Characterization, Purification and Immobilization …
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a nylon cloth to collect the extract (370 ml) which was centrifuged at 10,000 rpm to
obtain the clear supernatant (360 ml) which was subjected to ultra-filtration (using
PM 10 KDa membrane; Sartorius, Germany) and finally used as the source of crude
enzyme.
2.3 Determination of Enzyme Activity
Invertase activity was determined in 0.1 M acetate buffer, pH 5.0 at 50 °C using
2% (w/ v) sucrose as substrate. Reducing sugars formed were estimated by DNSA
method. One unit of enzyme activity was taken as the amount of enzyme that could
produce 1 μmole of reducing sugar/min under the assay conditions (Mukherjee et al.
2010). Protein was determined by coomassie brilliant blue protein assay reagent
(Bradford 1976).
2.4 Purification of the Enzyme
Enzyme was purified by (NH 4 ) 2 SO 4 precipitation (90% saturation), ion exchange
chromatography using DEAE-Sephadex (A50) column (3 cm × 25 cm), size exclusion chromatography using Sephacryl S-300-HR column (1.5 cm × 60 cm) and
HPLC [BioSep-SEC-S-2000 (300 mm × 7.8 mm)]. Purified protein sample (40 μg)
was subjected to denaturing SDS–PAGE. The gels were also subjected to Periodic
Acid-Schiff (PAS) stain.
2.5 Determination of Physicochemical Properties
of the Enzyme
The pH optimum and stability of plant invertase was determined in the pH range
of 3.0–9.0 using various buffer systems. The optimum temperature and thermostability of the enzyme was determined in the temperature range of 20–80 °C. Effect
of different chemical modifiers on enzyme activity was determined by incubating 20
U of purified invertase separately (at 37 °C) with iodoacetic acid (50 mM), iodoacetamide (50 mM), NEM (50 mM), PCMB (1 mM), DTNB (5 mM) and EDAC
(50 mM) at their respective recommended pH buffers (Mukherjee et al. 2010). Effect
of various metal ions on purified invertase was determined by incubating 5 U/ml of
enzyme (in 0.1 M acetate buffer, pH 5.0; 37 °C) in presence of different metal ions
161
a nylon cloth to collect the extract (370 ml) which was centrifuged at 10,000 rpm to
obtain the clear supernatant (360 ml) which was subjected to ultra-filtration (using
PM 10 KDa membrane; Sartorius, Germany) and finally used as the source of crude
enzyme.
2.3 Determination of Enzyme Activity
Invertase activity was determined in 0.1 M acetate buffer, pH 5.0 at 50 °C using
2% (w/ v) sucrose as substrate. Reducing sugars formed were estimated by DNSA
method. One unit of enzyme activity was taken as the amount of enzyme that could
produce 1 μmole of reducing sugar/min under the assay conditions (Mukherjee et al.
2010). Protein was determined by coomassie brilliant blue protein assay reagent
(Bradford 1976).
2.4 Purification of the Enzyme
Enzyme was purified by (NH 4 ) 2 SO 4 precipitation (90% saturation), ion exchange
chromatography using DEAE-Sephadex (A50) column (3 cm × 25 cm), size exclusion chromatography using Sephacryl S-300-HR column (1.5 cm × 60 cm) and
HPLC [BioSep-SEC-S-2000 (300 mm × 7.8 mm)]. Purified protein sample (40 μg)
was subjected to denaturing SDS–PAGE. The gels were also subjected to Periodic
Acid-Schiff (PAS) stain.
2.5 Determination of Physicochemical Properties
of the Enzyme
The pH optimum and stability of plant invertase was determined in the pH range
of 3.0–9.0 using various buffer systems. The optimum temperature and thermostability of the enzyme was determined in the temperature range of 20–80 °C. Effect
of different chemical modifiers on enzyme activity was determined by incubating 20
U of purified invertase separately (at 37 °C) with iodoacetic acid (50 mM), iodoacetamide (50 mM), NEM (50 mM), PCMB (1 mM), DTNB (5 mM) and EDAC
(50 mM) at their respective recommended pH buffers (Mukherjee et al. 2010). Effect
of various metal ions on purified invertase was determined by incubating 5 U/ml of
enzyme (in 0.1 M acetate buffer, pH 5.0; 37 °C) in presence of different metal ions