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Digestive enzymes secreted by the body are vital for digestion and nutrient absorption. Enzyme supplements are commonly used to support them in achieving optimal digestion. Herein, the efficacy of digestive enzyme supplement (DigeSEB Super) in digestion of complex food was assessed using INFOGEST simulated static and modified semi-dynamic in vitro digestion models. Digestive enzyme supplement was found to assist the endogenous digestive enzymes to disintegrate the food matrix. Hence, it reduced the viscosity of the gastric digesta by 2.75 fold ( p  = 0.04) compared to the control digestion (only endogenous digestive enzymes) during the first hour of digestion. Similarly, enzyme supplement showed statistically higher release of reducing sugars in the gastric digestion ( p  ≤ 0.05) indicating improved digestion of carbohydrates. Further, digestion of proteins and fats was also improved in the presence of enzyme supplement. The kinetic aspects of the semi-dynamic model (transient nature of gastric secretions and gradual acidification) was found to alter the macronutrient digestion compared to the static digestion. Thus, semi-dynamic model should be preferred for the in vitro studies. Overall, current study demonstrated the potential of a digestive enzyme supplement to improve digestion by aiding digestive action of the endogenous enzymes., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Swati Jadhav reports a relationship with Advanced Enzyme Technologies Limited that includes: employment. Abhijit Rathi reports a relationship with Advanced Enzyme Technologies Limited that includes: employment. Sneha Potale reports a relationship with Advanced Enzyme Technologies Limited that includes: employment. Rutuja Vaze reports a relationship with Advanced Enzyme Technologies Limited that includes: employment. Abhijeet Muley reports a relationship with Advanced Enzyme Technologies Limited that includes: employment. All the named authors are paid employees of Advanced Enzyme Technologies which has a corporate affiliation with Specialty Enzymes and Probiotics, USA. Specialty Enzymes and Probiotics had provided study material (DigeSEB Super) but had no role in study design and actual conduct of the study., (© 2024 The Authors.)

Transaminases capable of carrying out chiral selective transamination of 1-(3-methylphenyl)ethan-1-one to (1R)-(3-methylphenyl)ethan-1-amine were screened, and ATA-025 was the best enzyme, while dimethylsulfoxide (10% V/V) was the best co-solvent for said bioconversion. The variables such as enzyme loading, substrate loading, temperature, and pH for development of process displaying maximum conversion with good product formation and higher yield were optimized. The ambient processing conditions were 10% enzyme loading/50 g/L substrate loading/45 °C/pH 8.0, and 5% enzyme loading/36.78 g/L substrate loading/42.66 °C/pH 8.2 displaying maximum conversion 99.01 ± 2.47% and 96.115 ± 1.97%, and 76.93 ± 1.05% and 73.12 ± 1.04% yield with one factor at a time approach and numerical optimization with Box Behnken Design, respectively. In the final optimized reaction, ATA-025 showed the highest 99.22 ± 2.61% conversion, 49.55 g/L product formation, with an actual product recovery of 38.16 g corresponding to a product yield 77.03 ± 1.01% with respect to the product formed after reaction. The purity of recovered product (1R)-(3-methylphenyl)ethan-1-amine formed was ≥ 99% (RP-HPLC), and chiral purity ≥ 98.5% (Chiral-GC), and it was also confirmed and characterized with instrumental methods using boiling point, LC-MS, ATR-FTIR, and 1 H NMR. The findings of 'What If' studies performed by investigating timely progress of reaction on gram scale by drastically changing the process parameters revealed a substantial modification in process variables to achieve desired results. (1R)-(3-methylphenyl)ethan-1-amine synthesized by green, facile and novel enzymatic approach with an optimized process could be used for synthesis of different active pharma entities., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Nitrilases capable of performing hydroxylation of 2-chloroisonicotinonitrile to 2-chloroisonicotinic acid were screened, and ES-NIT-102 was the best nitrilase for said biotransformation. Nitrilase was immobilized as cross linked enzyme aggregates (nitrilase-CLEAs) by fractional precipitation with iso-propanol, and cross linked with glutaraldehyde. The nitrilase-CLEAs prepared with optimized 35 mM glutaraldehyde for 120 min cross linking time had 82.36 ± 4.45% residual activity, and displayed type-II structural CLEAs formation as confirmed by particle size, SEM, FTIR, and SDS-PAGE analysis. Nitrilase-CLEAs had superior pH and temperature stability, showed a shift in optimal temperature by 5 °C, and retained nearly 1.5 to 1.7 folds activity over free nitrilase at 50 °C and 55 °C after more than 9 h incubation. Nitrilase-CLEAs showed reduced affinity and decreased conversion of substrate as indicated by slightly higher K m values by 5.19% and reduced V max by 17%. Furthermore, these nitrilase-CLEAs showed 98% conversion, 94.72 g/L product formation, and 83.30% recovery after 24 h when used for hydroxylation of 2-chloroisonicotinonitrile to 2-chloroisonicotinic acid. Nitrilase-CLEAs were catalytically active for 3 cycles showcasing 81% conversion, 75.53 g/L product formation and 66.42% yield. The recovered product was confirmed by HPLC, FTIR, LC-MS, and 1 H NMR, and displayed > 99% purity., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Cross-linked enzyme aggregates (CLEAs) of lipase were prepared after fractional precipitation with 40-50% ammonium sulfate and then cross-linking with glutaraldehyde. The process variables for the preparation of lipase-CLEAs such as glutaraldehyde concentration, cross-linking period, and initial pH of medium were optimized. The optimized conditions for the preparation of lipase-CLEAs were 25 mM/80 min/pH 7.0, and 31.62 mM/90 min/pH 6.0 with one factor at a time approach and numerical optimization with central composite design, respectively. Lipase-CLEAs were characterized by particle size analysis, SEM, and FTIR. Cross-linking not only shifted the optimal pH and temperature from 7.0 to 7.5 and 40-45 to 45-50 °C, but also altered the secondary structure. Lipase-CLEAs showed an increase in K m by 7.70%, and a decrease in V max by 16.63%. Lipase-CLEAs presented better thermostability than free lipase as evident from thermal inactivation constants (t 1/2 , D and E d value), and thermodynamic parameters (E d , ΔH°, ΔG°, and ΔS°) in the range of 50-70 °C. Lipase-CLEAs retained more than 65% activity up to four cycles and showed good storage stability for 12 days when stored at 4 ± 2 °C. They were successfully utilized for the epoxidation of lemongrass oil which was confirmed by changes in iodine value, epoxide value, and FTIR spectra.

Zinc oxide particles (ZnOPs) of both nanometer and sub-micron sizes are important components of high demand consumer products such as sunscreen, paint, textile, food packaging, and agriculture. Their ultimate discharge in the aquatic ecosystem is nearly unavoidable. For sustainable use of ZnOPs, there is an urgent need to assess its ecotoxicity using ecological indicator organisms. Moina macrocopa, an important component of the aquatic ecosystem is one such less explored indicator organism. In the present investigation, ZnOPs of two different sizes (250 ± 20 and 500 ± 50 nm) were selected for risk assessment as most of the previous reports were based on the use of 10-100 nm ZnOPs. ZnOPs of 500 nm were more lethal than that of 250 nm size, with respective LC 50 of 0.0092 ± 0.0012 and 0.0337 ± 0.0133 mg/L against M. macrocopa after 48 h of exposure. We further used a sublethal concentration of 500 nm (0.00336 mg/L) and 250 nm (0.00092 mg/L) ZnOPs followed by measurement of enzymatic biomarkers of toxicity (acetylcholinesterase, digestive enzymes, antioxidant enzymes). A size-dependent variation in enzymatic response to 250 and 500 nm ZnOPs was seen. Exposure to ZnOPs inhibited acetylcholinesterase and digestive enzymes (trypsin, amylase), and elevated antioxidant enzymes (catalase, glutathione S-transferase) levels. The exposure also decreased the superoxide dismutase activity and increased that of β-galactosidase. Microscopic investigation revealed the accumulation of ZnOPs in the digestive tract of M. macrocopa that possibly disrupts enzyme activities. The present study will contribute to establishing regulatory policy on the maximum permissible limit of ZnOPs in different water bodies., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Chitosan and whey protein isolate (WPI) conjugate films were prepared as a novel matrix for encapsulating and extending the postharvest shelf life of strawberries. Film forming solutions of chitosan, WPI, and chitosan-WPI conjugate were mixed with glycerol, casted for films at 60 ± 2 °C and assessed for their colour, water vapour and oxygen transfer rate, textural, functional groups and secondary structure, thermal, crystallinity, and antioxidant properties. Chitosan-WPI conjugate films were applied as an edible coating on strawberries, and studied for storage stability at 5 °C and 20 °C by assessing physical and biochemical parameters. A considerable reduction in colour indices, weight loss, pH and titratable acidity, reducing sugars, ascorbic acid, total phenolics, DPPH and ABTS assay was noted in the coated strawberries over the control at both the studied temperatures. The control strawberries had a shelf life of 5 and 3 days, whereas coating enhanced the shelf life of strawberries to 8 and 5 days when stored at 5 °C and 20 °C, respectively., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

MYB transcription factors are one of the most important mediators for the survival of plants under multiple stress responses. In the present study, EaMYB18 , encoding a single R3 repeat MYB DNA binding domain was isolated from stress-tolerant wild relative species of sugarcane Erianthus arundinaceus . In silico analysis of 948 bp coding mRNA sequence of EaMYB18 exhibited the presence of four exons and three introns. Further, the EaMYB18 gene was transformed in tobacco and its stable inheritance was confirmed through antibiotic resistance screening, PCR amplification and Southern hybridization blotting. Results of the estimation of MDA, proline, total chlorophyll and antioxidant activities of EaMYB18 transgenic tobacco lines exhibited least oxidative damage under drought and cold stress over the untransformed ones, the over-expression of EaMYB18 has improved drought and cold stress tolerance ability in tobacco. The comparative physiological and biochemical analysis of transgenic tobacco plants overexpressing SoMYB18 , SsMYB18 and EaMYB18 , revealed that the EaMYB18 and SsMYB18 transgenic plants demonstrated effective tolerance to drought and cold stresses, while SoMYB18 showed improved tolerance to salt stress alone. Amongst these three genes, EaMYB18 displayed the highest potential for drought and cold stress tolerances as compared to SoMYB18 and SsMYB18 genes., Competing Interests: Conflict of interestThe authors declare that they have no conflict of interest., (© King Abdulaziz City for Science and Technology 2020.)

Acrylamidase produced by Cupriavidus oxalaticus ICTDB921 was recovered directly from the fermentation broth by ammonium sulfate (40-50%) precipitation and then stabilized by cross-linking with glutaraldehyde. The optimum conditions for the preparation of cross-linked enzyme aggregates of acrylamidase (acrylamidase-CLEAs) were using 60 mM glutaraldehyde for 10 min at 35 °C and initial broth pH of 7.0. Acrylamidase-CLEAs were characterized by SDS-PAGE, FTIR, particle size analyzer and SEM. Cross-linking shifted the optimal temperature and pH from 70 to 50 °C and 5-7 to 6-8, respectively. It also altered the secondary structure fractions, pH and thermal stability along with the kinetic constants, K m and V max , respectively. A complete degradation of acrylamide ~ 1.75 g/L in industrial wastewater was achieved after 60 min in a batch process under optimum operating conditions, and the kinetics was best represented by Edward model (R 2  = 0.70). Acrylamidase-CLEAs retained ~ 40% of its initial activity after three cycles for both pure acrylamide and industrial wastewater, and were stable for 15 days at 4 °C, retaining ~ 25% of its original activity.

Immobilization of enzymes on matrices like iron oxide magnetic nanoparticles provides a new technique against the stagnant conventional approaches for stabilization of enzymes. Along with ease in synthesis and modification, the advantages of using these iron oxide nano-metallic matrices as enzyme support matrices are three dimensional viz. structural and thermal stability, storage stability, and reusability. This book chapter details the protocol(s) for synthesis of iron oxide magnetic nanoparticles via chemical co-precipitation method, its surface modification and functionalization with 3-aminopropyl-triethoxysilane (APTES), followed by immobilization of enzymes using glutaraldehyde as a cross linker. A detailed protocol for instrumental characterization is also described. Additionally, the chapter also elucidates the basic characterization of enzyme(s), effect of operating conditions (pH, temperature) on the enzyme activity, thermal inactivation kinetics, thermodynamic parameters, storage stability, and reusability of the free and immobilized enzymes. Further, the results of all the indicated protocols and stability studies are thoroughly discussed with the findings on immobilization of cellulase in a tri-enzyme mixture on iron oxide magnetic nanoparticles. The optimum parameters for immobilization were 30mM glutaraldehyde with 3h incubation and 3:1 (w/w) ratio of nanoparticles:protein content. Immobilization altered the kinetic constants (K m and V max ) marginally but enhanced the thermal stability as evident from inactivation kinetic constants (k d , t 1/2 and D-value) and thermodynamic parameters (E d , ΔH°, ΔG° and ΔS°) within 55-75°C. The immobilized cellulase retained 71.68±3.48% activity during 21-day storage, and 81.15±5.27% activity till fifth reusability cycle., (© 2020 Elsevier Inc. All rights reserved.)

This paper reports on stabilization of serine cutinase activity by immobilizing it through cross linking with glutaraldehyde on magnetic nanoparticles (Fe-NPs) and intensification of catalytic activity by ultrasonic treatment. The optimum parameters were cross linking with 10.52 mM glutaraldehyde for 90 min using 1:2 (w/w) ratio of enzyme:Fe-NPs. The characterization of cutinase-Fe-NPs was done by different instrumental analysis. Ultrasonic power showed a beneficial effect on the activity of free and immobilized cutinase at 5.76 and 7.63 W, respectively, after 12 min. Immobilization and ultrasonic treatment led to increments in kinetic parameters (K m and V max ) along with noticeable changes in the secondary structural fractions of cutinase. Cutinase-Fe-NPs showed augmented pH (4-8) and thermal stability (40-60 °C). Considerably higher thermal inactivation kinetic constants (k d , t 1/2 and D-value) and thermodynamic constants (E d , ΔH°, ΔG° and ΔS°) highlighted superior thermostability of cutinase-Fe-NPs. Cutinase-Fe-NPs and ultrasound treated cutinase-Fe-NPs retained 61.88% and 38.76% activity during 21-day storage, and 82.82 and 80.69% activity after fifth reusability cycle, respectively., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Chitosan, a functional biopolymer, was irradiated with 100 kGy gamma irradiation and used to access its physical, antioxidant, plant growth promoting and antimicrobial properties. The molecular weight of chitosan reduced to 82.2 kDa from 337.7 kDa after irradiation. UV-Vis spectroscopy and FTIR results revealed slight changes in chitosan skeleton after irradiation but the degree of acetylation of both chitosan was ~18%. DSC profile indicated a prominent decline in enthalpy and energy for phase transition, TGA indicated shift in decomposition temperature, while XRD analysis showed a reduction in chitosan crystallinity after irradiation. DPPH and ABTS radical scavenging activity of chitosan (2-10 mg/mL) enhanced significantly by 1.25-1.45 and 1.80-3.14 folds after irradiation. There was a considerable improvement in morphological parameters (number of leaves, nodes, height, fresh weight and dry weight) and biochemical parameters (chlorophyll, total soluble sugars and soluble proteins content) of in vitro potato plant in chitosan supplemented medium at 75 mg/L concentration than the control. The minimum inhibitory concentration of normal and irradiated chitosan for Alternaria spp. was 2500 and 2000 mg/L and for Fusarium spp. was 1750 and 1500 mg/L, respectively. IC 50 value of normal and irradiated chitosan for Fusarium spp. was 1387.9 ± 9.2 and 954.3 ± 6.1 mg/L, and for Alternaria spp. was 1536.1 ± 24.3 and 1416.8 ± 3.5 mg/L, respectively., (Copyright © 2019 Elsevier B.V. All rights reserved.)

In the field of nanoecotoxicology, very few reports have focused on biochemical changes in non-target organisms after nanoexposure. A less explored aquatic non-target crustacean, Moina macrocopa, was used in the present study to analyze toxicity effects of gold nanoparticles (AuNPs), an emerging nanomaterial. AuNPs was fabricated using tannic acid and were 29 ± 2 nm in size. The 48 h LC 50 value of AuNPs was 14 ± 0.14 mg/L against M. macrocopa. The sub-lethal exposure of M. macrocopa juveniles to AuNPs (1.47 and 2.95 mg/L) decreased the activities of acetyl cholinesterase and digestive enzymes (trypsin and amylase). A concentration dependant increase in the activities of antioxidant enzymes such as catalase, superoxide dismutase and glutathione S-transferase suggested the generation of oxidative stress in M. macrocopa after AuNPs exposure. Changes in enzyme activity can be utilized as biomarker(s) for early detection of nanoparticle contamination in aquatic habitat. AuNPs accumulation in gut of M. macrocopa increased the metal bio burden (11 mg/L) and exhibited inhibitory action on digestive enzymes. Complete depuration of AuNPs was not observed after transferring nano-exposed M. macrocopa to normal medium without AuNPs. AuNPs tended to adhere on external body parts such as setae, carapace of M. macrocopa which interfered with swimming activity and also changed the behavioral pattern. AuNPs underwent agglomeration in the medium used for maintenance of M. macrocopa. As nanomaterials are emerging pollutants in aquatic systems, the present work highlights the hazardous effect of AuNPs and development of enzymatic biomarkers to curtail it at community level., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Oligo-chitosan (82.20 kDa) was prepared from chitosan (337.73 kDa) by application of 100 kGy γ-irradiation. UV-vis spectroscopy, FTIR, XRD, DSC and TGA analyses showed typical properties of chitosan with slight variations after γ-irradiation. Degree of deacetylation of chitosan and oligo-chitosan was 82%, while 1,1-diphenyl-2-picrylhydrzyl and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) radical scavenging activity were 10.01 ± 0.18 and 43.30 ± 3.75 μMTE/mL and 13.64 ± 0.16 and 79.93 ± 4.44 μMTE/mL, respectively. Chitosan and oligo-chitosan was applied as foliar spray on potato plants to analyze growth promoting and stress tolerance inducing effects. Improvement in shoot height and number of nodes was observed after foliar spray of chitosan and oligo-chitosan at 50-75 mg/L. Furthermore, membrane stability index and malondialdehyde reduced while chlorophyll, carotenoids, proline, reducing and total sugars, enhanced considerably. The antioxidant and defense enzymes CAT, POD, SOD, chitinase and chitosanase showed prominent increment. Overall results indicated that chitosan (75 mg/L) and oligo-chitosan (50 mg/L) can augment plant growth and induce defense mechanism for drought stress tolerance in potato., (Copyright © 2019. Published by Elsevier Ltd.)

Acrylamidase from Cupriavidus oxalaticus ICTDB921 was immobilized on magnetic nanoparticles (MNPs) for degradation of acrylamide (a group 2A carcinogen and an environmental contaminant) from industrial waste water. Acrylamidase-MNPs were prepared (maximum recovery ∼94%) at optimized process parameters viz. 1.5:1 (v/v) of acetone: crude acrylamidase/5 mM of glutaraldehyde/90 min/1.5:1 of enzyme: MNP ratio. MNPs and acrylamidase-MNPs were characterized by particle size analysis, FTIR, XRD, SEM and vibrating sample magnetometer. Acrylamidase-MNPs showed a shift in optimum pH (8-8.5) and temperature (60-65 °C) with higher pH/thermal stability vis-à-vis free enzyme. A significant increase in kinetic constants, thermal inactivation constants and thermodynamic parameters were noted for acrylamidase-MNPs. A complete degradation of acrylamide ∼2100 mg/L was achieved in industrial waste water under optimized conditions for batch process and the kinetics was best represented by Haldane model. Acrylamidase-MNPs retained >80% of its initial activity after 4 cycles for both pure acrylamide and industrial waste water., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

A tri-enzyme mixture of cellulase, pectinase and xylanase present in fermentation broth of Aspergillus niger culture was directly recovered by solvent precipitation and co-immobilized on magnetic nanoparticles (MNPs) by cross linking with glutaraldehyde. A 2:1 ratio of isopropanol:total protein in the fermentation broth gave a precipitate with maximal activity recovery of the three enzymes. The co-immobilization on MNPs was optimized at 30 mM glutaraldehyde for 3 h incubation, and 3:1 MNPs:enzyme ratio, yielding a maximum activity recovery of cellulase (80.25 ± 1.03%), pectinase (84.76 ± 1.71%) and xylanase (75.62 ± 0.76%), respectively. The tri-enzyme co-immobilized MNPs were characterized by particle size analysis, FTIR, XRD, VSM and SEM studies, and showed enhanced thermal stability as evident from lower k d , higher t 1/2 and D-value in the range of 55-75 °C. The tri-enzyme co-immobilized MNPs showed a shift in optimum temperature from 55 to 60 °C with improved pH tolerance along with a slender rise in kinetic constants and enhanced thermodynamic parameters. It was stable for 36 days at 5 °C, and retained >90% activity up to 4 cycles. The tri-enzyme co-immobilized MNPs were successfully utilized for extraction of piperine from black pepper, preparation of sugarcane cell protoplasts, and clarification of papaya juice with prominent yield and reusability vis-à-vis traditional methods., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The present article describes extraction of chitosan from prawn shells waste and its application in thermal stabilization of Fusarium sp. ICT SAC1 cutinase by non-covalent and covalent conjugation. Extracted chitosan represented 78.40% degree of deacetylation (DDA), a molecular weight of 173 kDa and was soluble in 1% acetic acid with 2.8 ± 0.15% insoluble matter. The structural (FTIR, NMR and XRD) and thermal characterization (DSC and TGA) indicated unique properties for chitosan. Plausible chitosan structure was also deduced. The water and fat binding capacities were 923% and 598.05% while 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) and 1,1-diphenyl-2-picrylhydrzyl radicals scavenging activity was 60.62 and 11.83 μM Trolox-Equivalent/ml. The K m and V max values of free cutinase were 0.82 mM and 20.64 mM/min which increased by 14.63 and 17.07%; and 27.18 and 43.94% after non-covalent and covalent conjugation, respectively. A marginal increment in thermal inactivation constants and energy (k d , t 1/2 , D and E d value) were also noticed for cutinase-chitosan conjugates. The enthalpy, free energy and entropy values increased marginally in covalent conjugate vis-à-vis non-covalent conjugated and free cutinase. A reduction in α-helix, random coils and β-sheets content was noted after conjugation., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Cutinase, a member of α/β-fold hydrolase family possess potentially diverse applications in several industrial processes and products. The present work aims towards thermo-stabilization of cutinase from novel source Fusarium sp. ICT SAC1 via non-covalent interaction with polysaccharides. Although all six polysaccharides chosen for study enhanced the thermal stability, pectin was found to be most promising. The interaction protocol for cutinase with pectin was optimized sequentially with respect to the ratio of enzyme to pectin, solution pH, and buffer strength. Cutinase-pectin conjugate under optimized conditions (1:12, pH-6.5, 50mM) showed enhanced thermal stability as evident from lower inactivation rate constant, higher half-life and D-value within the 40-55°C. A slender rise in K m and V max values and enhanced thermodynamic parameters of cutinase-pectin conjugate were observed after non-covalent interaction. Entropy values were 1.5-fold higher for cutinase-pectin conjugate at each temperature suggesting an upsurge in number of protein molecules in a transition activated state. Positive values of entropy for both forms of cutinase suggested a rise in disordered conformation. Noticeable conformational changes in cutinase after conjugation with pectin were confirmed by FTIR as well as fluorescence emission spectra. An increment in helix to turn conversion was observed in complexed cutinase vis-à-vis free cutinase., (Copyright © 2017 Elsevier B.V. All rights reserved.)

In the present work, optimization of pectinase-assisted and tri-solvent-mediated extraction of lycopene from waste tomato peels was carried out. The optimized parameters for enzymatic pre-treatment were 2% pectinase concentration, pH 5.5, 4-h incubation, 45 °C and 150 rpm. Maximum recovery of lycopene from tomato peels using optimized tri-solvent extraction was achieved at 45 °C, 120-min incubation and 200 rpm. The extracted lycopene was confirmed through functional and characteristic peaks in UV-Vis and FTIR spectra and with retention time in HPLC. The radical scavenging activity was 72.30 ± 2.70 and 43.40 ± 2.01 µg ascorbic acid equivalents (AAE)/ml for 1,1-diphenyl-2-picrylhydrzyl (DPPH) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) radicals, respectively. The optimized method resulted in 7.38, 4.65 and 1.59 times enhancement in lycopene extraction and recovery in correlation with single solvent, enzyme-treated and tri-solvent extraction methods, respectively.

Macromolecular cross-linked enzyme aggregates (M-CLEAs) of α-amylase were prepared by precipitation and subsequent cross-linking. The non-toxic, biodegradable, biocompatible, renewable polysaccharide based macromolecular cross-linkers viz. agar, chitosan, dextran, and gum arabic were used as a substitute for traditional glutaraldehyde to augment activity recovery toward macromolecular substrate. Macromolecular cross-linkers were prepared by periodate mediated controlled oxidation of polysaccharides. The effects of precipitating agent, concentration and different cross-linkers on activity recovery of α-amylase CLEAs were investigated. α-Amylase aggregated with ammonium sulphate and cross-linked by dextran showed 91% activity recovery, whereas glutaraldehyde CLEAs (G-CLEAs) exhibited 42% activity recovery. M-CLEAs exhibited higher thermal stability in correlation with α-amylase and G-CLEAs. Moreover, dextran and chitosan M-CLEAs showed same affinity for starch hydrolysis as of free α-amylase. The changes in secondary structures revealed the enhancements in structural and conformational rigidity attributed by cross-linkers. Finally, after five consecutive cycles dextran M-CLEAs retained 1.25 times higher initial activity than G-CLEAs., (Copyright © 2015 Elsevier B.V. All rights reserved.)