BtRgl11A(Rhamnogalacturonan lyase)
BtRgl11A(BT4175)
En-Rgl0173
(EC. 4.2.2.23) Rhamnogalacturonan lyase
CAZy Family: PL11
PROPERTIES
1. ELECTROPHORETIC PURITY
-Single band on SDS-gel electrophoresis (MW ~66 kDa)
Figure 1. Electrophoresis analysis of BtRgl11A. 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, BtRgl11A purified from Ni sepharose fast flow column.
2. SPECIFIC ACTIVITY
6.9 U/mg protein (on RG-I-AT from Adenophora tetraphylla(Thunb.)Fisch.) at pH 8.0 and 37°C.
One unit of RGL activity was defined as the amount of enzyme required to generate 1 μmol of 4,5-unsaturated galacturonic acid in 1 min.
3. RELATIVE RATES OF HYDROLYSIS OF SUBSTRATES
Table 1 Activity of BtRgl11A
Substrate |
kcat(min-1) |
Km (mM) |
kcat/Km(mM-1min-1) |
AT-RG-I |
658 ± 26.4 |
0.064 ± 0.007 |
1.0×104 ± 1.4x103 |
Sugar beet arabinan |
1.1×103 ± 42.5 |
0.230 ± 0.029 |
4.9×103 ± 804 |
P-RG-I |
222.8 ± 9.1 |
0.079 ± 0.013 |
2.8×103 ± 569 |
4. PHYSICOCHEMICAL PROPERTIES
pH Optima: 9.0
Temperature Optima: 37°C
Figure 2. Effect of pH on the activity of recombinant BtRgl11A. Assays were carried out at 37°C for 10 min in buffers ranging in pH from 5.5 to 11.0. The activity at optimum pH was defifined as 100%.
Figure 3. HILIC-UV ( UV235nm ) chromatograms of BtRgl11A oligosaccharides.
5. STORAGE CONDITIONS
The enzyme should be stored at -20°C. For assay, this enzyme should be diluted in phosphate buffer (50 mM) pH 8.0. Swirl to mix the enzyme immediately prior to use.
6. References
[1] Luís A S. Microbial pectin recognition and utilization of the mammalian gastrointestinal tract[D]. Newcastle University, 2017.
