3. Results and discussion3.1. Immob

3. Results and discussion
3.1. Immobilization of PLA1
3.1.1. Carrier screening
Immobilization of an enzyme yields the enzyme reusable, min- imizes product contamination by the enzyme, and simplifies separation of product and enzyme. The type of carrier is a crucial factor
in such an enzyme immobilization process. Table 1 shows the effect of the selected carriers on the fixation level (%) and specific activity (lmol/g protein/min) of the immobilized enzyme. Among
the eight carriers examined, Lewatit VP OC 1600, Accurel MP 1000, Amberlite XAD 4 and Octyl silica were hydrophobic. Since hydrophobic carriers are not easily suspended in the enzyme suspension, pre-wetting hydrophobic carriers with ethanol prior to immobilization
was suggested to solve this problem in the previous studies (Vikbjerg et al., 2007; Zhang, Hellgren, & Xu, 2007). In this study, Pre-wetting indeed made it possible to suspend these carriers in the enzyme suspension, resulting also in increased fixation level of PLA1. Lewatit VP OC 1600 (fixation level, 79.5%) exhibited the highest fixation, followed by Amberlite XAD 7HP (78.2%) and Duolite A568 (73.2%). The catalytic activities of the various immobilized PLA1 were
compared by measuring the specific activities based on its hydrolytic activity, according to Vikbjerg et al. (2007). Lewatit VP OC 1600 (specific activity, 6.7  10 3 lmol/g protein/min), Amberlite XAD7HP (6.6  103 lmol/g protein/min) and Duolite A568 (6.3  103 lmol/g protein/min) as carriers resulted not only in significantly higher fixation levels but also higher specific activities, compared with the other carriers tested. However, low fixation did not necessarily lead to low specific activity of the immobilized enzyme. For instance, in case of Accurel MP 1000, even though fixation level was low, specific activity was significantly higher, compared with Amberlite XAD 4, which showed very similar fixation level to Accurel MP 1000. Among the carriers,
Lewatit VP OC 1600 had the highest protein fixation as well as specific activity. Thus, Lewatit VP OC 1600 was judged a suitable carrier for further experimentation.
3.1.2. Protein concentration of initial enzyme suspension
The initial enzyme concentration is also an important factor to be considered for the efficiency of the immobilization. The initial PLA1 suspension, whose protein concentration ranged from 2.9
to 29.6 mg/mL, was tested for the immobilization of PLA1. Increased protein concentration in the PLA1 suspension increased the amount of protein immobilized (mg/g, Fig. 1a). Meanwhile,
the fixation level remained constant up to a protein concentration of 14.9 mg/g, but deceased significantly with further increased protein concentration. This indicated that, after the carrier was saturated with loaded protein, binding ability of the carrier decreased with the increasing protein concentration of the initial PLA1 suspension. The apparent activity increased steadily when protein amount in the carrier was increased from 16.9 to 147.9 mg/g, which corresponded to a protein concentration of 2.9–20.6 mg/mL, (Fig. 1b). However, there was no significant difference in apparent activity as the protein amount in the carrier increased further. Meanwhile,the specific activity of immobilized enzyme increased when the protein amount of the immobilized enzyme was increased from 16.9 to 56.9 mg/g and the specific activity remained constant when the protein amount of the immobilized enzyme was increased up to 147.9 mg/g. However, the specific activity decreased with further increase of protein amount in immobilized enzyme. The decline in specific activity was related to the increased steric restrictions and the mass-transfer limitations of the enzyme. Due to the lack of surface area, the enzyme tends to be packed into more than a monolayer on the surface, causing decrease in activity (Madoery, Gattone, & Fidelio, 1995). Therefore, an initial enzyme concentration of 20.6 mg/g was selected for the immobilization of PLA1 and the resulting immobilized enzyme preparation was
used for subsequent acidolysis reactions.