# Immobilization of Pectinolytic Enzymes on Nylon 6/6 Carriers
## 3 Results and Discussion
### Optimization of Immobilization Process  
Nylon 6/6 is a polyamide of which secondary amide groups can be converted into secondary imidate groups by O-alkylation with DMS. This process introduces a reactive functional group into nylon without any accompanying polymerization, and the formed functional groups react directly with amino groups of enzymes to produce a stable amidine link [38]. However, additional use of glutaraldehyde as a linker results in more stable immobilization of enzymes [28].  
Nylon threads were first treated with DMS at 60 \({}^{\circ}\)C, which has been previously reported as the optimum temperature for nylon activation [30]. We examined the effect of DMS treatment duration (1-7.5 min) on pectinase immobilization, as in addition to generating active groups, the mechanical strength of nylon 6/6 can decrease after a longer treatment over 5 min. Assuming that the thread with immobilized enzyme is homogenous, the activities of immobilized enzymes can be expressed in meters, which is extremely convenient for technological applications [39]. The effect of the length of DMS treatment on the activity of immobilized on nylon 6/6 threads pectinase is presented in Table 2.  
Increasing incubation time in DMS resulted in a decreased immobilized pectinase activity. In fact, over O-alkylation of nylon carrier can result in intra-cross-linkage of the amino groups with glutaraldehyde hindering the attachment of the enzyme [30]. For further enzyme immobilization procedure, we incubated nylon carriers in DMS for 2 min at 60 \({}^{\circ}\)C, as this treatment time resulted also in more stable immobilized enzymes (as measured after five working cycles). Two different concentrations of glutaraldehyde, 2.5% [40] and 12.5% [28], were used for enzyme immobilization. (Table 3). We did not use higher glutaraldehyde concentrations, as, in high rate immobilization processes, glutaraldehyde promotes crosslinking between enzyme molecules [41].  
Treatment with 12.5% glutaraldehyde solution resulted in higher immobilized pectinase activity and also residual pectinase activity after five cycles was 2.5 times higher compared to the threads treated with 2.5% glutaraldehyde solution. So, for further immobilization experiments, nylon carriers were treated with 12.5% glutaraldehyde solution for 1 h at room temperature.  
After the immobilization process, the carriers should be washed carefully for several times with buffer using an orbital shaker (150 rpm) for 5 to 15 min each time to remove all non-covalently attached enzymes.  
\begin{table}
\begin{tabular}{c c c c c} \hline \hline
**DMS Incubation Time (min)** & **1** & **2** & **2.5** & **5** & **7.5** \\ \hline Immobilized Pectinase Activity (mU/m thread) & \(72\pm 2\) * & \(64\pm 1\) & \(45\pm 2\) & \(43\pm 5\) & \(38\pm 4\) \\ \hline \hline \end{tabular}  
* All values are expressed as means \(\pm\) standard deviation (n = 3).  
\end{table}
Table 2: The effect of incubation time in DMS on the activity of immobilized pectinase.  
\begin{table}
\begin{tabular}{c c c} \hline \hline
**Glutaraldehyde Concentration (\%)** & **2.5** & **12.5** \\ \hline Immobilized Pectinase Activity (mU/m thread) & \(56\pm 1\)* & \(64\pm 1\) \\ \hline Residual activity after 5 cycles (mU/m thread) & \(14\pm 1\) & \(37\pm 2\) \\ \hline \hline \end{tabular}  
* All values are expressed as means \(\pm\) standard deviation (n = 3).  
\end{table}
Table 3: The effect of glutaraldehyde concentration on pectinase immobilization.