3.3 Catalytic characterization
The protein proportions in the nanoreactors were estimated by densitometry on an SDS-PAGE gel. According to the band’s intensity, the GOx enzyme in the unfunctionalized VLP-GOx corresponds to 3.58% of the total protein, while in the HSA functionalized preparations (VLP-GOx@HSA) the GOx represented only 0.82%, coat protein (CP) 22.8% and the cover shell of HSA 76.38% of the total protein in the nanoreactor.
The catalytic constants for GOx activity of nanoreactors were estimated using a Michaelis-Menten nonlinear regression. The assembly from 1:3 GOx:CP molar ratio showed the highest specific activity (1,208 U/g) and a Michaelis constant (KM) of 34.6 mM (Table 1). Thus, this molar ratio was used for further studies and for HSA functionalization. Considering the GOx content in the nanoreactor preparations, the Vmax of VLP-GOx, in enzyme activity basis, is estimated to be 33,743 U/g of GOx, representing 14% of the transformation rate of free enzyme. On the other hand, the Vmax of functionalized VLP-GOx@HSA en enzyme basis is 12,990 U/g of GOx, 5.5% of the value of the free enzyme. Using the amount of enzyme contained in the nanoreactors, the transformation rate constant (kcat) could be obtained. The free enzyme showed a kcat value of 634 s-1, while the preparations VLP-GOx and VLP-GOx@HSA showed values of 90 s-1 and 34 s-1, respectively. In addition, it is possible to estimate the number of GOx molecules (active dimer of 160,000 Da) per capsid or nanoreactor (180 monomers of 20,385 Da). The number of GOx molecules inside each VLP was estimated as an average of 0.85 molecules of GOx per VPL nanoreactor.