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Purification and characterization of Mn-peroxidase from Musa paradisiaca (banana) stem juice
Pratibha Yadav1, V K Singh, Meera Yadav
1Department of Chemistry, U P P G College, Varanasi 221002 (U. P.), India.
Abstract:
Mn-peroxidase (MnP), a biotechnologically important enzyme was purified for the first time from a plant source Musa paradisiaca (banana) stem, which is an agro-waste easily available after harvest of banana fruits. MnP was earlier purified only from the fungal sources. The enzyme was purified from stem juice by ultrafiltration and anion-exchange column chromatography on diethylamino ethylcellulose with 8-fold purification and purification yield of 65%. The enzyme gave a single protein band in SDS-PAGE corresponding to molecular mass 43 kDa. The Native-PAGE of the enzyme also gave a single protein band, confirming the purity of the enzyme. The UV/VIS spectrum of the purified enzyme differed from the other heme peroxidases, as the Soret band was shifted towards lower wavelength and the enzyme had an intense absorption band around 250 nm. The K(m) values using MnSO4 and H2O2 as the substrates of the purified enzyme were 21.0 and 9.5 microM, respectively. The calculated k(cat) value of the purified enzyme using Mn(II) as the substrate in 50 mM lactate buffer (pH 4.5) at 25 degrees C was 6.7s(-1), giving a k(cat)/K(m) value of 0.32 microM(-1)s(-1). The k(cat) value for the MnP-catalyzed reaction was found to be dependent of the Mn(III) chelator molecules malonate, lactate and oxalate, indicating that the enzyme oxidized chelated Mn(II) to Mn(III). The pH and temperature optima of the enzyme were 4.5 and 25 degrees C, respectively. The enzyme in combination with H2O2 liberated bromine and iodine in presence of KBr and KI respectively. All these enzymatic characteristics were similar to those of fungal MnP. The enzyme has the potential as a green brominating and iodinating agent in combination with KBr/KI and H2O2.
Insights
This study reports the first purification of manganese peroxidase (MnP) from banana stems, an abundant agro-waste. The plant-derived enzyme exhibits characteristics similar to fungal MnP and shows potential as a green brominating and iodinating agent.
Area of Science:
- Biochemistry
- Enzymology
- Plant biotechnology
Background:
- Manganese peroxidase (MnP) is a crucial enzyme primarily isolated from fungal sources.
- Agro-waste, such as banana stems, represents an underutilized resource for enzyme extraction.
- Investigating plant-derived enzymes offers novel biotechnological applications.
Purpose of the Study:
- To purify and characterize manganese peroxidase (MnP) from the plant source Musa paradisiaca (banana) stem.
- To compare the enzymatic properties of plant-derived MnP with its fungal counterparts.
- To explore the potential of plant-derived MnP as a green brominating and iodinating agent.
Main Methods:
- Enzyme purification using ultrafiltration and anion-exchange chromatography.
- Enzyme characterization via SDS-PAGE, Native-PAGE, UV/VIS spectroscopy, and kinetic analysis (K(m), k(cat)).
- Assessing enzyme activity with various substrates and chelators, and determining optimal pH and temperature.
Main Results:
- MnP was successfully purified from banana stem juice with 8-fold purification and 65% yield.
- The purified enzyme presented a single band on SDS-PAGE and Native-PAGE, with a molecular mass of 43 kDa.
- Kinetic parameters (K(m), k(cat)) and optimal conditions (pH 4.5, 25°C) were determined, and the enzyme demonstrated bromination and iodination capabilities.
Conclusions:
- Musa paradisiaca stem is a viable source for purifying manganese peroxidase (MnP).
- The purified plant MnP shares biochemical and catalytic properties with fungal MnP.
- This enzyme holds promise as an environmentally friendly agent for bromination and iodination reactions.
