Biochemical Engineering Journal 4 (2000) 207215

Lipase immobilisation: an equilibrium study of lipases immobilised on

hydrophobic and hydrophilic/hydrophobic supports
B. Al-Duri , Y.P. Yong
School of Chemical Engineering, University of Birmingham, Edgbaston, Birmingham, B1S 2TT, UK
Received 30 April 1999; accepted 8 September 1999

Abstract
This work investigates the enzyme-support equilibrium behaviour in immobilised lipase biocatalysts. Equilibrium data determines the
maximum enzyme up-take by unit weight of support. Four lipases were immobilised on two polymeric supports, respectively. They were
Lipase PS from Pseudomonas, Lipolase 100L from Humicola, SP871 from Rhizomucor miehel and QL from Alcaligenes. The supports were
Accurel EP100 (a polypropylene material) and 45SAA (a polypropylene/silica composite). Experimentally, equilibrium was expressed in
terms of lipase loading (LU/g support) versus residual lipase concentration (LU/dm3 ). Activity, efficiency and operational stability of the
immobilised lipases were assayed by solvent-free esterification of oleic acid and octanol.
Equilibrium data were modelled by the Langmuir, Freundlich and RedlichPeterson formulae. It was found that Lipolase 100L/Accurel,
PS/45SAA and SP871/45SAA systems conformed to the Langmuir behaviour, while Lipase PS/Accurel and SP871/Accurel systems
followed the Freundlich behaviour and Lipolase 100L/45SAA, QL/45SAA and QL/Accurel EP100 resembled RedlichPeterson behaviour.
Whereas immobilisation on Accurel EP100 resulted in classical equilibrium isotherms with all four lipases, immobilisation on support
45SAA resulted in two-plateau equilibrium curves which included a step change in the isotherm for all lipases studied, except for SP871.
Quantitatively, for 1 g lipase, Accurel and 45SAA had a maximum capacity of 140 and 260 kLU for PS, 112 and 550 kLU for Lipolase
100L, 320 and 800 kLU for SP871 and 18 and 29 kLU for QL, respectively. 2000 Elsevier Science S.A. All rights reserved.
Keywords: Lipase immobilisation; Equilibrium; Hydrophobic; Hydrophilic

1. Introduction
Structured triglycerides (ST) are developed by interesterification of long chain fats (or oils) with medium chain
triglycerides, to achieve a specific fatty acid profile. This resultant triglycerides have unique chemical, physical or physiological properties that are not observed by simply blending
mixtures of the starting fats and oils [1,2]. Other specialists
esters and fatty acids are produced by parallel modification
reactions all following the biochemical route.
The use of immobilised lipase is generally preferred due
to its reusability [3,4], its operational flexibility [5] and ease
of product recovery from the enzyme. On the other hand, immobilised lipase has its mass transfer limitations and therefore an insight into the immobilisation process and iminobilised lipase behaviour is required [5].
Lipase immobilisation was carried out by covalent attachment, entrapment [3,5], encapsulation and adsorption to
hydrophobic or hydrophilic surfaces. Among these meth

Corresponding author. Fax: +44-121-414-5324.

ods, adsorption being a simple and economical method, was

found to be most suitable for large scale lipase immobilisation [6]. Current literature, however, suffers from lack of
a systematic approach to lipase immobilisation. Selection
of a suitable support must be preceded by characterisation
studies that yield fundamental information which are used
as guidelines for other lipase/support systems. Modelling of
lipase adsorption equilibrium provides a more reliable approach than the random methods which would provide information merely on the system investigated.
The present work investigates the adsorption of four lipases from different sources, all immobilised on two supports which are hydrophobic and hydrophobic/hydrophilic,
respectively. Analytically, lipase/support systems were modelled by three adsorption isotherms namely the Langmuir,
Freundlich and RedlichPeterson. Furthermore, activity of
the immobilised lipases was assayed at various lipase loading values by means of solvent free synthesis of octyloleate.
Also, lipase stability (operational longevity) was tested by
a series of successive syntheses using the same sample of
immobilised lipase until activity was reduced to 60% of its

1369-703X/00/$ see front matter 2000 Elsevier Science S.A. All rights reserved.
PII: S 1 3 6 9 - 7 0 3 X ( 9 9 ) 0 0 0 5 0 - 9

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B. Al-Duri, Y.P. Yong / Biochemical Engineering Journal 4 (2000) 207215

Table 1
Lipases under investigation in the present study
Lipase

Source

Suppliers

Activity (LU/g)

Lipase PS
Lipolase 100L
Lipase SP871
Lipase QL

Pseudomonas
Humicola sp.
Rhizomucor miehei
Alcaligenes sp.

Amano Europe (Milton Keynes, England)

Novo-Nordisk (Copenhagen, Denmark)
Novo-Nordisk (Copenhagen, Denmark)
Meito-Sangyo (Japan)

30, 000
100000
65600
50800

original value. The stability tests were not continued beyond

this point due to the limiting time factor.

high range. The Langinuir isotherm reflects irreversible adsorption, where permanent bonds are formed between the
enzyme and support surface.

2. Background theory

2.1.2. The Freundlich isotherm

It describes equilibrium on energetically heterogeneous
surfaces with non identical adsorption sites and exponential
distribution of energy of adsorption on the support surface.
Mathematically it is characterised by the heterogeneity factor 1/n as shown below:

Although most commercial lipase preparations contain

various proteins, lipases were reported to show considerable
affinity towards hydrophobic supports and can be preferentially or selectively adsorbed onto hydrophobic surfaces
[7,8]. Therefore, when commercial lipase is adsorbed on hydrophobic support, the loading is expected to be mainly lipase. Other proteins and impurities will remain in solution.
2.1. The adsorption isotherm
Adsorption equilibrium is represented by an isotherm representing the enzyme loading (i.e. amount adsorbed) Qe
(mg/g) plotted against the concentration of the lipase residual in solution Ce (mg/din3 ) after immobilisation. Equilibrium analysis evaluates the affinity (or capacity) of the support particles for the lipase. In addition, the shape of the
isotherm provides a qualitative description of the nature of
the adsorbent surface (homogeneous or heterogeneous) [9].
There exist various adsorption isotherms according to the
basic assumptions, the nature of adsorbent surface and the
shape of isotherm. However, this is all well cited in literature [10] and therefore, the isotherms used in this work will
be briefly stated below:
2.1.1. The Langmuir isotherm
Since lipase molecules attach via multipoint-contact to the
adsorbent surface, high affinity Langmuir-type isotherm is
expected whereby the lipase molecules form a homogeneous
monolayer on the surface of the support (adsorbent). The
Langmuir isotherm is presented by
Qe =

KL Ce
1 + aL Ce

(1)

where KL and aL are the Langmuir constants. KL /aL represents the monolayer capacity of adsorbent.
This isotherm is based on the assumption of a structurally
homogeneous adsorbent where all adsorption sites are identical and energetically equivalent, hence they carry equal
numbers of molecules. This gives the Langinuir isotherm its
typical box shape characterised by a sharp and steep slope
at the lower concentration range, and a flat plateau at the

1/n

Qe = KF Ce

(2)

where KF and 1/n are the Freundlich constants; the larger

aF the higher the capacity and the smaller 1/n the stronger
the heterogeneity.
2.1.3. The RedlichPeterson isotherm
This isotherm incorporates the features of Langmuir and
Freundlich isotherms is the RedlichPeterson isotherm. It is
represented by
Qe =

KJ Ce

1 + bJ Ce

(3)

where KJ , bJ and are the RedlichPeterson constants. Generally, isotherm constants can be evaluated by linearisation
of isotherms.

3. Experimental section
3.1. Materials
3.1.1. Lipases
Four lipases (listed in Table 1) were investigated in this
study. The enzyme loading of the lipases are 30,000 LU/g;
50,800 LU/g; 65,600 LU/g and 100,000 LU/g for Lipase PS,
QL, SP871 and Lipolase 100L, respectively. These lipases
are commercially available and known for their high activities in catalysing esterification (ester synthesis) reactions
and were therefore selected for the present work.
3.1.2. Supports
Two supports, namely Accurel EP100 (supplied by Akzo
Nobel Faser, Obemburg, Germany) and 45SAA (supplied
by QDM Laboratories, Lisburn, Northern Ireland) were selected. Accurel is a polypropylene based hydrophobic support made in granules. 45SAA is a hydrophobic/hydrophilic

B. Al-Duri, Y.P. Yong / Biochemical Engineering Journal 4 (2000) 207215

Table 2
Physical properties of supports under investigation
Physical properties

Support
Accurel EP100 45SAA

Composition
Nature
Surface area (m2 /g)
Particle density (g/cm3 )
Particle voidage
Particle size (m)

polypropylene
hydrophobic
45
0.902
0.75
4001000

polypropylene/silica composite
hydrophobic/hydrophilic
70
0.708
0.70
5001200

support based on polypropylene, cross-linked with silica.

Physical properties of both are listed in Table 2.
3.1.3. Reagents
For preparation of lipase solutions, 0.01 M glycine buffer
(pH 10.5) was used for Lipolase 100L and SP871, while
0.1 M phosphate buffer (pH 7) was selected for lipases PS
and QL. Tributyrin (>98%) (purchased from Fluka) was required for free lipase assay, in addition to NaOH (0.025 M
or 0.01 M) analar grade reagent (supplied by Fisons). Ingredients for the emulsification reagent were glycerol, NaCl,
KH2 PO4 and gum Arabic (all supplied by Fisons). For assay of activity of immobilised lipase, oleic acid (90%) and
octan-1-ol (99%) were purchased from Aldrich and Sigma,
respectively. Hexane (95%, for biocatalyst washing) was
supplied by Fisons.
3.2. Methods
3.2.1. Assay of initial (free) lipase activity
Prior to immobilisation, the activity of free lipase solutions was assayed by the tributyrin hydrolysis assay method,
a standard Novo method [11]. Accordingly, a sample of
lipase solution was prepared to hydrolyse 40 ml of the
tributyrin emulsion. The tributyrin emulsion was prepared
by homogenising a mixture of 12 ml tributyrin, 188 ml
de-ionised water and 40 ml emulsification reagent 1 in an
Ultra-Turrax 25 homogeniser 25 at 21,500 rpm for 20 s. The
lipase-tributyrin emulsion mixture was stirred vigorously at
40 C, left to cool to room temperature (20 C), then diluted
up to 4000 ml using de-ionised water. During lipase assay,
as hydrolysis proceeded, butyric acid was being released
and continuously neutralised with a 0.025 M solution of
NaOH. This was done automatically using an Autotitrator
(Mettler-Toledo DL25). The Free lipase activity was defined in terms of lipase units where one lipase unit (LU) is
defined as the amount of lipase which is required to liberate
1 m of butyric acid under these conditions.

1 Prepared by dissolving sodium chloride (17.9 g), potassium dihydrogen

phosphate (0.41 g) in 400 ml deiomsed water. Glycerol (450 ml) was then
added with gum arabic (6.0 g) under gentle heating (50 C) and vigorous
mixing, and the mixture was made up to 1000 ml with deionised water.

209

3.2.2. Lipase immobilisation

For each lipase, 20 solutions of consecutively increasing
concentrations were prepared using buffer solutions (Section 3.1). In preparation for immobilisation, the support was
pre-wet with 50 ml ethanol for 30 min and pre-washed with
50% ethanolwater, to exclude the air within the support particles. The mixture was then washed with 50 ml de-ionised
water, decanted then washed with 100 ml fresh de-ionised
water (to remove any trace of unbound lipase) and filtered
ready for immobilisation.
Upon immobilisation, 1 g of prepared support was brought
into contact with 50 ml of lipase solution (of predetermined
initial concentration-see Table 3) in a sealed vessel, and
placed in an orbital shaker at 250 rpm and 30 C (optimum
temperature for all the lipases used in this study) for 48 h.
Then the lipase laden support particles were filtered from
solution, dried and stored at 4 C, ready for the assay of
immobilised lipase activity. The amount of lipase adsorbed
was defined in terms of lipase unit (LU, as defined in Section 3.2.1) per unit weight of support and was determined
by subtracting the residual free lipase activity (after immobilisation) from the original free lipase activity (before immobilisation).
3.2.3. Assay of immobilised lipase activity
The activity of the immobilised lipase was measured by
the rate of solvent-free esterification of oleic acid and octanol to produce octyloleate. The solvent-free esterification
reaction was chosen for this assay because the ultimate aim
of this study is to examine the feasibility of such reactions.
Before the reaction started, equimolar amounts of oleic
acid and octanol (34 and 15.7 g, respectively) were added to
0.6 g of de-ionised water and homogenised in a sealed vessel by an immersible magnetic stirrer at 330 rpm, in a 40 C
water bath. Then, 12 g of immobilised lipase were added
to the substrate mixture and the reaction was started. 0.5 ml
samples of the reaction mixture were withdrawn at 10 min
intervals, and treated with 5 ml of 1 : 1 acetone/ethanol mixture to stop further reaction and extract the oleic acid into the
aqueous phase. Each sample was then analysed titrimetrically with 0.1 M NaOH solution to determine the amount of
oleic acid remaining and hence the amount of ester formed
(stoichiometrically, the amount of oleic acid reacted would
be equivalent to that of ester produced). Activity of immobilised lipase was expressed as the number of micromoles
of ester produced per minute per milligram of biocatalyst,
while efficiency was defined as the lipase activity divided by
lipase loading.
3.2.4. Stability tests
Stability, or operational longevity of the biocatalysts was
carried out by conducting a series of successive esterifications (activity assays) using the same sample until lipase activity was reduced by 60% of its original value.
After each reaction the biocatalyst was washed with hexane

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B. Al-Duri, Y.P. Yong / Biochemical Engineering Journal 4 (2000) 207215

Table 3
r2 values for the linearisation of the equilibrium models
r2 value

Immobilised lipase system

Lipolase 100L/Accurel EP100: 150 LU/ml < Co < 3000 LU/ml Lipolase 100L/45SAA
150 LU/ml < Co < 1300 LU/ml
1300 LU/ml < Co < 11500 LU/ml PS/Accurel EPlOG:
150 LU/ml < Co < 3000 LU/ml PS/45SAA:
150 LU/ml < Co < 400 LU/ml
400 LU/ml < Co < 8000 LU/ml SP871/Accurel EP100
150 LU/ml < Co < 3000 LU/ml SP871/45SAA
200 LU/ml < Co < 20000 LU/ml QL/Accurel EP100
50 LU/ml < Co < 1000 LU/ml QL/45SAA
100 LU/ml < Co < 1520 LU/ml
1520 LU/ml < Co < 2500 LU/ml

and dried by air (to remove remaining trace of reaction

mixture).
4. Results and discussion
4.1. Adsorption equilibrium
Figs. 18 show the equilibrium isotherms of the eight
systems investigated. In principle, adsorption isotherms de-

Langmuir

Freundlich

RedlichPeterson

0.987
0.882
0.818
0.929
0.999
0.970
0.822
0.946
0.912
0.926
0.368

0.921
0.937
0.820
0.973
0.997
0.971
0.986
0.945
0.904
0.892
0.316

0.986
0.972
0.849
0.97
0.937
0.391
0.980
0.900
0.957
0.982
0.894

scribe the support affinity (or capacity) for lipase, while the
isotherm shape reflects the lipase distribution on the support surface. Figs. 1, 3, 5 and 7 (of lipases on Accurel) gave
conventional equilibrium curves that were described by the
Langmuir, Freundlich or RedlichPeterson models, respectively. On the other hand, Figs. 2, 4, 6 and 8 showed that
isotherms of 45SAA systems (except for SP871) contained
a step or two plateaux, over the range of lipase concentra-

Fig. 4. Equilibrium isotherm of Lipase PS immobilised on 45SAA.

Fig. 1. Equilibrium isotherm of Lipolase 100L immobilised on Accurel
EP100.

Fig. 2. Equilibrium isotherm of Lipolase 100L immobilised on 45SAA.

Fig. 5. Equilibrium isotherm of Lipase SP871 immobilised on Accurel

EP100.

Fig. 3. Equilibrium isotherm of Lipase PS immobilised on Accurel EP100.

Fig. 6. Equilibrium isotherm of Lipase SP871 immobilised on 45SAA.

B. Al-Duri, Y.P. Yong / Biochemical Engineering Journal 4 (2000) 207215

Fig. 7. Equilibrium isotherm of Lipase QL immobilised on Accurel EP100.

tions investigated. This phenomenon would be linked to the

hydrophobic-hydrophilic structure of 45SAA.
Norde and Lyklema [12] have previously reported the existence of steps in the adsorption isotherms of serum albumin and -globulin. They attributed the findings to the
occurrence of two different modes of adsorption, one operating at low, the other at higher protein concentrations. They
suggested that conformational flattening of the molecule
could occur at low protein concentration and low surface
coverage. However, at higher concentration and surface coverage, the protein would adsorbed in a more compact, apparently native configuration.In literature several researchers
have explained the occurrence of a step change in adsorption isotherms in terms of the structural orientation of the
adsorbed species [1214] as well as the manner of which
the adsorption occurred [15]. However, none had related the
support structure and composition to protein adsorption. The
present work suggested that, in addition to the explanations
offered, the isotherm shape is also related to the structure of
45SAA surface, which is a silica-polypropylene composite
with a hydrophobic/hydrophilic structure. While the lipases
bound to the hydrophobic surface (polypropylene) in a fashion that enhanced their unfolding and therefore their activities, the hydrophilic component would have a different effect
on the lipases. It would encouraged the formation of lipase
clusters on the surface, analogous to the two-dimensional
crystal proposed by Fair and Jamieson [15], thus resulting in
an apparent increase in lipase loading. In fact, this would
be an inactive complex that would not enhance, if not inhibit, the biocatalytic activity of the system. Such a complex
would be formed due to the unfavourable orientation of lipase on the support surface on the one hand, and the abundance of water in the hydrophilic environment. Both factors might well deactivate the lipase. Activity and efficiency
studies confirmed this hypothesis (see Section 4.3).

Fig. 8. Equilibrium isotherm of Lipase QL immobilised on 45SAA.

211

Fig. 9. Lipolase 100L/Accurel EP100: linearisation of the equilibrium

data for Langmuir analysis (150 LU/ml < Co < 3000 LU/ml).

Fig. 10. Lipolase 100L/45SAA: linearisation of the equilibrium data for

RedlichPeterson analysis (150 LU/ml < Co < 1300 LU/ml).

4.2. Modelling of equilibrium

Figs. 914 showed the linearisation of Eqs. (1)(3) applied
to the present experimental data.
Lipolase 100L/Accurel system (Fig. 9) was best described
by the Langmuir model which implied the formation of lipase monolayer on the support surface. Thermodynamically,
it indicated an energetically homogeneous surface where all
sites were identical. Similar results were obtained for Lipase
SP871/45SAA system.
PS/Accurel (Fig. 12), SP871/Accurel systems showed typical characteristics of the Freundlich isotherm. This implied
energetically heterogeneous support surface and nonequiva-

Fig. 11. Lipolase 100L/45SAA linearisation of the equilibrium data for

RedlichPeterson analysis (1300 LU/ml < Co < 3000 LU/ml).

Fig. 12. PS/Accurel EP100: linearisation of the equilibrium data for

Freundlich analysis (150 LU/ml < Co < 3000 LU/ml).

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B. Al-Duri, Y.P. Yong / Biochemical Engineering Journal 4 (2000) 207215

Fig. 13. PS/45SAA: linearisation of the equilibrium data for Freundlich

analysis (150 LU/ml < Co < 400 LU/ml).

Fig. 15. Lipolase 100L immobilised on Accurel EP100: Effects of enzyme

loading on the activity and the efficiency of the biocatalyst.

4.3. Activity and efficiency studies

lent sorption sites. Hence, molecular clusters would form
at the sorption sites of lower energy, without all sites being necessarily occupied. With PS/45SAA system (Figs. 13
and 14) two-plateau isotherm was formed over Ce ranges of
150800 LU/dm3 and 8008000 LU/dm3 , respectively. Each
range was successfully modelled by the Freundlich isotherm.
QL/Accurel system was described by the RedlichPeterson
formula. Both Lipolase 100L/45SAA (Figs. 10 and 11)
and QL/45SAA yielded the two-plateau isotherm. For
each system, both curves successfully conformed to the
RedlichPeterson behaviour.
While biological adsorption systems were mostly described by the Langmuir isotherm [3], a deeper insight
showed that the structural properties of the support contribute to the lipase distribution on the support surface.
Therefore, 45SAA (except with SP871) gave a two-plateau
isotherm, while Accurel yielded the conventional adsorption equilibrium. Both supports showed heterogeneous
behaviour with most lipases, and a high capacity.
The isotherm constants were evaluated by linearising
the Langmuir, Freundlich and RedlichPeterson equations
(Table 4). The ratio KL /aL represents the monolayer capacity of the support and 1/n represents the heterogeneity factor,
the lower the value the higher the heterogeneity effects.
In general, support 45SAA showed higher capacity for
lipase adsorption compared to Accurel EP100 (Table 5).
However, this would not necessarily imply higher activity of
the biocatalyst. Activity and stability investigations on the
immobilised lipase would therefore be required.

Fig. 14. PS/45SAA: linearisation of the equilibrium

Freundlich
analysis
(400 LU/ml < Co < 8000 LU/ml)
(1520 LU/ml < Co < 2500 LU/ml).

data for
analysis

Immobilised lipase activity was defined in terms of the

pinol of octyloleate produced per unit time per unit mass of
immobilised lipase (mole/(min mg catalyst). Furthermore,
lipase efficiency was given by the ratio of lipase activity to
lipase loading (mole/(kLU min)).It is generally acknowledged that water plays an important role in lipase activity. In
this study, a constant initial water concentration of 1% w/w
was maintained for all the experiments to provide hydration
water to the dried immobilised lipases. Since the activity
was a measurement of initial reaction rate, it was therefore
reasonable to assume that
1. the effects of water was the same in all cases because
the initial water concentration was kept constant and
2. the amount of water produced in the esterification reaction would not exert significant effects on the initial
reaction rate since the reaction equilibrium has yet to
be reached.
Figs. 1522 show results of lipase activity and efficiency versus lipase loading for the eight systems studied.

Fig. 16. Lipolase 100L immobilised on 45SAA: Effects of enzyme loading

on the activity and the efficiency of the biocatalyst.

Fig. 17. Lipase PS immobilised on Accurel EP100: Effects of enzyme

loading on the activity and the efficiency of the biocatalyst.

B. Al-Duri, Y.P. Yong / Biochemical Engineering Journal 4 (2000) 207215

213

Table 4
Isotherm constants for the immobilisation systems studied. KL /aL in LU/g
Lipase

Co range (LU/ml)

Langmuir constants
KL /aL

(104 )

Freundlich constants

RedlichPeterson constants
(101 )

KF (10)

1/n

4.364
12.82
1627.7
2560.4
2981.3
350.76
1809.1

9.080
8.790
3.960
4.740
4.36
2.810
1900

KJ

b (103 )

(101 )

2.157
3.980
232.6
27.10
30.03
0.319
0081

49400
31000
14300
1100
1000
9000
4.000

9.200
12.20
60.40
52.60
56.40
71.90
81.00

Support: 45SAA
PS
Lipolase 100L
SP871
QL

Lipase

150400
4008000
1501300
130011500
20020000
1001520
15202500

14.28
166.5
3.3318
100.0
1001
1.431
3.347

Co range (LU/ml)

Langmuir constants
KL /aL

Support: Accurel EP100

PS
1503000
Lipolase 100L
1503000
SP871
1503000
QL
501000

(104 )

1.000
10.00
20.01
1.427

Freundlich constants

RedlichPeterson constants
(101 )

KF (10)

1/n

308.7
362.6
170.0
9557

3.052
2.789
2.135
3940

KJ

b (103 )

(101 )

75.19
61.73
188.7
0498

24.00
17.00
11.00
5.000

6.950
7.210
7.870
6.060

Table 5
Maximum capacities of the supports towards the lipases Lipolase 100L, PS, SP871 and QL
Lipase

Accurel EP100
45SAA

Support
Lipase PS (kLU/g)

Lipolase 100L (kLU/g)

SP871 (kLU/g)

Lipase QL (kLU/g)

140
260

112
550

320
800

18
29

In all systems, lipase activity increased with increasing lipase loading to a certain value Amax , at loading Qm , then
stabilised or declined. Consequently, lipase efficiency decreased when lipase loading exceeded Qm . It suggested
that in order to operate at maximum lipase efficiency,

Fig. 18. Lipase PS inimobilised on 45SAA: Effects of enzyme loading

on the activity and the efficiency of the biocatalyst.

Fig. 19. Lipase SP871 immobilised on Accurel EP100: Effects of enzyme

loading on the activity and the efficiency of the biocatalyst.

loading need not exceed Qm . Corresponding to the maximum efficiency, an optimum loading Qopt and optimum
activity Aopt would be selected (Table 6). The identification of Qopt and Aopt has important economic and operational implications, in terms of the quantity of lipase

Fig. 20. Lipase SP871 immobilised on 45SAA: Effects of enzyme loading

on the activity and the efficiency of the biocatalyst.

Fig. 21. Lipase QL immobilised on Accurel EP100: Effects of enzyme

loading on the activity and the efficiency of the biocatalyst.

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B. Al-Duri, Y.P. Yong / Biochemical Engineering Journal 4 (2000) 207215

Fig. 23. Stability of lipases immobilised on Accurel EP100.

Fig. 22. Lipase QL immobilised on 45SAA: Effects of enzyme loading
on the activity and the efficiency of the biocatalyst.

purchased and the initial concentration of lipase solution

prepared.
For most systems, Qopt occurred at the lower lipase concentration range. For the two-plateau isotherms, Qopt occurred at the point of step change in Q where the second
layer would be formed. This suggested that multilayer lipase
formation did not enhance lipase activity (Section 4 and Figs.
16, 18, 20 and 22) and confirmed the earlier explanations
given on lipase cluster on support surfaces. The bi-modal
adsorption model proposed by Fair and Jamieson [15] would
be analogous to the hydrodynamic theory for a spherical
particle composed of a shell of small rigid close-packed
sub-units deposited on a large impermeable spherical core.
In this situation, only the lipase molecules on the surface
of the crystal (core) were active. As the initial lipase concentration increased, the increasing enzyme loading encouraged the growth of the crystal. This in turn caused a higher
proportion of the lipase molecules to be buried inside the
crystal, therefore rendering them inactive. Consequently, a
decrease in lipase efficiency was observed (Figs. 16, 18, 20
and 22).
Comparing the activity of each lipase on the two supports
revealed similar orders of magnitudes, however lipase efficiency varied. Lipase PS and QL showed much higher efficiency than Lipolase 100L and SP871. This implied that for
PS and QL less lipase would be required to achieve optimum
activity. Inevitably, this would have important economical
implications on the selection of lipases for industrial bioprocesses although more elaborate and detailed investigations
would be required prior to industrial scale-up.
Table 6
Activity and efficiency studies of the eight immobilised lipase systems.
Aopt is the optimum activity (mole/(min mg cat)), opt is the corresponding optimum efficiency (mole/(min kLU)) and Qopt is the lipase loading
(kLU/g)
Lipase Support
AccurelEP 100
Aopt
PS
L
SP871
QL

4
0.9
1.01
0.38

opt
88
8.35
7.2
38

Fig. 24. Stability of lipases immobilised on 45SAA.

4.4. Stability studies

To test the operational longevity, stability studies were
carried out by conducting a series of successive esterification
reactions using the same sample of immobilised lipase. The
biocatalysts retained their activity (at least 60%) for at least
six runs (except for SP871 immobilised on Accurel EP100)
over a period of three weeks (Table 7). The high stability
of the biocatalysts suggested that enzyme inactivation and
leaching were insignificant with these biocatalysts. Ideally,
the esterification reaction should be repeated until the biocatalysts reached their half-life i.e. 50% of their (respective)
original activities. However, in this instance the high stability of the biocatalysts would render any further tests beyond
60% of their original activities impractical with respect to
the time available.
Figs. 23 and 24 showed the profile of % activity retained.
The half-lives of Lipolase 100L, PS and SP871 immobilised
on Accurel were estimated to be 11, 10 and 3 h, respectively. Immobilising on 45SAA, the three lipases have estimated half-lives of 3, 5 and 3.5 h, respectively. From these
results it would seem that immobilisation on Accurel would
produce biocatalysts which have longer operational stability
compared to 45SAA. With QL immobilised on Accurel and
45SAA, the activity remained nearly 100% after 4 and 6 h
of reaction time, respectively.

45SAA
Qopt

Aopt

50
112
225
10

3.25
1.5
1.45
0.835

opt
58
7.0
7.2
55

Qopt
53.5
360
630
15

5. Conclusions
From the present study, the following points may be concluded:

B. Al-Duri, Y.P. Yong / Biochemical Engineering Journal 4 (2000) 207215

215

Table 7
Stability tests of the immobilised lipase systems
Support

Lipase
PS

Accurel EP100
45SAA

Lipolase 100L

SP871

QL

Run No.

% Activity retained

Run No.

% Activity retained

Run No.

% Activity retained

Run No.

% Activity retained

8
9

64
75

9
3

65
60

3
6

50
60

5
8

100
100

1. The equilibrium behaviour of the lipase/support in the

immobilisation systems would depend on the physical
and structural properties of the support, and the physical
and chemical properties of the lipase. Contrary to the
believe that the equilibrium behaviour of lipase adsorption would generally conform to the Langmuir model
[3], an insight analysis of the equilibrium behaviour
showed that the lipase distribution on the support surface could be best described by the Freundlich or even
the RedlichPeterson models.
2. Accurel EP100 and support 45SAA both showed considerable adsorption capacities for all the lipases investigated. In literature, however, compared to 45SAA immobilisation of lipases on Accurel had been more widely
reported [3].
3. Immobilisation with 45SAA produced equilibrium
isotherms which consisted of two plateaux. This phenomenon could be explained by the bi-modal adsorption
model proposed by Fair and Jamie son [15] and was
confirmed by the fact that the increased lipase loading
did not enhance the activities or the efficiencies of the
biocatalysts.
4. Lipolase 100L on Accurel EP100, lipases PS and
SP871 on 45SAA systems conformed to the Langmuir
equilibrium behaviour which implied monolayer adsorption. The Freundlich isotherm best described PS
and SP871 on Accurel EP100 systems. This suggested
heterogeneous adsorption and formation of multilayer
or clusters of lipase molecules. Lipolase 100L and lipase QL on 45SAA and QL on Accurel EP100 showed
RedlichPeterson behaviour which also implied multilayer or cluster formation.
5. Support 45SAA showed higher capacity for lipase adsorption, but it did not improve the lipase activity and
efficiency. This was related to the interactions inflicted
by its hydrophobic-hydrophilic nature.
6. All systems (except for SP871 immobilised on Accurel
EP100) maintained their activities for not less than 3-h
reaction time (or five successive batch of reaction).

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