318 Christensen et al. Eur. J. Lipid Sci. Technol.

105 (2003) 318–321

Industrial lipase immobilization with a minimum of enzyme leakage from the carrier. b)
The enzyme has to be stable during each step in the im-
Morten Würtz Christensena, Lotte Andersenb, mobilization process. c) The immobilization procedure
Tommy Lykke Husumb, Ole Kirkb should preferably be robust and reproducible. d) Cost-ef-
a Novozymes North America, Inc., North Carolina, USA fective immobilization process. e) Production logistics –
b Novozymes A/S, Denmark optimize production throughput. f) From a regulatory
standpoint, the materials and equipments should apply to
food grade regulations, if the immobilized lipase is to be
used in food applications. g) From an application stand-
1 Introduction
point, the immobilized lipase should be physically robust
Immobilization of enzymes plays an import role within ap- and preferably applicable in both batch and fixed bed
plied biotechnology. The main reason for immobilizing en- processes.
zymes is the ability to isolate the biocatalyst from the re-
action product and reuse it in order to increase its pro- Commercially available immobilized lipases are, in gener-
ductivity. For lipase(triacylglycerolhydrolase)-catalyzed al, immobilized by adsorption onto polymer-based matri-
reactions immobilization can help in providing non-aque- ces (for example the Candida antarctica lipase B immobi-
ous conditions necessary for ester synthesis and inter- lized onto polyacrylate type matrix, available as Novozym
esterification. 435 (Novozymes) or Chirazyme L-2 (Roche)). Some li-
pases are also available in cross-linked forms, such as
Different immobilization technologies have been devel-
cross-linked enzyme crystals (CLECs) offered by Altus.
oped over the past 30 years and can be classified in the
For some applications of immobilized lipases, the rela-
following general classes:
tively high cost of those biocatalysts becomes prohibitive
1. Deposition onto hydrophilic inorganic material, such as for further exploitation. The relative high cost is associat-
celite or silica gel. ed with the usage of an expensive carrier material. There-
2. Encapsulation (for example, in alginate or car- fore, further development of a less expensive immobiliza-
rageenan beads). tion process technology is necessary to emerge into rela-
3. Covalent linkage to carriers, for example using epoxy tive low margin applications such as interesterification of
functionalized polymer beads. fats as compared to relative high margin applications, for
4. Adsorption onto polymer-based carriers. example enantioselective synthesis of pharmaceutical in-
5. Cross-linking using for example glutaraldehyde. termediates.

Each technology has been further developed for example At Novozymes, researchers have recently developed a
new chemistry for covalent linkage or new materials for new, less expensive immobilization process that com-
adsorptions or encapsulations techniques. More recently, bines methodologies such as adsorption and encapsula-
methodologies such as adsorption and encapsulation into tion of lipases using silica granulation technology (Peder-
sol-gel material, Reetz [1], and Hsu et al. [2] and cross- sen et al. [4]). The use of inexpensive inorganic materials
linking of lipase enzyme aggregates, Lopez-Serrano et al. such as silica gel and diatomaceous earth has been pro-
[3] have been exploited. Several different lipases have posed as an alternative to the expensive organic sup-
been immobilized using the described methodologies. ports, Sonnet et al. [5], and Wisdom et al. [6]. Unfortu-
Examples are lipases from Candida rugosa, Candida nately, these materials are not available in a particle size-
antarctica, Pseudomonas cepacia, Rhizopus oryzae, Rhi- enabling its use in fixed bed reactors, and the mechanical
zopus delemar, Rhizomucor miehei, and Thermomyces properties in stirred batch reactors are poor. Furthermore,
lanuginosus. the adsorption or precipitation procedures used during
the immobilization would still be highly labor-demanding
When considering the choice of immobilization method-
when performed on a larger scale.
ologies for large scale immobilized lipase applications,
several issues have to be addressed: a) The immobiliza-
tion should preferably increase the stability of the lipase in Our focus has been to develop an immobilized lipase that
terms of higher temperature stability and also productivity, would be cost-efficient for the use within the oils and fats
industry. Main applications are the production of zero
trans-fat margarine/shortening base stocks and applica-
tions within oleochemicals (fatty acid esters in general).
Correspondence: Morten Würtz Christensen, 77 Perry Church
Lipase from Thermomyces lanuginosus was immobilized
Chapel Rd., Franklinton 27525, NC, USA. Phone: +1-919-494- and used for interesterification application, whereas Can-
3039, Fax: +1-919-494-3415; e-mail: [email protected] dida antarctica lipase B was used in ester synthesis.
Eur. J. Lipid Sci. Technol. 105 (2003) 318–321 Industrial lipase immobilization 319

2 Materials and methods glass vials and placed into a dessicator, containing satu-
rated NaCl solution for the control of water activity. The
2.1 Granulation immobilized lipase was allowed to gas phase equilibrate
2.1.1. Process for 24 h at room temperature.

The granulation was performed using a mechanical gran- Trilaurin/myristic acid solution (20 ml) was placed in a
ulator (Lödige Ploughshare Mixer FM50, Lödige, Pader- 100-ml conical flack with septum seal. A zero-sample of
born, Germany). The operation was batch-wise. Silica- 100 µl was withdrawn from the solution and diluted with
power (Sipernat 22, Degussa AG, Düsseldorf, Germany) 500-µl actone/acetonitrile (1:1). The flask was placed in a
and other dry materials were fed into the container manu- water-shaking bath at 40 °C and 145 rpm.
ally. Binder (Glucidex DE21, Roquette, Lestrem) dis-
solved in the aqueous Thermomyces lanuginosus con- Immobilized lipase (50 mg, equilibrated) was added to the
centrate (or Candida antarctica lipase B lipase concen- reaction. During 1 h a sample (100 µl) was withdrawn
trate) was pumped into the granulation chamber. The every 10 min from the reaction, diluted with 500 µl of the
fluffy silica was granulated to a more dense mixture, see dilution solvent and analyzed using HPLC. One activity
Pedersen, Christensen [7]. After the granulation had oc- unit (U) was defined as 1 µmol incorporated myristic
curred, the process was terminated and the granules acid/min at the above-defined conditions.
were dried in a Glatt Fluid Bed System (Glatt, Weimar,
Germany), until they contained 2–5% water. 2.5 Interesterification assay
Substrate is comprised of fully hydrogenated soybean oil
2.2 Characterization of the dry granulates
and soybean oil (27:73 w/w); enzyme dosage in assay: 10
Typical particles sizes were 300-1000 µm, with a mean wt-%; temperature: 70 °C, no co-solvent. The content of
particle diameter in the range of 500-600 µm. The surface tristearin (a major component in fully hydrogenated soy-
area of the granules obtained, was in the range of 40- bean oil) is determined by HPLC and the initial enzyme
60 m2/g. The lipase content of the granules was – based activity is expressed as 1 IUN (Interesterification Unit No-
on the amount of lipase activity units applied during the vo) is equivalent to 0.01% of tristearin converted per
granulation – calculated to 20 kLU/g. The surface area minute.
(for particle fraction 425-710 µm) was determined by N2-
adsorption (BET-isotherm) on a Micromeritics Gemini in- 2.6 Synthesis of isopropyl myristate – batch
strument (Gemini Instruments, Dunstable, UK). The parti- operation
cle size distribution was determined by laser diffraction
(Low angle light scattering, Malvern Instruments, Myristic acid (4.75 g, 20.8 mmol) was dissolved in iso-
Malvern, UK). For comparison: Novozym 435: particle di- propanol (5.01 g, 83.3 mmol) using a water shaker bath at
ameter: 300-900 µm; surface area: 80 m2/g. 40 °C (150 rpm). Immobilized Candida antarctica lipase B
(1.00 g; lipase content 73 kLU/g granules) was added to
the reaction mixture. Sampling was conducted after 0, 15,
2.3 Assay for free lipase activity – LU
30, 60, 120, and 180 min for NMR analysis.
Tributyrin was hydrolyzed under standard conditions at
30 °C, pH 7.0, and the activity was determined from the 2.7 Synthesis of isopropyl myristate – fixed
alkali consumption using a pH-stat (Radiometer). The ac- bed operation
tivity was given as LU (lipase unit), where 1 LU corre-
sponds to the amount of lipase that liberates 1 mmol titra- Granulated Candida antarctica lipase B (6.05 g, lipase
ble butyric acid per minute. 1 kLU = 1000 LU. Specific ac- content 73 kLU/g granules) was mixed with isopropanol in
tivity of Candida antarctica lipase B and Thermomyces vacuo to remove air from the granules and packed into a
lanuginosus lipase is 500 LU/mg and 5000 LU/mg en- jacketed BioRad (glass column (ID = 1.00 cm, BioRad,
zyme protein, respectively. Hercules, USA). Novozym 435 (2.13 g) was packed using
the same procedure and column. The temperatures of the
water jackets were set to 40 °C. Myristic acid was dis-
2.4 Acidolysis of trilaurin with myristic acid solved in isopropanol at a concentration of 29.69 wt-%.
The acidolysis activity of the immobilized lipase was de-
termined by the incorporation of myristic acid into trilaurin. The substrate was pumped up at different flow rates using
a peristaltic pump. The degree of myristic acid conversion
Trilaurin (2.00 g; 3.04 mmol) and myristic acid (5.72 g; was followed by fatty acid titration. In order to compare
24.5 mmol) were dissolved in 200 ml n heptane in a the two columns on equal enzyme weight basis, the con-
sealed flask. Immobilized lipase (50 mg) was weighed in version vs. F/w was plotted assuming first order reaction
320 Christensen et al. Eur. J. Lipid Sci. Technol. 105 (2003) 318–321

kinetics: y = K (1-exp(-k[w/F])), where (F = mass flow [g

substrate/h]; w = weight of enzyme in column [g])

3 Results

Granulation technology has been used to produce immo-

bilized Thermomyces lanuginosus lipase with different
base materials such as silica types and cellulose as seen
in Fig. 1. The hydrophobicity could be altered by incorpo-
rating methylated silica (Aerosil R 974), which increased
the relative activity in acidolysis reactions. Thus, relative
to the commercially available immobilized Rhizomucor
miehei lipase (Lipozyme RM IM, Novozymes, Bagsvaerd,
Denmark), the new silica lipase granules displayed com- Fig. 2. Interesterification activity of immobilized Ther-
parable or higher activities. Similar to chemical heteroge- momyces lanuginosus lipase (Lipozyme TL IM Batch
LA201016) in different sieve fraction. Number above each
bar indicates the wt-% of the specific sieve fraction of the
total batch.

neous catalysts, the physical characteristics (such as sur-

face area, pore volume and pore diameter) of the new sil-
ica lipase granules are of great importance as indicated in
Tab. 1. The influence of particle size on the observed en-
zyme activity is shown in Fig. 2, which displays the activi-
ty of each particle size fractions from a large-scale pro-
duction batch in an interesterification assay. Evidently, en-
zyme performance in terms of initial activity vs. physical
properties can be optimized.

Candida antarctica lipase B was immobilized using simi-

lar procedures and used in synthesis of iso-propylmyris-
tate (a common emollient in skin care formulations). Fig.
3 shows the time course of the batch operation experi-
Fig. 1. Activity of immobilized Thermomyces lanuginosus ment as compared to the commercially available immobi-
lipase using various silica. Particle sizes 425-710 µm; 162 lized Candida antarctica lipase B (Novozym 435). The
kLU/g dry material in granulation. Acidolysis assay: Tri- performance in fixed bed reactor was investigated and
laurin and myristic acid. compared to the performance of Novozym 435, see

Tab. 1. Characterization of granules with different granulation components. 1)BET-isotherm: Brunauer-Emmet-Teller-

isotherm 2) BHJ-isotherm: Barret-Joyner-Halenda-isotherm.
Granulate Surface area Pore volume Pore diameter Catalytic Lipase Components
1)BET [m2/g] 2) BHJ [ml/g] BHJ (Å) activity added
(17 – 3000 Å) Average [U/g] [KLU/g]
#1 55.70 0.4560 317 35 1062 Aerosil 200
Cellulose
#2 3.30 0.0069 128 16 519 Clarcel,
Cellulose, Glucidex
#3 3.09 0.0066 109 15 562 Clarcel, Microtalc,
Glucidex
#4 103 0.7550 280 51 162 Aerosil 200
Glucidex
Eur. J. Lipid Sci. Technol. 105 (2003) 318–321 Industrial lipase immobilization 321

Lipozyme TL IM and has been reported in several refer-

ences, Zhang et al. [8], Torres et al. [9] and Kim et al. [10],
and is now used in large scale modification of fats (com-
munication from Karlshamns AB [11]).

The silica-granulated lipases will be suitable in many non-

aqueous applications. However, in aqueous processes,
the granules will disintegrate, and other immobilization
methodologies are needed. For the application of silica
granulated Candida antarctica lipase B in ester produc-
tion, further process development is, therefore, neces-
sary, because water is produced during this process and
Fig. 3. Synthesis of iso-propylmyristate. Time course in needs to be effectively removed during the process to en-
batch operation. sure stability of the catalyst.

References
[1] M. T. Reetz: Entrapment of biocatalysts in hydrophobic sol-
gel materials for use in organic chemistry. Adv. Mater. 9, 12
(1997) 943-954.

[2] A. Hsu, T. A. Foglia, S. Shen: Immobilization of

Pseudomonas cepacia lipase in phyllosilicate sol-gel ma-
trix: Effectiveness as a biocatalyst. Biotech. App. Biochem.
31 (2000) 179-183.

[3] P. Lopez-Serrano, L. Cao, F. van Rantwijk, R. A. Sheldon:

Cross-linked enzyme aggregates with enhanced activity:
Application to lipases. Biotech. Lett. 24 (2002) 1379-1383.

[4] S. Pedersen, A. M. Larsen, P. Aasmul: United States Patent

5 776 741 (1998).
Fig. 4. Synthesis of iso-propylmyristate in fixed bed oper- [5] P. E. Sonnet, G. P. McNeill, W. Jun: Lipase of Geotrichum
ation. Conversion of myristic acid at different flow rates. candidum immobilized on silica gel. J. Am. Chem. Soc. 71
(1994) 1421-1423.

[6] R. A. Wisdom, P. Dunnill, M. D. Lilly: Enzymatic interesteri-

fication of fats: The effect of non-lipase material on immobi-
Fig. 4. The conversion of myristic acid was determined at lized enzyme activity. Enzyme Microb. Technol. 7 (1985)
different flow rates. Compared to the activity of Novozym 567-572.
435 (normalized to 100%), the granulated Candida
[7] S. Pedersen, M. W. Christensen: Immobilized Biocatalysts.
antarctica lipase B exhibited 33% activity in packed bed In: Applied Biocatalysis. Eds. A. J. J. Straathof, P. Adler-
operation, based on equal enzyme weight basis. creutz, Harwood Academic Publishers, Amsterdam (The
Netherlands) 2000, pp. 213-228.
4 Discussion [8] H. Zhang, X. Xu, J. Nilsson, H. L. Mu, J. Adler-Nissen, C. E.
Hoy: Production of margarine fats by enzymatic interesteri-
The industrial usage of immobilized lipases requires dif- fication with silica-granulated Thermomyces lanuginose li-
ferent qualities and characteristics of the biocatalyst de- pase in a large-scale study. J. Am. Chem. Soc. 78 (2001)
pending on the specific application. Therefore, a contin- 57-64.
ued effort within immobilization technology is necessary [9] C. F. Torres, F. Munir, R. M. Blanco, C. Otero, C. G. Hill:
to provide solutions for each application. Catalytic transesterification of corn oil and tristearin using
immobilized lipases from Thermomyces lanuginosa. J. Am.
The immobilized particles obtained by the silica granula- Chem. Soc. 79 (2002) 775-781.
tion process presented in this paper are suitable for both [10] I. H. Kim, H. Kim, K. T. Lee, S. H. Chung, S. N. Ko: Lipase-
batch and fixed bed operation, the latter being very im- catalyzed acidolysis of perila oil with caprylic acid to pro-
portant for industrial application and the described immo- duce structured lipids. J. Am. Chem. Soc. 79 (2002) 363-
bilization technology is a means to provide cost-effective 367.
immobilized biocatalysts. Silica granulated Thermomyces [11] Karlshamns International Oils & Fats Magazine. June 2002.
lanuginosus lipase, which is commercially available as Karlshamns AB (Sweden).