The gene encoding a thermoactive pullulanase from the hyperthermophilic anaerobic archaeon Desulfurococcus mucosus (apuA) was cloned in Escherichia coli and sequenced. apuA from D. mucosus showed 45.4% pairwise amino acid identity with the pullulanase from Thermococcus aggregans and contained the four regions conserved among all amylolytic enzymes. apuA encodes a protein of 686 amino acids with a 28-residue signal peptide and has a predicted mass of 74 kDa after signal cleavage. The apuA gene was then expressed in Bacillus subtilis and secreted into the culture fluid. This is one of the first reports on the successful expression and purification of an archaeal amylopullulanase in a Bacillus strain. The purified recombinant enzyme (rapuDm) is composed of two subunits, each having an estimated molecular mass of 66 kDa. Optimal activity was measured at 85°C within a broad pH range from 3.5 to 8.5, with an optimum at pH 5.0. Divalent cations have no influence on the stability or activity of the enzyme. RapuDm was stable at 80°C for 4 h and exhibited a half-life of 50 min at 85°C. By high-pressure liquid chromatography analysis it was observed that rapuDm hydrolyzed ␣-1,6 glycosidic linkages of pullulan, producing maltotriose, and also ␣-1,4 glycosidic linkages in starch, amylose, amylopectin, and cyclodextrins, with maltotriose and maltose as the main products. Since the thermoactive pullulanases known so far from Archaea are not active on cyclodextrins and are in fact inhibited by these cyclic oligosaccharides, the enzyme from D. mucosus should be considered an archaeal pullulanase type II with a wider substrate specificity.Pullulanases (pullulan-6-glucanohydrolase [EC 3.2.1.41]) are classified as type I or type II (amylopullulanase) depending on their ability to degrade ␣-1,4 glycosidic linkages in starch, amylopectin, and related oligosaccharides. Unlike type II, pullulanase type I is unable to attack ␣-1,4 glycosidic linkages. Both pullulanase type I and type II attack ␣-1,6 glycosidic linkages in pullulan, producing maltotriose. All pullulanases known to date are unable to degrade cyclodextrins. In contrast, pullulan hydrolase type I (neopullulanase) and type II (isopullulanase) are able to cleave ␣-1,4 glycosidic linkages in pullulan, releasing panose and isopanose, respectively, and are highly active on cyclodextrins (32). Enzymes that degrade cyclodextrins faster than starch are designated cyclodextrinases (EC 3.2.1.54; cyclomaltodextrinase) (30).In recent years, a large number of pullulanases have been isolated, particularly from thermophilic microorganisms (32). The research on pullulanases from thermophiles is interesting not only for understanding enzyme stability but also for discovering improved enzymes for application in the industrial starch hydrolysis process. The first step in the conversion of starch to glucose is liquefaction, which runs at temperatures of 95 to 105°C in the presence of a thermostable ␣-amylase. Due to the absence of thermoactive enzymes, saccharification follows at a temperature...
The gene encoding the type I pullulanase from the extremely thermophilic anaerobic bacterium Fervidobacterium pennavorans Ven5 was cloned and sequenced in Escherichia coli. The pulA gene from F. pennavoransVen5 had 50.1% pairwise amino acid identity with pulA from the anaerobic hyperthermophile Thermotoga maritima and contained the four regions conserved among all amylolytic enzymes. The pullulanase gene (pulA) encodes a protein of 849 amino acids with a 28-residue signal peptide. The pulA gene was subcloned without its signal sequence and overexpressed in E. coli under the control of the trc promoter. This clone, E. coli FD748, produced two proteins (93 and 83 kDa) with pullulanase activity. A second start site, identified 118 amino acids downstream from the ATG start site, with a Shine-Dalgarno-like sequence (GGAGG) and TTG translation initiation codon was mutated to produce only the 93-kDa protein. The recombinant purified pullulanases (rPulAs) were optimally active at pH 6 and 80°C and had a half-life of 2 h at 80°C. The rPulAs hydrolyzed α-1,6 glycosidic linkages of pullulan, starch, amylopectin, glycogen, α-β-limited dextrin. Interestingly, amylose, which contains only α-1,4 glycosidic linkages, was not hydrolyzed by rPulAs. According to these results, the enzyme is classified as a debranching enzyme, pullulanase type I. The extraordinary high substrate specificity of rPulA together with its thermal stability makes this enzyme a good candidate for biotechnological applications in the starch-processing industry.
Eighteen Pediococcus strains were screened for their potential as silage inoculants. Pediococcus acidilactici G24 was found to be the most suitable, exhibiting a short lag phase on both glucose and fructose, a rapid rate of acid production, a high sugar-to-lactate conversion efficiency, no detectable breakdown of proteins or lactic acid, and the ability to grow within a broad range of pH and temperature. When tested in laboratory silos using grass with a water-soluble carbohydrate content of 24 gfkg of aqueous extract, P. acidilactici G24 stimulated the natural LactobaciUus plantarum population and accelerated the rates of lactic acid production and pH decrease. After 6 days of fermentation, the inoculated silage exhibited a 12% decrease in ammonia nitrogen and an 11% increase in crude protein levels compared with uninoculated controls. The use of an L. plantarum inoculant at a rate of 104 bacteria per g of grass in conjunction with P. acidilactici G24 produced no additional beneficial effect. Inoculation of grass with a water-soluble carbohydrate level of 8 g/kg of aqueous extract with P. acidilactici G24 led to no acceleration in the rate of L. plantarum growth or pH decrease. However, after 7 days of fermentation the inoculated silage had a 14% lower ammonia nitrogen protein content than did uninoculated controls. The results suggest that P. acidilactici G24 may be useful as a silage inoculant for crops with a sufficiently high water-soluble carbohydrate level.
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