Biocatalysis and Biotransformation, 2002 Vol. 20 (1), pp. 63–71
Effect of the Immobilization Method of Lipase from Thermomyces lanuginosus on Sucrose Acylation MANUEL FERRERa, FRANCISCO J. PLOUa,*, GLORIA FUENTESa, M. ANGELES CRUCESa, LOTTE ANDERSENb, OLE KIRKb, MORTEN CHRISTENSENb and ANTONIO BALLESTEROSa a
Departamento de Biocata´lisis, Instituto de Cata´lisis, CSIC, Cantoblanco 28049, Madrid, Spain; bNovozymes A/S, Novo´ Alle´ 2880, Bagsvaerd, Denmark (Received 5 April 2001; Revised 7 May 2001)
Lipase from Thermomyces lanuginosus (formerly Humicola lanuginosa ) was immobilized using granulation by incubating low-particle-size silica with the lipase. Granules with a particle diameter in the range 0.3 – 1 mm were obtained. The immobilized lipase was tested in the acylation of sucrose with vinyl laurate in mixtures of tert-amyl alcohol: dimethyl sulfoxide. Results were compared with immobilization of enzyme by adsorption on polypropylene (Accurel EP100), deposition on Celite by precipitation, and covalent attachment to Eupergit C. Granulated lipase converted >95% of sucrose into 6-O-lauroylsucrose in 6 h. Accurel-lipase was also very active, converting 70% of sucrose into monoester in 2 h. The residual activity of granules after five reaction cycles under the best reaction conditions was 72%; this value was considerably higher than the one observed for the same lipase adsorbed on Accurel (15% residual activity after five cycles). Keywords: Immobilization; Adsorption; Granulation; Lipases; Sucrose esters; Lipase stability
INTRODUCTION Lipase-catalyzed modification of fats and oils has proven to be a feasible industrial process (Gandhi, 1997; Pandey et al., 1999). Immobilized lipases are being used in various applications, e.g. in the manufacturing of structured lipids for infant formula and cocoa butter substitutes (Xu, 2000). However, the industrial breakthrough of this technology has not yet emerged in the area of bulk commodities, mainly
due to the high ratio cost/efficiency of the biocatalysts commercially available today. Immobilization is a suitable approach that allows biocatalyst reuse, makes product recovery easier and is able to enhance resistance against inactivation by different denaturants (Tischer and Kasche, 1999). Several properties of immobilized lipases are important for potential industrial applications: mechanical strength, chemical and physical stability, hydrophobic/hydrophilic character, enzyme loading capacity, and cost. Silica-granulation is a new immobilization technique that makes use of inexpensive inorganic materials to get granules of 0.3 –1 mm particle size and high enzymatic efficiency (Pedersen and Christensen, 2000). The granules exhibit high-pressure resistance, good filterability and are therefore suitable for both batch and fixedbed reactors. Thus, silica-granulated Thermomyces lanuginosus lipase has proven useful for the interesterification of fats (Zhang et al., 2001). In addition, the hydrophobic character of the granules may be modulated by varying the source of silica. Among the applications of lipases, the acylation of carbohydrates is of significant importance. In particular, sucrose fatty acid esters are non-ionic surfactive agents with applications in the food industry (Nakamura, 1997; Watanabe, 1999), in cosmetics (Vermeire et al., 1996; Hill and Rhode, 1999) and as pharmaceuticals (e.g. with antimicrobial, antitumoural and/or antibiotic activity) (Sachiko et al., 1999). Regioselective acylation of sucrose is an arduous task due to its multifunctionality (Haines, 1976).
*Corresponding author. Tel.: þ34-91-5854869. Fax: þ34-91-5854760. E-mail:
[email protected] ISSN 1024-2422 q 2002 Taylor & Francis Ltd DOI: 10.1080/10242420290003191
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Sucrose esters can be synthesized using either chemical and/or enzymatic reactions. Current chemical production is usually base-catalyzed at high temperatures, has a low selectivity, and coloured by-products are formed (Nakamura, 1997). Selective chemical acylation, however, requires even more significant synthetic control via protecting-groups methodologies (Vlahov et al., 1997). The enzyme-catalyzed synthesis of sugar esters can provide regio- and stereoselective products (Soedjak and Spradlin, 1994; Riva et al., 1998). However, when using lipases, their low stability in the polar solvents required to solubilize sugars is a critical problem (Woudenberg et al., 1996; Plou et al., 1999). The use of solvents of intermediate polarity for lipase-catalyzed acylation of carbohydrates is now well documented (Woudenberg et al., 1996; Degn et al., 1999), but the yields obtained are low due to their low solubility. We recently developed a new and simple process for the regioselective acylation of sucrose (Ferrer et al., 1999) and other di- and trisaccharides (Ferrer et al., 2000), using mixtures of two miscible solvents. We employed mixtures of tert-amyl alcohol (a non-toxic and slightly polar solvent, where most lipases remain active) and a polar solvent (dimethyl sulfoxide, at a concentration not higher than 30%). We demonstrated that the use of co-solvents may become an excellent tool to increase the solubility of sugars and at the same time to modulate enzyme properties. In this work, we have studied the activity and stability of immobilized T. lanuginosus lipase (formerly Humicola lanuginosa ) in these reactions. Results using a silica-granulated lipase preparation were compared with the same enzyme adsorbed on Accurel, deposited on Celite, and covalently attached to Eupergit C. The test reaction was the synthesis of sucrose monolaurate in mixtures tert-amyl alcohol: dimethyl sulfoxide, using vinyl laurate as acyl donor.
2-methyl-2-butanol (tert-amyl alcohol) and Bial’s reagent were from Sigma. Sucrose and dimethyl sulfoxide were supplied by Merck. Vinyl laurate was from Fluka. Salts for water activity control were purchased from Sigma, Aldrich and Panreac, and were of the highest purity available. All other reagents and solvents were of the purest grade available. All solvents were stored prior to use over ˚ ), at least for 24 h. molecular sieves (3 A Enzyme Immobilization Granulation Silica-granulated lipases from T. lanuginosus were prepared according to a method previously described (Pedersen et al., 1995; Christensen et al., 1998; Pedersen et al., 1998). The granulation was performed using a mechanical granulator (Lo¨dige Ploughshare Mixer FM50, consisting of two vertical operating ploughs and one horizontal operating high shear mixer head, maximum capacity 50 l). The operation was done batch-wise and the filling was based on 10 kg dry silica powder. Silica-powder (Sipernatw 22) and other dry materials were fed into the container manually. Binder (Glucidex DE21) was dissolved in the T. lanuginosus lipase concentrate (maximum 20% w/w binder) and the mixture was then pumped from a holding tank to the granulation chamber through a nebulizer on the top of the granulation chamber. After spraying the desired amount of liquid lipase and binder mixture in the chamber, the silica (small size particles) was granulated to more dense agglomerates over a time period of 10 – 30 min. After the granulation had occurred, the process was terminated and the granules were dried in a Glatt Fluid Bed system to water contents of 2.5% w/w. After drying the granules were sieved in the fraction 300 –1000 mm. The activity of the resulting biocatalyst was
