## 3 Results
### Optimization of Reaction Conditions  
In this section, the reaction conditions such as reaction temperature, substrate molar ratio of FFA to glycerol, adding an amount of immobilized lipase, and 4 A molecular sieve (water absorbent) were investigated based on the reduction of oil acid values. In the temperature range of 45-85 \({}^{\circ}\)C (Figure 4a), the acid value of rapeseed oil was drastically decreased during a 3 h reaction time. Specifically, the final acid value was reduced from 3.1 mg KOH/g to 1.7 mg KOH/g when the temperature was increased from 45 \({}^{\circ}\)C to 65 \({}^{\circ}\)C and further increased in temperature, leading to a final acid value rise to 2.2 mg KOH/g at 75 \({}^{\circ}\)C and 2.5 mg KOH/g at 85 \({}^{\circ}\)C. The first-half increase in temperature can not only promote the lipase activity but also minimize the system viscosity to enhance the lipase-substrate contract for improved enzymatic reaction [57]. However, a second-half temperature increase from 65 \({}^{\circ}\)C to 85 \({}^{\circ}\)C could reduce the catalytic ability of lipase CALB, thus leading to the mitigated performance in enzymatic deacidification [58; 59]. Overall, lipase CALB presented the highest catalytic capacity at \(\sim\)65 \({}^{\circ}\)C, and this is also consistent with the previous literature [60]. Hence, the optimal reaction temperature of 65 \({}^{\circ}\)C was determined for CALB@MCM-41-C8 in this study. Glycerol is used as the acyl donor to facilitate the esterification with FFA, thus decreasing the acid value of rapeseed oil. The addition of glycerol significantly lowered down the acid values with the molar ratio of FFA to glycerol between 1:0.5 and 1:5 (Figure 4b). The lowest acid value of 1.7 mg KOH/g was from the reaction condition, where the molar ratio of FFA to glycerol was 1:4, suggesting that the inadequate or excessive amount of glycerol would diminish the enzymatic reactions. On the one hand, insufficient glycerol means limited FFA removal by the esterification between glycerol and FFA, leading to a relatively higher acid value. On the other hand, increasing the amount of glycerol can obviously boost the solution viscosity and delay the enzymatic catalysis. Moreover, the exceeded glycerol can partially cover the immobilized lipase to hinder the lipase-substrate contact and subsequently result in inefficient catalytic performance [61]. Thus, the optimal molar ratio of FFA to glycerol was identified to be 1:4. In addition, at the optimal reaction temperature of 65 \({}^{\circ}\)C and a molar ratio of FFA to glycerol of 1:4, the effects of adding an amount of CALB@MCM-41-C8 on the enzymatic deacidification were studied. As depicted in Figure 4c, it was apparent that the use of 0.1-3% immobilized lipase promoted the removal of FFA. Particularly, increasing the adding amount of CALB@MCM-41-C8 from 0.1% to 2% further lowered the final acid value from 8.6 mg KOH/g to 1.7 mg KOH/g, thus demonstrating the catalytic effectiveness of the self-made immobilized lipase. However, the 3% of CALB@MCM-41-C8 addition conversely resulted in poor deacidification efficiency, reflected by the slightly higher acid value of 5.7 mg KOH/g. The excessive addition of immobilized lipase tended to agglomerate together to form larger clusters, which prevented the mass transfer and minimized the catalytic reaction [62]. Thus, 2% of CALB@MCM-41-C8 is regarded as the proper amount to add for the catalytic deacidification. Figure 3: Effects of (**a**) initial protein concentration and (**b**) pH on the loading amount and immobilization efficiency of lipase CALB. To remove the byproduct of water generated during the esterified deacidification, a 4 A molecular sieve worked as the water absorbent was used. Compared to the reaction without the use of water sorbent (namely, 0% of 4 A molecular sieve addition), lower acid values were noticed with 1-8% addition of 4 A molecular sieve, thus validating its feasibility in water removal and enhancement for enzymatic esterification (Figure 4d).