Literature summary for 3.2.1.78 extracted from

Lu, H.; Luo, H.; Shi, P.; Huang, H.; Meng, K.; Yang, P.; Yao, B.
A novel thermophilic endo-beta-1,4-mannanase from Aspergillus nidulans XZ3: functional roles of carbohydrate-binding module and Thr/Ser-rich linker region (2014), Appl. Microbiol. Biotechnol., 98, 2155-2163.

Activating Compound

Activating Compound	Comment	Organism
acetone	activates the wild-type enzyme and mutant Man5DELTACBM at 40-60% v/v, no effect on mutant Man5DELTACL	Aspergillus nidulans
SDS	the wild-type enzyme slightly at 0.5-1.0% v/v, inhibits the mutant enzymes	Aspergillus nidulans
Triton X-100	activates the wild-type enzyme slightly at 0.5% v/v, inhibits the mutant enzymes	Aspergillus nidulans

Cloned(Commentary)

Cloned (Comment)	Organism
gene man5XZ3, sequence comparisons, cloning in Escherichia coli strain Trans1-T1, and recombinant expression of wild-type and mutant enzymes fused in frame to the DNA sequence for the Saccharomyces cerevisiae alpha-factor secretory signal peptide and under the control of the methanol-inducible alcohol oxidase promoter in Pichia pastoris strain GS115, induction by methanol, the proteins are secreted	Aspergillus nidulans

Cloned (Comment)

Organism

gene man5XZ3, sequence comparisons, cloning in Escherichia coli strain Trans1-T1, and recombinant expression of wild-type and mutant enzymes fused in frame to the DNA sequence for the Saccharomyces cerevisiae alpha-factor secretory signal peptide and under the control of the methanol-inducible alcohol oxidase promoter in Pichia pastoris strain GS115, induction by methanol, the proteins are secreted

Aspergillus nidulans

Protein Variants

Protein Variants	Comment	Organism
additional information	generation of two truncated enzyme mutants lacking the family 1 carbohydrate-binding module Man5DELTACBM or the family 1 carbohydrate-binding module and the Thr/Ser-rich linker region, Man5DELTACL, respectively. The mutant enzymes show significantly altered secondary structures compared to the wild-type enzyme, overview. Removal of the family 1 carbohydrate-binding module alone improves the thermostability of the enzyme, but additional removal of the linker region results in worse thermostability. The mutants are less stable in presence of acetone, SDS, Triton X-100, or urea compared to the wild-type enzyme	Aspergillus nidulans

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism	Structure
additional information	-	additional information	Km values with ocust bean gum are 0.9 mg/ml for wild-type enzyme, 0.8 mg/ml for mutant Man5DELTACBM, and 1.1 mg/ml for mutant Man5DELTACL	Aspergillus nidulans

Localization

Localization	Comment	Organism	GeneOntology No.	Textmining
extracellular	the enzyme is secreted	Aspergillus nidulans	-	-

Molecular Weight [Da]

Molecular Weight [Da]	Molecular Weight Maximum [Da]	Comment	Organism
38000	-	x * 63000, glycosylated recombinant wild-type enzyme, SDS-PAGE, x * 58000, glycosylated mutant lacking the CBM1 domain, SDS-PAGE, x * 41000, glycosylated mutant lacking the CBM1 domain and linker region, SDS-PAGE, x * 60000, deglycosylated recombinant wild-type enzyme, SDS-PAGE, x * 55000, deglycosylated mutant lacking the CBM1 domain, SDS-PAGE, x * 38000, deglycosylated mutant lacking the CBM1 domain and linker region, SDS-PAGE	Aspergillus nidulans
41000	-	x * 63000, glycosylated recombinant wild-type enzyme, SDS-PAGE, x * 58000, glycosylated mutant lacking the CBM1 domain, SDS-PAGE, x * 41000, glycosylated mutant lacking the CBM1 domain and linker region, SDS-PAGE, x * 60000, deglycosylated recombinant wild-type enzyme, SDS-PAGE, x * 55000, deglycosylated mutant lacking the CBM1 domain, SDS-PAGE, x * 38000, deglycosylated mutant lacking the CBM1 domain and linker region, SDS-PAGE	Aspergillus nidulans
55000	-	x * 63000, glycosylated recombinant wild-type enzyme, SDS-PAGE, x * 58000, glycosylated mutant lacking the CBM1 domain, SDS-PAGE, x * 41000, glycosylated mutant lacking the CBM1 domain and linker region, SDS-PAGE, x * 60000, deglycosylated recombinant wild-type enzyme, SDS-PAGE, x * 55000, deglycosylated mutant lacking the CBM1 domain, SDS-PAGE, x * 38000, deglycosylated mutant lacking the CBM1 domain and linker region, SDS-PAGE	Aspergillus nidulans
58000	-	x * 63000, glycosylated recombinant wild-type enzyme, SDS-PAGE, x * 58000, glycosylated mutant lacking the CBM1 domain, SDS-PAGE, x * 41000, glycosylated mutant lacking the CBM1 domain and linker region, SDS-PAGE, x * 60000, deglycosylated recombinant wild-type enzyme, SDS-PAGE, x * 55000, deglycosylated mutant lacking the CBM1 domain, SDS-PAGE, x * 38000, deglycosylated mutant lacking the CBM1 domain and linker region, SDS-PAGE	Aspergillus nidulans
60000	-	x * 63000, glycosylated recombinant wild-type enzyme, SDS-PAGE, x * 58000, glycosylated mutant lacking the CBM1 domain, SDS-PAGE, x * 41000, glycosylated mutant lacking the CBM1 domain and linker region, SDS-PAGE, x * 60000, deglycosylated recombinant wild-type enzyme, SDS-PAGE, x * 55000, deglycosylated mutant lacking the CBM1 domain, SDS-PAGE, x * 38000, deglycosylated mutant lacking the CBM1 domain and linker region, SDS-PAGE	Aspergillus nidulans
63000	-	x * 63000, glycosylated recombinant wild-type enzyme, SDS-PAGE, x * 58000, glycosylated mutant lacking the CBM1 domain, SDS-PAGE, x * 41000, glycosylated mutant lacking the CBM1 domain and linker region, SDS-PAGE, x * 60000, deglycosylated recombinant wild-type enzyme, SDS-PAGE, x * 55000, deglycosylated mutant lacking the CBM1 domain, SDS-PAGE, x * 38000, deglycosylated mutant lacking the CBM1 domain and linker region, SDS-PAGE	Aspergillus nidulans

Organism

Organism	UniProt	Comment	Textmining
Aspergillus nidulans	-	gene man5XZ3	-
Aspergillus nidulans XZ3	-	gene man5XZ3	-

Posttranslational Modification

Posttranslational Modification	Comment	Organism
glycoprotein	one potential N-glycosylation site, Asn98-Phe99-Thr100, the enzyme is deglycosylated by endo-beta-N-acetylglucosaminidase H. The Thr/Ser-rich linker region has numerous putative O-glycosylation sites	Aspergillus nidulans

Purification (Commentary)

Purification (Comment)	Organism
recombinant wild-type and mutant enzymes from Pichia pastoris strain GS115 culture supernatant by ultrafiltration and anion exchange chromatography to homogeneity	Aspergillus nidulans

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
guar gum + H2O	41.2% of the activity with locust bean gum	Aspergillus nidulans	?	-	?
guar gum + H2O	41.2% of the activity with locust bean gum	Aspergillus nidulans XZ3	?	-	?
locust bean gum + H2O	best substrate	Aspergillus nidulans	mannobiose + mannotriose + mannotetraose	hydrolysis products are 17.2% mannobiose, 37.4% mannotriose, 9.9% mannotetraose, and 35.5% other unidentified oligosaccharides	?
locust bean gum + H2O	best substrate	Aspergillus nidulans XZ3	mannobiose + mannotriose + mannotetraose	hydrolysis products are 17.2% mannobiose, 37.4% mannotriose, 9.9% mannotetraose, and 35.5% other unidentified oligosaccharides	?
additional information	no activity with beechwood xylan, 4-nitrophenyl beta-D-glucopyranoside, 4-nitrophenyl beta-D-mannoside, and carboxymethyl cellulose	Aspergillus nidulans	?	-	?
additional information	no activity with beechwood xylan, 4-nitrophenyl beta-D-glucopyranoside, 4-nitrophenyl beta-D-mannoside, and carboxymethyl cellulose	Aspergillus nidulans XZ3	?	-	?

Subunits

Subunits	Comment	Organism
?	x * 63000, glycosylated recombinant wild-type enzyme, SDS-PAGE, x * 58000, glycosylated mutant lacking the CBM1 domain, SDS-PAGE, x * 41000, glycosylated mutant lacking the CBM1 domain and linker region, SDS-PAGE, x * 60000, deglycosylated recombinant wild-type enzyme, SDS-PAGE, x * 55000, deglycosylated mutant lacking the CBM1 domain, SDS-PAGE, x * 38000, deglycosylated mutant lacking the CBM1 domain and linker region, SDS-PAGE	Aspergillus nidulans
More	the multimodular enzyme consists of a family 1 carbohydrate-binding module (CBM1), a Thr/Ser-rich linker region, and a catalytic domain	Aspergillus nidulans

Synonyms

Synonyms	Comment	Organism
Man5XZ3	-	Aspergillus nidulans

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
80	-	-	Aspergillus nidulans

Temperature Range [°C]

Temperature Minimum [°C]	Temperature Maximum [°C]	Comment	Organism
30	90	activity range, wild-type enzyme	Aspergillus nidulans

Temperature Stability [°C]

Temperature Stability Minimum [°C]	Temperature Stability Maximum [°C]	Comment	Organism
additional information	-	removal of the family 1 carbohydrate-binding module alone improves the thermostability of the enzyme, but additional removal of the linker region results in worse thermostability	Aspergillus nidulans
30	90	purified recombinant wild-type enzyme, pH 5.0, 1 h, more than 45% of maximal activity at 50-80°C and over 30% activity at 90°C	Aspergillus nidulans

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
5	-	-	Aspergillus nidulans

pH Range

pH Minimum	pH Maximum	Comment	Organism
4	9	activity range, wild-type enzyme, optimum at pH 5.0, 45% of maximal activity at pH 9.0	Aspergillus nidulans

pH Stability

pH Stability	pH Stability Maximum	Comment	Organism
1	2	purified recombinant wild-type enzyme, 37°C, 1 h, 50% activity remaining	Aspergillus nidulans
3	-	purified recombinant wild-type enzyme, 37°C, 1 h, 50% activity remaining	Aspergillus nidulans
4	9	purified recombinant wild-type enzyme, 37°C, 1 h, stable at	Aspergillus nidulans

General Information

General Information	Comment	Organism
evolution	the enzyme belongs to the glycosyl hydrolase family 5, GH5	Aspergillus nidulans