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  • br Materials Aspergillus oryzae GS U mg solid was

    2022-06-15


    Materials Aspergillus oryzae βGS (8.4 U mg−1 solid) was obtained from Sigma-Aldrich Chemicals Co. (USA). o-Nitrophenyl β-d-galactopyranoside (ONPG), sodium carbonate, zinc chloride, manganese chloride, calcium chloride and potassium chloride were procured from Sisco Research Laboratories (SRL), Mumbai, India. CS, silver nitrate, hydrazine hydrate, cetyl-trimethyl ammonium bromide as surfactant, ammonium persulphate and hydrochloric BIX 02565 were purchased from Fisher Scientific, India. All solvents and other reagents were of analytical grade.
    Methodology
    Results and discussion
    Conclusion Some metal ions are significantly found in milk. It is crucial to monitor their effect on the activity and structure of native and nano-support bound βGS as immobilized enzyme preparation has to be employed in assemblage of a nano-biosensor for lactose hydrolysis in milk. PANI-CS-Ag-NC immobilized βGS exhibited maximum increase in catalytic activity in the presence of combination of Zn2+ + K+ + Ca2+ + Mn2+ ions. In addition, major secondary structural changes were also noted in PANI-CS-Ag-NC bound enzyme incubated with corresponding metal ions. It is indicative of favorable changes in protein active site which thereby enhances the catalytic activity of the enzyme. This study reports an improvement in catalytic activity of PANI-CS-NCs immobilized βGS preparations in the presence of metal ions commonly found in milk which can be used for the improved hydrolysis of lactose in milk. Furthermore, the capsules of such enzymes can be prepared by entrapping in a polymeric gel and enclosing it in a semi-permeable membrane and these encapsulated enzymes may be useful for the lactose intolerant people. These nano-biocatalysts can also be effectively employed in an assemblage of a nano-biosensor for lactose detection. However, further trials which include in vivo experiments and human trials are required to arrive at an acceptable conclusion.
    Introduction With the discovery of operon model, the LacZ gene expressing β-galactosidase enzyme has always been an important aspect in molecular toxicology (Juers et al., 2012). Because of the large tetrameric structure of β-galactosidase enzyme and rapid induction by isopropyl β-d-1-thiogalactopyranoside (IPTG), toxic insults by certain chemicals may significantly affect its expression and enzymatic functions (Juers et al., 2012). As the interaction of reactive skin sensitizers with carrier proteins at the beginning of sensitization process, the interaction of β-galactosidase-expressing E. coli with reactive toxins might result in disturbance, slow or no production of β-galactosidase (Bitton and Koopman, 1992; Nepal et al., 2018a). Moreover, presence of nucleophilic pockets at Glu537 of β-galactosidase might support the possible interaction with reactive electrophiles (Yuan et al., 1994). As an animal model, local lymph node assay (LLNA) has been considered as a gold standard to determine chemicals as skin sensitizers and non-sensitizers, where test chemicals were applied repeatedly on animal’s ear to elicit chemical specific T-cell proliferation in the local lymph node (Kimber et al., 1991). After the OECD adopted 3Rs concept and European Union promulgated the rule of using non-animal models to test cosmetic ingredients, several in vitro models for skin sensitization, such as in chemico, hCLAT, and U-Sens were proposed as alternatives to in vivo (Ashikaga et al., 2006; Gerberick et al., 2007; Nepal et al., 2018b; Urbisch et al., 2015). When a test chemical is applied onto the skin, immature Langerhans cells (LC) present in epidermis would recognize the chemical and bind to the haptens to form LC-hapten complex (Gerberick et al., 2007). Then, LC-hapten complex would migrate from skin to draining lymph nodes where the chemical-specific T cells would proliferate and release into the circulation. After subsequent skin exposure to the same chemical, clinical manifestation of skin sensitization could be observed (Agüero et al., 2012; Nepal et al., 2018b). During the interaction of sensitizers with proteins, some haptens can directly bind to skin protein, whereas some need spontaneous air-oxidation or enzymatic metabolic activation to become reactive haptens, so called pre- or pro-haptens (Gerberick et al., 2009; Troutman et al., 2011). Although several in vitro approaches were successful in separating chemicals as skin sensitizers and non-sensitizers, none of the methods completely mimics the absolute in vivo scenario of skin sensitization, specifically to detect pre- or pro-haptens. Based on the previous reports, more than 25% of the total chemicals tested for skin sensitization would be pre- or pro-haptens, indicating that an alternative test incorporated with metabolic activation system would be required for improving predictability of the method (Patlewicz et al., 2016). Although an effort to incorporate peroxidase-peroxide oxidation system to convert chemicals to reactive haptens has been made, methods that can sufficiently categorize pre- or pro-haptens as sensitizers in simple and easy manners are still necessary (Gerberick et al., 2009).