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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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β- Xylosidase from Selenomonas ruminantium (SXA) is one of the most important enzyme for the hydrolysis of cell wall hemicellulose. SXA has potential utility in industrial processes especially production of bioethanol from bagasse. However, this xylosidase lose activ‌ity drastically above 50 °C. Each monomer of this homotetramer has four free buried cysteine. It seems that cysteine 286 has no role in protein function. In this study, to investigate effects of free buried cysteine on protein thermal stability, Cys 286 was replaced with the same size amino acid, valine. The mutant and native protein have expressed in Pichia pastoris. Kinetic and thermostability parameters of mutant were compared with the wild type enzyme. While pH optimum, temperature profile and catalytic efficiency of recombinant mutant were be found similar to native enzyme, mutant showed about 65% increase in thermostability respect to the wild type at 55 ˚C. Our results showed that free thiol group of cysteine caused the destabilization. Moreover, hydrophobic side chain of valine could involve in a hydrophobic interaction to stabilize SXA. Elimination of a free cysteine enhanced thermal stability without changing the catalytic efficiency of the enzyme that could be very important for biotechnological applications.

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Purpose: Evaluation of humoral and cellular immune responses of naturally infected dogs against type I (rCPB) (Recombinant cycsteine proteinase B), and II (rCPA) (Recombinant cycsteine proteinase A) recombinant cysteine proteinases and C-terminal extension (CTE) of Leishmania infantum (L. infantum). Materials and Methods: In this study, fourteen infected dogs (7 with symptoms, 7 asymptomatics) from an endemic area and three uninfected dogs from a nonendemic region were selected and their humoral and cellular responses against type I and II recombinant cysteine proteinases, C-terminal extension (CTE) and F/T of Leishmania infantum were evaluated using the ELISA and lymphocyte proliferation assay, respectively. The level of specific IgG isotypes (IgG1 and IgG2) and lymphocyte proliferative response against rCPA, rCPB, CTE and Freezed/Thawed lysate (F/T) of L. infantum were examined. Results and Discussion: The results showed that in both of the symptomatic and asymptomatic dogs there is a high lymphoproliferative response to F/T antigens and moderate responses were observed when rCPs (Recombinant cycsteine proteinase) (rCPA and rCPB) and CTE were used. The level of antibody (total IgG, IgG1 and IgG2) recognition toward rCPA was low in the both groups of the dogs. In contrast, the CTE stimulates similarly as the CPB both of the humoral and cellular responses of all the infected animals and the level of total IgG and IgG2 isotypes against these antigens compared to the IgG1 was higher in the asymptomatic dogs. Since, the CTE is the terminal fragment of the CPB, it seems that the immunogenicity of the CPB is dependent on the CTE. Conclusion: The results of our investigation indicates that the CPB and CTE stimulate both humoral and cellular immune responses of L. infantum infected dogs, wherase the CPA is a weaker immunogen.

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Iran is one of the most important fig producer countries around the world. Intermediate moisture fig is a processed product. One problem about intermediate moisture fig is that the color will be changed and transmute to brown during storage after processing. The purpose of this research was to study soaking temperature and time effects and to evaluate the effects of calcium chloride, cysteine, sodium metabisulphite and citric acid in various concentrations on prevention of semi-moisture fig browning at room temperature. Chemical compounds (protein, total sugar, fat, fiber and moisture) of fig samples were determined. In order to prepare product five different periods of time(3, 6, 9, 12, 15 min) and five different degrees of temperature (20, 40, 60, 80, 100°C) based on central composite rotational design were used. Samples were kept in room temperature for 2 weeks to evaluate the effects of soaking parameters on color and texture of product. After that moisture, color and texture of samples were determined. First different solution of calcium chloride (0.6, 1, 1.5, 2% w/w), citric acid (0.5, 1, 2, 3%w/w), cysteine (0.05, 0.07, 0.2, 0.5%w/w) and sodium metabisulphite (500, 800, 1000 and 1200 ppm ) were prepared. Dried figs were dipped in prepared solutions and water was used to evaluate control samples to optimize the time and temperature, then color of the samples were measured in specific period of time during four months. Results showed that temperature of 60°C and 3min interval provided 20% moisture in the product which assessed as the best moisture content for preserving color and texture. The most desirable L value was obtained using citric acid (1, 2, 3%w/w) and Calcium chloride (1.5%w/w). However sodium metabisulphite and cysteine in concentration of 0.07%, 0.05%, 0.2% showed significant difference with control, the results were not satisfactory. Results showed that using suitable temperature in rehydration of fig to inactivate poly phenol oxidase and chemical treatments to postpone and reduce the browning reaction rate were effective.    

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The impact of three different fabaceous host plants including cowpea (Vigna unguiculata), chickpea (Cicer arietinum), and mung bean (Vigna radiata) seeds was investigated using biochemical approaches on possible changes of gut proteolytic activity of the cowpea weevil, Callosobruchus maculatus at 30±1˚C and 70±5% RH and a photoperiod of 8:16 (L:D). Results revealed that pH of 4-5 and 9 was optimal for the activity of larval gut proteases using azocasein and hemoglobin as general substrates. Different serine (BApNA, SAAPFpNA, PMSF, TLCK, and TPCK) and cysteine (Z-Arg-Arg- pNA, Z-Phe-Arg-pNA and DTT) specific substrates inhibitors and activator were used as a further proof of the proteolytic profile in the gut of C. maculates. Although combinations of serine and cysteine proteases were observed, the cysteine proteases had the highest rate on the studied hosts. The protease activity, especially cystein protease, was the highest on cowpea, which was supported by hemoglobin (0.156±0.045 U mg^-1), Z-Phe-Arg-pNA (2.85 U mg^-1) substrates and DTT (90.00±0.10%) as an activator. Due to the importance and frequency of cysteine proteinases and their effects on biological and physiological process, it would be better to design pest management programs based on cysteine plant proteinase inhibitors as transgenic plants.