Literature summary for 3.2.1.57 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.

Cloned(Commentary)

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

Crystallization (Commentary)

Crystallization (Comment)	Organism
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

Crystallization (Comment)

Organism

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

Protein Variants	Comment	Organism
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
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
Y440A	site-directed mutagenesis, the mutant shows reduced thermal stability compared to the wild-type enzyme	Aspergillus brasiliensis

Organism

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

Posttranslational Modification

Posttranslational Modification	Comment	Organism
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)

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

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
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]	?
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

Synonyms	Comment	Organism
IPU	-	Aspergillus brasiliensis

Temperature Optimum [°C]

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

Temperature Stability [°C]

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

pH Optimum

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

General Information

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