**3. The immobilized lipases in biodiesel production**

The term "enzyme immobilization" was defined at the first Enzyme Engineering Conference held at Hennicker, NH, USA, in 1971. It describes "enzymes physically confined at or

The Immobilized Lipases in Biodiesel Production 401

with higher porosity and interconnectivity compared with other nanostructured carriers (Sakai et al., 2010), polymethacrylate (Salis et al., 2009), etc. The naturally occurring materials used as carriers for lipase immobilization include: activated carbon (Moreno-Parajàn & Giraldo, 2011; Naranjo et al., 2010) and carbon cloth (Naranjo et al., 2010), celite (Ji et al., 2010; Shah & Gupta, 2007); hydrotalcite (Yagiz et al., 2007; Zeng et al., 2009), zeolites (Yagiz et al., 2007), etc. The role of the nature of the support surface on the loading and the activity, as well as on the operational stability of the immobilized enzyme has been

> Séverac et al., 2011; Tongboriboon et al., 2010 Salis et al., 2008 ; Tongboriboon et al., 2010 Salis et al., 2008 ; Tongboriboon et al., 2010

Cheirsilp et al., 2008 Salis et al., 2008, 2009 Tongboriboon et al., 2010

Tongboriboon et al., 2010

Moreno-Parajàn & Giraldo, 2011

Naranjo et al., 2010

Shah & Gupta, 2007 Shah & Gupta, 2007 Ji et al., 2010 Shah & Gupta, 2007

Salis et al., 2008 Salis et al., 2008 Salis et al., 2008 Salis et al., 2008

**Carrier Immobilized lipase origin Reference** 

Pre-treated textile *Candida sp.* Chen et al., 2009; Li et al., 2010 ; Lu et al., 2007

Entrapment involves capture of the enzyme within a matrix of a polymer, although enzyme encapsulation refers to the formation of a membrane-like physical barrier around the enzyme preparation (Cao, 2005). The matrix is usually formed during the process of the immobilization. The enzyme entrapped in a gel matrix can be further encapsulated. Both processes require simple equipment and relatively inexpensive reagents. It is supposed that

and 2010

Chen et al., 2009 Salis et al., 2009

investigated in details.

Accurel *Candida Antarctica* 

Activated carbon *Candida Antarctica* 

Silica *Rhizomucor miehei* 

Celite *Candida rugosa* 

*Candida rugosa Pseudomonas cepacia Pseudomonas sp. Pseudomonas fluorescens Pseudomonas fluorescens Mucor javanicus Penicillium roqueforti Penicillium camembertii Rhizopus oryzae* 

*Candida rugosa* 

*Pseudomonas cepacia Pseudomonas aeroginosa Pseudomonas fluorescens* 

Polystyrene *Pseudomonas cepacia* Li & Yan, 2010 Carbon cloth *Pseudomonas cepacia* 23 Naranjo et al., 2010 Poly(acrylonitrile) *Pseudomonas cepacia* 16 Sakai et al., 2010 Ceramics *Pseudomonas cepacia* Shah & Gupta, 2007

Hydrophilic resins *Rhizomucor miehei* De Paola et al., 2009

**3.2 Lipases immobilization by entrapment and/or encapsulation** 

*Pseudomonas fluorescens* 

Mg-Al hydrotalcites *Saccharomyces cerevisiae* Zeng et al., 2009 Resin D4020 *Penicillium expansum* Li et al., 2009 Polymethacrylate *Pseudomonas fluorescens* Salis et al., 2009 Organosilicate *Pseudomonas fluorescens* Salis et al., 2009 Hydrotalcite *Thermomyces lanuginosus* Yagiz et al., 2007 Zeolites *Thermomyces lanuginosus* Yagiz et al., 2007 Table 1. Carrier used for lipases immobilization by adsorption.

*Thermomyces lanuginosus* 

localized in a certain region of space with retention of their catalytic activity and which can be used repeatedly and continuously" (Powel, 1996). It is considered that lipase immobilization induces the enzyme conformational change required to enable the free access of substrate to the active centre. Especially, hydrophobic supports allow the adsorption of the open form of the lipases via interfacial activation, mimicking the lipophilic substrate (Ahn et al., 2010; Rodrigues & Fernandez-Lafuente, 2010; Salis et al., 2008; Séverac et al., 2011).

The revision of the literature covering the period 2005-2011 demonstrates that a large variety of matrices have been used for lipases immobilization, and that the main methods applied include adsorption, entrapment and/or encapsulation, and covalent attachment.

#### **3.1 Lipases immobilization by adsorption**

Physical adsorption is considered as the simplest method for enzyme immobilization. Enzyme fixation is performed through hydrogen bonds, salt linkages, and Van der Waal's forces. The process is carried out in mild conditions, without or with minimum support activation and clean up procedures application, and in the absence of additional reagents. Thus, it is economic and allows preserving enzyme activity and specificity. The chemical composition of the carrier, the molar ratio of hydrophilic to hydrophobic groups, as well as the particle size and the surface area determine the amount of enzyme bound and the enzyme behaviour after immobilization. Some of the most commonly used carriers for lipases immobilization by adsorption are listed in Table 1.

Data shown in Table 1 indicate that among the variety of lipase immobilization supports, Accurel has found a large application. Accurel is the trade name of a group of macroporous polymers. As carriers for lipase immobilization are used the polypropylene based hydrophobic Accurel MP (MP1000 with particle size below 1500 m, Accurel MP1001 with particle size below 1000 m and Accurel MP1004 with particle size below 400 m), and Accurel EP-100. On the most hydrophobic support tested, Accurel MP1001, no glycerol adsorption was observed (Séverac et al., 2011). It has been demonstrated (Salis et al., 2009) comparing the catalytic efficiencies (activity/loading) of eight lipases, that they show a different level of adaptation to the support. Immobilized *Pseudomonas fluorescens* lipase is the most active biocatalyst, followed by immobilized *Pseudomonas cepacia* lipase. The other lipases tested (from *Rhizopus oryzae*, *Candida rugosa*, *Mucor javanicus*, *Penicillium roqueforti*, *Aspergillus niger*, *Penicillium camembertii*), are inactive toward biodiesel synthesis in the described conditions.

Enzyme immobilization on Accurel could be performed by direct contact between the lipase solution and the support (Cheirsilp et al., 2008). However, it has been confirmed that ethanol pre-treatment improves the immobilization process by inducing a better penetration of the enzyme solution inside the hydrophobic Accurel and by reducing the enzyme thermodynamic activity, thus forcing the adsorption process (Foresti, & Ferreira, 2004). Enzyme adsorption with previous ethanol treatment of the support is carried out via: (i) wetting the support with, sequentially: ethanol, aqueous ethanol solution and finally with water, with intermediary filtration, or (ii) a single wetting with ethanol and then a direct contact with the enzyme solution without removing ethanol.

Accurel, due to its hydrophobic properties, should stabilise the enzyme in its open (active) conformation. Thus, it is considered as efficient for lipase immobilization.

Other synthetic polymers used for lipases immobilization comprise: hydrophobic polystyrene macroporous resin (Li & Yan, 2010), electrospun polyacrylonitrile nanofibers

localized in a certain region of space with retention of their catalytic activity and which can be used repeatedly and continuously" (Powel, 1996). It is considered that lipase immobilization induces the enzyme conformational change required to enable the free access of substrate to the active centre. Especially, hydrophobic supports allow the adsorption of the open form of the lipases via interfacial activation, mimicking the lipophilic substrate (Ahn et al., 2010; Rodrigues & Fernandez-Lafuente, 2010; Salis et al., 2008; Séverac

The revision of the literature covering the period 2005-2011 demonstrates that a large variety of matrices have been used for lipases immobilization, and that the main methods applied

Physical adsorption is considered as the simplest method for enzyme immobilization. Enzyme fixation is performed through hydrogen bonds, salt linkages, and Van der Waal's forces. The process is carried out in mild conditions, without or with minimum support activation and clean up procedures application, and in the absence of additional reagents. Thus, it is economic and allows preserving enzyme activity and specificity. The chemical composition of the carrier, the molar ratio of hydrophilic to hydrophobic groups, as well as the particle size and the surface area determine the amount of enzyme bound and the enzyme behaviour after immobilization. Some of the most commonly used carriers for

Data shown in Table 1 indicate that among the variety of lipase immobilization supports, Accurel has found a large application. Accurel is the trade name of a group of macroporous polymers. As carriers for lipase immobilization are used the polypropylene based hydrophobic Accurel MP (MP1000 with particle size below 1500 m, Accurel MP1001 with particle size below 1000 m and Accurel MP1004 with particle size below 400 m), and Accurel EP-100. On the most hydrophobic support tested, Accurel MP1001, no glycerol adsorption was observed (Séverac et al., 2011). It has been demonstrated (Salis et al., 2009) comparing the catalytic efficiencies (activity/loading) of eight lipases, that they show a different level of adaptation to the support. Immobilized *Pseudomonas fluorescens* lipase is the most active biocatalyst, followed by immobilized *Pseudomonas cepacia* lipase. The other lipases tested (from *Rhizopus oryzae*, *Candida rugosa*, *Mucor javanicus*, *Penicillium roqueforti*, *Aspergillus niger*, *Penicillium camembertii*), are inactive toward biodiesel synthesis in the

Enzyme immobilization on Accurel could be performed by direct contact between the lipase solution and the support (Cheirsilp et al., 2008). However, it has been confirmed that ethanol pre-treatment improves the immobilization process by inducing a better penetration of the enzyme solution inside the hydrophobic Accurel and by reducing the enzyme thermodynamic activity, thus forcing the adsorption process (Foresti, & Ferreira, 2004). Enzyme adsorption with previous ethanol treatment of the support is carried out via: (i) wetting the support with, sequentially: ethanol, aqueous ethanol solution and finally with water, with intermediary filtration, or (ii) a single wetting with ethanol and then a direct

Accurel, due to its hydrophobic properties, should stabilise the enzyme in its open (active)

Other synthetic polymers used for lipases immobilization comprise: hydrophobic polystyrene macroporous resin (Li & Yan, 2010), electrospun polyacrylonitrile nanofibers

include adsorption, entrapment and/or encapsulation, and covalent attachment.

et al., 2011).

described conditions.

**3.1 Lipases immobilization by adsorption** 

lipases immobilization by adsorption are listed in Table 1.

contact with the enzyme solution without removing ethanol.

conformation. Thus, it is considered as efficient for lipase immobilization.

with higher porosity and interconnectivity compared with other nanostructured carriers (Sakai et al., 2010), polymethacrylate (Salis et al., 2009), etc. The naturally occurring materials used as carriers for lipase immobilization include: activated carbon (Moreno-Parajàn & Giraldo, 2011; Naranjo et al., 2010) and carbon cloth (Naranjo et al., 2010), celite (Ji et al., 2010; Shah & Gupta, 2007); hydrotalcite (Yagiz et al., 2007; Zeng et al., 2009), zeolites (Yagiz et al., 2007), etc. The role of the nature of the support surface on the loading and the activity, as well as on the operational stability of the immobilized enzyme has been investigated in details.


Table 1. Carrier used for lipases immobilization by adsorption.

#### **3.2 Lipases immobilization by entrapment and/or encapsulation**

Entrapment involves capture of the enzyme within a matrix of a polymer, although enzyme encapsulation refers to the formation of a membrane-like physical barrier around the enzyme preparation (Cao, 2005). The matrix is usually formed during the process of the immobilization. The enzyme entrapped in a gel matrix can be further encapsulated. Both processes require simple equipment and relatively inexpensive reagents. It is supposed that

The Immobilized Lipases in Biodiesel Production 403

activity, stability and reusability of the enzyme (Meunier & Legge, 2010). The three types of Celite considered (R633, R632, and R647) were compared to unsupported lipase sol–gels. It has been established that sol–gel immobilized lipase supported on Celite R632 allowed achieving an average conversion of 60% per gram of material for 6 h, and exhibited an average initial lipase activity comparable to that of the unsupported sol–gel formulation. Orçaire et al. (2006) report a technique for encapsulation of *Candida Antarctica* and *Burkholderia cepacia* lipases in silica aerogels reinforced with silica quartz fibre felt and dried by the CO2 supercritical technique. The aerogel encapsulation permits maintaining the enzymes in a dispersion state similar to the dispersion prevailing in an aqueous solution, even in organic media, while agglomeration of the lipase occurs if it is used directly in the organic solvent. At present, sol-gel enzyme entrapment/encapsulation is considered to be

Covalent attachment is a result of a chemical reaction between the active amino acid residues outside the active catalytic and binding site of the enzyme, and the active functionalities of the carrier (Cao, 2005). Although drastic and complicated, and strongly affected by the carriers' properties, covalent attachment is the most efficient technique for enzyme immobilization. Some carriers used for covalent lipase immobilization are

**Carrier Immobilized lipase origin References** 

Polymers *Thermomyces lanuginosus* Dizge et al., 2008, 2009a, 2009b Polyurethane foam *Thermomyces lanuginosus* Dizge & Keskinler, 2008 Nb2O5 and SiO2-PVA *Burkholderia cepacia* Da Rуs et al., 2010 Chitosan *Candida rugosa* Shao et al., 2008

Yücel (2011) reports a method for *Thermomyces lanuginosus* lipase covalent binding on polyglutaraldehyde-activated olive pomace powder. The technique is cost effective, because of the low price of the support and because of the strong covalent bond formed, leading to enzyme stabilization without loss of activity, allowing the multiple reuse of the enzyme. Immobilized lipase was stable for 10 batches of pomace oil transesterification retaining more

Among the other naturally occurring materials, chitosan is considered as appropriate for enzyme binding. Its membrane forming and adhesion ability, high mechanical strength and facility of forming insoluble in water thermally and chemically inert films make it suitable for lipase immobilization. For instance, *Candida rugosa* type VII lipase was fixed onto chitosan beads using a binary method consisting in the follows: (i) lipase

Mendes et al., 2011 Mendes et al., 2011

Ting et al., 2008

Lee and al., 2008 Kumari et al., 2009

Xie & Ma, 2010

Dussan et al., 2007, 2010

the most successful immobilization technique for lipase immobilization.

Olive pomace *Thermomyces lanuginosus* Yücel, 2011

*Pseudomonas fluorescens* 

Lewatit *Thermomyces lanuginosus* Rodrigues et al., 2010

*Enterobacter aerogenes* 

Table 3. Carrier used for lipases immobilization by covalent attachment

*Thermomyces lanuginosus* 

**3.3 Lipases immobilization by covalent attachment** 

Resins *Thermomyces lanuginosus* 

Silica *Rhizopus orizae+Candida rugosa* 

Magnetic nanostructures *Candida rugosa* 

than 80% residual activity.

displayed in Table 3.

enzymes immobilized by entrapment and/or encapsulation are more stable than the physically adsorbed ones. At the same time the immobilized enzymes maintain their activity and stability.

Numerous materials and techniques have been used for lipases entrapment and/or encapsulation. Some of the immobilization matrices developed during the last years (2005- 2011) are enumerated in Table 2.


Table 2. Carrier used for lipases immobilization by entrapment and/or encapsulation.

For instance, a simple technique for lipase encapsulation in -carrageenan by co-extrusion was suggested by Jegannathan et al. (2009, 2010). Carrageenan has been selected because of its availability, biodegradability, low cost, and lack of toxicity. It was found that at optimized reaction conditions a methyl ester conversion up to 100% could be achieved in transesterification of palm oil using the liquid core encapsulated lipase PS from *Burkholderia cepacia.* The immobilized lipase was stable and retained 82% relative transesterification activity after five cycles.

Another technique for lipase immobilization by entrapment and/or encapsulation, which has received a considerable attention in recent years, is the sol-gel process. The method involves an aqueous solution of the enzyme, a catalyst (NaOH, NaF, HCl), and an inorganicorganic matrix precursor (alkoxysilane). The hydrolysis and condensation of the precursor result in an amorphous silica matrix that covers the enzyme. The method has been applied for *R. miehei* lipase encapsulation within the micellar phase of a surfactant that is selfassembled with silica (Macario et al., 2009). It has been demonstrated that the enzyme preserves its mobility and activity. More over, because of the activation of the enzyme catalytic centre by the hydrophobic groups of the surfactant, the immobilized lipase was more active than its free form. In addition, the obtained ordered mesoporous structure improved the stability of the enzyme and decreased the rate of leaching.

Comprehensive characterization of sol–gel immobilized lipase has been performed by Noureddini et al. (2005). Lipase PS was entrapped within a sol–gel polymer matrix, prepared by polycondensation of hydrolyzed tetramethoxysilane and isobutyltrimethoxysilane. The immobilized lipase was stable and more active than the free lipase toward the transesterification of soybean oil.

Various supports could be used to improve the stability of the entrapped/encapsulated enzymes. Celite supported lipase sol–gels were investigated aiming such problems as

enzymes immobilized by entrapment and/or encapsulation are more stable than the physically adsorbed ones. At the same time the immobilized enzymes maintain their activity

Numerous materials and techniques have been used for lipases entrapment and/or encapsulation. Some of the immobilization matrices developed during the last years (2005-

**Carrier Immobilized lipase origin References** 

Jegannathan et al., 2010 Jegannathan et al., 2010 Jegannathan et al., 2009, 2010 Jegannathan et al., 2010 Jegannathan et al., 2010

Meunier & Legge, 2010 Meunier & Legge, 2010

Nassreddine et al., 2008 Orçaire et al., 2006 Orçaire et al., 2006

Khor et al., 2010 Macario et al., 2009 Noureddini et al., 2005

*Candida rugosa Burkholderia cepacia Pseudomonas fluorescens Aspergillus niger* 

*Pseudomonas cepacia*

Lipase NS44035

*Candida Antarctica Burkholderia cepacia* 

improved the stability of the enzyme and decreased the rate of leaching.

lipase toward the transesterification of soybean oil.

Table 2. Carrier used for lipases immobilization by entrapment and/or encapsulation.

For instance, a simple technique for lipase encapsulation in -carrageenan by co-extrusion was suggested by Jegannathan et al. (2009, 2010). Carrageenan has been selected because of its availability, biodegradability, low cost, and lack of toxicity. It was found that at optimized reaction conditions a methyl ester conversion up to 100% could be achieved in transesterification of palm oil using the liquid core encapsulated lipase PS from *Burkholderia cepacia.* The immobilized lipase was stable and retained 82% relative transesterification

Another technique for lipase immobilization by entrapment and/or encapsulation, which has received a considerable attention in recent years, is the sol-gel process. The method involves an aqueous solution of the enzyme, a catalyst (NaOH, NaF, HCl), and an inorganicorganic matrix precursor (alkoxysilane). The hydrolysis and condensation of the precursor result in an amorphous silica matrix that covers the enzyme. The method has been applied for *R. miehei* lipase encapsulation within the micellar phase of a surfactant that is selfassembled with silica (Macario et al., 2009). It has been demonstrated that the enzyme preserves its mobility and activity. More over, because of the activation of the enzyme catalytic centre by the hydrophobic groups of the surfactant, the immobilized lipase was more active than its free form. In addition, the obtained ordered mesoporous structure

Comprehensive characterization of sol–gel immobilized lipase has been performed by Noureddini et al. (2005). Lipase PS was entrapped within a sol–gel polymer matrix, prepared by polycondensation of hydrolyzed tetramethoxysilane and isobutyltrimethoxysilane. The immobilized lipase was stable and more active than the free

Various supports could be used to improve the stability of the entrapped/encapsulated enzymes. Celite supported lipase sol–gels were investigated aiming such problems as

*R. miehei* 

and stability.

2011) are enumerated in Table 2.


Celite supported sol-gel *Candida Antarctica* 

Silica aerogel *Candida Antarctica* 

activity after five cycles.

Silica gel *Thermomyces lanuginosus* 

activity, stability and reusability of the enzyme (Meunier & Legge, 2010). The three types of Celite considered (R633, R632, and R647) were compared to unsupported lipase sol–gels. It has been established that sol–gel immobilized lipase supported on Celite R632 allowed achieving an average conversion of 60% per gram of material for 6 h, and exhibited an average initial lipase activity comparable to that of the unsupported sol–gel formulation.

Orçaire et al. (2006) report a technique for encapsulation of *Candida Antarctica* and *Burkholderia cepacia* lipases in silica aerogels reinforced with silica quartz fibre felt and dried by the CO2 supercritical technique. The aerogel encapsulation permits maintaining the enzymes in a dispersion state similar to the dispersion prevailing in an aqueous solution, even in organic media, while agglomeration of the lipase occurs if it is used directly in the organic solvent. At present, sol-gel enzyme entrapment/encapsulation is considered to be the most successful immobilization technique for lipase immobilization.
