BtGlu31A(exo-α-Glucosidase,BT3299)

BtGlu31A

Ex-Glu0003

(EC.3.2.1.20) exo-α-Glucosidase

CAZy Family: GH31

PROPERTIES

1.ELECTROPHORETIC PURITY

-Single band on SDS-gel electrophoresis (MW ~76kDa)

Figure 1. Electrophoresis analysis of BtGlu31A. 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, BtGlu31A purified from Ni sepharose fastflow column.


2.SPECIFIC ACTIVITY

0.08 U/mg protein (on pNP-α-glu) at pH7.0 and 37oC

One Unit of pNP-α-glu activity is defined as the amount of enzyme required to release 1 μmol of glucose per minute from pNP-α-glu (5 mM) in phosphate buffer(10mM) pH7.0.


3.RELATIVE RATES OF HYDROLYSIS OF SUBSTRATES

Fig.2.Comparison of the hydrolytic activity of a-glucosidases BT0339 (black bars) and BtGlu31A(grey bars). Substrates assessed were maltose (1), isomaltase (2), maltotriose (3), maltotetraose (4), maltopentaose (5), maltohexaose (6), maltoheptaose (7), lactose (8) and sucrose (9).

Fig. 3.Table of experimentally derived Ki and BtGlu31A specific activity values. Ki values were determined for secondary substrates added to the BtGlu31A kinetic experiment usingpNP-glucose as a substrate. The specific activity was also generated in order to monitor the effect of the secondary substrate on the enzyme activity with pNP-glucose.

4.PHYSICOCHEMICAL PROPERTIES

pH Optima:6.0

       pH Stability:4.0-9.0

Figure4. pH profile ofBtGlu31Aactivity using pNP glucose as a substrate. ThepNP-glucose assay was performed under different pH conditions using a variety of buffers (pH 4.0-9.0). The pH at which the optimal activity occurred (pH 6.0) was assigned an activity value of 100% and all other activity values were assigned relative to this value.



5.STORAGE CONDITIONS

The enzyme should be stored at -20°C. For assay, this enzyme should be diluted in phosphate buffer (10 mM) pH7.0. Swirl to mix the enzyme immediately prior to use.



6. REFERENCES

[1]Jacobs, Jenny-Lyn L. I..Characterization of BT3299 :a Family GH31 Enzyme from a Prominent Gut Symbiont, Bacteroides thetaiotaomicron [J]. Library and Archives Canada = Bibliothèque et Archives Canada,2012.

[2] Marcia M. Chaudet,Jenny-Lyn Allen,David R. Rose.Expression and purifification of two Family GH31 a-glucosidases from Bacteroides thetaiotaomicron[J].Protein Expression and Purifification[J].2012, 86:135-141.

糖苷酶库 | 学校主页

糖苷酶库

当前位置: 网站首页 \ 糖苷酶库 \ α-葡萄糖苷酶 \ 正文

α-葡萄糖苷酶

BtGlu31A(exo-α-Glucosidase,BT3299)

BtGlu31A

Ex-Glu0003

(EC.3.2.1.20) exo-α-Glucosidase

CAZy Family: GH31

PROPERTIES

1.ELECTROPHORETIC PURITY

-Single band on SDS-gel electrophoresis (MW ~76kDa)

Figure 1. Electrophoresis analysis of BtGlu31A. 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, BtGlu31A purified from Ni sepharose fastflow column.


2.SPECIFIC ACTIVITY

0.08 U/mg protein (on pNP-α-glu) at pH7.0 and 37oC

One Unit of pNP-α-glu activity is defined as the amount of enzyme required to release 1 μmol of glucose per minute from pNP-α-glu (5 mM) in phosphate buffer(10mM) pH7.0.


3.RELATIVE RATES OF HYDROLYSIS OF SUBSTRATES

Fig.2.Comparison of the hydrolytic activity of a-glucosidases BT0339 (black bars) and BtGlu31A(grey bars). Substrates assessed were maltose (1), isomaltase (2), maltotriose (3), maltotetraose (4), maltopentaose (5), maltohexaose (6), maltoheptaose (7), lactose (8) and sucrose (9).

Fig. 3.Table of experimentally derived Ki and BtGlu31A specific activity values. Ki values were determined for secondary substrates added to the BtGlu31A kinetic experiment usingpNP-glucose as a substrate. The specific activity was also generated in order to monitor the effect of the secondary substrate on the enzyme activity with pNP-glucose.

4.PHYSICOCHEMICAL PROPERTIES

pH Optima:6.0

       pH Stability:4.0-9.0

Figure4. pH profile ofBtGlu31Aactivity using pNP glucose as a substrate. ThepNP-glucose assay was performed under different pH conditions using a variety of buffers (pH 4.0-9.0). The pH at which the optimal activity occurred (pH 6.0) was assigned an activity value of 100% and all other activity values were assigned relative to this value.



5.STORAGE CONDITIONS

The enzyme should be stored at -20°C. For assay, this enzyme should be diluted in phosphate buffer (10 mM) pH7.0. Swirl to mix the enzyme immediately prior to use.



6. REFERENCES

[1]Jacobs, Jenny-Lyn L. I..Characterization of BT3299 :a Family GH31 Enzyme from a Prominent Gut Symbiont, Bacteroides thetaiotaomicron [J]. Library and Archives Canada = Bibliothèque et Archives Canada,2012.

[2] Marcia M. Chaudet,Jenny-Lyn Allen,David R. Rose.Expression and purifification of two Family GH31 a-glucosidases from Bacteroides thetaiotaomicron[J].Protein Expression and Purifification[J].2012, 86:135-141.