The practical use of free lipase in reaction systems suffers from technological difficulties such as contamination of the products with residual enzymatic activity and economic difficulties such as the use of enzyme for a single reactor pass.

Hence, part of the overall potential enzymatic activity is lost. If the lipase is immobilized, it becomes an independent phase within the reaction system, which may easily be retained in the reactor with concomitant advantages of preventing contamination of the products and extending its useful active life. Furthermore, as mentioned in Section 6.7, immobilization provides a more rigid external backbone for lipase molecule, allowing it to maintain its activity at higher temperatures than if it is in free-form. Therefore, the reaction optimum temperature is expected to increase, which results in faster rate of reaction. In addition, immobilization of lipases has been proposed as a countermeasure to the high water content usually present in WO (Fukuda et al., 2001). Furthermore, by immobilization, the enzyme is dispersed over a large surface, which results in an enhanced catalytic performance, especially in organic media, which is the case in biodiesel production. It was shown that lipase from C. antarctica performed better when immobilized on ceramic beads than in free-form (Al-Zuhair et al., 2008). Similar results were also found using lipase from P. cepacia (Shah and Gupta, 2006).

The main advantage of immobilization of lipase, however, is the ability of repeated use. The ability to use the immobilized enzyme repeatedly is actually the factor that determines its effectiveness. Due to the negative effect caused by by-product glycerol adsorption on the surface of the immobilized lipase, a loss in activity is inevitable with repeated uses. However, the immobilized lipase retained more than 70% of its initial activity even after more than ten cycles. This was found when using different lipases immobilized on different solid surfaces, such as Novozym 435 (Wei et al., 2004) and P. fluorescens lipase immobilized on toyonite (Iso et al, 2001). Organic solvents are usually used to dissolve the by-product glycerol, which clogs the active sites of the immobilized lipase. By using t-butanol as solvent, Wang et al. (2006) showed that there is no obvious loss in biodiesel yield even after immobilized lipase from T. Lanuginosa was used for 120 cycles. Even better results were found by Li et al (2006) using immobilized lipase from T. Lanuginosa and Novozyme 435; with the number of cycles reaching 200. However, as mentioned earlier, the addition of organic solvent has inherent problems, such as, diluting substrates and requiring additional solvent recovery unit.

From an economical point of view, a continuous reaction process without the use of any organic solvent is needed for the industrial production of biodiesel. It has been shown that the activity of immobilized lipase could be significantly increased and deactivated enzyme could be regenerated when t-butanol was used for an immersion pretreatment of the enzyme (Chen and Wu, 2003). It was shown that the activity of pretreated Novozyme 435 increased about tenfold in comparison to the enzyme not subjected to pretreatment. In addition, following complete deactivation by methanol, washing the enzyme with t-butanol successfully regenerated the enzyme and restored up to 75% of its original activity level. Recently, it was found that activity, methanol tolerance and operational stability of immobilized lipase from Candida sp. 99-125 can be significantly enhanced by pretreatment with 1 mM salts solutions of CaCl2 and MgCl2 (Lu et al, 2010). The reason might be that these
salts incorporate with the protein to form a more stable molecule that resists conformational change induced by high methanol concentration.