Lipase, one of the most important and versatile industrial enzymes, has been isolated, identified, and immobilized onto three novel supports prepared based on our US patent (US20110076737). Nine fungal isolates were cultivated, and maximum lipase activity of 285 U/mL was achieved from the fungal isolate identified as Rhizopus oryzae GF1. The enzyme was shown to be thermally stable at 50°C for 210 min. Three different environmentally friendly biopolymers prepared according to our US patent have been used to immobilize covalently the lipase from Rhizopus oryzae GF1. The structures of the gel beads; grafted alginate, carrageenan and alginate-carrageenan; have been proved by the FTIR. The best formulation, alginatecarrageenan, covalently immobilized 183.5 U/g lipase and was further optimized to load 223 U/g lipase. The immobilization process increased the operational temperature from 30 to 50°C compared to the free enzyme. The hydrolysis of oil using the free and the immobilized lipase was achieved at the same time, 90 min, which reflects no diffusion limitation. The shelf stability showed that the immobilized enzyme retained full activity for over 9 weeks at 4°C, whereas the free enzyme lost 80% of its initial activity after 4 weeks. The reusability test proved the durability of the grafted beads for 20 cycles with a retention of 97% of the immobilized enzyme activity compared to 23% by other authors after the 10th use.

This study aim to isolate, identify a bacterial isolate and optimize production medium using frying oil waste for lipase production. Nine strains were isolated from an Egyptian soil samples. Among the isolates, a potent bacterial candidate ASSCRC-P1 was found to be the most potent lipase producer strain at 60 °C. Genotypic identification of ASSCRC-P1 showed 94% similarity with Bacillus sp. strains. Phylogenetic tree confirmed that ASSCRC-P1 was nearly similar to Bacillus cereus. Therefore, it was given the name Bacillus cereus ASSCRC-P1 and its 16S rRNA nucleotide has been deposited in the GenBank Data Library under the accession number: KJ531440. A sequential optimization strategy, based on statistical experimental designs, was employed to enhance the lipase production by this strain. A 2-level Plackett-Burman design was applied to differentiate between the bioprocess parameters that significantly influence lipase production followed by Box-Behnken design to optimize the amounts of variables which have the highest positive significant effect on lipase production. Overall more than 2.15-fold improvement in lipase production was achieved due to optimization compared to that obtained using the basal medium.

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Objective: L-asparaginase (L-asp) is a vital enzyme used as a therapeutic agent in combination with other drugs in the treatment of acute lymphoma, melanosarcoma and lymphocytic leukemia. Immobilization of enzymes through loading on nanoemulsion (NE) results in some advantages such as enhancing their stability and increasing their resistance to proteases. Aim of the present study is to formulate L-asp loaded nanoemulsion to enhance its efficiency and thermal stability. Methods: Nanoemulsion loaded with L-asp crude extract (specific activity 13.23U/mg protein) was prepared employing oleic acid as oil, tween 20/tween 80 as surfactants and propylene glycol (PG) as co-surfactant. L-asp loaded NE underwent several thermodynamic stability studies and the optimized formulae were further examined for their biochemical properties and thermal stability. Results The developed formulations were spherical in shape and their sizes were in the nanometric dimensions with negatively charged zeta potential values. Upon comparing the enzyme activity of L-asp loaded NE employing tween 20 (F1) or tween80 (F4) at different concentrations, the results revealed that F4 NE showed higher enzymatic activity [323 U/ml] compared to F1 NE [197 U/ml] at the same concentration. The nanosized immobilized L-asp was more stable in the pH range from 8 to 8.5 as compared to free L-asp. The immobilized enzyme preserved about 59.11% of its residual activity at 50 °C; while free L-asp preserved about 33.84%. Conclusion: In the view of these results, NE composed of oleic acid, tween 80 and PG represents a promising dosage form for enhancing the activity and stability of Streptomyces griseoplanus L-asp.

A b s t r a c tGamma irradiation is used on Penicillium cyclopium in order to obtain mutant cells of high L-asparaginase productivity. Using gamma irradiation dose of 4 KGy, P. cyclopium cells yielded L-asparaginase with extracellular enzyme activity of 210.8 ± 3 U/ml, and specific activity of 752.5 ± 1.5 U/mg protein, which are 1.75 and 1.53 times, respectively, the activity of the wild strain. The enzyme was partially purified by 40-60% acetone precipitation. L-asparaginase was immobilized onto Amberlite IR-120 by ionic binding. Both free and immobilized enzymes exhibited maximum activity at pH 8 and 40°C. The immobilization process improved the enzyme thermal stability significantly. The immobilized enzyme remained 100% active at temperatures up to 60°C, while the free asparaginase was less tolerant to high temperatures. The immobilized enzyme was more stable at pH 9.0 for 50 min, retaining 70% of its relative activity. The maximum reaction rate (V max ) and Michaelis-Menten constant (K m ) of the free form were significantly changed after immobilization. The K m value for immobilized L-asparaginase was about 1.3 times higher than that of free enzyme. The ions K + , Ba 2+ and Na + showed stimulatory effect on enzyme activity with percentages of 110%, 109% and 106% respectively. K e y w o r d s: Penicillium cyclopium, Amberlite IR-120, gamma irradiation, ionic binding immobilization, L-asparaginase