## 2 Results and Discussion  
### Influence of Contact Time on the Adsorption Process: Kinetic and Mass Transfer Studies  
In this study, the adsorption process of lipase Eversa(r) Transform 2.0 on PSty-DVB beads in a batch mode was performed under moderate experimental conditions--pH 5.0 at low ionic strength (5 mmol-L-1--buffer sodium acetate), 25 \({}^{\circ}\)C, mechanical stirring of 200 rpm and an initial protein loading of 40 mg:g\({}^{-1}\) of support for providing a heterogeneous biocatalyst with high immobilized protein amount and, thus, catalytic performance to produce valuable industrial esters [28; 29]. According to Figure 1A-C, a linear profile of lipase adsorption can be observed during the first 180 min of contact time due to the high surface area and pore size of PSty-DVB beads, followed by a slight decrease of the adsorption rate due to possible reduction of the available surface area in the support and/or restrict access of the enzyme to the internal part of the pores (reduction of the pore diameter by the previously adsorbed lipase molecules). The maximum adsorbed protein amount of 31 mg:g\({}^{-1}\) of support was achieved after 480 min of contact time, which corresponds to 77.5% of the protein loading initially offered (40 mg:g\({}^{-1}\) of support). The immobilization yield, based on the units of activity (hydrolytic activity) lost during the adsorption process, was very similar (80.4%), thus showing that the soluble lipase used in this study (Eversa(r) Transform 2.0) has few contaminant proteins in its formulation. A control test by incubating the lipase under the same experimental conditions (lipase solution in the absence \begin{table}
\begin{tabular}{c c c} \hline \hline
**Ester** & **Chemical Structure** & **Flavor Profiles** \\ \hline Methyl octanoate & & Fruit, orange, wax, wine a \\ Propyl octanoate & & Herbal and flower b \\ Isoamyl octanoate & & Fruit c \\ (Fusel oil) & & Fruit a \\ Isoamyl octanoate & & Green, herbal, and oil a \\ Hexyl octanoate & & Herbal and fruit d \\ Benzyl octanoate & & Herbal and fruit d \\ \hline \hline \end{tabular} a
[FOOTNOTE:a]Footnote a: Reference [30]: b
[FOOTNOTE:b]Footnote b: footnotetext: Reference [31]: c
[ENDFOOTNOTE] Reference [32]: d  
\end{table} 
Table 1: Technical information of the different flavor esters from octanoic acid produced in this study. of support) was also performed, and it retained all of its original activity after 540 min. These results clearly showed that all units of activity that disappeared in the supernatant were due to adsorption of the enzyme on the support surface. SEM images show that the support has a spherical geometry with non-uniform particle size distribution varying from 250 to 850 \(\mathrm{\SIUnitSymbolMicro m}\) (magnification of 30\(\times\)--Figure 2A), and large pores (magnification of 20,000\(\times\)--Figure 2B). In fact, an apparent decrease of pore size and uniform distribution of the enzyme on the support surface after the adsorption process can be observed (magnification of 20,000\(\times\)--Figure 2C). These images show that lipase Eversa(r) Transform 2.0 can be immobilized on both external and internal support surfaces. Three non-linear empirical adsorption kinetic models such as pseudo-first-order (PFO), pseudo-second-order (PSO) and Elovich were used to fit these experimental adsorption data for determining of adsorption rate of the lipase on the support surface and better understanding on the adsorption process [34]. According to results in Figure 1A-C, PFO and PSO kinetic models gave the highest and very similar coefficient correlation (R\({}^{2}\)) values-0.9948 and 0.9945, respectively, followed by the Elovich kinetic model (R\({}^{2}\) = 0.9641). However, the theoretical q\({}_{\mathrm{e}}\) value obtained for PFO kinetic value (q\({}_{\mathrm{e-theor}}\) = 34.09 mg\(\cdot\)g\({}^{-1}\) of support--Figure 1A) was consistent with the experimental value (q\({}_{\mathrm{e-exp}}\) = 31 mg\(\cdot\)g\({}^{-1}\) of support).