2.5 Enzyme assay
AGL activity was measured by the release of glucose from maltose[46]. Each reaction mixture (100 μL) containing 25 mM maltose, 100 mM sodium acetate buffer (pH 4.0), and 10 μg purified enzyme (or 20 μL culture supernatant) was incubated for 10 min at 37 °C. After heating at 100 °C for 7 min, the glucose of 10 μL reaction mixture was assayed using the D-glucose GOD-POD colorimetric assay kit[47-48]. One activity unit was defined as the amount of enzyme that liberate 2 μmol of glucose from maltose per minute under the above assay conditions.
The pH optimum of rAGL was measured in the range of pH 3.0–7.0 using 100 mM sodium citrate buffer (pH 3.0–3.5), sodium acetate buffer (pH 4.0–5.5) and sodium phosphate buffer (pH 6.0–7.0), respectively. To determine the pH stability, rAGL (2.12 mg/mL) was preincubated in the various buffers described above at 4 °C for 24 h and 48 h, and then the residual activity was assayed under the standard assay conditions[28].
The optimal temperature of rAGL was measured at pH 4.0 and temperatures range from 37 °C to 70 °C, and maltose was used as substrate in 100 mM acetate buffer. The thermal stability was determined by incubating rAGL (2.12 mg/mL) in 100 mM acetate buffer (pH 4.0) at different temperatures (30–70 °C). At each temperature, the buffer and rAGL were preincubated for 15 min, 30 min, 45 min and 1 h, respectively. The reaction was initiated by adding maltose, and allowed to proceed for another 10 min. At different time intervals, samples were collected and the residual activity was assayed under the standard assay conditions[28, 37].
To determine the influence of metal ions (Cu2+, Ca2+, Mg2+, Mn2+, Zn2+, Fe3+, Fe2+) on enzyme activity, rAGL (2.12 mg/mL) was preincubated with each metal ion (10 mM) in 100 mM acetate buffer (pH 4.0) at 37 °C for 1 h. The residual enzyme activity was detected at 37 °C.
For determination of the Michaelis-Menten constants of rAGL, reactions were carried out under the standard assay conditions with different concentrations of maltose (5–100 mM). After incubation for 10 min, the reaction mixture was heated to 100 °C for 7 min and analyzed by the GOD-POD method. On the basis of Michaelis-Menten kinetic model, theKm value of rAGL for maltose was determined by non-linear curve-fitting.
All reactions were conducted in triplicate. The relative activity (%) was calculated in terms of that of the maximum activity (100%).