2.3 Immobilization of Candida rugosa lipase
The aqueous
lipase solution was centrifuged
at 4025 xg for 10 min at 4°C to
remove the insoluble material to obtain a homogeneous semi-purified
lipase. The resulting supernatant was separated and used for subsequent
experiments.
As different supports have their suitable immobilization conditions, it
is reasonable to compare the immobilization effect of the supports on
this basis. 0.1 g of modified magnetic diatomite was added to 5 mL of
semi-purified CRL with different concentrations and different pH values
(6.0-8.0) and then stirred at 25-40°C for 4-12 h. After filtration under
reduced pressure, washing, and freeze drying, the immobilized enzyme
product was obtained. Enzyme
loading and immobilization efficiency are important parameters that
define the immobilization process. Enzyme loading is an important
parameter to examine the loading capacity of the support, and
immobilization efficiency (immobilization yield) is usually employed to
define the percentage of the enzyme that is immobilized on or in the
support.
The BCA method was used to detect the protein content in the
supernatant. All the measurements were done in triplicate and the error
was less than 5% in each case,
and the following formula was used to calculate CRL loading and
immobilization efficiency.
\begin{equation}
\begin{matrix}\text{CRL\ loading}\left(\frac{\text{mg}}{g}\right)=\frac{C_{i}\times M_{i}-C_{f}\times V_{f}}{M_{f}}\#\left(1\right)\\
\end{matrix}\nonumber \\
\end{equation}\begin{equation}
\begin{matrix}\text{Immobilization\ efficiency}\left(\%\right)=\frac{C_{i}\times M_{i}-C_{f}{\times V}_{f}}{C_{i}\times M_{i}}\ \times 100\#\left(2\right)\\
\end{matrix}\nonumber \\
\end{equation}where \(C_{i}\) (mg/g) is the initial protein concentration in CRL
before immobilization, \(C_{f}\) (mg/mL) is the final protein
concentration in supernatant and washing solution after immobilization,\(M_{i}\) (mg) is the mass of CRL added to the buffer solution,\(V_{f}\) (mL) is the total volume of the supernatant and washing
liquid, and \(M_{f}\) (g) is the mass of the support.
2.4 Enzyme
activity assay
The expressed activity gives the
enzyme activity expressed by the immobilized enzyme itself and the
specific activity shows the effect of the immobilization process on the
enzyme activity, these two parameters together with the enzyme loading
and the immobilization efficiency form the parameters required to define
the immobilization process[32].
The p-nitrophenyl palmitate
(p-NPP) assay has been widely used to analyze lipase activity[33,
34]. One unit (1 U) is defined as the amount of enzyme required to
catalyze the hydrolysis of p-nitrophenyl palmitate to produce 1 μmol of
p-nitrophenol per minute at 40°C and pH 7.0. All the measurements were
done in triplicate and the error was less than 5% in each
case.
The activity of CRL (immobilized
CRL) and activity recovery were calculated by the following equation:
\begin{equation}
\begin{matrix}\text{Expressed\ activity\ }\left({Ug}^{-1}\right)=\frac{A\times
10^{6}\times V_{t}}{\varepsilon\times t\times m_{1}}\#\left(3\right)\\
\end{matrix}\nonumber \\
\end{equation}\begin{equation}
\begin{matrix}\text{Specific\ activity}\ \left({Ug}^{-1}\right)=\frac{A\times 10^{6}\times V_{t}}{\varepsilon\times t\times m_{2}}\#\left(4\right)\\
\end{matrix}\nonumber \\
\end{equation}\begin{equation}
\begin{matrix}\text{Activity\ recovery\ }\left(\%\right)=\frac{\text{SA}_{1}}{\text{SA}_{0}}\times 100\#\left(5\right)\\
\end{matrix}\nonumber \\
\end{equation}Where \(A\) is the absorbance of the samples, \(V_{t}\) (L) is the
volume of the solution, \(\varepsilon\)(L·mol-1·cm-1) is the molar
extinction coefficient of p-nitrophenol, \(t\) (min) is the reaction
time, \(m_{1}\) (g) is the mass of immobilized CRL and\(m_{2}\) (g) is the protein mass
of immobilized CRL,\(\text{SA}_{1}\)(U·g-1) is the
specific activity of immobilization CRL, \(\text{SA}_{0}\)(U·g-1) is the
specific activity of free CRL.
The following formula was used to calculate relative activity and
residual activity.
\begin{equation}
\begin{matrix}\text{Relative\ activity\ }\left(\%\right)=\frac{A_{t}}{A_{\max}}\times 100\#\left(6\right)\\
\end{matrix}\nonumber \\
\end{equation}\begin{equation}
\begin{matrix}\text{Residual\ activity\ }\left(\%\right)=\frac{A_{t}}{A_{i}}\times 100\#\left(7\right)\\
\end{matrix}\nonumber \\
\end{equation}Where \(A_{t}\) is the absorbance of the samples, \(A_{\max}\) is the
maximum absorbance, and \(A_{i}\) is the initial absorbance value
of the samples.