Swati B. Jadhav

Conjugation of α-amylase with dextran for enhanced stability: process details, kinetics and structural analysis.

The influence of enzyme polysaccharide interaction on enzyme stability and activity was elucidated by covalently binding dextran to a model enzyme, α-amylase. The conjugation process was optimized with respect to concentration of oxidizing agent, pH of enzyme solution, ratio of dextran to enzyme concentration, temperature and time of conjugate formation, and was found to affect the stability of α-amylase. α-Amylase conjugated under optimized conditions showed 5% loss of activity but with enhanced thermal and pH stability. Lower inactivation rate constant of conjugated α-amylase within the temperature range of 60-80 °C implied its better stability. Activation energy for denaturation of α-amylase increased by 8.81 kJ/mol on conjugation with dextran. Analysis of secondary structure of α-amylase after covalent binding with dextran showed helix to turn conversion without loss of functional properties of α-amylase. Covalent bonding was found to be mandatory for the formation of conjugate.

Laccase–gum Arabic conjugate for preparation of water-soluble oligomer of catechin with enhanced antioxidant activity

Catechin was oligomerized using free laccase and laccase-gum Arabic conjugate. The process of oligomerization was optimized with respect to solvent, ratio of solvent to buffer (0.2:10 to 1:10), pH of buffer (3-10), enzyme (575-18,400 U/mg) and substrate concentration (1-7mM). Maximum production of oligomer was observed in methanol at ratio 0.6:10 of methanol:buffer of pH 5 using 2300 U/mg of laccase and 5mM of catechin. The laccase-gum Arabic conjugate showed lower activity but higher stability than free laccase in methanol. Free laccase produced cross linked water-insoluble oligomer, whereas conjugated laccase produced linear water-soluble oligomer. The linear water-soluble oligomer showed higher antioxidant activity, as determined by the DPPH assay, and reducing power as compared to monomer making it suitable for biological applications. The molecular weight of the linear oligomer was found to be 13.14kDa, which suggested it to be composed of 45 monomer units. Further characterizations of linear and cross linked oligomer were done using FTIR and differential scanning calorimetry.

Screening of polysaccharides for preparation of α-amylase conjugate to enhance stability and storage life

Nine polysaccharides differing in structure and chemical nature were screened for their ability to conjugate with α-amylase by covalent binding for enhancing the thermal and pH stability of α-amylase. Among these polysaccharides, agar, dextran, pectin and xanthan showed better results but dextran stood out among all the polysaccharide for providing both thermal and pH stability to α-amylase. α-Amylase conjugated with agar, dextran, pectin and xanthan showed antimicrobial property with added preservative (0.2% sodium benzoate) in liquid formulation of α-amylase challenged with Bacillus subtilis and Escherichia coli. Dextran was the only polysaccharide which showed significant reduction of microbial growth of challenged organisms and aerobic flora without any preservative added. Aerobic flora could flourish well in the liquid α-amylase, but low temperature (4 °C), dextran, and preservative showed synergistic effect in efficiently increasing the storage life of liquid α-amylase.

Pullulan-complexed α-amylase and glucosidase in alginate beads: Enhanced entrapment and stability

Enhanced entrapment of the enzymes, α-amylase and glucoamylase, was found in alginate beads on addition of pullulan in the enzyme mixture. Under optimized process conditions of entrapment, enzymes-pullulan complex showed an entrapment of 85% in the alginate beads as opposed to 25% for the free enzymes. Beads of enzymes-pullulan complex showed lower inactivation rate constant and higher half life than corresponding beads of free enzymes. Activation energy of beads of enzymes-pullulan was increased by 6.81kJ/mole compared to beads of free enzymes. This implies better stability the enzymes in enzymes-pullulan beads along with increased immobilization yield. Moreover, enzymes-pullulan beads also showed pH stability at extreme acidic and alkaline pH. Addition of pullulan in the enzymes mixture lowered the Km and increased the Vmax as compared to beads of free enzymes. Hydrolysis of starch and reusability study showed better applicability of beads of enzymes-pullulan as compared to free enzymes.

Co-conjugation vis-à-vis individual conjugation of α-amylase and glucoamylase for hydrolysis of starch.

Two enzymes, α-amylase and glucoamylase have been individually and co-conjugated to pectin by covalent binding. Both the enzyme systems showed better thermal and pH stability over the free enzyme system with the complete retention of original activities. Mixture of individually conjugated enzymes showed lower inactivation rate constant with longer half life than the co-conjugated enzyme system. Individually conjugated enzymes showed an increase of 56.48 kJ/mole and 38.22 kJ/mole in activation energy for denaturation than the free enzymes and co-conjugated enzymes, respectively. Km as well as Vmax of individually and co-conjugated enzymes was found to be higher than the free enzymes. SDS-polyacrylamide gel electrophoresis confirmed the formation of conjugate and co-conjugate as evident by increased molecular weight. Both the enzyme systems were used for starch hydrolysis where individually conjugated enzymes showed highest release of glucose at 60 °C and pH 5.0 as compared to free and co-conjugated enzyme.

A green process for the production of butanol from butyraldehyde using alcohol dehydrogenase: process details

Depletion of energy sources has drawn attention towards production of bio-butanol by fermentation. However, the process is constrained by product inhibition which results in low product yield. Hence, a new strategy wherein butanol was produced from butyraldehyde using alcohol dehydrogenase and NADH as a cofactor was developed. Butyraldehyde can be synthesized chemically or through fermentation. The problem of cofactor regeneration during the reaction for butanol production was solved using substrate coupled and enzyme coupled reactions. The conventional reaction produced 35% of butanol without regeneration of cofactor using 300 μM NADH. The process of substrate coupled reaction was optimized to get maximum conversion. NADH (30 μM) and 100 μg per ml of alcohol dehydrogenase (320 U mg−1) could convert 17.39 mM of butyraldehyde to butanol using ethanol (ratio of butyraldehye to ethanol 1 : 4) giving a maximum conversion of 75%. The enzyme coupled reaction under the same conditions showed only 24% conversion of butyraldehyde to butanol using the glutamate dehydrogenase-L-glutamate enzyme system for the regeneration of cofactor. Hence, substrate coupled reaction is suggested as a better method over the enzyme coupled reaction for the cost effective production of butanol.

Enzyme-Assisted Extraction of Bioactives

The use of enzymes in food, pharmaceutical, and other industries has long been recognized for its effectiveness, both technologically and economically. It encompasses to achieve high product yields, reduce by-product formation, and avoid severe operational conditions. Hence, enzymes are also being used in the extraction of important bioactive compounds. The use of enzymes for efficient extraction of biomolecules is a recent and environmentally friendly ‘green’ extraction technique. Enzyme-assisted extraction technique uses specific enzymes to disrupt the cell wall of source material to improve its extraction yield. This technique can be combined with various other techniques to enhance the overall recovery of bioactives from source materials. The present chapter discusses the prospects of enzyme-assisted extraction techniques over conventional extraction techniques of bioactives. The mechanism of enzyme-assisted extraction and structural modifications of biomolecules during extraction are also detailed vividly. Besides, different bioactives extracted from plant and non-plant sources are also summarized comprehensively. Finally, various challenges and opportunities to improve the current knowledge on the enzyme-assisted extraction of bioactives are also considered.

Interaction of polyphenol oxidase of Solanum tuberosum with β-cyclodextrin: Process details and applications.

Polysaccharides differing in structure and chemical nature were screened for their ability to bind non-covalently with polyphenol oxidase (PPO) from potato (as a model) and their effect on enzyme activity. All the polysaccharides selected inhibited the PPO but β-cyclodextrin showed maximum inhibition under optimum conditions. Process details for the inhibition of PPO were studied with respect to concentration of β-cyclodextrin, temperature, pH, and time. Higher inhibition constant and lower half life was obtained at 40 °C than at 30 °C in the presence of inhibitor. β-Cyclodextrin showed mixed type of inhibition of PPO. β-Cyclodextrin was further exploited as anti-browning agent in selected fruit juices. It not only showed a significant anti-browning effect on freshly prepared potato juice but was also effective in other fruit juices. Better effect was seen in pineapple, apple and pear as compared to banana, sugarcane and guava fruit juices.