U.S. patent application number 10/434958 was filed with the patent office on 2003-10-30 for process for immobilization of enzymes.
This patent application is currently assigned to Novozymes A/S. Invention is credited to Christensen, Morten Wurtz, Kirk, Ole, Pedersen, Christian.
Application Number | 20030203457 10/434958 |
Document ID | / |
Family ID | 27221327 |
Filed Date | 2003-10-30 |
United States Patent
Application |
20030203457 |
Kind Code |
A1 |
Christensen, Morten Wurtz ;
et al. |
October 30, 2003 |
Process for immobilization of enzymes
Abstract
A process for producing an immobilized enzyme preparation for
use in a mainly organic medium devoid of free water comprising
using a fluid bed.
Inventors: |
Christensen, Morten Wurtz;
(Lyngby, DK) ; Kirk, Ole; (Virum, DK) ;
Pedersen, Christian; (Roedovre, DK) |
Correspondence
Address: |
NOVOZYMES NORTH AMERICA, INC.
500 FIFTH AVENUE
SUITE 1600
NEW YORK
NY
10110
US
|
Assignee: |
Novozymes A/S
Bagsvaerd
DK
|
Family ID: |
27221327 |
Appl. No.: |
10/434958 |
Filed: |
May 9, 2003 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10434958 |
May 9, 2003 |
|
|
|
09649942 |
Aug 29, 2000 |
|
|
|
6582942 |
|
|
|
|
09649942 |
Aug 29, 2000 |
|
|
|
09210192 |
Dec 11, 1998 |
|
|
|
6156548 |
|
|
|
|
60074189 |
Feb 10, 1998 |
|
|
|
Current U.S.
Class: |
435/134 ;
435/177; 435/198 |
Current CPC
Class: |
C12N 11/00 20130101;
C12N 11/14 20130101; C12N 11/082 20200101 |
Class at
Publication: |
435/134 ;
435/198; 435/177 |
International
Class: |
C12P 007/64; C12N
011/02; C12N 009/20 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 23, 1997 |
DK |
1527/97 |
Claims
What is claimed is:
1. A process for producing an immobilized enzyme preparation for
use in a mainly organic medium devoid of free water, comprising: a)
fluidizing a particulate porous carrier in a fluid bed, b)
introducing an enzyme containing liquid medium by atomization into
the fluid bed, so as to fixate the enzyme on the carrier, and c)
removing volatile components of the liquid medium from the carrier
in the fluidized bed.
2. A process for producing an immobilized enzyme preparation for
use in a mainly organic medium devoid of free water, comprising: a)
contacting an enzyme containing liquid medium with a particulate
porous carrier having a substantially hydrophobic surface, so as to
adsorb the enzyme on the carrier, b) introducing a hygroscopic
substance, so as to suppress agglomeration of the carrier by
absorbing excess liquid, and c) removing volatile components of the
liquid medium and the hygroscopic substance from the resulting
product in a fluidized bed.
3. A process for producing an immobilized enzyme preparation for
use in a mainly organic medium devoid of free water, comprising: a)
introducing an enzyme containing liquid medium enzyme by
atomization onto a particulate porous carrier having a
substantially hydrophilic surface, so as to fixate the enzyme on
the carrier, wherein the liquid is introduced in an amount such
that substantially no agglomeration of the carrier occurs and b)
removing volatile components of the liquid medium from the
resulting product in a fluidized bed.
4. The process of claim 1, wherein the carrier comprises an
inorganic material having a substantially hydrophilic surface,
which is essentially insoluble in hydrophilic or hydrophobic
liquids or mixtures thereof.
5. The process of claim 4, wherein the carrier is selected from the
group consisting of silicas, zeolites, aluminas and kaolins.
6. The process of claim 1, wherein the carrier comprises an
inorganic material selected from the group consisting of silicas,
zeolites, aluminas and kaolins coated with organic moieties so as
to provide a substantially hydrophobic surface.
7. The process of claim 1, wherein the carrier comprises an organic
polymer resin.
8. The process of claim 7, wherein the resin is an adsorbent
resin.
9. The process of claim 8 wherein the adsorbent resin is a
polyacrylate, a polymethacrylate, polystyrene cross-linked with
divinylbenzene, polyurethane or polypropylene.
10. The process of claim 7 wherein the resin is an ion exchange
resin.
11. The process of claim 10, wherein the ion exchange resin is an
anion exchange resin.
12. The process of claim 11, wherein the ion exchange resin is a
weakly basic anion exchange resin.
13. The process of claim 1, wherein the carrier has an average pore
size of 10-500 nm.
14. The process of claim 1, wherein the carrier has a surface area
of 20-1000 m2/g.
15. The process of claim 1, wherein the carrier has a particle size
of 200-1000 .mu.m.
16. The process of claim 15, wherein the carrier has a particle
size of 400-700 .mu.m.
17. The process of claim 2, wherein the hygroscopic substance is
particulate having a particle size which is smaller than the
particle size of the carrier, and wherein the hygroscopic substance
can be removed in step (c) by inserting at the top of the fluidized
bed a filter having a pore size which will allow the hygroscopic
substance to pass through.
18. The process of claim 17, wherein the hygroscopic substance is a
silica material.
19. The process of claim 1, wherein said liquid medium is an
aqueous medium.
20. The process of claim 1, wherein the enzyme is a lipase.
21. The process of claim 20, wherein the lipase is derived from a
strain of the genus Humicola (also known as Thermomyces),
Pseudomonas, Candida, or Rhizomucor.
22. The process of claim 21, wherein the lipase is derived from the
species H. lanuginosa (also known as Thermomyces lanuginosa), C.
antarctica or R. miehei.
23. A process for enzymatic modification of an organic compound
comprising contacting in a reaction medium essentially devoid of
free water said organic compound with an immobilized enzyme
produced by the process of claim 1.
24. The process according to claim 23, wherein the modification is
a trans-esterification reaction comprising contacting a first
reactant which is a fatty acid ester, and a second reactant which
is another fatty acid ester, an alcohol or a free fatty acid with
an immobilized lipase produced by the process of claim 1.
25. The process of claim 24, wherein the first reactant is a
triglyceride.
26. The process of claim 24, wherein the second reactant is a fatty
acid ester, and the lipase positionally specific.
27. The process of claim 24, wherein the first and the second
reactants are different triglycerides or different mixtures of
triglycerides, and the lipase is positionally 1,3-specific.
28. The process of claim 24, wherein the reaction medium consists
essentially of triglycerides.
29. The process of claim 24, wherein the reaction medium comprises
an organic solvent.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Divisional of application Ser. No.
09/649,942 filed on Aug. 29, 2000, which is a Divisional of
application Ser. No. 09/210,192 filed on Dec. 11, 1998 (Now U.S.
Pat. No. 6,156,548), which claims priority under 35 U.S.C. 119 of
U.S. provisional application 60/074,189 filed Feb. 10, 1998 and
Danish application 1527/97 filed Dec. 23, 1997, which are hereby
incorporated by reference.
FIELD OF THE INVENTION
[0002] The invention relates to a process for producing an
immobilized enzyme preparation for use in a mainly organic medium
essentially devoid of free water, and use of the immobilized enzyme
preparation for organic synthesis.
BACKGROUND OF THE INVENTION
[0003] Immobilized enzymes are known to be used for organic
synthesis.
[0004] The most commonly immobilized enzymes are lipases used for
esterification reactions in mainly organic media essentially devoid
of free water.
[0005] EP 140542 B2 describes a process, wherein an enzyme
containing liquid is brought in contact with a weak anion exchange
resin carrier by dispersing the carrier in the liquid and mixing by
stirring with a magnetic stirrer, whereby the enzyme is immobilized
on the carrier. The immobilization is subsequently followed by
vacuum drying of the enzyme-carrier.
[0006] WO 95/22606 describes a process, wherein an enzyme
containing liquid is brought in contact with a porous silica
carrier by atomizing the liquid onto the carrier in a mixer,
subsequently followed by drying overnight at ambient
conditions.
[0007] In industrial immobilization processes described in prior
art, the carrier or support material is placed in a column shaped
adsorption vessel and an enzyme containing liquid is recirculated
until sufficient adsorption of the enzyme on the carrier has been
obtained. Following the adsorption step the column is emptied by
manually shoveling the enzyme-carrier product into trays. The
product is then dried by placing the trays under vacuum at room
temperature for a period of 14-16 hours.
[0008] WO 94/26883 describes a process for producing dust-free
enzyme granules by absorbing the enzyme on a porous material, said
material including NaCl, Soda, and silica, and optionally coating
the product with a protective outer layer. Generally immobilization
of enzymes should not be compared with granulation of enzymes as
granulation serves a completely different purpose, viz. to provide
a preferably non-dusting delivery material from which an enzyme may
be delivered to an aqueous solution by disintegration of the
granule and/or dissolution of the enzyme in the aqueous phase.
Enzyme immobilization concerns immobilizing an enzyme product on a
carrier on which the enzyme is fixed and yet functional and for
which the enzyme is not liberated to the solvent to which it is
applied.
[0009] Immobilization processes known to the art are limited in
capacity as they involve laborious and manual steps and require
heavy equipment investments (e.g. vacuum rooms), which, in turn,
means inflexible production and expensive products.
SUMMARY OF THE INVENTION
[0010] The present invention provides alternative processes for
industrial immobilization of enzymes, which significantly increases
capacity and reduces labor costs, by means of standard
multi-purpose process equipment.
[0011] Thus the invention provides processes for producing an
immobilized enzyme preparation for use in a mainly organic medium
essentially devoid of free water, which in a first aspect
comprises:
[0012] a) fluidizing a particulate porous carrier in a fluid
bed,
[0013] b) introducing an enzyme containing liquid medium by
atomization into the fluid bed, so as to adsorb the enzyme on the
carrier, and
[0014] c) removing volatile components of the liquid medium from
the carrier in the fluidized bed.
[0015] In a second aspect the process comprises:
[0016] a) contacting an enzyme containing liquid medium with a
particulate porous carrier having a substantially hydrophobic
surface, so as to adsorb the enzyme on the carrier,
[0017] b) introducing a particulate hygroscopic substance, so as to
suppress agglomeration of the carrier, and
[0018] c) removing volatile components of the liquid medium and the
hygroscopic substance from the resulting product in a fluidized
bed.
[0019] Finally in a third aspect the process comprises:
[0020] a) introducing an enzyme containing liquid medium by
atomization onto a particulate porous carrier having a
substantially hydrophilic surface, so as to adsorb the enzyme on
the carrier, wherein the liquid is introduced in an amount such
that substantially no agglomeration of the carrier occurs and
[0021] b) removing volatile components of the liquid medium from
the resulting product in a fluidized bed.
DETAILED DESCRIPTION OF THE INVENTION
[0022] The Carrier
[0023] In the embodiment of the invention the carrier is a
particulate porous material. The particles may suitably be of a
diametrical size of 200-1000 .mu.m, preferably 400-700 .mu.m; have
a surface area of 20-1000 m.sup.2/g, preferably 100-700 m.sup.2/g
and have a pore size of 10-500 nm, preferably 100-300 nm.
[0024] The carrier particles may comprise inorganic, organic or
both inorganic and organic material. Said carrier may further have
a hydrophilic or hydrophobic surface.
[0025] In a first embodiment of the invention the carrier particles
comprise an inorganic material with a substantially hydrophilic
surface, which is essentially insoluble in hydrophilic or
hydrophobic liquids or mixtures thereof. Preferred carriers may be
based on silicas (e.g. Celite from Manville, USA), zeolites (e.g.
Wessalith MS330 from Degussa, Germany), aluminas, ceramics (e.g. as
disclosed in Yoshihiko Hirose et Al. (Proceedings from 3.sup.rd
International Symposium on Biocatalysis and Biotransformations, La
Grande Motte, France, 1997, p 238) and kaolins (e.g. kaolin's
subjected to acid, hydrothermal and baking treatment as disclosed
in U.S. Pat. No. 5,614,401).
[0026] In a second embodiment of the invention the carrier
particles comprise a hydrophilic inorganic material as described in
the first embodiment coated with organic moieties thus having a
substantially hydrophobic surface, e.g. as described in JP
09000257-A, wherein an acid treated kaolin carrier is coated with
N-phenyl-gamma-aminopropyltrimethox- ysilane. Further carriers are
described in JP 08126489-A, wherein a water insoluble carrier is
coated with a polymer forming a disulphide linkage with enzymes. A
third type of carrier is described in Biotechnology Techniques vol.
3 No 5 345-348, wherein a ceramic carrier is coated with
polyethylene amine, polyethylene imine or
3-aminopropyltriethoxysilane, all three surface types allowing an
enzyme to be covalently bound via glutaraldehyde coupling.
[0027] In a third embodiment of the invention the carrier particles
comprise an organic polymer resin with a substantially hydrophobic
surface. The resin may be an adsorbent resin, preferably a
polyacrylate, a polymethacrylate (e.g. polymethyl methacrylate),
polystyrene cross-linked with divinylbenzene, polyurethane or
polypropylene or the resin may be an ion exchange resin, preferably
an anion exchange resin, e.g. a weakly basic anion exchange resin.
A preferred anion exchange resin is a phenolic type Duolite resin
from Rohm & Haas.
[0028] Further the carrier may be made from regenerated chitosan as
disclosed in DE 4429018-A.
[0029] The Enzyme
[0030] The enzyme to be immobilized according to the invention may
be any enzyme suitable for use in media essentially devoid of free
water. The most commonly used enzymes are lipases and in a specific
embodiment of the invention the lipase may be derived from a strain
of the genus Humicola (also known as Thermomyces), Pseudomonas,
Candida, or Rhizomucor, preferably the species H. lanuginosa (also
known as Thermomyces lanuginosa as described in U.S. Pat. No.
4,810,414 and EP 305216 which are hereby included by reference), C.
antarctica or R. miehei.
[0031] Further the lipase may be positionally site specific (i.e.
1,3 specific) or non-specific, upon interaction with triglycerides
as substrates.
[0032] The enzyme may further be covalently cross-linked by
glutaraldehyde treatment during the immobilization process.
[0033] The Enzyme Containing Liquid Medium
[0034] The enzyme containing liquid medium is a hydrophilic medium,
preferably aqueous. It may thus contain more than 20% water,
preferably more than 40% water, more preferably more than 50%
water, more preferably more than 60% water, more preferably more
than 70% water, more preferably more than 80% water, more
preferably more than 90% water, e.g. more than 95% water. It may
contain other organic or biological matter. Thus it may be a
fermentation broth or an enzyme concentrate solution obtainable by
purifying a fermentation broth by e.g. ultra filtration or by
protein precipitation, separation and re-dissolution in an other
aqueous medium. It may further be substantially pure enzyme
dissolved in an aqueous medium. In a special embodiment of the
invention the enzyme containing aqueous liquid has not been
subjected to costly processing steps prior to immobilization to
remove water such as evaporation nor has it been subjected to
addition of non aqueous solvents, e.g. organic solvents such as
alcohols, e.g. (poly)ethylene glycol and/or (poly) propylene
glycol.
[0035] The Immobilization Process
[0036] Immobilization of Enzyme on Carriers with a Hydrophilic
Surface
[0037] Without being bound to the theory it is contemplated that
immobilization of enzyme on carriers having a substantially
hydrophilic surface involves no adsorption of the enzyme, but is a
deposition of enzyme in surface pores as a result of removal of the
liquid, in which the enzyme is dissolved.
[0038] i. In one embodiment of the invention the immobilization of
enzyme on a carrier having a substantially hydrophilic surface may
thus be conducted in standard mixing equipment (e.g. Lodiger,
Germany), wherein an enzyme containing liquid is introduced by
atomization to the dry porous and particulate carrier during
mixing, e.g. using a nebulizer connected to a pump (e.g. a standard
peristaltic Watson-Marlow pump).
[0039] The liquid should be added in such amounts that
substantially no agglomeration of the carrier occurs, thus enabling
the subsequent drying of the enzyme-carrier product by fluidizing
said product in standard fluid bed equipment, e.g. a Uni-Glatt
fluidized bed apparatus (Glatt, Germany), thereby removing volatile
components.
[0040] ii. In a second embodiment of the invention the
immobilization of enzyme on a carrier having a substantially
hydrophilic surface may alternatively be conducted in standard
fluid bed equipment, e.g. a Uni-Glatt fluidized bed apparatus
(Glatt, Germany), wherein the dry porous and particulate carrier is
fluidized and an enzyme containing liquid at ambient temperature is
introduced by atomization to the fluidized carrier, e.g. using a
nebulizer connected to a pump (e.g. a standard peristaltic
Watson-Marlow pump). In this embodiment immobilization and drying
are conducted simultaneously.
[0041] Immobilization on Carriers with a Hydrophobic Surface
[0042] Without being bound to the theory it is contemplated that
immobilization of enzyme on carriers having a substantially
hydrophobic surface involves adsorption of the enzyme on the
surface. The immobilization may be enabled by the enzyme forming
hydrogen bonds, ionic bonds or covalent bonds with moieties in the
surface.
[0043] iii. In a third embodiment of the invention the
immobilization of enzyme on a carrier having a substantially
hydrophobic surface may thus be conducted in standard mixing
equipment, wherein an enzyme containing liquid is introduced to the
dry porous and particulate carrier in an amount sufficient to form
a paste or a slurry. The paste or slurry is mixed for a period of
time in which the enzyme is adsorbed. Following the adsorption step
a hygroscopic particulate substance of a particle size smaller than
the carrier is introduced to the slurry or paste. Said substance
substantially prevents agglomeration of the enzyme-carrier by
adsorption of excess liquid, thereby enabling the subsequent drying
of the enzyme-carrier product by fluidizing said product in
standard fluid bed equipment, e.g. a Uni-Glatt fluidized bed
apparatus (Glatt, Germany), thereby removing volatile components
and if necessary the hygroscopic substance. The hygroscopic
substance may be any particulate fine ground hydrophilic components
capable of absorbing excess liquid such as silicas (e.g. Hyper Flow
Celite), kaolin, aluminas, zeolites or ceramics. Removal of the
hygroscopic substance may be achieved by inserting a filter at the
top of the fluidized bed with a suitable pore size which will allow
the hygroscopic substance to pass but will retain the
enzyme-carrier. A pore size of 100-900 .mu.m, preferably 200-400
.mu.m may be employed.
[0044] iv. In a fourth embodiment of the invention the
immobilization of enzyme on a carrier having a substantially
hydrophobic surface may alternatively be conducted in standard
fluid bed equipment, e.g. a Uni-Glatt fluidized bed apparatus
(Glatt, Germany), wherein the dry porous and particulate carrier is
fluidized and an enzyme containing liquid at ambient temperature is
introduced by atomization to the fluidized carrier, e.g. using a
nebulizer connected to a pump (e.g. a standard peristaltic
Watson-Marlow pump). In this embodiment immobilization and drying
are conducted simultaneously.
[0045] Common Features for i and iii Mixing Step
[0046] Immobilizing the enzyme on the carrier in a mixer may
suitably be conducted at ambient temperature. Mixing times may for
this size of equipment suitably be 5-60 minutes, preferably 10-30
minutes.
[0047] Common Features for i, ii, iii and iv Fluid Bed Step
[0048] A suitable air inlet flow in the fluid bed equipment will
depend on the size and density of the enzyme-carrier, the amount of
carrier and the fluid bed equipment. Further the air inlet flow has
an upper limit, as the flow should be sufficient to keep the
enzyme-carrier fluidized, but not so powerful as to "blow off" the
enzyme-carrier.
[0049] Suitable temperatures of the inlet air for removing volatile
components will primarily depend of the thermal stability of the
enzyme (the inactivation temperature). The temperature may be
40-90.degree. C., preferably 50-70.degree. C., e.g. 60.degree. C. A
higher temperature provides shorter immobilization and drying
times.
[0050] Further, time consumption for immobilization and/or drying
of the enzyme-carrier when equipment, air inlet flow and air
temperature are fixed will depend on the quantity of
enzyme-carrier. The immobilization/drying process may be monitored
by measuring the air inlet temperature and the air outlet
temperature. While the enzyme-carrier is moist the outlet
temperature is lower than the inlet temperature due to the heat
absorption and evaporation of volatile components. Typically a
steady state evaporation occurs during the immobilization/drying
process where the outlet temperature stabilizes on a temperature
lower than the inlet temperature indicating that evaporation of
volatile components (i.e. heat absorption) occurs at a constant
rate. At the end of the immobilization/drying process the outlet
temperature begins to rise and approach the inlet temperature
indicating that the heat absorption has decreased and thus the
moisture of the enzyme-carrier has been removed. Using a fluid bed
for immobilization and drying simultaneously the drying process
will occur for as long as the enzyme containing liquid is atomized
into the fluid bed, and may suitably be extended for 5-30 minutes
after inlet of the enzyme containing liquid has ended.
[0051] An important aspect of the invention is that the
immobilization processes can be easily scaled up by applying other
larger standard equipment. Thus the equipment setting ranges given
vide supra may be adjusted to optimize larger scale equipment.
[0052] Uses of Immobilized Enzyme Preparation
[0053] Immobilized enzyme prepared in context of the invention may
be used for hydrolysis, synthesis or modification of organic
substances in a medium essentially devoid of free water. Said
substances may be comprised in food products like plant or animal
oils/fats.
[0054] Accordingly the invention encompasses a process for
enzymatic modification of an organic compound comprising contacting
in a reaction medium essentially devoid of free water said organic
compound with an immobilized enzyme produced according to the
invention.
[0055] In a preferred embodiment of the invention an immobilized
lipase enzyme, e.g. a 1,3 specific lipase, is used for an
trans-esterification process in a medium essentially devoid of free
water. The trans-esterification may be used for fatty acid
substitution, comprising contacting a first reactant and a second
reactant with said immobilized lipase by which a substitution
reaction occurs, e.g. as shown in FIGS. 1-3.
[0056] The first reactant may be a fatty acid ester, preferably a
triglyceride or a mixture of triglycerides.
[0057] The second reactant may be another fatty acid ester
different from the first reactant, preferably a triglyceride or a
mixture of triglycerides. Further the second reactant may be an
alcohol or a free fatty acid.
[0058] The medium in this preferred embodiment of the invention
comprises an organic solvent, or it may consist essentially of
triglycerides.
[0059] Said use of the invention may be applied in production of
food products e.g. margarine or cocoa-butter substitutes.
[0060] The invention is illustrated by the following non-limiting
examples.
EXAMPLES
[0061] Lipase Assay:
[0062] The lipolytic activity may be determined using tributyrine
as substrate. This method is based on the hydrolysis of tributyrine
by the enzyme, and the alkali consumption is registered as a
function of time.
[0063] One Lipase Unit (LU) is defined as the amount of enzyme
which, under standard conditions (i.e. at 30.0.degree. C.; pH 7.0;
with Gum Arabic as emulsifier and tributyrine as substrate)
liberates 1 mmole titrable butyric acid per minute.
[0064] A folder AF 95/5 describing this analytical method in more
detail is available upon request to Novo Nordisk A/S, Denmark.
[0065] Trans-esterification Assay
[0066] a) 200 mg Trilaurin (Fluka) and 571 mg of myristic acid (8
molar equivalents) of myristic acid (Merck) was dissolved in 20 ml
heptane. 3 ml saturated NaCl solution was added and the mixture was
stirred in a closed bottle for 24 hours at ambient temperature.
[0067] b) The immobilized enzyme (50 mg) was water equilibrated in
a dissicator (hermetically closed vessel) for 24 hours, using gas
phase equilibrium with a saturated NaCl solution (water
activity=0.75).
[0068] c) At T=0 minutes the water equilibrated immobilized enzyme
and substrate were mixed in a closed bottle, which was placed in a
shaking bath at 40.degree. C. 100 .mu.l samples were withdrawn from
the closed bottle using a syringe at T=0, 10, 20, 30, 40, 50 and 60
minutes. The samples were diluted (1:5 vol:vol) with a 50/50 (%
v/v) mixture of acetone/acetonitrile and analyzed on an HPLC
system.
[0069] Analysis on the HPLC System:
[0070] d) The HPLC system was equipped with a LiChrosphere 100
RPC18 endcapped 5 .mu.m (125.times.4 mm) column (Merck). A 50/50 (%
v/v) isocratic acetonitrile/acetone solution was selected as the
mobile phase with a flow of 1 ml/minute.
[0071] e) 20 .mu.l of sample was injected and the formed products
(1,2-dilauroyl-3-myristoyl-glycerol (product 1) and
1,3-dimyristoyl-2-lauroyl-glycerol (product 2)) were measured by
evaporative light scattering detection (Sedex 55, Sedere, France)
at 2 bar pressure and a temperature at 30.degree. C.
[0072] f) The amounts of formed products were estimated by
comparing sample measurements to external standard curves of
1,2-dilauroyl-3-myristoyl-glycerol and
1,3-dimyristoyl-2-lauroyl-glycerol- .
[0073] The rate of the trans-esterification process may be
calculated in units, where 1 unit is defined as 1 .mu.mole myristic
acid incorporated in trilaurin per minute.
[0074] Alternatively the effect of the inter-esterification process
may be stated in % conversion of trilaurin to product 1 and product
2, which is calculated by: 1 % Conversion T = i = [ mole product 1
+ mole product 2 mole trilaurin at T = 0 ] T = i 100 %
[0075] where i indicates at which time the sample is withdrawn from
the incubation bottle (step c).
Example 1
[0076] Lipase adsorption onto zeolite based carrier in fluid bed
with simultaneous removal/evaporation of volatile liquids
[0077] 400 g of a solution of Humicola lanuginosa lipase (693
kLU/ml) was atomized onto 1 kg zeolite (Wessalith MS330; 0.5-0.9
mm; Degussa, Germany) using a two-way nebulizer in a Uni-Glatt
(Glatt, Germany) fluidized bed apparatus. The lipase solution was
applied via a peristaltic pump (Watson-Marlow). Inlet air
temperature was 60.degree. C. and product temperature was
40.degree. C. with an air flow at 100 m3/hours. After the
immobilization was finished the product was dried for an additional
5 minutes in the fluid bed.
[0078] The immobilization process was tested on the
inter-esterification assay, which measured 53% conversion of
trilaurin after T=24 hours
Example 2
[0079] Lipase adsorption onto silica based carrier in fluid bed
with simultaneous removal/evaporation of volatile liquids.
[0080] 94 g of a solution of Humicola lanuginosa lipase (693
kLU/ml) fermentation solution was diluted with 100 g of
demineralized water and atomized onto 200 g of Celite R648
(Manville, USA) in a Uni-Glatt (Glatt, Germany) fluidized bed
apparatus, using a two-way nebulizer (made in-house). The lipase
solution was applied via a peristaltic pump (Watson-Marlow) (flow
rate 238 g/hour). Inlet air temperature and product temperature
were identical with those indicated in example 1. After the
immobilization was finished the product was dried for an additional
5 minutes in the fluid bed.
[0081] The immobilization process was tested on the
inter-esterification assay, which measured 12% conversion of
trilaurin after T=24 hours
Example 3
[0082] Lipase adsorption onto adsorbent resin in mixer and
subsequent drying in fluid bed using Hyper Flow Celite (HFC) as
drying aid.
[0083] 94 g of a solution of Humicola lanuginosa lipase (693
kLU/ml) was diluted with 260 g demineralized water. The solution
was added to 250 g adsorbent resin (a macro-porous divinylbenzene
cross-linked polystyrene, Purolite AP 1090; from Purolite, UK) in a
5 1 mixer (Lodiger, Germany). The suspension was mixed for 20
minutes at ambient temperature and with RPM of 30. 200 g Hyper Flow
Celite (HFC) was added to absorb residual liquid enabling the
mixture to be fluidized in the Uni-Glatt apparatus. Using the same
conditions as described in example 1, the mixture was dried for 20
minutes. In this period, the HFC was separated from the adsorbent
resin using a 300 .mu.m filter on the top of the fluid bed to
retain the resin particles while HFC was blown off.
[0084] Activity of Product:
[0085] The rate for T=0-60 minutes was measured to 3.9 U/g
product.
Example 4
[0086] Lipase adsorption onto adsorbent resin in fluid bed with
simultaneous drying of volatile liquids.
[0087] 94 g of a solution of Humicola lanuginosa lipase (693
kLU/ml) was diluted with 200 g of demineralized water and atomized
onto 200 g of adsorbent resin (macro-porous divinylbenzene
cross-linked polystyrene, Purolite AP 1090; from Purolite, UK) in a
Uni-Glatt (Glatt, Germany) fluidized bed apparatus, using a two-way
nebulizer. The lipase solution was applied via a peristaltic pump
(Watson-Marlow) (flow rate 238 g/hour). Inlet air temperature and
product temperature were 50.degree. C. and 30.degree. C.,
respectively. After the immobilization was finished the product was
dried for an additional 5 minutes in the fluid bed (flow rate 100
m3/hour).
[0088] The immobilization process was tested on the
inter-esterification assay, which measured 36% conversion of
trilaurin after T=24 hours.
Example 5
[0089] Lipase adsorption onto a kaolin based carrier in fluid bed
with simultaneous drying of volatile liquids.
[0090] 94 g of a solution of Humicola lanuginosa lipase (693
kLU/ml) was diluted with 300 g of demineralized water and atomized
onto 200 g of kaolin carrier (BIOFIX SC (500-250); from ECC, UK) in
a Uni-Glatt (Glatt, Germany) fluidized bed apparatus, using a
two-way nebulizer (made in-house). The lipase solution was applied
via a peristaltic pump (Watson-Marlow) (flow rate 238 g/hour).
Inlet air temperature and product temperature were 50.degree. C.
and 30.degree. C., respectively. After the immobilization was
finished the product was dried for an additional 5 minutes in the
fluid bed (flow rate 100 m3/hour).
[0091] The immobilization process was tested on the
inter-esterification assay, which measured 34% conversion of
trilaurin after T=24 hours.
* * * * *