BtMan76E (endo-α-1,6-Mannanase)

BtMan76E

Endo-Man0104

(EC.3.2.1.101)endo-α-1,6-Mannanase

CAZy Family: GH76


PROPERTIES

1.ELECTROPHORETIC PURITY

-Single band on SDS-gel electrophoresis (MW ~57 kDa)

Figure 1. Electrophoresis analysis ofBtMan76E. M, molecular weight marker (PageRuler Prestained Protein Ladder, Thermo Scientific); lane 1, culture lysate before IPTG induction; lane 2, culture lysate after IPTG induction; lane 3, BtMan76E purified from Ni sepharose fastflow column.


2.SPECIFIC ACTIVITY


3.RELATIVE RATES OF HYDROLYSIS OF SUBSTRATES

Figuer.2 (A)Representative structures and branching patterns of different α-mannans fromS. cerevisiaeandS. pombestrains. Legend for sugars and linkages is displayed at right .(B) Thin layer chromatography showing the mobility of different YM substrates and products released in the presence of BtMan76E. The mobility patterns of mannose, mannobiose (M 2), and mannotriose (M 3) standards are displayed on left. (C) Initial velocity measurements for BtMan76E in the presence of different YM substrates. Error is SD from three technical replicates. Significant differences in activity compared to WT S. cerevisiae YM is shown with ** (P ≤ 0.01). (D) Activity of BtMan76E on wild-type S. cerevisiae YM from pH 3.0-11.0, error bars represent SD over at least three replicates.

Figure 3. Structure of YM products released by BtMan76E. HPAEC-PAD chromatograms of (A) S. cerevisiae and (B) S. pombe YM products in the presence and absence of the GH125 exo-mannanase BT3781. Inserts show TLC separation and visualization of products with mannose, mannobiose (M2), and mannotriose (M3) standards. GC-MS total ion current chromatograms from linkage analysis of (C) S. cerevisiae and (D) S. pombe oligosaccharides and reduced (E) S. cerevisiae and (F) S. pombe oligosaccharide products. Peaks are labeled with monosaccharide linkages.


4.PHYSICOCHEMICAL PROPERTIES

pH Optima:5.8

Figuer4.pH Optima of BtMan76E. Activity of BtMan76E on wild-type S. cerevisiae YM from pH 3.0-11.0, error bars represent SD over at least three replicates.


5.STORAGE CONDITIONS

The enzyme should be stored at -20°C. For assay, this enzyme should be diluted in phosphate buffer (20 mM) pH7.0. Swirl to mix the enzyme immediately prior to use.


6. REFERENCES

[1]Jones D R, Xing X, Tingley J P, et al. Analysis of active site architecture and reaction product linkage chemistry reveals a conserved cleavage substrate for an endo-alpha-mannanase within diverse yeast mannans[J]. Journal of Molecular Biology, 2020, 432(4): 1083-1097.

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BtMan76E (endo-α-1,6-Mannanase)

BtMan76E

Endo-Man0104

(EC.3.2.1.101)endo-α-1,6-Mannanase

CAZy Family: GH76


PROPERTIES

1.ELECTROPHORETIC PURITY

-Single band on SDS-gel electrophoresis (MW ~57 kDa)

Figure 1. Electrophoresis analysis ofBtMan76E. M, molecular weight marker (PageRuler Prestained Protein Ladder, Thermo Scientific); lane 1, culture lysate before IPTG induction; lane 2, culture lysate after IPTG induction; lane 3, BtMan76E purified from Ni sepharose fastflow column.


2.SPECIFIC ACTIVITY


3.RELATIVE RATES OF HYDROLYSIS OF SUBSTRATES

Figuer.2 (A)Representative structures and branching patterns of different α-mannans fromS. cerevisiaeandS. pombestrains. Legend for sugars and linkages is displayed at right .(B) Thin layer chromatography showing the mobility of different YM substrates and products released in the presence of BtMan76E. The mobility patterns of mannose, mannobiose (M 2), and mannotriose (M 3) standards are displayed on left. (C) Initial velocity measurements for BtMan76E in the presence of different YM substrates. Error is SD from three technical replicates. Significant differences in activity compared to WT S. cerevisiae YM is shown with ** (P ≤ 0.01). (D) Activity of BtMan76E on wild-type S. cerevisiae YM from pH 3.0-11.0, error bars represent SD over at least three replicates.

Figure 3. Structure of YM products released by BtMan76E. HPAEC-PAD chromatograms of (A) S. cerevisiae and (B) S. pombe YM products in the presence and absence of the GH125 exo-mannanase BT3781. Inserts show TLC separation and visualization of products with mannose, mannobiose (M2), and mannotriose (M3) standards. GC-MS total ion current chromatograms from linkage analysis of (C) S. cerevisiae and (D) S. pombe oligosaccharides and reduced (E) S. cerevisiae and (F) S. pombe oligosaccharide products. Peaks are labeled with monosaccharide linkages.


4.PHYSICOCHEMICAL PROPERTIES

pH Optima:5.8

Figuer4.pH Optima of BtMan76E. Activity of BtMan76E on wild-type S. cerevisiae YM from pH 3.0-11.0, error bars represent SD over at least three replicates.


5.STORAGE CONDITIONS

The enzyme should be stored at -20°C. For assay, this enzyme should be diluted in phosphate buffer (20 mM) pH7.0. Swirl to mix the enzyme immediately prior to use.


6. REFERENCES

[1]Jones D R, Xing X, Tingley J P, et al. Analysis of active site architecture and reaction product linkage chemistry reveals a conserved cleavage substrate for an endo-alpha-mannanase within diverse yeast mannans[J]. Journal of Molecular Biology, 2020, 432(4): 1083-1097.