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Literature summary extracted from

  • Miyazaki, T.; Yashiro, H.; Nishikawa, A.; Tonozuka, T.
    The side chain of a glycosylated asparagine residue is important for the stability of isopullulanase (2015), J. Biochem., 157, 225-234.
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
3.2.1.57 expression of wild-type and mutant enzymes in Pichia pastoris strain GS115 Aspergillus brasiliensis

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
3.2.1.57 purified deglycosylated recombinant enzyme mutant N448A complexed with substrate isopanose, hanging drop vapour diffusion method, 20°C, mixing of 0.001 ml protein in 10 mM sodium acetate, pH 3.5, with 0.001 ml of reservoir solution containing 10-13% w/w PEG 8000, and 50 mM sodium acetate, pH 4.5-5.0, cryoprotection with 20% v/v glycerol in reservoir solution, X-ray diffraction structure determination and analysis at 2.2 A resolution, molecular replacement method, the crystal belongs to the P21 space group and contains four molecules in the asymmetric unit, substrate binding structure, overview Aspergillus brasiliensis

Protein Variants

EC Number Protein Variants Comment Organism
3.2.1.57 N448A site-directed mutagenesis, glycan-deficient variant, Asn448 is not N-glycosylated, the mutant shows reduced thermal stability compared to the wild-type enzyme Aspergillus brasiliensis
3.2.1.57 S450A site-directed mutagenesis, glycan-deficient variant, by replacing Ser450 with Ala, Asn448 is not N-glycosylated, but the Asn448 side chain remains intact, unlike the Asn448Ala mutation, the mutant shows reduced thermal stability compared to the wild-type enzyme Aspergillus brasiliensis
3.2.1.57 Y440A site-directed mutagenesis, the mutant shows reduced thermal stability compared to the wild-type enzyme Aspergillus brasiliensis

Organism

EC Number Organism UniProt Comment Textmining
3.2.1.57 Aspergillus brasiliensis O00105 formerly Aspergillus niger ATCC 9642
-
3.2.1.57 Aspergillus brasiliensis ATCC 9642 O00105 formerly Aspergillus niger ATCC 9642
-

Posttranslational Modification

EC Number Posttranslational Modification Comment Organism
3.2.1.57 glycoprotein highly N-glycosylated enzyme with 15 potential N-glycosylation sites (Asn-X-Ser/Thr), the side chain of a glycosylated asparagine residue is important for the stability of the enzyme, enzyme deglycosylation results in a decrease in thermostability. Enzyme deglycosylated with peptide-N-glycosidase F shows virtually no enzymatic activity, while a single GlcNAc residue remains following digestion with Endo H, and the activity of the Endo H-treated wild-type enzyme is 65% compared to intact, non-deglycosylated, wild-type enzyme Aspergillus brasiliensis

Purification (Commentary)

EC Number Purification (Comment) Organism
3.2.1.57 recombinnat wild-type and mutant enzymes from Pichia pastoris strain GS115 by hydrophobic interaction chromatography Aspergillus brasiliensis

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
3.2.1.57 pullulan + H2O the enzyme cleaves the alpha(1,4) glucosidic linkage using an inverting mechanism and liberating D-glucose Aspergillus brasiliensis isopanose trisaccharide [Glc-alpha(1,4)-Glc-alpha(1,6)-Glc] ?
3.2.1.57 pullulan + H2O the enzyme cleaves the alpha(1,4) glucosidic linkage using an inverting mechanism and liberating D-glucose Aspergillus brasiliensis ATCC 9642 isopanose trisaccharide [Glc-alpha(1,4)-Glc-alpha(1,6)-Glc] ?

Synonyms

EC Number Synonyms Comment Organism
3.2.1.57 IPU
-
 Aspergillus brasiliensis

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
3.2.1.57 25
-
 assay at Aspergillus brasiliensis

Temperature Stability [°C]

EC Number Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
3.2.1.57 46.6
-
 50% activity remaining for deglycosylated mutant N448A and for mutant Y440A Aspergillus brasiliensis
3.2.1.57 49.3
-
 50% activity remaining for mutant N448A Aspergillus brasiliensis
3.2.1.57 52
-
 50% activity remaining for deglycosylated mutant S450A Aspergillus brasiliensis
3.2.1.57 53
-
 50% activity remaining for the deglycosylated wild-type enzyme Aspergillus brasiliensis
3.2.1.57 54
-
 50% activity remaining for mutant S450A Aspergillus brasiliensis
3.2.1.57 55
-
 50% activity remaining for the wild-type enzyme Aspergillus brasiliensis

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
3.2.1.57 3.5
-
 assay at Aspergillus brasiliensis

General Information

EC Number General Information Comment Organism
3.2.1.57 evolution the enzyme belongs to the glycoside hydrolase family 49 (GH49) Aspergillus brasiliensis