FsGlu3A（exo-β-Glucosidase）

FsGlu3A

Ex-Glu0327

(EC.3.2.1.21) exo-β-1,3-Glucosidase

CAZy Family: GH3


PROPERTIES

1. ELECTROPHORETIC PURITY

-Single band on SDS-gel electrophoresis (MW ~84kDa)

Figure 1. Electrophoresis analysis of FsGlu3A. M, molecular weight marker (PageRuler Prestained Protein Ladder, Thermo Scientific); lane 1, culture lysate before IPTG induction; lane 2, culture lysate of after IPTG induction; lane 3，Crude enzyme of FsGlu3A；lane 4, FsGlu3A purified from Ni sepharose fastflow column.


2. SPECIFIC ACTIVITY

78.7 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 p-nitrophenyl per minute from pNP-β-glu (5 mM) in phosphate buffer (20 mM) pH 7.0.


3. RELATIVE RATES OF HYDROLYSIS OF SUBSTRATES

Table 1. Relative activity of FsGlu3A on different substrates

Substrate

Enzyme activity（U/mg）

pNPβGlc

78.7±5.2

pNPβGal

-

pNPβMan

-

pNPβXyl

-

pNPαGlc

-

pNPαGal

-

pNPαMan

-

pNPαXyl

-

pNPαArap

-

pNPαAraf

-

pNPαRha

-

pNPαFuc

-


Table2. Kinetic parameters for FsGlu3A on pNP-β-glu.

Enzyme

Km(mM)

Vmax(mmol/min/mg)

kcat（s-1）

FsGlu3A

1.93±0.16

20.34±1.03

27.38±1.40


Figure 2. TLC analyses of biotransformation of Rb1,Rb2,Rb3,Rc and Rd by recombinant FsGlu3A.


4. PHYSICOCHEMICAL PROPERTIES

pH Optima: 6.0

pH Stability: 6.0-9.0

Temperature Optima: 45°C

Temperature Stability: 30-35°C


Figure 3. Effect of pH on activity (a) and stability (b) of FsGlu3A using pNP-Glu as substrate. The optimal pH (a) was determined at different pH from 2 to 11. The maximum activity obtained was defined as 100% activity. Thermal stability was determined by incubating the enzyme for 3 h at different pH. The activity of the enzyme before incubation was defined as 100%. Results are presented as means ± standard deviations(n = 3).

Figure 4. Effect of temperature on activity (a) and stability (b) of FsGlu3A using pNP-Glu as substrate. The optimal temperature (a) was determined at different temperatures from 20 to 90℃. The maximum activity obtained was defined as 100% activity. Thermal stability was determined by incubating the enzyme for 3 h at different temperatures. The activity of the enzyme before incubation was defined as 100%. Results are presented as means ± standard deviations(n = 3).


5. STORAGE CONDITIONS

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


6. REFERENCES

[1]刘洋.嗜糖黄杆菌中β-葡萄糖苷酶重组表达及功能研究[D]. 东北师范大学, 2022.

[2]董博妍,原野.嗜糖黄杆菌β-葡萄糖苷酶的功能及在稀有人参皂苷制备中的应用.微生物学通报, 2023, 50(09): 3833-3847.

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β-葡萄糖苷酶

FsGlu3A（exo-β-Glucosidase）

FsGlu3A

Ex-Glu0327

(EC.3.2.1.21) exo-β-1,3-Glucosidase

CAZy Family: GH3


PROPERTIES

1. ELECTROPHORETIC PURITY

-Single band on SDS-gel electrophoresis (MW ~84kDa)

Figure 1. Electrophoresis analysis of FsGlu3A. M, molecular weight marker (PageRuler Prestained Protein Ladder, Thermo Scientific); lane 1, culture lysate before IPTG induction; lane 2, culture lysate of after IPTG induction; lane 3，Crude enzyme of FsGlu3A；lane 4, FsGlu3A purified from Ni sepharose fastflow column.


2. SPECIFIC ACTIVITY

78.7 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 p-nitrophenyl per minute from pNP-β-glu (5 mM) in phosphate buffer (20 mM) pH 7.0.


3. RELATIVE RATES OF HYDROLYSIS OF SUBSTRATES

Table 1. Relative activity of FsGlu3A on different substrates

Substrate

Enzyme activity（U/mg）

pNPβGlc

78.7±5.2

pNPβGal

-

pNPβMan

-

pNPβXyl

-

pNPαGlc

-

pNPαGal

-

pNPαMan

-

pNPαXyl

-

pNPαArap

-

pNPαAraf

-

pNPαRha

-

pNPαFuc

-


Table2. Kinetic parameters for FsGlu3A on pNP-β-glu.

Enzyme

Km(mM)

Vmax(mmol/min/mg)

kcat（s-1）

FsGlu3A

1.93±0.16

20.34±1.03

27.38±1.40


Figure 2. TLC analyses of biotransformation of Rb1,Rb2,Rb3,Rc and Rd by recombinant FsGlu3A.


4. PHYSICOCHEMICAL PROPERTIES

pH Optima: 6.0

pH Stability: 6.0-9.0

Temperature Optima: 45°C

Temperature Stability: 30-35°C


Figure 3. Effect of pH on activity (a) and stability (b) of FsGlu3A using pNP-Glu as substrate. The optimal pH (a) was determined at different pH from 2 to 11. The maximum activity obtained was defined as 100% activity. Thermal stability was determined by incubating the enzyme for 3 h at different pH. The activity of the enzyme before incubation was defined as 100%. Results are presented as means ± standard deviations(n = 3).

Figure 4. Effect of temperature on activity (a) and stability (b) of FsGlu3A using pNP-Glu as substrate. The optimal temperature (a) was determined at different temperatures from 20 to 90℃. The maximum activity obtained was defined as 100% activity. Thermal stability was determined by incubating the enzyme for 3 h at different temperatures. The activity of the enzyme before incubation was defined as 100%. Results are presented as means ± standard deviations(n = 3).


5. STORAGE CONDITIONS

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


6. REFERENCES

[1]刘洋.嗜糖黄杆菌中β-葡萄糖苷酶重组表达及功能研究[D]. 东北师范大学, 2022.

[2]董博妍,原野.嗜糖黄杆菌β-葡萄糖苷酶的功能及在稀有人参皂苷制备中的应用.微生物学通报, 2023, 50(09): 3833-3847.