Modares Journal of Biotechnology

Abstract Researchers are currently directing their efforts toward developing new enzyme stabilization and enhancement strategies to broaden their application in various industries. This study utilized a unified platform to stabilize and safeguard proteins in industrial settings. Despite the wide-ranging industrial applications of lipases, their utility in industrial processes is limited by their susceptibility to degradation under harsh environmental conditions. In our study, we used a dual-purpose strategy that involved both enzyme stabilization and the shielding of an organosilica protective layer. After expressing and purifying the recombinant lipase enzyme, we immobilized it onto silica nanoparticles and shielded it with an organosilica nanolayer to protect the enzyme. We meticulously examined the optimal thickness of the protective layer and its influence on enzyme stabilization against environmental stressors. Our research findings demonstrate that the immobilized enzyme exhibited a remarkable level of stability compared to its free enzyme when subjected to various factors, such as fluctuations in temperature and exposure to chemical agents. Furthermore, the immobilized samples displayed optimal activity across a broad range of temperatures, highlighting this approach's adaptability and efficacy. Notably, the organosilica layer significantly bolstered the reactivity recovery of denatured proteins with SDS and urea, highlighting the versatile applications of this method. These findings indicated that our present platform has great potential to improve the efficiency and stability of industrial enzymes against various environmental challenges.

Abstract Enzymes of marine organisms are ideal candidates for biomonitoring of pollution in marine environments. For the widespread use of enzymes in industrial processes, carried out under certain physico-chemical conditions, their stability must be improved. In this study, for the first time, chitosan nanoparticles were used as matrices for augmenting the stability of Penaeus vannamei (Whiteleg shrimp)-derived purified proteases against metallic ions. For the electrostatic binding of the enzyme to the chitosan nanoparticles, the protein solution at a concentration of 7mg/ml was added to the nanoparticles, and incubated for 4 hours at 10°C. After 3 times rinsing with phosphate buffer of pH=7.5, the nano-enzyme was dissolved in 1ml phosphate buffer, and used for further studies. The results of this study showed that Fe²⁺ and Mn²⁺ significantly increased the enzyme activity, whereas a strong inhibitory effect was observed in the presence of Cd²⁺, Hg²⁺, Co²⁺, Ni²⁺, Cu²⁺ and Zn²⁺, and a weak inhibitory effect in the presence of Na⁺ and K⁺. The immobilized enzyme exhibited greater resistance to metal ions than its free counterpart. The free enzyme was susceptible to the presence of metal ions, and with the increment of their concentrations, enzyme activity declines. From this nexus, it could be inferred that the high stability of immobilized enzyme is due to the presence of chitosan nanoparticles. Stability retention of the immobilized enzyme at high concentrations of metal ions indicates the efficacy and utility of the immobilization method in industrial enzyme technology.

Abstract Cellulase enzyme has shown their potential application in different industry. cellulase immobilization is one of the different methods for enzymatic stabilization. An advantage of immobilization is enzymatic reusability, which have an economical advantage for enzyme using in industry. Properties of Chitosan as a support for enzyme immobilization are always considerable. Due to its unique biological properties such as biocompability, biodegradability and non-toxicity, chitosan is an attractive support for immobilization. In this investigation Aa-cel9A endoglucanase gene was cloned in pET28 (+) expression vector. Sequencing result had been proved gene cloning in vector. Then the constructed vector was transformed to Eshershia.Coli (BL21) cells and enzyme production was induced. The result obtained from SDS-PAGE analysis and enzymatic assay showed the recombinant protein has been expressed and protein purification was done with Ni-NTA column. Chitosan macrobeads were prepared by precipitation procedure. After immobilization of enzyme with glutaraldehyde as linker, enzyme immobilization has been proved with FTIR and Bradford analysis. The obtained result showed optimum condition for covalent immobization on support are 0.7% of glutaraldehyde concentration and sodium phosphate buffer with pH 7. Bradford analysis and enzymatic activity assay have proved 85% of enzyme molecules immobilized on support.

Abstract Papain (EC2.22.4.3) is a thiol protease with high level of activity that has widespread industrial applications. The use of immobilized papain provides many advantages over its free form. In many applications, cysteine must be added as an activator. On the other hand, certain bivalent metal ions including Ca²⁺ behave as the inhibitors of papaein. In the present study, after preparation of Sepharose 6B with CNBr, a 5 mg/ml-protein solution was added to activate the gel for covalent attachment of enzyme and, subsequently, 2M glycine solution was added to block the remaining active groups on the gel. The immobilization process brought about significant enhancement of storage, thermal stability, stability at extreme pHs, and resistance against the inhibitory effect of bivalent metal ions with respect to papain. The optimum temperature of papain was increased by 20 °C (from 60 to 80 °C) and its optimum pH was shifted from 7 to 8.0 upon immobilization. Also k_m and k_cat of the enzyme altered due to the immobilization process.These results are important in particular if one considers that the major problem in enzyme immobilization is the loss of enzyme activity and catalytic efficiency.