2.1 Catalytic activity of LTv in aqueous media before and after UVB irradiation of the enzyme.
The oxidation reaction of SYR catalyzed by laccase generates the product TMAMQ (Scheme 1), which absorbs in the visible region with a maximum at 530 nm (Manole et al. 2008; Prasetyo et al. 2010).
In Fig. 1A (main), the presence of the product TMAMQ as a consequence of the enzymatic reaction can be observed in parallel to a decrease in the SYR absorption at 350 nm. In order to perform a kinetic analysis, the decrease in the absorption of SYR at 350 nm was registered as a function of the reaction time (Inset, Fig. 1a).
An analogous experiment was carried out after irradiation of the enzyme with UVB light. The variation of the absorbance of SYR at 350 nm as a function of the reaction time, before and after photolysis of the enzyme, and the initial rate value have been shown representatively for SYR concentration of 8.9 µM in Fig. 1b.
The changes in the reaction rate, after irradiation, indicate that the photolysis of the enzyme alters the catalytic process. The rate of the reaction in the presence of the photolyzed LTv seems to be lower by a factor of about 35%.
For the determination of the kinetic parameters KM, kCAT and kCAT/KM, the rate of the enzymatic reaction at pH 6 and 40°C was evaluated before and after photolysis of LTv at different initial concentrations of SYR and at fixed enzyme concentration, pH and temperature.
The experimental data obtained were interpreted according to the model described by Michaelis-Menten for enzymatic reactions (Johnson and Goody 2011). Laccase catalyze reactions for a diversity of substrates and reaction media are known to agree to this model (Feng et al. 2017; Iark et al. 2019; Kelbert et al. 2020; Cacciari et al. 2020).
A typical Michaelis-Menten plot of initial rate variation of the reaction vs. substrate concentration is shown in Fig. 2 (main) for SYR oxidation before and after UVB irradiation of LTv. The Hanes-Woolf equation was used to linearize the Michaelis-Menten graph (Inset, Fig. 2) with the purpose to obtain the kinetic parameters shown in Table 1.
Table 1
Summary of experimental kinetic parameters data
|
|
kCAT
min− 1
|
KM
µM
|
\({K}_{M}^{corr }\)
µM
|
kCAT / KM
|
kCAT /\({K}_{M}^{corr }\)
|
Aqueous media
|
Before LTv photolysis
|
0.43 ± 0.02
|
2.5 ± 0.1
|
-
|
0.170 ± 0.008
|
-
|
After LTv photolysis
|
0.32 ± 0.02
|
3.7 ± 0.2
|
-
|
0.086 ± 0.004
|
-
|
Micellar media
|
Before LTv photolysis
|
0.050 ± 0.002
|
8.6 ± 0.4
|
0.70 ± 0.03
|
0.0058 ± 0.0003
|
0.069 ± 0.003
|
After LTv photolysis
|
0.030 ± 0.001
|
5.9 ± 0.3
|
0.48 ± 0.02
|
0.0053 ± 0.0003
|
0.064 ± 0.003
|
As can be seen in Table 1, for aqueous media all the kinetic parameters are altered after UVB irradiation of the enzyme. The KM value increases, which allows inferring about a decrease in the enzyme affinity for the substrate, in parallel the kCAT decreases, which indicates a decrease in the capacity of the enzyme to catalyze the transformation from substrate to product. Consequently, the catalytic efficiency (kCAT/KM) is reduced by 50% approximately, which refers to the global alteration of the enzymatic process.
These results were expected by our working group since we previously reported the effect of UVB irradiation on the catalytic activity of laccase at pH 6 and 30°C, using the azine SYR and the aromatic compounds rifampicin and bromothymol blue as substrate (Cacciari et al. 2020). In the present contribution, the temperature at which the experiments were carried out was 10°C higher than previously reported, this explains why the values of the kinetic parameters that depend on the reaction rate, are slightly different. Usually, in the reactions catalyzed by enzymes it is observed an increase in the reaction rate by increasing temperature and opposite trend is found at very high temperatures attributable to enzyme thermal denaturation (Punekar 2018). For laccase, the enzymatic reaction rate increase as the temperature increases from 10°C to 50°C (Margot et al. 2013; Kang et al. 2019). Likewise, the increment in temperature did not modify the clear tendency between the results obtain in this work at 40°C and the previously report at 30°C, that is the kinetic parameters are significantly altered after irradiation of the enzyme with UVB light.
The alteration of the kinetic parameters as a consequence of the irradiation of LTv is due to an autosensitization process in which the enzyme absorbs UVB light and the reactive singlet oxygen (O2(1Δg)) is generated. This species leads to the photooxidation of susceptible amino acids present in the protein structure that can be involve in the catalytic site of LTv or close to it (Cacciari et al. 2020). The kinetics and mechanism of photodegradation of LTv in aqueous buffer media was exhaustively analyzed and reported by our group in the mentioned previous work (Cacciari et al. 2020).
2.2 Catalytic activity of LTv in micellar media before and after UVB photolysis of the enzyme.
Several authors have evaluated the catalytic activity of laccase against aromatics substrate in micellar media and investigated optimal conditions for their oxidative degradation (Michizoe et al. 2001, 2005; Okazaki et al. 2002; Chhaya and Gupte 2013; Xu et. al. 2020). However, there are not many reports for the oxidation of SYR, which is the substrate chosen in the present contribution, in micellar media. On the other hand, in this work we emphasize about the effect of UVB irradiation on the catalytic activity of the enzyme encapsulated in the RMs. As shown above, LTv is sensitive to direct irradiation with UVB light in an aqueous medium and its catalytic efficiency is drastically altered. The encapsulation of the enzyme in RMs systems could prevent this fact.
Changes in the absorption spectra of SYR in the presence of LTv hosted in RMs of 0.15 M AOT/isooctane at W0 = 30, were evaluated as a function of reaction time at 40°C and pHext=6, before (Fig. 3a) and after UVB irradiation of LTv (Inset, Fig. 3b).
A decrease in the absorbance of SYR at 353 nm can be seen in Fig. 3a which indicates that the enzyme remains active when it is included in the AOT RMs. This result is very important since the nano-aggregate system confer a solubilization media in the presence of organic solvent, both for the enzyme and the substrate, in which the catalytic function of the enzyme is preserved. Water-soluble enzymes such as laccase do not have activity in pure organic solvents since they undergo denaturation processes (Michizoe et al. 2001).
The variation of the absorbance of SYR at 353 nm as a function of reaction time and the initial rate value are showed representatively for 10 µM of SYR in main Fig. 3b, before and after UVB irradiation of the enzyme.
The kinetic parameters were determined in the same way as in the aqueous media. The Michaelis-Menten graphics and Hanes-Wolf plots before and after photolysis of LTv are shown in Fig. 4 (main and inset, respectively) and the results obtained are summarized in Table 1.
Analyzing and comparing the values of the parameters in Table 1, it is clear that the aqueous buffer medium seems to provide the best conditions for the oxidation of the substrate in terms of enzyme-substrate affinity, capacity of the enzyme to transform substrate into product per unit of time and catalytic efficiency with respect to the micellar medium.
In order to correctly compare the catalytic efficiency in both media, it is necessary to consider that the substrate could be distributed between the nonpolar organic phase and the micellar pseudo-phase, namely a fraction of substrate could be bound to the micelles and another could be free in the organic solvent.
The spectral properties of SYR in a series of solvents including cyclohexane, dioxane, acetonitrile, etc. have been summarized by Rajendiran N. and Balasubramanian T. (Rajendiran and Balasubramanian 2007). In their work they also inform that the SYR molecule can enter into the nonpolar cavity of β- cyclodextrin, which demonstrates de ability of SYR to be solved in organic media.
A preferential distribution of SYR by one of the phases could leading to a lower effective (local) substrate concentration in the site in which the reaction takes place, presumably the micellar pseudo-phase. This fact could affect the values of the kinetic parameter KM which is defined numerically as the substrate concentration at which the reaction rate is half its maximal value.
In order to correct the kinetic parameters, the SYR binding constant (Kb) was determined using the methodology detailed in the section 1.5. The SYR absorbance was recorded at different concentrations of AOT/isooctane W0 = 30 (inset, Fig. 5), then plotting 1/(A-Aiso) vs 1/[AOT] according to Ketelaar equation (main, Fig. 5), the Kb value of 74.3 ± 0.5 M-1 was determined. This Kb value reveals that a substrate fraction is bound to the micellar pseudo-phase (Silber et al. 1999, Correa et al. 2001).
The value of KM was corrected taking into account the partition of the substrate using two pseudo-phases model (Lissi and Abuin 2000; Aguilar et al. 2001). For this, the initial KM value obtained must be divided by the fraction of substrate that remains in the organic phase according to the following equation:
$${K}_{M}^{corr }= \frac{{K}_{M}}{\left(1+ {K}_{b} \left[AOT\right]\right)} Equation 4$$
The corrected KM (\({K}_{M}^{corr}\)) values are also summarized in Table 1.
In Table 1 it can be seen that the catalytic efficiency in the RMs, now defined as kCAT / \({K}_{M}^{corr }\), remains considerably above the value corresponding to the aqueous media in the absence of micelles. This is merely due to a significant decrease in \({K}_{M}^{corr }\) value. It can be observed that in the presence of interfaces some enzymes suffer conformational changes that slightly reduce kCAT but that notably increases its affinity for the substrate, rendering a noticeable decrease in the Michaelis-Menten constant (Aguilar et al. 2001).
A similar behavior in the kinetic parameters observed in the present contribution, for the oxidation of SYR by LTv in RMs of AOT/isooctane, was previously detected by other authors for the hydrolysis of 2-napthyl acetate catalyzed by the enzyme lipase in RMs of AOT/heptane at various AOT concentration and W0 values (Aguilar et al. 2001).
To support the fact that the enzyme could interact with the micellar interface and modify its conformation, fluorescent anisotropy measurement where performed. The anisotropy values obtained (Table 2) suggest that the mobility of the enzyme is restricted by attachment to RMs of AOT. <r > is higher in the micellar system, where the protein is confined in the aggregate, compared with aqueous medium, where there will be free rotation of the protein.
Table 2
Stationary fluorescence anisotropy, <r>, of LTv in aqueous media (buffer solution) and in micellar media (RMs of 0.15 M AOT/isooctane W = 30) at pH 6.
|
Aqueous media
|
Micellar media
|
<r>
|
0.0823 ± 0.005
|
0.102 ± 0.004
|
Although the catalytic efficiency in the micellar system is lower in relation to the aqueous media, in Table 1 it is possible to note that this kinetic parameter does not seem to vary significantly before and after enzyme direct photolysis in RMs while in the aqueous media the change observed is considerably marked. This fact allows inferring about the photo-protective effect of the nano-structure in which LTv is included.
The explanation for this phenomenon may be associated with multiple factors. One of them is the existence of an inner filter effect. Both the enzyme and the micellar system have absorption at the irradiation wavelength in the photolysis experiments. Figure 6 shows the absorption spectra of the micellar system with and without the enzyme, in which a slight contribution of inner filter by the absorption of the micelle must be taken into account.
Shapovalova et al. previously reported a work in which they entrapment the enzymes carbonic anhydrase, acid phosphatase and horseradish peroxidase in alumina sol-gel matrices (Shapovalova et al. 2016). In this type of structures, an increase in stability against UV radiation was observed for all enzymes since the ceramic rigid matrix itself absorbs light in the UV range. In line with this, very recently, Mena-Giraldo and Orozco reported a polymeric micro-carrier with the capacity to protect horseradish peroxidase, laccase and catalase enzymes from ultraviolet light, preserving their activity (Mena-Giraldo and Orozco 2022). In these cases, the absorption of light by one of the components co-encapsulate in the system is responsible for the photo-protective effect observed.
However, we consider that in the system presented in this work, the main photo-protective effect is due to the confinement of the biomolecule inside the micelle. The nano-environment that the enzyme has inside the micelle is essentially different from that the enzyme has in the aqueous media. For example, it is known that the macroscopic dielectric constant in the RMs water pool is lower than that of free water (Luiz et al. 2004; Biswas et al. 2008). Physical changes such as the polarity of the medium surrounding of the enzyme inside the micelle can modify photochemical reactions, such as those between O2(1Δg) and photooxidizable amino acids presents in the protein structure (Solterman et al 1999; Luiz et al. 2004).
In previous works carried out by our research group, we studied the O2(1Δg)-mediated photooxidation of bovine serum albumin and α-chymotrypsin proteins in reverse micellar systems (Reynoso 2008, 2019). In these investigations, it was observed that the reaction of biomolecules with the photogenerated species decreased markedly in the aforementioned system in comparison to the reaction in aqueous medium. The value of the O2(1Δg) quenching constant by the proteins determinate in the micellar medium was significantly lower than that found in aqueous medium.
This phenomenon which we consider, it is also present in the system studied in this contribution, was attributed to the formation of an in excited state encounter complex, with partial charge-transfer character between the oxygen reactive specie and amino acids like Tyr and Trp. This complex is affected by changes in the medium polarity.