162
A. Mukherjee
(Hg
2+ , Ca
2+ , Mg
2+ , Zn
2+ , Fe
2+ , Fe
3+ , Cd
2+ , Cu
2+ , Ag
+ and Mn
2+ ) of 1 mM concentration. Effect of proteolytic enzymes on the stability of invertase was determined by
pre-incubating (at 37 °C) purified invertase (25 U/ml) separately with pepsin (150
U/ml), trypsin (200 U/ml), chymotrypsin (132 U/ml) and proteinase-K (40 U/ml) in
their respective buffers.
Activity of purified invertase on various substrates (sucrose, maltose, raffinose,
melezitose, trehalose, inulin, levan, isomaltose) was determined in 0.1 M acetate
buffer, pH 5.0 at 50 °C. The kinetics of sucrose hydrolysis was studied in terms of
liberation of reducing sugars from the reaction mixtures (20 ml) containing 3–6 U/ml
of purified invertase and 1%–8% (w/v) sucrose in 0.1 M acetate buffer, pH 5.0 at
50 °C.
2.6 Immobilization of Mentha spicata Invertase on Bagasse
Dialdehyde Cellulose
Bagasse (1 kg), collected from the local market, was boiled thrice in distilled water,
dried in a hot air oven (60 °C), chopped into small pieces (0.2–0.5 cm long), treated
alternatively with 1.25% (v/v) sulphuric acid and 1.25% (w/v) sodium hydroxide,
followed by thorough washing with distilled water and 95% (v/v) ethanol. The
biomass was finally dried in a hot air oven to obtain purified bagasse which was
oxidized (Varavinit et al. 2002) by soaking 40 g of biomass in 1 L of 0.03 M periodic
acid (pH-3.0; 90 °C), followed by washing (with distilled water) and oven-drying
to obtain cellulose-diadlehyde. 200 ml of crude concentrated enzyme solution (300
± 50 U/ml) in 0.1 M phosphate buffer, pH 6.0 was coupled with 24 g cellulosedialdehyde in four steps (using 6 g biomass each step). The treated bagasse was then
vacuum filtered, washed twice in distilled water, press-dried on a blotting paper and
stored at 4 °C in 0.1 M phosphate buffer, pH 6.0.
3 Results and Discussion
3.1 Yield and Storage Stability
Though invertase activity has been reported in plants, like potato tuber (Bracho and
Whitaker 1990), carrot (Lee and Sturm 1996), apple fruit (Pan et al. 2005), cherry
fruit (Krishnan and Pueppke 1990), chicory root (Van Den Ende and Van Laere
1993), Tinospora cordifolia stem (Mukherjee et al. 2012; Sengupta et al. 2013) etc.,
yet, except in Tinospora cordifolia (4500 ± 500 U/100 g fresh stem), the presence of
appreciable amount of enzyme has not been reported. Mentha spicata stem yielded
1900 ± 100 U of acid stable invertase/100 g when extracted with 0.1 M phosphate
buffer, pH 6.0 at 37 °C. Lyophilized stem powder yielded 8800 ± 400 U/100 g
A. Mukherjee
(Hg
2+ , Ca
2+ , Mg
2+ , Zn
2+ , Fe
2+ , Fe
3+ , Cd
2+ , Cu
2+ , Ag
+ and Mn
2+ ) of 1 mM concentration. Effect of proteolytic enzymes on the stability of invertase was determined by
pre-incubating (at 37 °C) purified invertase (25 U/ml) separately with pepsin (150
U/ml), trypsin (200 U/ml), chymotrypsin (132 U/ml) and proteinase-K (40 U/ml) in
their respective buffers.
Activity of purified invertase on various substrates (sucrose, maltose, raffinose,
melezitose, trehalose, inulin, levan, isomaltose) was determined in 0.1 M acetate
buffer, pH 5.0 at 50 °C. The kinetics of sucrose hydrolysis was studied in terms of
liberation of reducing sugars from the reaction mixtures (20 ml) containing 3–6 U/ml
of purified invertase and 1%–8% (w/v) sucrose in 0.1 M acetate buffer, pH 5.0 at
50 °C.
2.6 Immobilization of Mentha spicata Invertase on Bagasse
Dialdehyde Cellulose
Bagasse (1 kg), collected from the local market, was boiled thrice in distilled water,
dried in a hot air oven (60 °C), chopped into small pieces (0.2–0.5 cm long), treated
alternatively with 1.25% (v/v) sulphuric acid and 1.25% (w/v) sodium hydroxide,
followed by thorough washing with distilled water and 95% (v/v) ethanol. The
biomass was finally dried in a hot air oven to obtain purified bagasse which was
oxidized (Varavinit et al. 2002) by soaking 40 g of biomass in 1 L of 0.03 M periodic
acid (pH-3.0; 90 °C), followed by washing (with distilled water) and oven-drying
to obtain cellulose-diadlehyde. 200 ml of crude concentrated enzyme solution (300
± 50 U/ml) in 0.1 M phosphate buffer, pH 6.0 was coupled with 24 g cellulosedialdehyde in four steps (using 6 g biomass each step). The treated bagasse was then
vacuum filtered, washed twice in distilled water, press-dried on a blotting paper and
stored at 4 °C in 0.1 M phosphate buffer, pH 6.0.
3 Results and Discussion
3.1 Yield and Storage Stability
Though invertase activity has been reported in plants, like potato tuber (Bracho and
Whitaker 1990), carrot (Lee and Sturm 1996), apple fruit (Pan et al. 2005), cherry
fruit (Krishnan and Pueppke 1990), chicory root (Van Den Ende and Van Laere
1993), Tinospora cordifolia stem (Mukherjee et al. 2012; Sengupta et al. 2013) etc.,
yet, except in Tinospora cordifolia (4500 ± 500 U/100 g fresh stem), the presence of
appreciable amount of enzyme has not been reported. Mentha spicata stem yielded
1900 ± 100 U of acid stable invertase/100 g when extracted with 0.1 M phosphate
buffer, pH 6.0 at 37 °C. Lyophilized stem powder yielded 8800 ± 400 U/100 g