A new thermoactive pullulanase from Desulfurococcus mucosus: cloning, sequencing, purification, and characterization of the recombinant enzyme after expression in Bacillus subtilis.
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 degrees 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 degrees C for 4 h and exhibited a half-life of 50 min at 85 degrees C. By high-pressure liquid chromatography analysis it was observed that rapuDm hydrolyzed alpha-1,6 glycosidic linkages of pullulan, producing maltotriose, and also alpha-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.
Duffner
F
F
Enzyme Research, Novo Nordisk A/S, 2880 Bagsvaerd, Denmark.
Bertoldo
C
C
Andersen
J T
JT
Wagner
K
K
Antranikian
G
G
D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics*; D001412:Bacillus subtilis / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D017124:Conserved Sequence; D003505:Cyclodextrins / Q000378:metabolism; D019620:Desulfurococcaceae / Q000201:enzymology / Q000235:genetics*; D005798:Genes, Bacterial; D005936:Glucans / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008320:Maltose / Q000032:analysis; D008969:Molecular Sequence Data; D008970:Molecular Weight; D013213:Starch / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature; D014312:Trisaccharides / Q000032:analysis
D019843:Archaeal Proteins; D001426:Bacterial Proteins; D003505:Cyclodextrins; D005936:Glucans; D014312:Trisaccharides; C008317:maltotriose; D008320:Maltose; C009109:pullulan; D013213:Starch; D006026:Glycoside Hydrolases; C082507:amylopullulanase