Characterization, Purification
and Immobilization of an Acid Invertase
from Mentha Spicata (Pudina)
for the Production of Invert Syrup
Abhishek Mukherjee
Abstract Invertase (β-fructofuranosidase), a commercially important enzyme, for
the preparation of invert syrup, has gained importance due to its various biotechnological applications in food and pharmaceutical industry. The present study reports
that a 68 kDa invertase (specific activity = 230 ± 25 U/mg protein), appreciably
present in Mentha spicata stem (1900 ± 100 U/100 g fresh wt.), was purified by
(NH 4 ) 2 SO 4 precipitation, ion exchange chromatography, size exclusion chromatography and HPLC. The enzyme was stable in the pH range of 3.5–7.0 up to 55 °C,
with a K m of 7.9 mM sucrose. Hg
2+ > PCMB > Ag
+ > Pb
2+ > Cd
2+ inhibited the
enzyme activity. Iodoacetic acid, iodoacetamide, DTNB and N-ethylmaleimide did
not affect the invertase activity suggesting the non-thiol nature of the enzyme. The
enzyme hydrolyzed sucrose, raffinose and inulin (slightly) with no maltose or levan
hydrolyzing activities. The enzyme (3U/mL) completely hydrolyzed sucrose (8%
w/v) to invert syrup in 5 h at 50 °C. Immobilization of the enzyme on oxidized
bagasse (dialdehyde cellulose) increased its temperature optima (by 10 °C) and thermostability (retaining 45 ± 2% and 30 ± 1% activities at 70 and 80 °C, respectively). Immobilized enzyme system efficiently produced invert syrup from sucrose,
remaining 80 ± 1% active after 20th cycle.
Keywords Mentha spicata · Invertase · Sucrose · Enzyme immobilization ·
Bagasse
1 Introduction
Invertase (EC 3.2.1.26) or β-fructofuranosidase catalyses the cleavage of sucrose to
an equimolar mixture of glucose and fructose, known as invert syrup. Invert syrup
is sweeter than sucrose and does not crystallize easily (remains in liquid form), and
therefore widely used in food (as fondant filling for chocolates, confectioneries, jams
A. Mukherjee (B)
Department of Biotechnology, Heritage Institute of Technology, Chowbaga Road
P.O.-Anandapur, East Kolkata Township, Kolkata, West Bengal 700107, India
e-mail: abhi.india79@gmail.com
© Springer Nature Singapore Pte Ltd. 2021
D. Ramkrishna et al. (eds.), Advances in Bioprocess Engineering and Technology,
Lecture Notes in Bioengineering,
https://doi.org/10.1007/978-981-15-7409-2_16
159
and Immobilization of an Acid Invertase
from Mentha Spicata (Pudina)
for the Production of Invert Syrup
Abhishek Mukherjee
Abstract Invertase (β-fructofuranosidase), a commercially important enzyme, for
the preparation of invert syrup, has gained importance due to its various biotechnological applications in food and pharmaceutical industry. The present study reports
that a 68 kDa invertase (specific activity = 230 ± 25 U/mg protein), appreciably
present in Mentha spicata stem (1900 ± 100 U/100 g fresh wt.), was purified by
(NH 4 ) 2 SO 4 precipitation, ion exchange chromatography, size exclusion chromatography and HPLC. The enzyme was stable in the pH range of 3.5–7.0 up to 55 °C,
with a K m of 7.9 mM sucrose. Hg
2+ > PCMB > Ag
+ > Pb
2+ > Cd
2+ inhibited the
enzyme activity. Iodoacetic acid, iodoacetamide, DTNB and N-ethylmaleimide did
not affect the invertase activity suggesting the non-thiol nature of the enzyme. The
enzyme hydrolyzed sucrose, raffinose and inulin (slightly) with no maltose or levan
hydrolyzing activities. The enzyme (3U/mL) completely hydrolyzed sucrose (8%
w/v) to invert syrup in 5 h at 50 °C. Immobilization of the enzyme on oxidized
bagasse (dialdehyde cellulose) increased its temperature optima (by 10 °C) and thermostability (retaining 45 ± 2% and 30 ± 1% activities at 70 and 80 °C, respectively). Immobilized enzyme system efficiently produced invert syrup from sucrose,
remaining 80 ± 1% active after 20th cycle.
Keywords Mentha spicata · Invertase · Sucrose · Enzyme immobilization ·
Bagasse
1 Introduction
Invertase (EC 3.2.1.26) or β-fructofuranosidase catalyses the cleavage of sucrose to
an equimolar mixture of glucose and fructose, known as invert syrup. Invert syrup
is sweeter than sucrose and does not crystallize easily (remains in liquid form), and
therefore widely used in food (as fondant filling for chocolates, confectioneries, jams
A. Mukherjee (B)
Department of Biotechnology, Heritage Institute of Technology, Chowbaga Road
P.O.-Anandapur, East Kolkata Township, Kolkata, West Bengal 700107, India
e-mail: abhi.india79@gmail.com
© Springer Nature Singapore Pte Ltd. 2021
D. Ramkrishna et al. (eds.), Advances in Bioprocess Engineering and Technology,
Lecture Notes in Bioengineering,
https://doi.org/10.1007/978-981-15-7409-2_16
159