Continuous fast Fourier transforms cyclic voltammetry as a new approach for investigation of skim milk k-casein proteolysis, a comparative study.

Cheese production is relied upon the action of Rennet on the casein micelles of milk. Chymosin assay methods are usually time consuming and offline. Herein, we report a new electrochemical technique for studying the proteolysis of K-casein. The interaction of rennet and its substrate was studied by fast Fourier transform continuous cyclic voltammetry (FFTCCV) based on a determination of k-casein in aqueous solution. FFTCCV technique is a very useful method for studying the enzymatic procedures. Fast response, no need of modified electrodes or complex equipment is some of FFTCCV advantages. Various concentrations of enzyme and substrate were selected and the increase in the appearance of charged species in solution as a result of the addition of rennet was studied. Data obtained using FFTCCV technique were also confirmed by turbidity analysis. The results show that rennet proteolysis activity occurs in much shorter time scales compare with its aggregation. Hence, following the appearance of charged segments as a result of proteolysis could be under consideration as a rapid and online method.

Acetylcholinesterase immobilization on polyacrylamide/functionalized multi-walled carbon nanotube nanocomposite nanofibrous membrane.

In this work, polyacrylamide/multi-walled carbon nanotubes (MWCNT) solution is electrospun to nanocomposite nanofibrous membranes for acetylcholinesterase enzyme immobilization. A new method for enzyme immobilization is proposed, and the results of analysis show successful covalent bonding of enzymes on electrospun membrane surface besides their non-covalent entrapment. Fourier transform infrared spectroscopy, mechanical and thermal investigations of nanofibrous membrane approve successful cross-linking and enzyme immobilization. The enzyme relative activity and kinetic on both pure and nanocomposite membranes is investigated, and the results show proper performance of designed membrane to even improve the enzyme activity followed by immobilization compared to free enzyme. Scanning electron microscopy images show nanofibrous web of 3D structure with a low shrinkage and hydrogel structure followed by enzyme immobilization and cross-linking. Moreover, the important role of functionalized carbon nanotubes on final nanofibrous membrane functionality as a media for enzyme immobilization is investigated. The results show that MWCNT could act effectively for enzyme immobilization improvement via both physical (enhanced fibers' morphology and conductivity) and chemical (enzyme entrapment) methods.

Covalent immobilization of Drosophila acetylcholinesterase for biosensor applications.

The synthesized cDNA coding for AChE (acetylcholinesterase) was subcloned in pENTR/D-TOPO plasmid and expressed using baculovirus expression vector and Sf9 insect cells as host. Purified enzyme (specific activity 36374 micromol x min(-1) x mg(-1)) was immobilized on pre-activated perlite (a porous silica matrix) by silanization and glutaraldehyde treatment. The total enzyme immobilized was then measured, and total and specific activity of immobilized AChE was compared with that of soluble enzyme. Using this perlite support not only resulted in a great amount of maintained immobilized enzyme activity (more than 70%, specific activity 26238 micromolx min(-1) x mg(-1)), but also significantly improved stability against temperature (8.7- and 17.7-fold at 50 and 60 degrees C respectively), urea (2.7-fold) and acetonitrile (1.7-fold). Kinetic studies showed that the K(m) value for immobilized enzyme is very similar to the soluble one (0.088 and 0.081 mM respectively). In addition, immobilized enzymes retained 80% of their initial activity after 16 consecutive reactor batch cycles. A comparison of the inhibitory effect of paraoxon on soluble and immobilized AChE showed that immobilization increased the linearity of the inhibition plot particularly in the range 0.1 nM-0.1 microM.