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.