Immobilized lipase

Adsorption on solid matrices, which improves (at optimal protein/support ratios) enzyme dispersion, reduces diffusion limitations and favors substrate access to individual enzyme molecules. Immobilized lipases with excellent activity and stability were obtained by entrapping the enzymes in hydrophobic sol-gel materials [20]. Finally, in order to minimize substrate diffusion limitations and maximize enzyme dispersion, various approaches have been attempted to solubilize the biocatalysts in organic solvents. The most widespread method is the one based on the covalent linking of the amphiphilic polymer polyethylene glycol (PEG) to enzyme molecules [21]. 

Recently, an interesting example of the enzymatic kinetic resolution of a-acetoxyamide 8 was demonstrated using native wheat germ lipase and immobilized lipase PS (AMANO) (Scheme 5.6). 

Tbyonife-immobilized lipase-catalyzed resolution of solketal ( )-3 (vinyl butylate)... 

Effect of organic bridges on the royonite-immobilized lipase-cataiyzed resolution of ( )-4... 

Practical resolution of azirine 1 by the low-temperature method combined with Toyowifc-immobilized lipase and optimized acylating... 

These results indicate that the low-temperature method increases the enantioselectivity, at least above inversion temperature, and the enantioselectivity and reaction rate can be optimized by the use of Toyon/te-immobilized lipase and a suitable acylating agent. 

The catalysts was added after the reactants were fed in the tank reactor and pressure and temperature were set to the target values [84]. The study was performed using an immobilized lipase, Novozym-435 , as biocatalyst. The temperature was set to 65-75 °C and the pressure was reduced (60 mmHg). A catalyst concentration of 1-5% with an acid alcohol ratio of 1 3, 1 1 or 3 1 was used. 

Immobilized lipase Covalent binding on poly- Olive oil hydrolysis... 

Scheme 5.9 DKR using Shvo s catalyst as racemization catalyst and immobilized lipase TL to yield...

Enzyme activity for the polymerization of lactones was improved by the immobilization on Celite [93]. Immobilized lipase PF adsorbed on a Celite showed much higher catalytic activity than that before the immobilization. The catalytic activity was further enhanced by the addition of a sugar or poly(ethylene glycol) in the immobilization. Surfactant-coated lipase efficiently polymerized the ring-opening polymerization of lactones in organic solvents [94]. 

One of the most promising applications of enzyme-immobilized mesoporous materials is as microscopic reactors. Galameau et al. investigated the effect of mesoporous silica structures and their surface natures on the activity of immobilized lipases [199]. Too hydrophilic (pure silica) or too hydrophobic (butyl-grafted silica) supports are not appropriate for the development of high activity for lipases. An adequate hydrophobic/hydrophilic balance of the support, such as a supported-micelle, provides the best route to enhance lipase activity. They also encapsulated the lipases in sponge mesoporous silicates, a new procedure based on the addition of a mixture of lecithin and amines to a sol-gel synthesis to provide pore-size control. 

Biodiesel production by immobilized lipase on zeolites and related materials... 

In this communication a study of the catalytic behavior of the immobilized Rhizomucor miehei lipase in the transesterification reaction to biodiesel production has been reported. The main drawbacks associated to the current biodiesel production by basic homogeneous catalysis could be overcome by using immobilized lipases. Immobilization by adsorption and entrapment have been used as methods to prepare the heterogeneous biocatalyst. Zeolites and related materials have been used as inorganic lipase supports. To promote the enzyme adsorption, the surface of the supports have been functionalized by synthesis procedures or by post-treatments. While, the enzyme entrapping procedure has been carried out by sol-gel method in order to obtain the biocatalyst protected by a mesoporous matrix and to reduce its leaching after several catalytic uses. 

Table 2. Catalytic activity of the immobilized lipase in the transesterification reaction of triolein with methanol (18 hours of reaction, 40°C, trioleimmethanol molar ratio 1 3).

The high catalyst loading typical of sol gel entrapped catalysts ensures a desirably high substrate/catalyst (S/C) ratio as the major part of the heterogeneous catalyst weight originates from the silicate matrix. For example, in a preparative-scale reaction of the alcohol raol-(2-naphthyl)-ethanol only 250 mg of sol-gel CaLB immobilizate could be used per 10 g of substrate. For comparison, all this makes the process based on sol-gel immobilized lipase very competitive with the commercial BASF process using lipase immobilized on Amberlite to produce the amine at a scale of 1000 tons per year. 

Monoglyceride (MG) is one of the most important emulsifiers in food and pharmaceutical industries [280], MG is industrially produced by trans-esterification of fats and oils at high temperature with alkaline catalyst. The synthesis of MG by hydrolysis or glycerolysis of triglyceride (TG) with immobilized lipase attracted attention recently, because it has mild reaction conditions and avoids formation of side products. Silica and celite are often used as immobilization carriers [281], But the immobilized lipase particles are difficult to reuse due to adsorption of glycerol on this carriers [282], PVA/chitosan composite membrane reactor can be used for enzymatic processing of fats and oils. The immobilized activity of lipase was 2.64 IU/cm2 with a recovery of 24%. The membrane reactor was used in a two-phase system reaction to synthesize monoglyceride (MG) by hydrolysis of palm oil, which was reused for at least nine batches with yield of 32-50%. 

Dynamic Kinetic Resolution of 1-Phenylethanol by Immobilized Lipase Coupled with In Situ Racemization over Zeolite Beta... 

The immobilized lipase was prepared by adsorption of the hpase onto Amberlite XAD7. The lipase solution (50 mL) was prepared by dissolving 2.2 g of cmde lipase in 50 mL of phosphate buffer solution, pH 7. The lipase solution was gently stirred for a few minutes until dissolved. 

Procedure 3 Enzymatic Kinetic Resolution of (/ ,S)-2-Ethoxyethyl Ibuprofen Ester with Immobilized Lipase... 

The immobilized lipase (0.1 g) in pH 7 phosphate buffer (25 mL) was added to 25 mL (20 mM) of ester stock solution in a 250 mL Erlenmeyer flask (reaction flask). The reaction flask was incubated in an incubator shaker at 40 °C with the agitation speed set to 200 rpm. Samples from the organic phase and aqueous phase were withdrawn at 24 h intervals over a 5-day reaction period. The samples collected were filtered using 0.45 pm nylon filter and injected into the HPLC system to determine the rate of resolution by monitoring both substrate ((/ ,5)-2-ethoxyethyl ibuprofen ester) and product (5-ibuprofen acid concentration). 

To isooctane (25 mL) was added (7 ,5)-ibuprofen ester (0.27 g, 40 mM), 0.5 m sodium hydroxide (25 mL) and immobilized lipase (0.2 g). The reaction medium consisted of two layers of solution, namely ibuprofen ester in isooctane and aqueous NaOH, where the reaction only occurred at the interface between these two layers. The mixture was agitated in an orbital shaker at constant temperature of 45 °C and at 200 rpm. 

The procedure shows that it is feasible to combine racemization with the kinetic resolution process (hence the DKR) of R,S)- ethoxyethyl ibuprofen ester. The chemical synthesis of the ester can be applied to any esters, as it is a common procedure. The immobilized lipase preparation procedure can also be used with any enzymes or support of choice. However, the enzyme loading will need to be optimized first. The procedures for the enzymatic kinetic resolution and DKR will need to be adjusted accordingly with different esters. Through this method, the enantiopurity of (5)-ibuprofen was found to be 99.4 % and the conversion was 85 %. It was demonstrated through our work that the synthesis of (5)-ibuprofen via DKR is highly dependent on the suitability of the reaction medium between enzymatic kinetic resolution and the racemization process. This is because the compatibility between both processes is crucial for the success of the DKR. The choice of base catalyst will vary from one reaction to another, but the basic procedures used in this work can be applied. DKRs of other profens have been reported by Lin and Tsai and Chen et al. ... 

An example of an industrial membrane bioreactor is the hollow-fiber membrane system for the production of (-)-MPGM (3-(4-methoxyphenyl)glycidic acid methyl ester), which is an important intermediate for the production of diltiazem hydrochloride [81, 82]. For the enantiospecific hydrolysis of MPGM a hollow-fiber ultrafiltration membrane with immobilized lipase from Serratia marcescens is used. (-f)-MPGM is selectively converted into (2S,3J )-(-F)-3-(4-methoxyphenyl)glyci-dic acid and methanol. The reactant is dissolved in toluene, whereas the hydrophilic product is removed via the aqueous phase at the permeate side of the membrane, see Fig. 13.9. EnantiomericaUy pure (-)-MPGM is obtained from the to-... 

In a similar investigation, transesterification reactions of vinyl acetate with alcohols in [BMIM]BF4 and [BMIM]PF6 in the presence of immobilized lipases CALB and PS-C were found to proceed with higher enantioselectivities than in THF or toluene, with the best result again being observed with [BMIM]PF6 (280). 

The catalysts were found to be stable up to 323 K without any deactivation. An activation energy of 45 kJ/mol was found for the free enzyme, 41 kJ/mol for the immobilized lipase, indeed confirming the absence of mass transfer limitations. 

---