164
A. Mukherjee
3.3 Physicochemical Properties
Purified invertase was found to be optimally active in the pH range of 4.0–5.5, with
optimal activity at 5.0, similar to that of Tinospora codifolia invertase (Sengupta et al.
2013), potato tuber invertase (Bracho and Whitaker 1990) and cherry fruit invertase
(Krishnan and Pueppke 1990). The enzyme retained 46 ± 2%, 75 ± 2% and 83 ± 1%
of its activity at pH 3.0, 4.0 and 5.0, respectively, after 3 h of incubation, displaying
maximum stability at pH 6.0 but lost 62 ± 2% of its activity at pH 8.0. Enzyme
activity increased with the increase in temperature till 60°C, being optimally active
in the range of 50–60°C and stable in the range of 0–55°C. pH and thermal stability
of Mentha spicata invertase makes it an effective remedy (oral enzyme therapy) for
sucrose intolerance. The enzyme need not be purified and can be used as a crude
concentrated powder or liquid as the source plant is completely non-toxic (Güney
et al. 2006; Kumar et al. 2008) and has been used since a long time in various
traditional flavoured drinks and recipes throughout the Indian subcontinent.
Mentha spicata invertase was completely inhibited by 1 mM PCMB (within
10 min of incubation). On the other hand, DTNB, iodoacetamide, iodoacetic acid and
NEM had no significant effect on enzyme activity thereby suggesting its non-thiol
nature unlike that of papain, T. cordifolia amylase, bromelain and ficin (which are all
thiol enzymes). However, water-soluble carbodimide decreased invertase activity
by 49 ± 2% and 69 ± 1% in 2 and 4 h, respectively, suggesting the requirement of carboxyl group for its catalytic activity. The enzyme efficiently hydrolyzed
sucrose, displayed much less activity on raffinose, hydrolyzed inulin slightly but was
completely inactive on maltose, melezitose, trehalose, isomaltose and levan. Purified enzyme (3–6 U/ml) efficiently hydrolyzed 93–95% of 8% (w/v) sucrose in 5 h
(Fig. 2a). Purified invertase displayed K m and V max of 7.9 mM (2.7 mg/ml) and 0.90
μmole of reducing sugar/min, respectively, using sucrose as substrate. Invertase was
found to be strongly inhibited by heavy metal ions (Hg
2+ > Ag
+ > Pb
2+ > Cd
2+ ) at
1 mM concentration while Ca
2+ , Mg
2+ , Zn
2+ had no significant effect on enzyme
activity. However, Mn
2+ slightly increased the enzyme activity. It was observed that
the proteolytic enzymes (trypsin, chymotrypsin, proteinase-K) at their optimal pH
did not lower the activity of Mentha spicata invertase till 2 h of incubation.
3.4 Immobilization
Immobilization of invertase (Fig. 1) on oxidized bagasse (% activity yield = 49 ± 2%)
improved its temperature optima (by 10 °C) and thermostability. Immobilized invertase was found to be optimally active at 70 °C (instead of at 60 °C for soluble enzyme)
and displayed 24 ± 1% more activity than soluble enzyme at 80 °C. Soluble invertase was sharply inactivated beyond 65 °C while the immobilized enzyme system
remained fairly stable retaining 45 ± 2% and 30 ± 1% of its activity at 70 and 80 °C,
respectively. The immobilized enzyme system could efficiently run for 20 successive
A. Mukherjee
3.3 Physicochemical Properties
Purified invertase was found to be optimally active in the pH range of 4.0–5.5, with
optimal activity at 5.0, similar to that of Tinospora codifolia invertase (Sengupta et al.
2013), potato tuber invertase (Bracho and Whitaker 1990) and cherry fruit invertase
(Krishnan and Pueppke 1990). The enzyme retained 46 ± 2%, 75 ± 2% and 83 ± 1%
of its activity at pH 3.0, 4.0 and 5.0, respectively, after 3 h of incubation, displaying
maximum stability at pH 6.0 but lost 62 ± 2% of its activity at pH 8.0. Enzyme
activity increased with the increase in temperature till 60°C, being optimally active
in the range of 50–60°C and stable in the range of 0–55°C. pH and thermal stability
of Mentha spicata invertase makes it an effective remedy (oral enzyme therapy) for
sucrose intolerance. The enzyme need not be purified and can be used as a crude
concentrated powder or liquid as the source plant is completely non-toxic (Güney
et al. 2006; Kumar et al. 2008) and has been used since a long time in various
traditional flavoured drinks and recipes throughout the Indian subcontinent.
Mentha spicata invertase was completely inhibited by 1 mM PCMB (within
10 min of incubation). On the other hand, DTNB, iodoacetamide, iodoacetic acid and
NEM had no significant effect on enzyme activity thereby suggesting its non-thiol
nature unlike that of papain, T. cordifolia amylase, bromelain and ficin (which are all
thiol enzymes). However, water-soluble carbodimide decreased invertase activity
by 49 ± 2% and 69 ± 1% in 2 and 4 h, respectively, suggesting the requirement of carboxyl group for its catalytic activity. The enzyme efficiently hydrolyzed
sucrose, displayed much less activity on raffinose, hydrolyzed inulin slightly but was
completely inactive on maltose, melezitose, trehalose, isomaltose and levan. Purified enzyme (3–6 U/ml) efficiently hydrolyzed 93–95% of 8% (w/v) sucrose in 5 h
(Fig. 2a). Purified invertase displayed K m and V max of 7.9 mM (2.7 mg/ml) and 0.90
μmole of reducing sugar/min, respectively, using sucrose as substrate. Invertase was
found to be strongly inhibited by heavy metal ions (Hg
2+ > Ag
+ > Pb
2+ > Cd
2+ ) at
1 mM concentration while Ca
2+ , Mg
2+ , Zn
2+ had no significant effect on enzyme
activity. However, Mn
2+ slightly increased the enzyme activity. It was observed that
the proteolytic enzymes (trypsin, chymotrypsin, proteinase-K) at their optimal pH
did not lower the activity of Mentha spicata invertase till 2 h of incubation.
3.4 Immobilization
Immobilization of invertase (Fig. 1) on oxidized bagasse (% activity yield = 49 ± 2%)
improved its temperature optima (by 10 °C) and thermostability. Immobilized invertase was found to be optimally active at 70 °C (instead of at 60 °C for soluble enzyme)
and displayed 24 ± 1% more activity than soluble enzyme at 80 °C. Soluble invertase was sharply inactivated beyond 65 °C while the immobilized enzyme system
remained fairly stable retaining 45 ± 2% and 30 ± 1% of its activity at 70 and 80 °C,
respectively. The immobilized enzyme system could efficiently run for 20 successive