Lipase immobilization commercialization

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. 

The support obtained by the sol-gel technique was used to immobilize commercial CRL following three procedures. In the first, the lipase was immobilized on PS by ADS in the second, the enzyme was covalently bonded on the support previously silanized and activated with glutaralde-hyde (SPS) in the absence (CB1) and presence of an additive (CB2) and, in the third, the enzyme was encapsulated in the absence (EN1) and presence of an additive (EN2). 

Various lipase immobilization methods were tested with different silica matrices, and the immobilized enzyme samples were examined by morphologic, physicochemical, and biochemical characterization methods. The results allowed correlation of the activity-coupling yield of different immobilization methods in relation to the incorporation of lipase in the silica gels and showed that the most active biocatalyst resulted from the encapsulation of commercial CRL in the presence of PEG. 

For industrial applications, thermostable lipases are desirable in order to work in melted fat. Thermostable, positional specific lipases are well known. One example is Mucor miehei lipase which we have developed into an immobilized commercial product (Lipozyme)... 

The most important factor for adsorption is the choice of immobilization matrix. Several commercially available matrices commonly employed for nonaqneous enzymology are listed in Table 8.3. They consist of macroporons millimeter-sized particulates that are hydrophilic with the exception of Accnrel (polypropylene). Celite (diatomaceons earth) is probably, the most commonly employed matrix of those listed in Table 8.3. In addition, a few enzymes are commercially available in immobilized forms, inclnding Lipozyme IM and Novozyme from Novo-Nordisk, the Chirazyme prodnct line from Roche Molecular Biochemicals, and Pseudomonas cepacia lipase immobilized in Sol-Gel AK (Fluka). 

Commercially available lipase PS immobilized on Celite. Toyonite without organic bridges. 

In conventional synthetic transformations, enzymes are normally used in aqueous or organic solvent at moderate temperatures to preserve the activity of enzymes. Consequently, some of these reactions require longer reaction times. In view of the newer developments wherein enzymes can be immobilized on solid supports [183], they are amenable to relatively higher temperature reaction with adequate pH control. The application of MW irradiation has been explored with two enzyme systems namely Pseudomonas lipase dispersed in Hyflo Super Cell and commercially available SP 435 Novozym (Candida antarctica lipase grafted on an acrylic resin). 

To understand the potential of pressure application to enzyme processes and to help elucidate the reaction mechanism as well as a rational design of alcoholysis reactors for future scale-up, we investigated the influence of temperature, pressure, exposure times, and decompression rates on the activity of a commercial immobilized lipase (Novozym 435) activity in high-pressure C02 medium. 

The commercial lipase Candida antarctica (Novozym 435), immobilized on a macroporous anionic resin (0.12 U/g, 1.4% water, and diameter... 

We conclude that a commercial immobilized lipase from C. antarctica (Novozym 435) was stable in SCC02 for all experimental conditions investigated. Based on the results obtained here and comparison of them with the results obtained by other investigators, it can be concluded that the magnitude of pressure, temperature, decompression rate, and exposure time needed to inactivate the enzyme strongly depends on the nature and the source of enzyme and, primarily, whether the enzyme is in its native or immobilized form. For the purpose of using this enzyme to catalyze the transesterification reaction of vegetable oils in order to produce esters, the results obtained herein are relevant, because the immobilized lipase can be used with low activity loss at typical conditions of temperature and pressure employed in many biotransformations of raw materials. 

The objective of the present work was to study the synthesis of monolaurin by direct lipase-catalyzed esterification between glycerol and lauric acid without any solvent or surfactant. The effects of lauric acid/ glycerol molar ratio, enzyme concentration, and temperature were studied using an experimental design. The reuse of the commercial immobilized lipase, to reduce the process cost, was also investigated. 

Two commercial immobilized lipases were kindly supplied by Novozymes Brazil (Araucaria, PR, Brazil) Mucor miehei (Lipozyme IM) immobilized on a macroporous anion-exchange resin (0.15 U/g and 4 wt% water) and Candida antarctica (Novozym 435) immobilized on a macro-porous anionic resin (0.12 U/g and 1.4 wt% water). 

Interesterification of blends of milk fat and palm kernel olein by a mycelium-bound lipase from Rhizomucor miehei or a commercially immobilized enzyme preparation resulted in a lower slip melting point and solid fat content. An interesterified product made from a 70 30 mixture of palm kernel olein and anhydrous milk fat was considered to be suitable for use in ice cream (Liew et al., 2001). 

Shieh et al. (2003) indicated a biodiesel transesterification using soybean oil and methanol and commercial immobilized lipase from R. miehei (Lipozyme IM-77). The response surface analysis showed that the following variables were important reaction time, temperature, enzyme amount, molar ratio of methanol to soybean oil, and added water content on percentage weight conversion to soybean oil methyl ester by transesterification. The optimum yield based on ridge max analysis gave a 92.2% weight conversion. 

The enzymatic esterification of oleic acid and oleyl alcohol to obtain oleyl oleate, which is a synthetic analogue of jojoba oil, was studied. The reaction was catalyzed by a commercially available immobilized lipase from Rhizomucor miehei. As solvents, carbon dioxide and liquid n-butane were used. Reactions were performed in a batch and in continuously operating high pressure reactors. 

Myristic acid was purchased from Sigma (St Louis, MO) and ethanol (99.85 %) from Prolabo (France). Ultra pure carbon dioxide (99.995 %) was purchased from Airgaz (France). The lipase (E C. 3.1.1.3.) was a commercial enzyme from Mucor miehei kindly supplied by Novo Nordisk (Denmark). This lipase (Lipozyme TM) is immobilized on Duolite A568 (Rohm and Hass). The resin particles have a size comprised between 300 to 600 pm. In order to see if a phenomenon of internal mass transfer occurs during the enzymatic esterification, we sieved the support into different size series. The average granulometry was determined by Coulzer Sizer method (Table 1). 

Figure 32 includes results illustrating the performance of lipase/car-bon monolith systems in an acylation reaction. For comparison, the free lipase and a commercial immobilized lipase (Novozyme) were also tested. As expected, in all cases the specific activity of immobilized lipase was foimd to be lower than that of the free enzyme. Such a difference is usually ascribed to conformational changes of the enzyme, steric effects, or denaturation. For the monolithic biocatalysts, the activity of the immobilized catalyst relative to that of the pure enzyme was found to be 30-35%, and for the Novozyme catalyst about 80% in the first rim. However, the Novozyme catalyst underwent significant deactivation, in contrast to the carbon monolith-supported catalysts. The deactivation of the Novozyme catalyst in consecutive runs is probably a consequence of the instability of the support matrix under reaction conditions (101,102). 

The application of enzymes as catalysts in organic chemistry is closely linked to their immobilization. Indeed, many enzymes are only available in an immobilized form. The immobilized enzymes can be used as received, greatly easing their application. Numerous of these readily available immobilized enzymes are now the working horses of biocatalysis. This has even led to the incorrect use of the abbreviation of an enzyme name for a specific enzyme preparation, that is CALB for the immobilized form of Candida antarctica lipase B on cross-linked polymethacrylate (also known as Novozym 435). Vice versa the commercial name of an enzyme preparation-Amano PS-has taken the place of the enzyme (Burkhdderia cepacia lipase on dextrin or diatomaceous earth). Surprisingly, often no attention is paid to the fact that the enzyme is immobilized [1]. 

Lipase B from Candida antarctica (CALB) has been shown to be an excellent enantioselective biocatalyst for the stereo-selective acylation of racemic alcohols [14, 15]. The most often used commercial preparation of CALB is Novozym 435, where the enzyme is immobilized on a macroporous acrylic resin and the matrix presents about 90% of the total mass. 

In this research the kinetic resolution of 1-phenylethanol catalyzed by commercially available immobilized lipase from CALB was assayed in non-aqueous conditions in SC-CO2 and IL/SC-CO2 systems with the aim of studying the enan-tioselectivity of Novozym 435. The influence of different reaction parameters, such as pressure, the acyl donor/alcohol molar ratio and different ILs, on the enantio-merically pure compound (R)-l-phenylethyl acetate formation via kinetic resolution of 1-phenylethanol was investigated. 

The influence of different ILs, based on N, N dialkyUmidazolium cations as reaction media, on the enantioselectivity of commercially available immobilized lipase from CALB for the kinetic resolution of 1-phenylethanol was studied. Further, the performance of the enzymatic kinetic 1-phenylethanol resolution by CALB in the system [bmim][PF6]/SC-C02 was studied. 

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