resistance to 4 M simvastatin were isolated. The transformants exhibited growth in the presence of 20 M simvastatin, and we observed a 30-fold increase in intracellular HMG-CoA reductase activity. The expected gene disruption via double-crossover recombination occurred at the target locus, but we also observed recombination events at the hmg Tk locus when the endogenous hmg Tk gene was used. This could be avoided by using the corresponding gene from Pyrococcus furiosus (hmg Pf ) or by linearizing the plasmid prior to transformation. While both gene disruption strains displayed normal growth on amino acids or pyruvate, cells without the sugar transporter genes could not grow on maltooligosaccharides or polysaccharides, indicating that the gene cluster encodes the only sugar transporter involved in the uptake of these compounds. The ⌬apu Tk strain could not grow on pullulan and displayed only low levels of growth on amylose, suggesting that Apu Tk is a major polysaccharide-degrading enzyme in T. kodakaraensis.

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“…Furthermore, these data illustrate that the extracellular amylopullulanase, encoded by apuB of B. breve UCC2003, is capable of hydrolyzing ␣-1,4 and ␣-1,6 linkages at different active sites and can therefore be classified as a so-called type II bifunctional amylopullulanase (15,21). Disruption of the apuB gene in B. breve UCC2003.…”

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Characterization of ApuB, an Extracellular Type II Amylopullulanase from Bifidobacterium breve UCC2003

Motherway¹,

Fitzgerald

Neirynck³

et al. 2008

Appl Environ Microbiol

101

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The apuB gene of Bifidobacterium breve UCC2003 was shown to encode an extracellular amylopullulanase. ApuB is composed of a distinct N-terminally located ␣-amylase-containing domain which hydrolyzes ␣-1,4-glucosidic linkages in starch and related polysaccharides and a C-terminally located pullulanase-containing domain which hydrolyzes ␣-1,6 linkages in pullulan, allowing the classification of this enzyme as a bifunctional class II pullulanase. A knockout mutation of the apuB gene in B. breve UCC2003 rendered the resulting mutant incapable of growth in medium containing starch, amylopectin, glycogen, or pullulan as the sole carbon and energy source, confirming the crucial physiological role of this gene in starch metabolism.The study of the human gut microbiota in the context of health maintenance or improvement has in recent years enjoyed an exponentially increasing interest (reviewed in references 35 and 58). It is known that several pathogenic bacteria can cause acute gastroenteritis, while representatives of other bacterial genera, such as the lactobacilli, have the potential to provide protection against infection (11). It has also become apparent that elements of the colonic microbiota are capable of influencing the incidence and severity of gastrointestinal diseases and disorders, such as ulcerative colitis, bowel cancer, and pseudomembranous colitis (41). There have also been many studies focusing on prebiotics, which are "selectively fermented food ingredients that allow specific changes, both in the composition and/or activity in the gastrointestinal microbiota that confer benefits upon host well being and health" (33; see also references 28 and 42 for reviews). Carbohydrates that have been shown to exert prebiotic effects include those from whole-grain wheat, fructooligosaccharides, galactooligosaccharides, and lactulose (12,17,20,33). The development of functional foods containing prebiotics and/or probiotics which can change the composition or activity of the microbiota such that it prevents or ameliorates gastrointestinal disorders is a key goal for both the pharma and food industries. Achieving this goal may be facilitated by the availability of an increasing number of genome sequences of gastrointestinal microbes (1,9,10,26,40,48,49), since the genomic data should allow the selection of novel, perhaps more-selective prebiotics and would also be pivotal in attaining a fundamental understanding of the probiotic effect.Although starch and related compounds are digested and absorbed in the stomach and small intestine, a significant fraction that is resistant to digestion may represent valuable carbon and energy sources for colonic bacteria, such as bifidobacteria, and which thus may act as prebiotics (57). Plant cells and seeds are a rich source of starch where it is deposited as granules in the cytoplasm. Starch is composed of two highmolecular-weight components: amylose, which comprises 15 to 25% of starch, is a linear polymer consisting of ␣-1,4-linked glucopyranose, while the predominant component...

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Pullulan Degrading Enzymes of Bacterial Origin

Cited by 117 publications

References 108 publications

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

Disruption of a Sugar Transporter Gene Cluster in a Hyperthermophilic Archaeon Using a Host-Marker System Based on Antibiotic Resistance

Characterization of ApuB, an Extracellular Type II Amylopullulanase from Bifidobacterium breve UCC2003

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