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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.

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Sugar beet molasses is a well-known, inexpensive and available carbon source for microbial cell growth. Its sugar components are used to produce energy for microbial growth and non-sugar components, especially nitrogen components, have important roles in improvement of cell growth. On the other hand, immobilization of whole cell is establishment and physical limitation of intact cells in specific space that keeps their catalytic activity and provides the possibility of reuse of the cells. This technique allows continuous and accelerated biological processes. It also improves production efficiency and quality and simplifies recycling of product. Immobilized living cells, as controlled catalysts, are able to perform one-step enzymatic reaction and continuous fermentative processes. In this research, E.coli cells were immobilized in calcium alginate hydrogels and using sugar beet molasses as carbon source, were applied for tryptophan production reaction in the presence of its precursors, serine and indole. In comparison between free biocatalysts and immobilized bacterial cells that entrapped in alginate gels, indicated that larger amounts of amino acids (about 42/9%) can produce in calcium alginate. Also the production reaction was followed up for 9 sequential cycles, and results showed that the cells could produce tryptophan amino acid under above conditions. Use of sugar beet molasses (by-product of agriculture industries) for growth of microbial cells and tryptophan production, causes decrease in production cost and more economical production of tryptophan by immobilized E. coli.

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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.

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Immobilization of biosignal molecules including growth factors and cytokines is important for developing biologically active materials, because these materials will have important effects in targetted cell culture, photo- immobilization of visible-light induced crosslinkable biosealant in direct pulp capping material in the dental field, biosealants in tissue engineering and anti-adhesive agents for preventing tissue adhesions after surgery and design and fabrication of biological scaffold contributed to tissue engineering, photolithography. The photo-immobilization of biosignal molecules has more meanings than only immobilization of an enzyme in a bioreactor or ligand-receptor interactions, because the immobilized biosignal molecules work on cells which have very complex structures and functions. This review discusses so far progresses in immobilization of biosignal molecules including growth factors and cytokines with biological and medical applications. At first we will study on photolithography and cell patterning. Then biosignal molecules, photo-immobilization process and co-immobilization will be reviewed. Since material properties of surfaces directly affect the cellular functions and thereby affecting growth patterns, we will study on biological properties of surfaces such as cell adhesion, cell migration and cell growth. Finally different photo reactive biosystems including UV, visible and laser bio systems will be discussed.

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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.

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Phenols are organic and highly toxic compounds commonly found in the effluents of various industries due to their wide range of applications. The inhibitory effect of phenol at high concentrations, as well as the high salinity of industrial effluents, poses a serious challenge for treatment by microorganisms. One of the most common approaches to overcome this problem is the immobilization of phenol-degrading microorganisms. The aim of this study was to study the immobilization effect on the phenol removal efficiency of native Janibacter halotolerant bacterium. For this purpose, mica was used as a carrier for bacterial immobilization and the protein concentration assay was applied to determine the immobilization efficiency. The phenol removal by free and immobilized cells was studied as well as the effect of different parameters on phenol removal efficiency. The immobilization efficacy on mica was %68.75, based on protein concentration measurements. The removal time of 100 mg/L phenol by suspended cells was 88 h, while the immobilized cells degraded it in 40 h. Immobilized cells, unlike free cells, were able to remove phenol at lowered temperatures up to 16℃ , salt concentrations greater than 7/5%, and pH levels below 7/5 and above 8/5. Similar results regarding the superior performance of immobilized cells have been obtained in other studies. As a result, the immobilization process considerably improves the efficiency of phenol removal and makes the cells resistant to harsh environmental conditions by protecting the cells from the toxic effects of phenol.
 

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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.
 

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Objective: Organophosphorus (OPs) compounds are widely used in many pesticides, insecticides and chemical nerve agents. These compounds are hazardous for humans and the environment. Organophosphate hydrolase (OPH) is a homodimeric protein initially isolated from Pseudomonas diminuta MG and Flavobacterium species. This enzyme is able to degrade a broad spectrum of toxic OPs compounds. Using immobilized OPH commonly presents a variety of advantages versus the free form of the enzyme. Advantages include an increase in stability, cost reduction by simple recovery and reutilization of the enzyme, quick and easy separation of the reactant and product in the reaction medium. Methods: Plasmid pET-26b (+) was used to generate the OPH protein under the control of the T7lac promoter. E. coli BL21 (DE3) pLysS was used as the host for expression of the OPH enzyme. Recombinant OPH was secreted into the extracellular medium and the purified enzyme was immobilized on the surface of Bacillus subtilis spores by the adsorption method, for the first time. Results: Approximately 42% to 45% enzymatic activity was determined to be associated with spores. Optimal pH and temperature of the enzyme were not altered by the presence of the spores. Thermo and pH stabilities of the immobilized enzyme was higher than the free form of the enzyme. Conclusion: Bacillus subtilis spores are safe for humans and the environment. Therefore this system can be considered an environmentally friendly biocatalyst for degradation of OPs. 

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This study was carried out with the aim of covalent immobilization of Aspergillus oryzae beta-galactosidase and Bacillus licheniformis protease on multi-walled amino-carbon nanotubes. In this method, fractional 2k design was used to study the effect of seven continuous factors (activation pH, glutaraldehyde molarity, activation time, buffer solution pH, buffer solution molarity, MWCNT-NH3-glutaraldehyde amount and stabilization time) on the stabilization efficiency and enzyme activity. . Design-expert software was used to analyze data and draw graphs. The results showed that the aforementioned factors predict the level of enzyme activity of Bacillus licheniformis protease and Aspergillus oryzae beta-galactosidase with correlation coefficients of 0.80 and 0.92 at the rate of 77 and 88%, respectively. Also, the correlation coefficient of the covalent fixation efficiency model of Aspergillus oryzae beta-galactosidase and Bacillus licheniformis protease on multi-walled carbon nanotubes was 0.89 and 0.82, respectively, and the studied factors were able to determine the covalent fixation beta efficiency, respectively. Aspergillus oryzae galactosidase and Bacillus licheniformis protease on multi-walled amino-carbon nanotubes predict 83 and 77%, respectively.