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.