PsGlu3A (exo-β-Glucosidase,BglPC28）


PsGlu3A (BglPC28)

Ex-Glu0014

(EC.3.2.1.21) exo-β-Glucosidase

CAZy Family: GH3


PROPERTIES

1.ELECTROPHORETIC PURITY

-Single band on SDS-gel electrophoresis (MW ~79kDa)

Figure 1. Electrophoresis analysis of PsGlu3A. 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, PsGlu3A purified from Ni sepharose fastflow column.


2.SPECIFIC ACTIVITY

11 U/mg protein (on pNP-β-glu) at pH 7.0 and 37°C

One Unit of pNP-β-glu activity is defined as the amount of enzyme required to release 1 μmol of glucose per minute from pNP-β-glu (5 mM) in phosphate buffer (10 mM) pH 7.0.



3.RELATIVE RATES OF HYDROLYSIS OF SUBSTRATES

Table 1. Relative activity of PsGlu3A on different substratesa

a Final concentration, 2.0 mM.

b Activity toward pNP-β-D-glucopyranoside was set as 100%.

Figure2. Thin layer chromatography (TLC) analyses of biotransformation of Rb1, Rb2, Rb3, Rc and Rd by recombinantPsGlu3A. The sampling times were 10 min and 24 h. Developing solvent: CHCl3-CH3OH-H2O (65:35:10, v/v, lower phase). S, ginsenoside standards (PPD type ginsenoside mixtures).

Figure3. TLC analyses of time course of ginsenosides bioconversion byPsGlu3A. (a), transformation ofginsenoside Re; (b), transformation of ginsenoside Rg1. Developing solvent: CHCl3-CH3OH-H2O (65:35:10, v/v, lower phase). Lanes S, ginsenosidestandards (PPT type ginsenoside mixtures).


4.PHYSICOCHEMICAL PROPERTIES

pH Optima:7.0

pH Stability:5.0-8.0

Temperature Optima:37°C

Temperature Stability:<30°C

Figure 5. Effects of temperature (a) and pH (b) on the activity and stability of recombinantPsGlu3A.


5.STORAGE CONDITIONS

The enzyme should be stored at -20°C. For assay, this enzyme should be diluted in phosphate buffer (10 mM) pH 7.0. Swirl to mix the enzyme immediately prior to use.



6. REFERENCES

[1] Juan Du,Chang-Hao Cui, Sung Chul Park,et al. Identification and Characterization of a GinsenosideTransforming b-Glucosidase from Pseudonocardia sp. Gsoil 1536 and Its Application for Enhanced Production of Minor Ginsenoside Rg2(S) [J].Plos one,2014,9(6):e96914.

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PsGlu3A (exo-β-Glucosidase,BglPC28）


PsGlu3A (BglPC28)

Ex-Glu0014

(EC.3.2.1.21) exo-β-Glucosidase

CAZy Family: GH3


PROPERTIES

1.ELECTROPHORETIC PURITY

-Single band on SDS-gel electrophoresis (MW ~79kDa)

Figure 1. Electrophoresis analysis of PsGlu3A. 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, PsGlu3A purified from Ni sepharose fastflow column.


2.SPECIFIC ACTIVITY

11 U/mg protein (on pNP-β-glu) at pH 7.0 and 37°C

One Unit of pNP-β-glu activity is defined as the amount of enzyme required to release 1 μmol of glucose per minute from pNP-β-glu (5 mM) in phosphate buffer (10 mM) pH 7.0.



3.RELATIVE RATES OF HYDROLYSIS OF SUBSTRATES

Table 1. Relative activity of PsGlu3A on different substratesa

a Final concentration, 2.0 mM.

b Activity toward pNP-β-D-glucopyranoside was set as 100%.

Figure2. Thin layer chromatography (TLC) analyses of biotransformation of Rb1, Rb2, Rb3, Rc and Rd by recombinantPsGlu3A. The sampling times were 10 min and 24 h. Developing solvent: CHCl3-CH3OH-H2O (65:35:10, v/v, lower phase). S, ginsenoside standards (PPD type ginsenoside mixtures).

Figure3. TLC analyses of time course of ginsenosides bioconversion byPsGlu3A. (a), transformation ofginsenoside Re; (b), transformation of ginsenoside Rg1. Developing solvent: CHCl3-CH3OH-H2O (65:35:10, v/v, lower phase). Lanes S, ginsenosidestandards (PPT type ginsenoside mixtures).


4.PHYSICOCHEMICAL PROPERTIES

pH Optima:7.0

pH Stability:5.0-8.0

Temperature Optima:37°C

Temperature Stability:<30°C

Figure 5. Effects of temperature (a) and pH (b) on the activity and stability of recombinantPsGlu3A.


5.STORAGE CONDITIONS

The enzyme should be stored at -20°C. For assay, this enzyme should be diluted in phosphate buffer (10 mM) pH 7.0. Swirl to mix the enzyme immediately prior to use.



6. REFERENCES

[1] Juan Du,Chang-Hao Cui, Sung Chul Park,et al. Identification and Characterization of a GinsenosideTransforming b-Glucosidase from Pseudonocardia sp. Gsoil 1536 and Its Application for Enhanced Production of Minor Ginsenoside Rg2(S) [J].Plos one,2014,9(6):e96914.