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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 1H 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.

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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 Km values by 5.19% and reduced Vmax 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 1H NMR, and displayed > 99% purity.

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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 Km by 7.70%, and a decrease in Vmax by 16.63%. Lipase-CLEAs presented better thermostability than free lipase as evident from thermal inactivation constants (t1/2, D and Ed value), and thermodynamic parameters (Ed, 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.

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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.

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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.