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    • Nano biocatalyst prepared by assembling

    2021-10-14

    Nano-biocatalyst, prepared by assembling an enzyme on nanomaterial (NM) carriers is an emerging innovation in the field of nanobiotechnology. Some of the advantages of using NM as supports in 550 24 to conventional porous supports are effective enzyme loading due to large surface area with reduced internal diffusional limitations (Ansari and Husain, 2012, Cipolatti et al., 2016). Their surface is easily modifiable to improve enzyme stability, capability, and engineering performances. There have been many reports on enhancing enzyme activity and stability associated with nano-biocatalysts (Ali et al., 2018, Khan et al., 2016, Khan et al., 2017, Khan et al., 2018). However, there are also some drawbacks associated with nano-immobilization like cost of fabrication process and limitation in large scale application (Cipolatti et al., 2016). With the recent advancements in nanotechnology, the need for polymers and their composites with unique properties for specific applications have gained much attention (Khan et al., 2011). Among metal nanoparticles (NPs), silver NPs have attracted greater attention due to their antimicrobial property. They are widely applied in biological and medical fields such as biosensors, wound healing, treat burns and cancer therapeutics (Ahmad et al., 2018). Recently, our group has developed a nano-biocatalytic system involving βGS immobilized on polyaniline chitosan nanocomposite (PANI-CS-NC) and polyaniline chitosan silver nanocomposite (PANI-CS-Ag-NC). Both preparations were non-toxic, biocompatible, and highly stable with excellent reuse characteristics. Activity yield of βGS adsorbed on silver containing NC was surprisingly enhanced (Khan et al., 2018, Khan et al., 2018). In the current past, several researchers have used different combinations of PANI, Ag and CS with other nanomaterials for biosensor applications (Klein et al., 2016, Paithankar et al., 2018). Immobilization strategy employed in this study involved physical adsorption of βGS on the two supports. This method mainly involves van der Waals forces, hydrophobic and ionic interactions (Jesionowski et al., 2014). Ionic exchange between the chitosan chains containing positive charges and the negative charges on the protein led to efficient adsorption. Recently, a study has shown the importance of pH during ion exchange on the final properties of βGS. The bound enzyme at pH 5 was found to be about 2 and 6 folds more stable than the soluble counterpart at pH 5 and 9 respectively (Albuquerque et al., 2016). Enzymes often use metal ions as co-catalysts. Though βGS is active in the absence of metals, recent studies have demonstrated βGS exhibits enhanced activity in the presence of Mg2+, Mn2+, Co2+, Ni2+, Zn2+, K+, Ba2+, Ca2+ and Fe2+ ions (Adalberto et al., 2010, Liu et al., 2015). However, metal ions such as Ca2+, Zn2+, Mn2+, and Cu2+, have also been reported to decrease the activity of thermostable βGS from Thermotoga naphthophila and Lactobacillus delbrueckii (Kong et al., 2014). In some cases, the effect of addition of metal ion on the activity of thermostable βGS obtained from Bacillus coagulans is not significant (Batra et al., 2002). βGS from Saccharopolyspora rectivirgula consists of eight binding sites for divalent metals per monomer, placed in three classes, according to their affinities; there is a Ca2+-specific site in Class I; a Ca2+-specific site and a Mn2+-specific site in Class II; and four Mn2+ and Mg2+-specific sites in Class III. The removal of Ca2+ ion from the Class I site irreversibly deactivates the enzyme (Adalberto et al., 2010). In this study, an effort has been made to investigate the role of various metals ions significantly present in milk on the activity of free, PANI-CS-NC and PANI-CS-Ag-NC bound βGS. The effect of independent metal ions and their different combinations on the structure of native and immobilized βGS was carried out with the help of UV–visible (UV–vis), fluorescence, Fourier transform infrared (FTIR) and circular dichroism (CD) spectroscopy. In this study, we report that metal ions and their combinations significantly enhance the catalytic activity of Aspergillus oryzae βGS physically adsorbed to PANI-CS-NC and PANI-CS-Ag-NC. We also observed that some sort of conformational changes take place in or around the active site of the immobilized enzyme which leads to this enhanced activity.