U.S. patent application number 11/918150 was filed with the patent office on 2009-02-12 for immobilized enzymes and methods of using thereof.
Invention is credited to Jaroslav A. Kralovec, Weijie Wang.
Application Number | 20090042263 11/918150 |
Document ID | / |
Family ID | 38475222 |
Filed Date | 2009-02-12 |
United States Patent
Application |
20090042263 |
Kind Code |
A1 |
Kralovec; Jaroslav A. ; et
al. |
February 12, 2009 |
Immobilized Enzymes and Methods of Using Thereof
Abstract
The disclosed matter relates to immobilized enzymes and methods
of use thereof.
Inventors: |
Kralovec; Jaroslav A.;
(Halifax, CA) ; Wang; Weijie; (Dartmouth,
CA) |
Correspondence
Address: |
Ballard Spahr Andrews & Ingersoll, LLP
SUITE 1000, 999 PEACHTREE STREET
ATLANTA
GA
30309-3915
US
|
Family ID: |
38475222 |
Appl. No.: |
11/918150 |
Filed: |
June 13, 2006 |
PCT Filed: |
June 13, 2006 |
PCT NO: |
PCT/IB2006/003999 |
371 Date: |
December 18, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60691061 |
Jun 16, 2005 |
|
|
|
Current U.S.
Class: |
435/134 ;
435/174; 435/180 |
Current CPC
Class: |
C12N 11/06 20130101;
C12P 7/6436 20130101; C12N 11/082 20200101; C12N 9/20 20130101;
C12N 11/08 20130101; C12P 7/6472 20130101; C12P 7/6454
20130101 |
Class at
Publication: |
435/134 ;
435/174; 435/180 |
International
Class: |
C12P 7/64 20060101
C12P007/64; C12N 11/00 20060101 C12N011/00 |
Claims
1. Immobilized enzyme comprising an esterification,
transesterification, or interesterification/intraesterification
enzyme immobilized in a food grade matrix.
2-9. (canceled)
10. The immobilized enzyme of claim 1, wherein the food grade
matrix comprises less than 2% by weight non-polymerizable
impurities.
11. (canceled)
12. The immobilized enzyme of claim 1, wherein the food grade
matrix comprises a copolymer of divinylbenzene.
13. The immobilized enzyme of claim 1, wherein the food grade
matrix comprises a copolymer of (1) divinylbenzene and (2) acrylic
acid, methacrylic acid, methyl acrylate, methyl methacrylate, ethyl
vinyl benzene, or styrene.
14. The immobilized enzyme of claim 1, wherein the food grade
matrix comprises a copolymer of divinylbenzene and styrene.
15-16. (canceled)
17. The immobilized enzyme of claim 1, wherein the amount of enzyme
immobilized in the matrix is from 7.5 to 35 KLU/g matrix based on
the difference in the enzyme concentration in the solution used to
interact with the matrix as measured at 280 nm.
18. The immobilized enzyme of claim 1, wherein enzyme is derived
from Candida antarctica and the food grade matrix comprises a
copolymer of divinylbenzene and styrene.
19. A method for transesterifying an ester, comprising reacting the
ester with an alcohol in the presence of an immobilized enzyme in
claim 1, wherein the ester comprises an ester of an unsaturated
fatty acid.
20. (canceled)
21. The method of claim 19, wherein the unsaturated fatty acid is
derived from fish oil.
22-23. (canceled)
24. The method of claim 19, wherein the unsaturated fatty acid is
an omega-3 fatty acid.
25-26. (canceled)
27. The method of claim 19, wherein the unsaturated fatty acid is
linoleic acid, linolenic acid, gamma-linolenic acid, arachidonic
acid, mead acid, stearidonic acid, alpha-eleostearic acid,
eleostearic acid, pinolenic acid, docosadienic acid,
docosatetraenoic acid, octadecadienoic acid, octadecatrienoic acid,
eicosatetraenoic acid, or any combination thereof.
28. The method of claim 19, wherein the unsaturated fatty acid
comprises eicosapentaenoic acid 20:5.omega.3 (EPA), docosahexaenoic
acid 22:6.omega.3 (DHA), docosapentaenoic acid 22:5.omega.3 (DPA),
or any mixture thereof.
29-30. (canceled)
31. The method of claim 19, wherein the alcohol comprises
glycerol.
32-33. (canceled)
34. The method of claim 19, wherein the enzyme is from 0.1% to 20%
by weight of the reaction.
35-41. (canceled)
42. The method of claim 19, wherein after the reaction between the
ester and the alcohol, the amount of ester that is transesterified
is from 70 to 100%.
43. The method of claim 19, wherein the ester comprises an ethyl
ester of eicosapentaenoic acid 20:5.omega.3 (EPA), docosahexaenoic
acid 22:6.omega.3 (DHA), docosapentaenoic acid 22:5.omega.3 (DPA),
or any mixture thereof, and the alcohol comprises glycerol, wherein
the ester and the alcohol are present in a molar ratio of from 2:1
to 5:1, wherein the reaction is stirred in the presence of the
immobilized enzyme at a temperature of from 70.degree. C. to
90.degree. C. for 20 hours to 24 hours, wherein the immobilized
enzyme comprises an enzyme derived from Candida antarctica
immobilized in a food grade matrix comprising a copolymer of
divinylbenzene and styrene.
44. A method for esterifying a carboxylic acid, comprising reacting
the acid with an alcohol in the presence of an immobilized enzyme
in claim 1, wherein the carboxylic acid comprises an unsaturated
fatty acid.
45. (canceled)
46. The method of claim 44, wherein the unsaturated fatty acid is
derived from fish oil.
47-48. (canceled)
49. The method of claim 44, wherein the unsaturated fatty acid is
an omega-3 fatty acid.
50-51. (canceled)
52. The method of claim 44, wherein the unsaturated fatty acid is
linoleic acid, linolenic acid, gamma-linolenic acid, arachidonic
acid, mead acid, stearidonic acid, alpha-eleostearic acid,
eleostearic acid, pinolenic acid, docosadienic acid,
docosatetraenoic acid, octadecadienoic acid, octadecatrienoic acid,
eicosatetraenoic acid, or any combination thereof.
53. The method of claim 44, wherein the unsaturated fatty acid
comprises eicosapentaenoic acid 20:5.omega.3 (EPA), docosahexaenoic
acid 22:6.omega.3 (DHA), docosapentaenoic acid 22:5.omega.3 (DPA),
or any mixture thereof.
54-55. (canceled)
56. The method of claim 44, wherein the alcohol comprises
glycerol.
57-58. (canceled)
59. The method of claim 44, wherein the enzyme is from 0.1% to 20%
by weight of the reaction.
60-66. (canceled)
67. The method of claim 44, wherein after the reaction between the
carboxylic acid and the alcohol, the amount of carboxylic acid that
is esterified is from 70 to 100%.
68. The method of claim 44, wherein the carboxylic acid comprises
eicosapentaenoic acid 20:5.omega.3 (EPA), docosahexaenoic acid
22:6.omega.3 (DHA), docosapentaenoic acid 22:5.omega.3 (DPA), or
any mixture thereof, and the alcohol comprises glycerol, wherein
the ester and the alcohol are present in a molar ratio of from 2:1
to 5:1, wherein the reaction is stirred in the presence of the
immobilized enzyme at a temperature of from 60.degree. C. to
90.degree. C. for 2 hours to 24 hours, wherein the immobilized
enzyme comprises an enzyme derived from Candida antarctica
immobilized in a food grade matrix comprising a copolymer of
divinylbenzene and styrene.
69-71. (canceled)
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of priority to U.S.
Provisional Patent Application 60/691,061, filed Jun. 16, 2005,
which is incorporated by reference herein in its entirety.
FIELD
[0002] The disclosed matter relates to immobilized enzymes and
methods of use thereof.
BACKGROUND
[0003] The beneficial effects of the long-chain polyunsaturated
fatty acids (PUFA) that are characteristic of marine lipids,
especially cis-5,8,11,14,17-eicosapentaenoic acid (EPA) and
cis-4,7,10,13,16,19-docosahexaenoic acid (DHA) on lowering serum
triglycerides are now well established. These compounds are also
known for other cardioprotective benefits and other biological
effects. Among the most frequently mentioned benefits are those
related to the prevention of and the treatment of inflammation,
neurogenerative diseases, and cognitive development abnormalities.
The public is becoming increasingly aware of the health benefits of
fish oil and DHA and EPA concentrates, as it is evidenced from
global sales of polyunsaturated fatty acids (PUFA). For instance,
the sales of PUFA rose by 50% in 2002, and they were the only ones
out of all categories of nutraceuticals that made a significant
increase in sales.
[0004] Several methods of producing PUFA concentrates from marine
oils are known, for example, selective lipase hydrolysis, PUFA
complexation using urea (or more sophisticated molecular guest-host
frameworks involving metric control), and a physical removal of
unwanted components by fractionation. U.S. Publication No.
2004/0236128 describes the separation of EPA from DHA by
precipitating EPA magnesium salt.
[0005] Fractionation involving molecular distillation is usually
conducted on ethyl esters prepared from the starting triglycerides
since they are more volatile than corresponding triglycerides.
However, there is a controversy as to whether ethyl esters of PUFA
are as bioavailable as their triglyceride counterparts. Therefore,
there is a need to re-esterify the esters to the corresponding
triglycerides.
[0006] The reaction describing the formation of triglycerides from
the ethyl esters of fish oil is transesterification.
Transesterification is a process where an ester is converted into
another ester through interchange of the alkoxy moieties.
##STR00001##
[0007] The reaction is an equilibrium process and the
transformation occurs essentially by simply mixing the two
components. However, it has been shown that the reaction is
accelerated by Lewis acid catalysts (such as BBr.sub.3, AlCl.sub.3
etc., embedded in polystyrene-divinylbenzene), Bronsted acid
catalysts such as HCl, H.sub.3PO.sub.4, H.sub.2SO.sub.4, p-TosOH,
or basic catalysts such as metal alkoxides, metal hydroxides, and
metal carbonates.
[0008] It has been apparent, however, that the transesterification
reaction under these conditions does not meet in many cases the
requirements of modern synthetic chemistry, i.e., highly efficient
and regioselective reaction conditions. Thus efforts have continued
to conduct transesterifications under milder conditions and to
control randomization. Among the chemical catalysts developed,
distanoxane was found to be effective for transesterification of
various types of esters; however, this catalyst is difficult to
prepare and is not commercially available, at least not in large
quantities. The titanate-mediated transesterification method is
extremely mild but fails to achieve certain kinds of
transesterifications. Similarly, DMAP catalyzed reactions or
reactions in the presence of tin-based superacids have different
profiles of reaction selectivity. Successful attempts have also
been made to perform transesterifications using zeolites, neutral
chromatographic alumina, or kaolinites.
[0009] In spite of the advances in modern synthetic chemistry, the
most popular industrial catalysts are strong bases. They are
inexpensive, but because of their nature they generate a
significant degree of side products, particularly at high
temperatures necessary to achieve the desirable yields. In
addition, they are not regioselective and lead to side reactions.
Although they may be good choices for stable and structurally
suitable chemical entities, they are not preferred for complex
products such as, for example, fish oils. In the case of fish oils,
enzymatically catalyzed transesterifications are a viable
alternative, since they are regioselective and generate virtually
no side products. However, their drawback is generally their
cost.
[0010] Therefore, there is a need to transesterify complex esters
such as, for example, polyunsaturated fatty acid esters to more
useful esters. There is also a need for the efficient
esterification of complex carboxylic acids such as fatty acids. The
procedures should have a high efficiency with respect to
transesterification and esterification and yet not produce
undesirable side-products. The process should also be relatively
inexpensive for commercial applications. Finally, the esters
produced by the methods described herein should be ready to be
incorporated into numerous food products without the requirement of
removing additional impurities from the transesterification
process. The immobilized enzymes and methods of use described
herein satisfy these long-felt needs.
SUMMARY
[0011] In accordance with the purposes of the disclosed materials,
compounds, compositions, articles, and methods, as embodied and
broadly described herein, the disclosed subject matter, in one
aspect, relates to compounds and compositions and methods for
preparing and using such compounds and compositions. In a further
aspect, the disclosed subject matter relates to immobilized enzymes
and their methods of use.
[0012] Additional advantages will be set forth in part in the
description that follows, and in part will be obvious from the
description, or may be learned by practice of the aspects described
below. The advantages described below will be realized and attained
by means of the elements and combinations particularly pointed out
in the appended claims. It is to be understood that both the
foregoing general description and the following detailed
description are exemplary and explanatory only and are not
restrictive.
BRIEF DESCRIPTION OF FIGURES
[0013] The accompanying figures, which are incorporated in and
constitutes a part of this specification, illustrate several
aspects described below.
[0014] FIG. 1 is a schematic of a process for the enzymatic
transformation of ethyl, esters (or free fatty acids) to
triglycerides. In the figure, 1 represents a stirred heated holding
vessel, 2 represents a shear pump, 3 represents a fixed-enzyme bed
reactor (FEBR), 4 represents an evaporation tank, and 5 represents
a condenser. The reactor 3 can be of varying geometry (e.g.,
length>height or length<height).
[0015] FIG. 2 is a graph of an enzymatic conversion of free fatty
acids to triglyceride with a CALB enzyme immobilized on a food
grade matrix (AMBERLITE.TM. XAD16HP). The same enzyme bed was used
for 40 runs.
DETAILED DESCRIPTION
[0016] The materials, compounds, compositions, articles, and
methods described herein may be understood more readily by
reference to the following detailed description of specific aspects
of the disclosed subject matter and the Examples included therein
and to the Figures.
[0017] Before the present materials, compounds, compositions,
articles, and methods are disclosed and described, it is to be
understood that the aspects described below are not limited to
specific synthetic methods or specific reagents, as such may, of
course, vary. It is also to be understood that the terminology used
herein is for the purpose of describing particular aspects only and
is not intended to be limiting.
[0018] Also, throughout this specification, various publications
are referenced. The disclosures of these publications in their
entireties are hereby incorporated by reference into this
application in order to more fully describe the state of the art to
which the disclosed matter pertains. The references disclosed are
also individually and specifically incorporated by reference herein
for the material contained in them that is discussed in the
sentence in which the reference is relied upon.
GENERAL DEFINITIONS
[0019] In this specification and in the claims that follow,
reference will be made to a number of terms, which shall be defined
to have the following meanings:
[0020] Throughout the description and claims of this specification
the word "comprise" and other forms of the word, such as
"comprising" and "comprises," means including but not limited to,
and is not intended to exclude, for example, other additives,
components, integers, or steps.
[0021] As used in the description and the appended claims, the
singular forms "a," "an," and "the" include plural referents unless
the context clearly dictates otherwise. Thus, for example,
reference to "a compound" includes mixtures of two or more such
compounds, reference to "an unsaturated fatty acid" includes
mixtures of two or more such unsaturated fatty acids, reference to
"the matrix" includes mixtures of two or more such matrixes, and
the like.
[0022] "Optional" or "optionally" means that the subsequently
described event or circumstance can or cannot occur, and that the
description includes instances where the event or circumstance
occurs and instances where it does not.
[0023] Ranges can be expressed herein as from "about" one
particular value, and/or to "about" another particular value. When
such a range is expressed, another aspect includes from the one
particular value and/or to the other particular value. Similarly,
when values are expressed as approximations, by use of the
antecedent "about," it will be understood that the particular value
forms another aspect. It will be further understood that the
endpoints of each of the ranges are significant both in relation to
the other endpoint, and independently of the other endpoint. It is
also understood that there are a number of values disclosed herein,
and that each value is also herein disclosed as "about" that
particular value in addition to the value itself. For example, if
the value "10" is disclosed, then "about 10" is also disclosed. It
is also understood that when a value is disclosed, then "less than
or equal to" the value, "greater than or equal to the value" and
possible ranges between values are also disclosed, as appropriately
understood by the skilled artisan. For example, if the value "10"
is disclosed, then "less than or equal to 10" as well as "greater
than or equal to 10" is also disclosed. It is also understood that
throughout the application, data are provided in a number of
different formats and that these data represent endpoints and
starting points and ranges for any combination of the data points.
For example, if a particular data point "10" and a particular data
point "15" are disclosed, it is understood that greater than,
greater than or equal to, less than, less than or equal to, and
equal to 10 and 15 are considered disclosed as well as between 10
and 15. It is also understood that each unit between two particular
units are also disclosed. For example, if 10 and 15 are disclosed,
then 11, 12, 13, and 14 are also disclosed.
[0024] References in the specification and concluding claims to
parts by weight of a particular element or component in a
composition denotes the weight relationship between the element or
component and any other elements or components in the composition
or article for which a part by weight is expressed. Thus, in a
compound containing 2 parts by weight of component X and 5 parts by
weight component Y, X and Y are present at a weight ratio of 2:5,
and are present in such ratio regardless of whether additional
components are contained in the compound.
[0025] A weight percent of a component, unless specifically stated
to the contrary, is based on the total weight of the formulation or
composition in which the component is included.
[0026] Unless stated to the contrary, a formula with chemical bonds
shown only as solid lines and not as wedges or dashed lines
contemplates each possible isomer, e.g., each enantiomer and
diastereomer, and a mixture of isomers, such as a racemic or
scalemic mixtures.
[0027] Reference will now be made in detail to specific aspects of
the disclosed materials, compounds, compositions, articles, and
methods, examples of which are illustrated in the accompanying
Examples and Figures.
[0028] Disclosed herein are materials, compounds, compositions, and
components that can be used for, can be used in conjunction with,
can be used in preparation for, or are products of the disclosed
methods and compositions. These and other materials are disclosed
herein, and it is understood that when combinations, subsets,
interactions, groups, etc. of these materials are disclosed that
while specific reference of each various individual and collective
combinations and permutation of these compounds may not be
explicitly disclosed, each is specifically contemplated and
described herein. For example, if a compound is disclosed and a
number of modifications that can be made to a number of components
or residues of the compound are discussed, each and every
combination and permutation that are possible are specifically
contemplated unless specifically indicated to the contrary. Thus,
if a class of components or residues A, B, and C are disclosed as
well as a class of components or residues D, E, and F and an
example of a combination compound A-D is disclosed, then even if
each is not individually recited, each is individually and
collectively contemplated. Thus, in this example, each of the
combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are
specifically contemplated and should be considered disclosed from
disclosure of A, B, and C; D, E, and F; and the example combination
A-D. Likewise, any subset or combination of these is also
specifically contemplated and disclosed. Thus, for example, the
sub-group of A-E, B-F, and C-E are specifically contemplated and
should be considered disclosed from disclosure of A, B, and C; D,
E, and F; and the example combination A-D. This concept applies to
all aspects of this disclosure including, but not limited to, steps
in methods of making and using the disclosed compositions. Thus, if
there are a variety of additional steps that can be performed it is
understood that each of these additional steps can be performed
with any specific aspect or combination of aspects of the disclosed
methods, and that each such combination is specifically
contemplated and should be considered disclosed.
[0029] Certain materials, compounds, compositions, and components
disclosed herein can be obtained commercially or readily
synthesized using techniques generally known to those of skill in
the art. For example, the starting materials and reagents used in
preparing the disclosed compounds and compositions are either
available from commercial suppliers such as Ocean Nutrition Canada,
Ltd. (Dartmouth, Canada), Aldrich Chemical Co., (Milwaukee, Wis.),
Acros Organics (Morris Plains, N.J.), Fisher Scientific
(Pittsburgh, Pa.), or Sigma (St. Louis, Mo.) or are prepared by
methods known to those skilled in the art following procedures set
forth in references such as Fieser and Fieser's Reagents for
Organic Synthesis, Volumes 1-17 (John Wiley and Sons, 1991); Rodd's
Chemistry of Carbon Compounds, Volumes 1-5 and Supplementals
(Elsevier Science Publishers, 1989); Organic Reactions, Volumes
1-40 (John Wiley and Sons, 1991); March's Advanced Organic
Chemistry, (John Wiley and Sons, 4th Edition); and Larock's
Comprehensive Organic Transformations (VCH Publishers Inc.,
1989).
Immobilized Enzymes
[0030] Disclosed herein, in one aspect, are immobilized enzymes
that comprise an esterification, transesterification, or
interesterification/intraesterification enzyme immobilized in a
food grade matrix. Each component of the disclosed immobilized
enzymes is described below.
[0031] Enzymes
[0032] The enzymes useful herein are any naturally-occurring or
synthetic enzymes that can be used to esterify a carboxylic acid or
transesterify an ester. The term "esterify" is defined herein as
the conversion of a carboxylic acid to the corresponding ester by
reacting the carboxylic acid with an alcohol to produce the ester
(e.g. RCOOH+R.sup.1OH.fwdarw.RCOOR.sup.1+H.sub.2O). The term
"transesterify" is defined herein as the conversion of one ester to
another by reacting the ester with an alcohol to produce a
different ester (e.g.,
RCOOR.sup.1+R.sup.2OH.fwdarw.RCOOR.sup.2+R.sup.1OH). The term
"interesterify" is defined herein as the switching of ester
moieties between two or more separate, independent esters.
Interesterification between two esters is depicted in scheme 1A,
where the R.sup.2 and R.sup.4 groups are switched in the starting
materials (i.e., R.sup.1COOR.sup.2 and R.sup.3COOR.sup.4). Scheme
1B depicts the interesterification between a carboxylic acid
(R.sup.1COOH) and an ester (R.sup.3COOR.sup.4), which produces a
new carboxylic acid and ester. The term "intraesterify" is defined
herein as the switching of ester moieties within the same molecule.
Intraesterification is depicted in scheme 1C, where the R.sup.2 and
R.sup.3 groups are switched in the triester. Scheme 1D depicts the
intraesterification between a carboxylic acid group and an ester
within the same molecule, where hydrogen of the carboxylic acid
switches with R.sup.3 of the ester group.
##STR00002##
[0033] Suitable enzymes can be derived from a microorganism.
Examples of microorganisms that can produce enzymes useful herein
include, but are not limited to, Burkholderia sp., Candida
antarctica B, Candida rugosa, Candida cylindracea, Pseudomonas sp.,
Candida antarctica A, Porcine pancreas, Humicola sp., Humicola
lanuginose, Mucor miehei, Rhizopus javan., Pseudomonas fluor,
Pseudomonas cepacia, Candida cylindrcae, Aspergillus niger,
Rhizopus oryzae, Mucor jaanicus, Mucor javanicus, Rhizopus sp.,
Rhizopus japonicus, Rhizomucor miehi, Rhizopus niveus, or
penicillium camembertii (also Rhizopus delemar, Pseudonomas,
aeruginosa).
[0034] In one example, the enzyme is produced from Candida
antarctica. NOVOZYME.TM. CALB L is a lipase (lipase B) from Candida
antarctica produced by submerged fermentation of a genetically
modified Aspergillus oryzae microorganism. NOVOZYME.TM. CALB L is a
highly versatile catalyst with activity towards a great variety of
different substrates. The enzyme is used in particular as a
powerful enantioselective catalyst in the synthesis of optically
active alcohols, amines, and carboxylic acids. Candida antarctica
lipase B is known to effectively convert ethyl esters or free fatty
acids to triglycerides. This enzyme is a protein with 317 amino
acid residues and molecular weight of 33,008 Daltons. The amino
acids are assembled into 14.alpha.-helixes and 9.beta.-sheets. The
sequence and secondary structure of Candida antarctica lipase B are
provided in SEQ ID NO:1.
[0035] It is also contemplated that derivatives of enzymes produced
from microorganisms can be immobilized and used in the methods
described herein. It is understood that the structure of many
enzymes, as disclosed herein, are known and can be found, for
example, at Genbank, and are herein incorporated by reference.
[0036] As all microbial lipases, CALB belongs to .alpha./.beta.
hydrolases, the fold of which comprises of eight-stranded
.beta.-sheets sandwiched between two layers of amphiplilic
.alpha.-helices. The mechanism of ester hydrolysis of these enzymes
generally involves binding to the ester substrate, formation of the
first tetrahedral intermediate by nucleophilic attack of the
catalytic serine with the oxyanion stabilized by two or three
H-bonds, the so-called oxyanion hole. The ester bond is cleaved and
the acylated enzyme is hydrolyzed in the final step. The
nucleophilic attack by the catalytic serine is mediated by the
catalytic histidine and aspartic or glutamic acid residue. In
certain examples, the longest fatty acid chain that completely
binds inside the binding pocket of CALB is C13; thus, the scisille
fatty acid binding site of this enzyme is relatively short (13.5
.ANG.). The binding site of CALB is relatively short and has a
small hydrophobic area located at the wall of the binding funnel.
Structure of CALB has been published in the Protein Data Bank (The
Protein Data Bank: a computer-based archival file for
macromolecular structures. Bernstein et al., J. Mol. Biol.
112:525-542, 1977). It is also understood that the disclosed
enzymes can be defined by their conserved catalytic cores which are
understood in the art and are herein disclosed.
[0037] Sequence Similarities
[0038] It is understood that as discussed herein the use of the
terms "homology" and "identity" mean the same thing as similarity.
Thus, for example, if the use of the word homology is used between
two non-natural sequences, it is understood that this is not
necessarily indicating an evolutionary relationship between these
two sequences but, rather, is looking at the similarity or
relatedness between their sequences. Many of the methods for
determining homology between two evolutionarily related molecules
are routinely applied to any two or more nucleic acids or proteins
for the purpose of measuring sequence similarity, regardless of
whether they are evolutionarily related or not.
[0039] In general, it is understood that one way to define any
known variants and derivatives or those that might arise of the
disclosed genes and proteins herein, such as SEQ ID NO:1, is
through defining the variants and derivatives in terms of homology
to specific known sequences. This identity of particular sequences
disclosed herein is also discussed elsewhere herein. In general,
variants of genes and proteins herein disclosed typically have at
least about 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,
64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80,
81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,
98, or 99 percent homology to the stated sequence or the native
sequence. Those of skill in the art readily understand how to
determine the homology of two proteins or nucleic acids, such as
genes. For example, the homology can be calculated after aligning
the two sequences so that the homology is at its highest level.
[0040] Another way of calculating homology can be performed by
published algorithms. Optimal alignment of sequences for comparison
can be conducted by the local homology algorithm of Smith and
Waterman, Adv. Appl. Math. 2:482, 1981, by the homology alignment
algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443, 1970, by
the search for similarity method of Pearson and Lipman, Proc. Natl.
Acad. Sci. U.S.A. 85:2444, 1988, by computerized implementations of
these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin
Genetics Software Package, Genetics Computer Group, 575 Science
Dr., Madison, Wis.), or by inspection.
[0041] The same types of homology can be obtained for nucleic acids
by, for example, the algorithms disclosed in Zuker, Science
244:48-52, 1989, Jaeger et al., Proc. Natl. Acad. Sci. U.S.A.
86:7706-7710, 1989, Jaeger et al., Methods Enzymol. 183:281-306,
1989, which are herein incorporated by reference for at least
material related to nucleic acid alignment. It is understood that
any of the methods typically can be used and that in certain
instances the results of these various methods may differ, but the
skilled artisan understands if identity is found with at least one
of these methods, the sequences would be said to have the stated
identity and be disclosed herein.
[0042] For example, as used herein, a sequence recited as having a
particular percent homology to another sequence refers to sequences
that have the recited homology as calculated by any one or more of
the calculation methods described above. For example, a first
sequence has 80 percent homology, as defined herein, to a second
sequence if the first sequence is calculated to have 80 percent
homology to the second sequence using the Zuker calculation method
even if the first sequence does not have 80 percent homology to the
second sequence as calculated by any of the other calculation
methods. As another example, a first sequence has 80 percent
homology, as defined herein, to a second sequence if the first
sequence is calculated to have 80 percent homology to the second
sequence using both the Zuker calculation method and the Pearson
and Lipman calculation method even if the first sequence does not
have 80 percent homology to the second sequence as calculated by
the Smith and Waterman calculation method, the Needleman and Wunsch
calculation method, the Jaeger calculation methods, or any of the
other calculation methods. As yet another example, a first sequence
has 80 percent homology, as defined herein, to a second sequence if
the first sequence is calculated to have 80 percent homology to the
second sequence using each of calculation methods (although, in
practice, the different calculation methods will often result in
different calculated homology percentages).
[0043] Hybridization/Selective Hybridization
[0044] It is also understood that the enzymes disclosed herein,
such as SEQ ID NO:1, can be classified by the ability of the
nucleic acids encoding them to hybridize with other nucleic acids.
The term "hybridization" typically means a sequence driven
interaction between at least two nucleic acid molecules, such as a
primer or a probe and a gene. The phrase "sequence driven
interaction" means an interaction that occurs between two
nucleotides or nucleotide analogs or nucleotide derivatives in a
nucleotide specific manner. For example, G interacting with C or A
interacting with T are sequence driven interactions. Typically,
sequence driven interactions occur on the Watson-Crick face or
Hoogsteen face of the nucleotide. The hybridization of two nucleic
acids is affected by a number of conditions and parameters known to
those of skill in the art. For example, the salt concentrations,
pH, and temperature of the reaction all affect whether two nucleic
acid molecules will hybridize.
[0045] Parameters for selective hybridization between two nucleic
acid molecules are well known to those of skill in the art. For
example, in some examples selective hybridization conditions can be
defined as stringent hybridization conditions. For example,
stringency of hybridization is controlled by both temperature and
salt concentration of either or both of the hybridization and
washing steps. For example, the conditions of hybridization to
achieve selective hybridization may involve hybridization in high
ionic strength solution (6.times.SSC or 6.times.SSPE) at a
temperature that is about 12 to about 25.degree. C. below the
T.sub.m (the melting temperature at which half of the molecules
dissociate from their hybridization partners), followed by washing
at a combination of temperature and salt concentration chosen so
that the washing temperature is about 5.degree. C. to about
20.degree. C. below the T.sub.m. The temperature and salt
conditions are readily determined empirically in preliminary
experiments in which samples of reference DNA immobilized on
filters are hybridized to a labeled nucleic acid of interest and
then washed under conditions of different stringencies.
Hybridization temperatures are typically higher for DNA-RNA and
RNA-RNA hybridizations. The conditions can be used as described
above to achieve stringency, or as is known in the art. (Sambrook
et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold
Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989; Kunkel et
al. Methods Enzymol. 1987:154:367, 1987, which is herein
incorporated by reference for material at least related to
hybridization of nucleic acids). A preferable stringent
hybridization condition for a DNA:DNA hybridization can be at about
68.degree. C. (in aqueous solution) in 6.times.SSC or 6.times.SSPE
followed by washing at 68.degree. C. Stringency of hybridization
and washing, if desired, can be reduced accordingly as the degree
of complementarity desired is decreased, and further, depending
upon the G-C or A-T richness of any area wherein variability is
searched for. Likewise, stringency of hybridization and washing, if
desired, can be increased accordingly as homology desired is
increased, and further, depending upon the G-C or A-T richness of
any area wherein high homology is desired, all as known in the
art.
[0046] Another way to define selective hybridization is by looking
at the amount (percentage) of one of the nucleic acids bound to the
other nucleic acid. For example, in some examples selective
hybridization conditions would be when at least about 60, 65, 70,
71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,
88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 percent of the
limiting nucleic acid is bound to the non-limiting nucleic acid.
Typically, the non-limiting primer is in, for example, 10, or 100,
or 1000 fold excess. This type of assay can be performed at under
conditions where both the limiting and non-limiting primer are, for
example, 10 fold, or 100 fold, or 1000 fold below their k.sub.d, or
where only one of the nucleic acid molecules is 10 fold, or 100
fold, or 1000 fold, or where one or both nucleic acid molecules are
above their k.sub.d.
[0047] Another way to define selective hybridization is by looking
at the percentage of primer that gets enzymatically manipulated
under conditions where hybridization is required to promote the
desired enzymatic manipulation. For example, in some examples
selective hybridization conditions would be when at least about 60,
65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85,
86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 percent
of the primer is enzymatically manipulated under conditions which
promote the enzymatic manipulation; for example, if the enzymatic
manipulation is DNA extension, then selective hybridization
conditions would be when at least about 60, 65, 70, 71, 72, 73, 74,
75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, 100 percent of the primer molecules
are extended. Preferred conditions also include those suggested by
the manufacturer or indicated in the art as being appropriate for
the enzyme performing the manipulation.
[0048] Just as with homology, it is understood that there are a
variety of methods herein disclosed for determining the level of
hybridization between two nucleic acid molecules. It is understood
that these methods and conditions may provide different percentages
of hybridization between two nucleic acid molecules, but unless
otherwise indicated meeting the parameters of any of the methods
would be sufficient. For example, if 80% hybridization was required
and as long as hybridization occurs within the required parameters
in any one of these methods it is considered disclosed herein.
[0049] It is understood that those of skill in the art understand
that if a composition or method meets any one of these criteria for
determining hybridization either collectively or singly it is a
composition or method that is disclosed herein.
[0050] Peptides
[0051] As discussed herein there are numerous variants and strain
derivatives of the disclosed enzymes, such as SEQ ID NO:1 are known
and herein contemplated. Enzymes can be made from proteins or
peptides. Protein variants and derivatives are well understood to
those of skill in the art and can involve amino acid sequence
modifications. For example, amino acid sequence modifications
typically fall into one or more of three classes: substitutional,
insertional, or deletional variants. Insertions include amino
and/or carboxyl terminal fusions as well as intrasequence
insertions of single or multiple amino acid residues. Insertions
ordinarily will be smaller insertions than those of amino or
carboxyl terminal fusions, for example, on the order of one to four
residues. Immunogenic fusion protein derivatives, such as those
described in the examples, are made by fusing a polypeptide
sufficiently large to confer immunogenicity to the target sequence
by cross-linking in vitro or by recombinant cell culture
transformed with DNA encoding the fusion. Deletions are
characterized by the removal of one or more amino acid residues
from the protein sequence. Typically, no more than about from 2 to
6 residues are deleted at any one site within the protein molecule.
These variants ordinarily are prepared by site specific mutagenesis
of nucleotides in the DNA encoding the protein, thereby producing
DNA encoding the variant, and thereafter expressing the DNA in
recombinant cell culture. Techniques for making substitution
mutations at predetermined sites in DNA having a known sequence are
well known, for example M13 primer mutagenesis and PCR mutagenesis.
Amino acid substitutions are typically of single residues but can
occur at a number of different locations at once; insertions
usually will be on the order of from about 1 to 10 amino acid
residues; and deletions will range from about 1 to 30 residues.
Deletions or insertions preferably are made in adjacent pairs,
i.e., a deletion of 2 residues or insertion of 2 residues.
Substitutions, deletions, insertions or any combination thereof can
be combined to arrive at a final construct. The mutations must not
place the sequence out of reading frame and preferably will not
create complementary regions that could produce secondary mRNA
structure. Substitutional variants are those in which at least one
residue has been removed and a different residue inserted in its
place. Such substitutions generally are made in accordance with the
following Tables A and B and are referred to as conservative
substitutions.
TABLE-US-00001 TABLE A Amino Acid Abbreviations Amino Acid
Abbreviations alanine Ala (A) alloisoleucine AIle arginine Arg (R)
asparagine Asn (N) aspartic acid Asp (D) cysteine Cys (C) glutamic
acid Glu (E) glutamine Gln (Q) glycine Gly (G) histidine His (H)
isolelucine Ile (I) leucine Leu (L) lysine Lys (K) phenylalanine
Phe (F) proline Pro (P) pyroglutamic acid Glu serine Ser (S)
threonine Thr (T) tyrosine Tyr(Y) tryptophan Trp (W) valine Val
(V)
TABLE-US-00002 TABLE B Amino Acid Substitutions Original Residue
Exemplary Conservative Substitutions, others are known in the art.
Ala ser Arg lys or gln Asn gln or his Asp glu Cys ser Gln asn or
lys Glu asp Gly pro His asn or gln Ile leu or val Leu ile or val
Lys arg or gln; Met Leu or ile Phemet leu or tyr Ser thr Thr ser
Trp tyr Tyr trp or phe Val ile or leu
[0052] Substantial changes in function or immunological identity
are made by selecting substitutions that are less conservative than
those in Table B, i.e., selecting residues that differ more
significantly in their effect on maintaining: (a) the structure of
the polypeptide backbone in the area of the substitution, for
example as a sheet or helical conformation; (b) the charge or
hydrophobicity of the molecule at the target site; or (c) the bulk
of the side chain. The substitutions that are generally expected to
produce the greatest changes in the protein properties will be
those in which (a) a hydrophilic residue, e.g., seryl or threonyl,
is substituted for (or by) a hydrophobic residue, e.g., leucyl,
isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine or proline
is substituted for (or by) any other residue; (c) a residue having
an electropositive side chain, e.g., lysyl, arginyl, or histidyl is
substituted for (or by) an electronegative residue, e.g., glutamyl
or aspartyl; or (d) a residue having a bulky side chain, e.g.,
phenylalanine, is substituted for (or by) one not having a side
chain, e.g., glycine, in this case, (e) by increasing the number of
sites for sulfation and/or glycosylation.
[0053] For example, the replacement of one amino acid residue with
another that is biologically and/or chemically similar is known to
those skilled in the art as a conservative substitution. For
example, a conservative substitution would be replacing one
hydrophobic residue for another, or one polar residue for another.
The substitutions include combinations such as, for example, Gly,
Ala; Val, Ile, Leu; Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg; and
Phe, Tyr. Such conservatively substituted variations of each
explicitly disclosed sequence are included within the mosaic
polypeptides provided herein.
[0054] Substitutional or deletional mutagenesis can be employed to
insert sites for N-glycosylation (Asn-X-Thr/Ser) or O-glycosylation
(Ser or Thr). Deletions of cysteine or other labile residues also
may be desirable. Deletions or substitutions of potential
proteolysis sites, e.g., Arg, are accomplished for example by
deleting one of the basic residues or substituting one by
glutaminyl or histidyl residues.
[0055] Certain post-translational derivatizations are the result of
the action of recombinant host cells on the expressed polypeptide.
Glutaminyl and asparaginyl residues are frequently
post-translationally deamidated to the corresponding glutamyl and
asparyl residues. Alternatively, these residues are deamidated
under mildly acidic conditions. Other post-translational
modifications include hydroxylation of proline and lysine,
phosphorylation of hydroxyl groups of seryl or threonyl residues,
methylation of the .omega.-amino groups of lysine, arginine, and
histidine side chains (T. E. Creighton, Proteins: Structure and
Molecular Properties, W. H. Freeman & Co., San Francisco pp
79-86 [1983]), acetylation of the N-terminal amine and, in some
instances, amidation of the C-terminal carboxyl.
[0056] It is understood that one way to define the variants and
derivatives of the disclosed proteins herein is through defining
the variants and derivatives in terms of homology/identity to
specific known sequences. For example, SEQ ID NO:1 sets forth a
particular sequence of a lipase. Specifically disclosed are
variants of these and other proteins herein disclosed which have at
least about 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,
64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80,
81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,
98, or 99 homology to the stated sequence. Those of skill in the
art readily understand how to determine the homology of two
proteins. For example, the homology can be calculated after
aligning the two sequences so that the homology is at its highest
level.
[0057] Another way of calculating homology can be performed by
published algorithms. Optimal alignment of sequences for comparison
may be conducted by the local homology algorithm of Smith and
Waterman, Adv. Appl. Math. 2:482, 1981, by the homology alignment
algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443, 1970, by
the search for similarity method of Pearson and Lipman, Proc. Natl.
Acad. Sci. U.S.A. 85:2444, 1988, by computerized implementations of
these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin
Genetics Software Package, Genetics Computer Group, 575 Science
Dr., Madison, Wis.), or by inspection.
[0058] The same types of homology can be obtained for nucleic acids
by, for example, the algorithms disclosed in Zuker, Science
244:48-52, 1989, Jaeger et al., Proc. Natl. Acad. Sci. U.S.A.
86:7706-7710, 1989, Jaeger et al., Methods Enzymol. 183:281-306,
1989, which are herein incorporated by reference for at least for
their material related to nucleic acid alignment.
[0059] It is understood that the description of conservative
mutations and homology can be combined together in any combination,
such as embodiments that have at least 70% homology to a particular
sequence wherein the variants are conservative mutations.
[0060] As this specification discusses various proteins and protein
sequences it is understood that the nucleic acids that can encode
those protein sequences are also disclosed. This would include all
degenerate sequences related to a specific protein sequence, i.e.,
all nucleic acids having a sequence that encodes one particular
protein sequence as well as all nucleic acids, including degenerate
nucleic acids, encoding the disclosed variants and derivatives of
the protein sequences. Thus, while each particular nucleic acid
sequence may not be written out herein, it is understood that each
and every sequence is in fact disclosed and described herein
through the disclosed protein sequence. It is also understood that
while no amino acid sequence indicates what particular DNA sequence
encodes that protein within an organism, where particular variants
of a disclosed protein are disclosed herein, the known nucleic acid
sequence that encodes that protein in the particular strain from
which that protein arises is also known and herein disclosed and
described.
[0061] It is understood that there are numerous amino acid and
peptide analogs which can be incorporated into the disclosed
compositions. For example, there are numerous D amino acids or
amino acids which have a different functional substituent then the
amino acids shown in Table A and Table B. The opposite stereo
isomers of naturally occurring peptides are disclosed, as well as
the stereo isomers of peptide analogs. These amino acids can
readily be incorporated into polypeptide chains by charging tRNA
molecules with the amino acid of choice and engineering genetic
constructs that utilize, for example, amber codons to insert the
analog amino acid into a peptide chain in a site specific way
(Thorson et al, Meth. Mol. Biol. 77:43-73, 1991; Zoller, Curr.
Opinion Biotechnol. 3:348-354, 1992; Ibba, Biotechnol. Gen. Eng.
Rev. 13:197-216, 1995; Cahill et al., TIBS 14 (10):400-403, 1989;
Benner, TIB Tech 12:158-163, 1994; Ibba and Hennecke,
Bio/technology 12:678-682, 1994, all of which are herein
incorporated by reference at least for their material related to
amino acid analogs).
[0062] Molecules can be produced that resemble peptides, but which
are not connected via a natural peptide linkage. For example,
linkages for amino acids or amino acid analogs can include, but are
not limited to, CH.sub.2NH--, --CH.sub.2S--,
--CH.sub.2--CH.sub.2--, --CH.dbd.CH-- (cis and trans),
--COCH.sub.2--, --CH(OH)CH.sub.2--, and --CHH.sub.2SO-- (These and
others can be found in Spatola, in Chemistry and Biochemistry of
Amino Acids, Peptides, and Proteins, B. Weinstein, eds., Marcel
Dekker, New York, p. 267, 1983; Spatola, Vega Data (March 1983),
Vol. 1, Issue 3, Peptide Backbone Modifications (general review);
Morley, Trends Pharm. Sci. 463-468, 1980; Hudson et al., Int. J.
Pept. Prot. Res. 14:177-185, 1979 (--CH.sub.2NH--,
CH.sub.2CH.sub.2--); Spatola et al., Life Sci. 38:1243-1249, 1986
(--CH.sub.2--S); Hann, J. Chem. Soc Perkin Trans. I 307-314, 1982
(--CH.dbd.CH--, cis and trans); Almquist et al., J. Med. Chem.
23:1392-1398, 1980 (--COCH.sub.2--); Jennings-White et al.,
Tetrahedron Lett. 23:2533, 1982 (--COCH.sub.2--); Szelke et al.,
European App. No. EP 45665 CA (1982): 97:39405 (1982)
(--CH(OH)CH.sub.2--); Holladay et al., Tetrahedron. Lett.
24:4401-4404, 1983 (--C(OH)CH.sub.2--); and Hruby, Life Sci.
31:189-199, 1982 (--CH.sub.2--S--); each of which is incorporated
herein by reference. A particularly preferred non-peptide linkage
is --CH.sub.2NH--. It is understood that peptide analogs can have
more than one atom between the bond atoms, such as .beta.-alanine,
.gamma.-aminobutyric acid, and the like.
[0063] Amino acid analogs and analogs and peptide analogs often
have enhanced or desirable properties, such as, more economical
production, greater chemical stability, enhanced pharmacological
properties (half-life, absorption, potency, efficacy, etc.),
altered specificity (e.g., a broad-spectrum of biological
activities), reduced antigenicity, and others.
[0064] D-amino acids can be used to generate more stable peptides,
because D-amino acids are not recognized by peptidases and such.
Systematic substitution of one or more amino acids of a consensus
sequence with a D-amino acid of the same type (e.g., D-lysine in
place of L-lysine) can be used to generate more stable peptides.
Cysteine residues can be used to cyclize or attach two or more
peptides together. This can be beneficial to constrain peptides
into particular conformations. (Rizo and Gierasch, Ann. Rev.
Biochem. 61:387, 1992, which is incorporated herein by
reference).
Food Grade Matrix
[0065] The term "food grade matrix" is any material that can form a
matrix to immobilize an enzyme and that is cleared by the U.S. Food
and Drug Administration as a Secondary Direct Food Additive under
21 C.F.R. .sctn. 173. Sections 5-165 of 21 C.F.R. .sctn. 173
provide representative examples of materials useful as the food
grade matrix as well as permissible amounts of impurities to be
considered a food grade matrix useful herein. For example, the
material used to produce the food grade matrix comprises less than
10%, less than 8%, less than 6%, less than 4%, or less than 2% by
weight non-polymerizable impurities.
[0066] In certain examples, the food grade matrix can comprise an
acrylate-acrylamide resin (173.5), a polyacrylamide resin (173.10),
an ion exchange resin (173.25), a perfluorinated ion exchange
membrane (173.21), an ion exchange membrane (173.20), a molecular
sieve resin (173.40), polymaleic acid or the sodium salt thereof
(173.45), polyvinylpolypyrrolidone (173.50), polyvinylpyrrolidone
(173.55), dimethylamine-epichlorohydrin copolymer (173.60),
chloromethylated aminated styrene-divinylbenzene resin (173.70),
sodium polyacrylate (173.73), or sorbitan monooleate (173.75),
where the number in parenthesis is the federal registration section
number that provides information with respect to the requirements
of the material to be a secondary direct food additive.
[0067] In other examples, the food grade matrix can comprise a
copolymer of divinylbenzene. For example, the food grade matrix can
comprise a copolymer of (1) divinylbenzene and (2) acrylic acid,
methacrylic acid, methyl acrylate, methyl methacrylate, ethyl vinyl
benzene, or styrene. Title 21 C.F.R. .sctn. 173.65 provides the
requirements for the use of divinylbenzene copolymers as a
secondary direct food additive. For example, the divinylbenzene
copolymer must have at least 79 weight percent divinylbenzene and
no more than 4 weight percent nonpolymerizable impurities. Examples
of divinylbenzene copolymers useful herein as food grade matrices
include, but are not limited to, AMBERLITE.TM. XAD16HP,
AMBERLITE.TM. FPX600, AMBERLITE.TM. FPX66, and DUOLITE.TM. A7 all
of which are manufactured by Rohm and Haas (Philadelphia, Pa.).
AMBERLITE.TM. XAD16HP and FPX600 are crosslinked, macroporous
polystyrene/divinylbenzene copolymers.
[0068] In one specific example, the enzyme is Candida antarctica
lipase B and the food grade matrix is AMBERLITE.TM. XAD16HP.
Production of Immobilized Enzymes
[0069] The production of the immobilized enzyme generally involves
admixing the enzyme with the material used to produce the food
grade matrix in a solvent such as, for example, water. In one
aspect, a surfactant is not used in the preparation of the
immobilized enzyme. Immobilization parameters such as pH,
temperature, duration of immobilization, etc. will vary depending
upon the selection of the enzyme and food grade matrix material.
After immobilization is complete, the solution is drained and the
resultant enzyme-matrix can be washed with water or other suitable
solvents. The selection of solvents for the preparation of the
immobilized enzyme should be a material that is compatible with
human consumption. Thus, water is a preferred solvent. After the
enzyme has been immobilized, the enzyme-matrix can be dried under
reduced pressure and/or at elevated temperature, or using other gas
or liquid media (N.sub.2, Ar, oil etc.).
[0070] The enzyme can be immobilized on the matrix via covalent or
no-covalent (e.g., electrostatic, ionic, hydrogen bonding,
adsorption, entrapment, encapsulation, etc.) attachments or bonds
depending upon the selection of the enzyme and food grade matrix,
as well as immobilization conditions. Not wishing to be bound by
theory, it is believed that immobilization of the enzyme on the
matrix renders the enzyme significantly more stable due to the
fixation of the enzyme conformation. If possible, the enzyme should
not leach from the matrix, which will increase the efficiency of
the esterification, transesterification, or
interesterification/intraesterification process as well as prolong
the life of the immobilized enzyme for future use. It is also
desirable that the enzymes attached to the surface of the matrix be
such that the enzymes expose their catalytic center.
[0071] The amount of enzyme immobilized on the matrix can vary and
will depend upon the enzyme and matrix selected and the end-use of
the immobilized enzyme. In one aspect, the amount of enzyme
immobilized in the matrix is from about 7.5 to about 35 KLU (kilo
lipase units) per gram of matrix based on the difference in the
enzyme concentration in the solution used to interact with the
matrix, as measured at 280 nm. In other examples, the amount of
enzyme immobilized on the matrix can be about 7.5, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,
29, 30, 31, 32, 33, 34, or 35 KLU/g matrix, where any value can
form an end-point of a range. In another example, the amount of
enzyme immobilized in the matrix is from about 28 to about 32 KLU/g
matrix.
[0072] The immobilized matrix can be prepared by methods disclosed
herein. In certain examples, the disclosed immobilized matrix can
be prepared on a laboratory, pilot, or manufacturing scale. For
example, the system shown in FIGS. 1 and 3 can be used for the
preparation of pilot and manufacturing scale immobilized enzymes.
Further, more than one bed of enzyme can be used. That is, one can
used processes where more than one fixed enzyme bed reactor
containing the disclosed immobilized enzymes are connected in
serial or parallel. The number of reactors will depend on the
desired amounts of production and starting materials. Various
geometries of enzyme reactors can also be used. Examples of
suitable reactor geometries are shown in FIG. 4.
Use of Immobilized Enzymes
[0073] Described herein are methods for esterifying a carboxylic
acid that comprise reacting the carboxylic acid with an alcohol in
the presence of any of the immobilized enzymes described herein. In
a further aspect, described herein are methods for transesterifying
an ester that comprise reacting the ester with an alcohol in the
presence of any of the immobilized enzymes described herein. In a
still further aspect, described herein are methods for
interesterifying two or more different carboxylic acids or esters
thereof that comprise reacting the carboxylic acids or esters with
each other in the presence of any of the immobilized enzymes
described herein. In yet a still further aspect, described herein
are methods for intraesterifying a compound comprising at least two
ester groups or a compound comprising at least one carboxylic acid
group and one ester group, that comprise contacting the compound
with any of the immobilized enzymes described herein. A schematic
of the transesterification of an ethyl ester (EE) to a triglyceride
or an esterification of a free fatty acid (FFA) to a triglyceride
is shown below.
##STR00003##
[0074] Although the esterification of any carboxylic acid, the
transesterification of any ester, the interesterification of two or
more different carboxylic acids/esters, or the intraesterification
of a compound is contemplated using the methods described herein,
in many examples, a fatty acid or the ester there of can be used in
any of the methods. In certain examples, the ester of the fatty
acid is a C.sub.1-C.sub.6 branched or straight chain alkyl ester
such as, for example, methyl, ethyl, propyl, butyl, pentyl, and the
like.
[0075] In other specific examples, a fatty acid or the ester
thereof can be used in the methods described herein. By "fatty
acid" is meant a carboxylic acid with at least 10 carbon atoms. In
one aspect, the fatty acid or the ester thereof can comprise at
least 10, at least 12, at least 14, at least 16, at least 18, or at
least 20 carbon atoms. In some specific examples, the fatty acid or
the ester thereof can contain 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,
36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 carbon atoms, where any
of the stated values can form an upper or lower endpoint when
appropriate. In other examples, the fatty acid or the ester thereof
can comprise a mixture of fatty acids or the esters thereof having
a range of carbon atoms. For example, the fatty acid or the ester
thereof can comprise from about 10 to about 40, from about 12 to
about 38, from about 14 to about 36, from about 16 to about 34,
from about 18 to about 32, or from about 20 to 30 carbon atoms.
[0076] The fatty acids or esters thereof can be saturated,
unsaturated, or a mixture of saturated and unsaturated fatty acids.
By "saturated" is meant that the molecule or residue contains no
carbon-carbon double or triple bounds. By "unsaturated" is meant
that the molecule or residue contains at least one carbon-carbon
double or triple bond.
[0077] In one specific example, the fatty acids or the esters
thereof can be derived from marine oils, such as fish oil, prior to
esterification. Such oils typically contain mixtures of saturated
and unsaturated fatty acids, but can be processed to result in a
particular mixture of fatty acids (e.g., containing all saturated,
all unsaturated, mixtures of both, or mixtures with fatty acids of
a certain chain length or range of chain lengths). Any fish oil can
be used in the disclosed compounds and methods. Examples of
suitable fish oils include, but are not limited to, Atlantic fish
oil, Pacific fish oil, Mediterranean fish oil, light pressed fish
oil, alkaline treated fish oil, heat treated fish oil, light and
heavy brown fish oil, bonito oil, pilchard oil, tuna oil, sea bass
oil, halibut oil, spearfish oil, barracuda oil, cod oil, menhaden
oil, sardine oil, anchovy oil, capelin oil, Atlantic cod oil,
Atlantic herring oil, Atlantic mackerel oil, Atlantic menhaden oil,
salmonid oil, and shark oil, including mixtures and combinations
thereof. Non-alkaline treated fish oil is also suitable. Other
marine oils suitable for use herein include, but are not limited
to, squid oil, cuttle fish oil, octopus oil, krill oil, seal oil,
whale oil, and the like, including mixtures and combinations
thereof. Any marine oil and combination of marine oil can be used
in the disclosed compositions and in the disclosed methods to
prepare them. Further oils include, microbial oil, algal oil (e.g.,
oil from a dinoflagellate such as Crypthecodinium cohnii), fungal
oil (e.g., oil from Thraustochytrium, Schizochytrium, or a mixture
thereof), and/or plant oil, including mixtures and combinations
thereof.
[0078] Examples of specific saturated fatty acids or esters thereof
useful herein include, but are not limited to, capric acid (C10),
lauric acid (C12), myristic acid (C14), palmitic acid (C16),
margaric acid (C17), stearic acid (C18), arachidic acid (C20),
behenic acid (C22), lignoceric acid (C24), cerotic acid (C26),
montanic acid (C28), and melissic acid (C30), including branched
and substituted derivatives thereof.
[0079] The unsaturated fatty acids or esters thereof suitable for
the methods disclosed herein can comprise at least one unsaturated
bond (i.e., a carbon-carbon double or triple bond). In one example,
the unsaturated fatty acid or ester thereof can comprise at least
2, at least 3, at least 4, at least 5, at least 6, at least 7, at
least 8, at least 9, or at least 10 carbon-carbon double bonds,
triple bonds, or any combination thereof. In another example, the
unsaturated fatty acid or ester thereof can comprise 1, 2, 3, 4, 5,
6, 7, 8, 9, or 10 unsaturated bonds, where any of the stated values
can form an upper or lower endpoint when appropriate.
[0080] In one example, the unsaturated fatty acids or esters
thereof can comprise one carbon-carbon double bond (i.e., a monoene
acid or residue). Examples of unsaturated fatty acids or esters
thereof that are suitable for the methods disclosed herein include,
but are not limited to, those in the following Table 1.
TABLE-US-00003 TABLE 1 Examples of Monoene Acids Total number of
Carbon number where double bond begins. carbon atoms in the ("c"
denotes a cis double bond; "t" denotes a fatty acid chain trans
double bond) 10 4c 12 4c 14 4c and 9c 16 3t, 4c, 5t, 6c, 6t, 9c
(palmitooleic), and 11c 18 3t, 5c, 5t, 6c (petroselinic), 6t, 9c
(oleic), 10c, 11c (cis-vaccenic), 11t (vaccenic), and 13c 20 5c, 9c
(gadolenic), 11c, 13c, and 15c 22 5c, 11c (cetoleic), 13c (erucic),
and 15c 24 15c (selacholeic, nervonic) 26 9c, and 17c (ximenic) 28
9c, 19c (lumequic) 30 21c
[0081] In other examples, the unsaturated fatty acids or esters
thereof can comprise at least two unsaturated bonds (e.g., polyene
acids or residues). In some examples, the unsaturated fatty acids
or esters thereof can comprise at least one pair of methylene
interrupted unsaturated bonds. By "methylene interrupted
unsaturated bond" is meant that one carbon-carbon double or triple
bond is separated from another carbon-carbon double or triple bond
by at least one methylene group (i.e., CH.sub.2). Specific examples
of unsaturated fatty acids or esters thereof that contain at least
one pair of methylene interrupted unsaturated bonds include, but
are not limited to, the n-1 family derived from 9, 12, 15-16:3; n-2
family derived from 9, 12, 15-17:3, 15:3, 17:3, 17:4, 20:4; n-3
family derived from 9, 12, 15-18:3, 15:2, 15:3, 15:4, 16:3, 16:4,
18:3 (.alpha.-linolenic), 18:4, 18:5, 20:2, 20:3, 20:4; 20:5 (EPA),
21:5, 22:3, 22:5 (DPA), 22:6 (DHA), 24:3, 24:4, 24:5, 24:6, 26:5,
26:6, 28:7, 30:5; n-4 family derived from 9, 12-16:2, 16:2, 16:3,
18:2, 18:3; n-5 family derived from 9, 12-17:2, 15:2, 17:2, 17:3,
19:2, 19:4, 20:3, 20:4 21:4, 21:5; n-6 family derived from 9,
12-18:2, 15:2, 16:2, 18:2 (linoleic acid), 18:3 (.gamma.-linolenic
acid); 20:2, 20:3, 20:4 (arachidonic acid), 22:2, 22:3, 22:4
(adrenic acid), 22:5, 24:2, 24:4, 25:2, 26:2, 30:4; n-7 family
derived from 9-16:1, 15:2, 16:2, 17:2, 18:2, 19:2; n-8 family
derived from 9-17:1, 15:2, 16:2, 17:2, 18:2, 19:2; n-9 family
derived from 9-18:1, 17:2, 18:2, 20:2, 20:3, 22:3, 22:4; n-11
family 19:2, and the n-12 family 20:2.
[0082] In the above paragraph, the compounds are identified by
referring first to the "n-x family," where x is the position in the
fatty acid where the first double bond begins. The numbering scheme
begins at the terminal end of the fatty acid where, for example,
the terminal CH.sub.3 group is designated position 1. In this
sense, the n-3 family would be an omega-3 fatty acid, as described
herein. The next number identifies the total number of carbon atoms
in the fatty acid. The third number, which is after the colon,
designates the total number of double bonds in the fatty acid. So,
for example, in the n-1 family, 16:3, refers to a 16 carbon long
fatty acid with 3 double bonds, each separated by a methylene,
wherein the first double bond begins at position 1, i.e., the
terminal end of the fatty acid. In another example, in the n-6
family, 18:3, refers to an 18 carbon long fatty acid with 3
methylene separated double bonds beginning at position 6, i.e., the
sixth carbon from the terminal end of the fatty acid, and so
forth.
[0083] Some other examples are fatty acids or esters thereof that
contain at least one pair of unsaturated bonds interrupted by more
than one methylene group. Suitable examples of these acids and
esters include, but are not limited to, those in the following
Table 2:
TABLE-US-00004 TABLE 2 Examples of Polyene Acids Total number of
Carbon number where double bond begins. carbon atoms in the ("c"
denotes a cis double bond; "t" denotes a trans fatty acid chain
double bond) 18 5, 9 5, 11 2t, 9, 12 3t, 9, 12 5t, 9, 12 5, 9, 12
5, 11, 14 3t, 9, 12, 15 5, 9, 12, 15 20 5, 11 5, 13 7, 11 7, 13 5,
11, 14 7, 11, 14 5, 11, 14, 17 22 5, 11 5, 13 7, 13 7, 15 7, 17 9,
13 9, 15
[0084] Still other examples of unsaturated fatty acids or esters
thereof that are suitable for use in the methods disclosed herein
are those that contain at least one conjugated unsaturated bond. By
"conjugated unsaturated bond" is meant that at least one pair of
carbon-carbon double and/or triple bonds are bonded together,
without a methylene (CH.sub.2) group between them (e.g.,
--CH.dbd.CH--CH.dbd.CH--). Specific examples of unsaturated fatty
acids or esters thereof that contain conjugated unsaturated bonds
include, but are not limited to, those in the following Table
3.
TABLE-US-00005 TABLE 3 Examples of Conjugated Polyene Acids Total
number of Carbon number where double bond begins. carbon atoms in
the ("c" denotes a cis double bond; "t" denotes a trans fatty acid
chain. double bond) 10 2t, 4t, 6c 2c, 4t, 6t 3t, 5t, 7c 3c, 5t, 7t
12 3, 5, 7, 9, 11 14 3, 5, 7, 9, 11 18 10t, 12t 8c, 10t, 12c
(jacaric) 8t, 10t, 12c (calendic) 8t, 10t, 12t 9t, 11t, 13c
(catalpic) 9c, 11t, 13t (.alpha.-eleostearic) 9c, 11t, 13c
(punicic) 9t, 11t, 13t (.beta.-eleostearic) 9c, 11t, 13t, 15c
(.alpha.-parinaric) 9t, 11t, 13t, 15t (.beta.-parinaric)
[0085] Omega-3 fatty acids and esters thereof are also useful in
the methods described herein. Omega-3 fatty acids are unsaturated
fatty acids that are particularly useful in the compounds and
methods disclosed herein. Omega-3 fatty acids not only exhibit
proven effects on lowering serum triglyceride levels, but they have
strong connection to diabetes. For instance, docosahexaenoic acid
(DHA) also has a strong insulin permeability enhancement effect,
and it is viewed as a potential absorption enhancer for intestinal
delivery of insulin (Onuki et al., Int. J. Pharm. 198:147-56,
2000). DHA intake prevents certain biochemical processes that
originate from insulin deficiency (Ovide-Bordeaux and Grynberg, Am.
J. Physiol. Regul. Integr. Comp. Physiol. 286:R519-27, 2003) and
both DHA and EPA (eicosapentaenoic acid) significantly increase
fasting insulin levels (Mori et al., Am. J. Clin. Nutr. 71:1085-94,
2000).
[0086] An omega-3 fatty acid is an unsaturated fatty acid that
contains as its terminus CH.sub.3--CH.sub.2--CH.dbd.CH--. Specific
examples of omega-3 fatty acids and esters thereof that are
suitable for use herein include, but are not limited to, linolenic
acid (18:3.omega.3), octadecatetraenoic acid (18:4.omega.3),
eicosapentaenoic acid (20:5.omega.3) (EPA), docosahexaenoic acid
(22:6.omega.3) (DHA), docosapentaenoic acid (22:6.omega.3) (DPA),
derivatives thereof and mixtures thereof.
[0087] In still other examples, unsaturated fatty acids and esters
thereof can be derived from a compound comprising the following
formula:
##STR00004##
wherein R.sup.1 is a C.sub.3-C.sub.40 alkyl or alkenyl group
comprising at least one double bond. The term "alkane" or "alkyl"
as used herein is a saturated hydrocarbon group. The term "alkene"
or "alkenyl" as used herein is a hydrocarbon group of at least 2
carbon atoms with a structural formula containing at least one
carbon-carbon double bond. Asymmetric structures such as
(AB)C.dbd.C(CD) are intended to include both the E and Z isomers
(cis and trans). This may be presumed in structural formulae herein
wherein an asymmetric alkene is present, or it may be explicitly
indicated by the bond symbol C.dbd.C. In a further example, R.sup.1
can be a C.sub.5-C.sub.38, C.sub.6-C.sub.36, C.sub.8-C.sub.34,
C.sub.10-C.sub.32, C.sub.12-C.sub.30, C.sub.14-C.sub.28,
C.sub.16-C.sub.26, or C18-C.sub.24 alkenyl group. In yet another
example, the alkenyl group of R.sup.1 can have from 2 to 6, from 3
to 6, from 4 to 6, or from 5 to 6 double bonds. Still further, the
alkenyl group of R.sup.1 can have from 1, 2, 3, 4, 5, or 6 double
bonds, where any of the stated values can form an upper or lower
endpoint when appropriate.
[0088] Some specific examples of unsaturated fatty acids and esters
thereof that can be used in the methods disclosed herein include,
but are not limited to, linoleic acid, linolenic acid,
.gamma.-linolenic acid, arachidonic acid, mead acid, stearidonic
acid, .alpha.-eleostearic acid, eleostearic acid, pinolenic acid,
docosadienic acid, docosatetraenoic acid, docosapentaenoic acid,
docosahexaenoic acid, octadecadienoic acid, octadecatrienoic acid,
eicosatetraenoic acid, eicosapentaenoic, or any combination
thereof. In one aspect, the unsaturated fatty acid ester can be
derived from linolenic acid (18:3.omega.3), octadecatetraenoic acid
(18:4.omega.3), eicosapentaenoic acid (20:5.omega.3) (EPA),
eicosatetraenoic acid (20:40.omega.3), henicosapentaenoic acid
(21:5.omega.3), docosahexaenoic acid (22:6.omega.3) (DHA),
docosapentaenoic acid (22:5.omega.3) (DPA), including derivatives
and mixtures thereof.
[0089] Additional examples of suitable unsaturated fatty acid and
esters thereof that are suitable in the methods include, but are
not limited to, allenic and acetylenic acids, such as C14: 2, 4, 5;
C18: 5, 6 (laballenic); 5, 6, 16 (lamenallenic); C18: 6a (tarinic);
9a; 9a, 11t (ximenynic); 9a, 11a; 9a, 11a, 13c (bolekic); 9a, 11a,
13a, 15e, 8a, 10t (pyrulic) 9c, 12a (crepenynic); 9c, 12a, 14c
(dehydrocrepenynic acid); 6a, 9c, 12c; 6a, 9c, 12c, 15c, 8a, 11c,
14c and corresponding .DELTA.17e derivatives, 8-OH derivatives, and
.DELTA.17e, 8-OH derivatives.
[0090] Branched-chain acids and esters thereof, particularly
iso-acids and anteiso acids, polymethyl branched acids, phytol
based acids (e.g., phytanic, pristanic), furanoid acids are also
suitable fatty acids, for use in the methods disclosed herein.
[0091] Still further, suitable fatty acids and esters thereof
include, but are not limited to, cyclic acids, such as cyclopropane
fatty acids, cyclopropene acids (e.g., lactobacillic), sterulic,
malvalic, sterculynic, 2-hydroxysterculic, aleprolic, alepramic,
aleprestic, aleprylic alepric, hydnocarpic, chaulmoogric hormelic,
manaoic, gorlic, oncobic, cyclopentenyl acids, and
cyclohexylalkanoic acids.
[0092] Hydroxy acids and esters thereof, particularly butolic,
ricinoleic, isoricinoleic, densipolic, lesquerolic, and auriolic
are also suitable fatty acids that upon esterification can be used
in the methods disclosed herein.
[0093] Epoxy acids and esters, particularly epoxidated C18:1 and
C18:2, and furanoid acids and esters are further examples that can
be used in the disclosed methods.
[0094] The alcohol used in any of the methods disclosed herein can
be any alcohol. In one example, the alcohol is a polyol, which is
defined as a compound having two or more hydroxyl groups. Examples
of polyols useful herein include, but are not limited to,
pentaerythritol, dipentaerythritol, tripentaerythritol,
tetrapentaerythritol, tris(hydroxymethyl)ethane, or
tris(hydroxymethyl)propane. In other examples, the alcohol is a
sugar such as, for example, a glucosamine, a methyl glucoside, or
other sugars such as, for example, sucrose. In another example, the
polyol is glycerol.
[0095] The amount of carboxylic acid/ester and alcohol used will
vary depending upon the acid, ester, and alcohol selected. In one
example, a stoichiometric amount of carboxylic acid or ester
relative to number of hydroxyl groups present on the alcohol can be
used. For example, if the alcohol is a diol, then two molar
equivalents of carboxylic acid or ester can be esterified or
transesterified, respectively, with one molar equivalent of diol.
An excess of alcohol can be used to achieve maximum esterification
or transesterification as well as decrease the overall reaction
time. In one aspect, when the alcohol is glycerol, the molar ratio
of carboxylic acid or ester to alcohol is from 1:1 to 10:1, from
1:1 to 9:1, from 1:1 to 8:1, from 1:1 to 7:1, from 1:1 to 6:1, from
1:1 to 5:1, from 1:1 to 4:1, or from 1:1 to 3:1.
[0096] The amount of the immobilized enzyme (enzyme and matrix
together) can also vary as well. In one example, the immobilized
enzyme is from 0.1% to 20% by weight of total weight of carboxylic
acid/ester and alcohol. In other examples, the enzyme is 0.1, 0.2,
0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20% by weight of the total
reaction, where any value can form an endpoint of a range.
[0097] The carboxylic acid/ester, the alcohol, and the immobilized
enzyme can be admixed with one another in any order. Depending upon
the selection of the carboxylic acid/ester and the alcohol, it can
be desirable to conduct the esterification or transesterification
while the reaction mixture is stirred. For example, a solution of
ester and alcohol can be added to one another under stirring
followed by the addition of the immobilized enzyme. Also the
reaction mixture could be forced to pass through the bed of
immobilized enzyme and this could be executed either in continuous
or batch (single or recycled) process.
[0098] In certain aspects, the esterification, transesterification,
and interesterification/intraesterification reactions can take
place at an elevated temperature. The precise elevated temperature
can depend on the particular carboxylic acid or ester being used,
the particular alcohol being used, the amount or concentration of
the reagents, preference, and the like. Suitable temperatures at
which the esterification and transesterification reactions can
occur include, but are not limited to, from about 50.degree. C. to
about 100.degree. C., from about 70.degree. C. to about 90.degree.
C., from about 80.degree. C. to about 90.degree. C., or about
85.degree. C. In another example, the esterification temperature
can be from about 60.degree. C. to about 70.degree. C., or about
65.degree. C. By varying the temperature it is possible to reduce
reaction times depending upon the concentration of starting
materials. Thus, reaction times can vary from 2 hours to 72 hours,
10 hours to 48, 10 hours to 36, 10 hours to 24 hours, 15 hours to
24 hours, 20 hours to 24 hours, or 22 hours.
[0099] In other examples, the method involves esterifying
eicosapentaenoic acid 20:5.omega.3 (EPA), docosahexaenoic acid
22:6.omega.3 (DHA), docosapentaenoic acid 22:5.omega.3 (DPA), or
any mixture thereof with glycerol, wherein the acid and the alcohol
are present in a molar ratio of from about 2:1 to about 5:1,
wherein the reaction is stirred in the presence of the immobilized
enzyme at a temperature of from about 60.degree. C. to about
90.degree. C. for about 2 hours to about 24 hours, wherein the
immobilized enzyme comprises an enzyme derived from Candida
antarctica immobilized in a food grade matrix comprising a
copolymer of divinylbenzene and styrene.
[0100] In another aspect, the method involves transesterifying an
ethyl ester of eicosapentaenoic acid 20:5.omega.3 (EPA),
docosahexaenoic acid 22:6.omega.3 (DHA), docosapentaenoic acid
22:5.omega.3 (DPA), or any mixture thereof with glycerol, wherein
the ester and the alcohol are present in a molar ratio of from
about 2:1 to about 5:1, wherein the reaction is stirred in the
presence of the immobilized enzyme at a temperature of from about
70.degree. C. to about 90.degree. C. for about 20 hours to about 24
hours, wherein the immobilized enzyme comprises an enzyme derived
from Candida antarctica immobilized in a food grade matrix
comprising a copolymer of divinylbenzene and styrene.
[0101] As discussed above, esterification and transesterification
enzymes can be expensive. Thus, ways to reuse the enzyme would be
of commercial significance. The immobilized enzymes described
herein can be reused several times. For example, after the
esterification, transesterification, or interesterification
reaction is complete, the solution can be filtered off and the
immobilized enzyme can be and reused. The immobilized enzyme can be
washed optionally with a suitable solvent and water, which can be
desirable depending upon the selection of the ester and alcohol
used. For example, if the acid/ester and/or alcohol clog the
immobilized enzyme, then it would be desirable to wash off the
ester or alcohol in order to increase the efficiency of the
immobilized enzyme. In one example, water can be used to wash the
immobilized enzyme. For the purpose of storage, the product could
be stored in the presence of food preservatives, e.g., sodium
benzoate, potassium sorbate, etc.
[0102] The methods described herein are efficient with respect to
producing esters from carboxylic acids or other esters. For
example, after the esterification, transesterification, or
interesterification/intraesterification reaction is complete, the
yield of ester relative to starting material is from about 70 to
about 100%. In addition, because food grade matrices are used, the
resultant transesters produced by the methods herein described do
not require additional purification steps, do not produce unwanted
side-products, and can be readily incorporated into food products,
nutraceuticals, and pharmaceutical formulations, which are
additional advantages of the disclosed methods and compositions.
Still further, using the disclosed enzymatic methods to produce
triglycerides of fatty acids, which are often more bioavailable
than corresponding ethyl esters, can result in triglycerides that
have better color and lower levels of trans-isomers, CDs, polymers,
and side products. The disclosed methods can also result in
esterified, transesterified, interesterified, and/or
intraesterified products that have better sensory properties.
EXAMPLES
[0103] The following examples are set forth below to illustrate the
methods and results according to the disclosed subject matter.
These examples are not intended to be inclusive of all aspects of
the subject matter disclosed herein, but rather to illustrate
representative methods and results. These examples are not intended
to exclude equivalents and variations of the present invention,
which are apparent to one skilled in the art.
[0104] Efforts have been made to ensure accuracy with respect to
numbers (e.g., amounts, temperature, etc.) but some errors and
deviations should be accounted for. Unless indicated otherwise,
parts are parts by weight, temperature is in .degree. C. or is at
ambient temperature, and pressure is at or near atmospheric. There
are numerous variations and combinations of reaction conditions,
e.g., component concentrations, desired solvents, solvent mixtures,
temperatures, pressures and other reaction ranges and conditions
that can be used to optimize the product purity and yield obtained
from the described process. Only reasonable and routine
experimentation will be required to optimize such process
conditions.
[0105] X03020EE (30% EPA/20% DHA ethyl esters), XO0355EE (3%
EPA/55% DHA ethyl esters), EE4020 (40% EPA/20% DHA ethyl esters),
FFA4020 (40% EPA/20% DHA free fatty acids) were obtained from Ocean
Nutrition Canada, Mulgrave Nova Scotia. The starting ethyl esters
X03020EE and XO0355EE should be stored in airtight container with
Teflon lining under nitrogen. Glycerol was at a purity of 99.5%
purity. In Examples 2-5, NOVOZYME.TM. 435 manufactured by NOVOZYMES
(Bagsvaerd, Denmark) was used. In the following examples, reactions
with NOVAZYM.TM. 435 are considered comparative.
Example 1
Preparation of Immobilized Enzyme
[0106] The food grade matrix AMBERLITE.TM. XAD16HP (10 g, about 60%
moisture) manufactured Rohm and Haas, was washed with water and
suspended in 15 mL of solution containing 0.1% sorbic acid and 0.1%
benzoic acid, pH 4.8 (prepared by mixing benzoic acid 1 g, sorbic
acid 1 g, sodium hydroxide and distilled water (998 g)). A solution
of Candida antarctica lipase B (30 mL at a 4.5:1 w/w ratio to
matrix) was added and the mixture was gently stirred for 18 h at
20.degree. C. The liquid was drained, the solid material washed
with water, and finally dried at room temperature under vacuum at a
pressure of less than 1 Torr for 16 hours.
[0107] Further immobilized enzymes were prepared as just described
except that AMBERLITE.TM. FPX66 was used as the food grade
matrix.
Example 2
Analysis of Esterification/Transesterification
[0108] These immobilized enzymes produced in Example 1 were tested
to determine their ability to esterify fatty acids and to
transesterify ethyl esters into triglycerides. The immobilized
enzymes were loaded into a lab scale reaction vessel and the fatty
acid or ethyl ester and alcohol were added to the immobilized
enzyme. The amounts of triglyceride (TG) as well as di- and
mono-glyceride (DG and MG, respectively) produced were analyzed.
Lipid class was measured by normal phase high performance liquid
chromatography (HPLC) coupled with evaporative light scattering
detector (ELSD) using a YMC pack PVA-Sil column (150.times.4.6 mm,
5.mu.) or a Waters Spherisorb S3CN column (150.times.2 mm). The
results are shown in Tables 4-7.
TABLE-US-00006 TABLE 4 FFA to TG conversion with enzyme immobilized
on AMBERLITE .TM. XAD16HP at 65-70.degree. C. for 22 h. Components
after reaction (%) Trial FFA TG DG MG 1 4.3 91.0 4.7 0.0 2 4.8 91.7
3.5 0.0 3 3.5 93.1 3.5 0.0 4 3.7 92.9 3.4 0.0
TABLE-US-00007 TABLE 5 EE to TG conversion with enzyme immobilized
on AMBERLITE .TM. XAD16HP for 24 hours Components after reaction
(%) Trial Temp. EE TG DG MG 1 65-70.degree. C. 9.9 77.0 12.5 0.6 2
75-80.degree. C. 10.0 78.0 11.5 0.5 3 75-80.degree. C. 8.1 83.8 7.7
0.4 4 75-80.degree. C. 10.0 82.8 7.0 0.2
TABLE-US-00008 TABLE 6 FFA to TG conversion with enzyme immobilized
on AMBERLITE .TM. FPX66 at 65-70.degree. C. for 22 hours Components
after reaction (%) Trial FFA TG DG MG 1 11.9 78.6 9.6 0.0 2 9.0
86.7 4.3 0.0 3 8.5 87.0 4.6 0.0
TABLE-US-00009 TABLE 7 EE to TG conversion with enzyme immobilized
on AMBERLITE .TM. FPX66 at 75-80.degree. C. for 23-24 hours
Components after reaction (%) Trial EE TG DG MG 1 1.7 96.9 1.4 0.0
2 2.4 96.1 1.4 0.0 3 0.8 98.5 0.7 0.0 4 2.3 96.1 1.6 0.0
[0109] In further manufacturing scale example, the immobilized
enzymes were loaded into fixed enzyme bed reactor and the fatty
acid or ethyl ester and alcohol were pumped into the immobilized
enzyme.
Example 3
Preparation of Large Scale Immobilized Enzyme
[0110] A large scale reactor system as shown in FIG. 1 was used.
LIPOZYME.TM. CALB and distilled water were added to the holding
vessel. AMBERLITE.TM. FPX66 food grade matrix was added to the
fixed-bed enzyme reactor(s) (4000 kg and batch 8000 kg batches were
run). The liquid enzyme mixture was recirculated through the matrix
bed in the enzyme reactor for 24 hours or until protein samples
leveled out and became consistent. The remaining enzyme solution
was returned to the holding vessel. Filtered water was then pumped
through the enzyme reactors to wash the immobilized enzymes. The
remaining liquid enzyme was removed from the holding vessel.
Example 4
Large Scale Conversion of FFA to Triglycerides
[0111] FFA fish oil was added to the holding vessel of the system
described in Example 3. The holding vessel was heated to 70.degree.
C..+-.5.degree. C. While heating the vessel, agitation was started
and glycerol was added. Once the addition of glycerol was complete,
the contents of the holding vessel were agitated for 15 minutes.
The enzyme reactor(s) containing the immobilized enzyme solution
were prepared as in Example 3, and these can be re-used for several
enzymatic conversions. The conversion cycle sequence began at this
point for 24 hours. During this period, samples were taken and
tested for % FFA every 3 hours. The conversion was complete when
the % FFA was Max 15%. The oil was then pumped out and transferred
to a feed tank where it awaited to undergo FFA stripping in an
evaporation tank.
Example 5
Assays of Immobilized Enzyme Performance
[0112] Various other immobilized enzymes were prepared as described
in Examples 1 and 3. These immobilized enzymes, as well as the
immobilized enzyme produced in Examples 1 and 3, were tested to
determine their ability to esterify fatty acids. The immobilized
enzyme was loaded into a fixed enzyme bed reactor and the fatty
acid and alcohol were pumped through the immobilized enzyme. The
amounts of triglyceride (TG) as well as di- and mono-glyceride (DG
and MG, respectively) produced were analyzed. The results are shown
in Tables 8-10.
TABLE-US-00010 TABLE 8 (Comparative Example) Conversion of FFA 4020
to triglyceride (TG) with 2% immobilized NOVOZYME .TM. CALB L on
XAD-1180 Reaction conditions: ~3:1 FFA 4020 to glycerol at
65-70.degree. C. for 21 hours No. of Immobilized Enzyme Components
after reaction % (average) Trials (enzyme:matrix) FFA TG DG MG 2
3:1 lyophilized 21.0 73.5 3.60 2.00 2 3:1 dried at 50.degree. C.
11.6 85.6 2.35 0.55 2 1.5:1 dried 17.8 65.9 16.3 0.00 2 1:1 dried
12.9 78.5 8.60 0.00 7 3:1 7.63 86.4 5.34 0.16 11 5:1 6.69 87.3 5.01
1.41
TABLE-US-00011 TABLE 9 (Comparative Example) Conversion of EE4020
to triglyceride (TG) with 2% immobilized NOVOZYME .TM. CALB L on
XAD-1180 Reaction conditions: ~3:1 EE4020 to glycerol at
80-85.degree. C. for 22 hours No. of Immobilized Enzyme Components
after reaction % (average) Trials (enzyme:matrix) FFA TG DG MG 1
5:1 11.7 62.4 25.4 0.5 4 1.5:1 39.3 6.03 43.8 10.7
TABLE-US-00012 TABLE 10 Conversion of FFA 4020 to triglyceride (TG)
with 2% immobilized NOVOZYME .TM. CALB L on various matrixes
Reaction conditions: ~3:1 FFA4020 to glycerol at 65-70.degree. C.
for 22 hours Enzyme/ Enzyme/ water wet beads No. of Components
after reaction % (average) Matrix (W/W) (W/W) Trials FFA TG DG MG
XAD16HP 1.5:1 3:1 4 6.9 87.88 4.78 0.00 XAD761 2:1 3:1 2 18.2 59.8
22.1 0.05 XAD4 2.25:1 3:1 1 76.0 0.9 10.4 10.5 Comparative Examples
XAD16 2:1 3.5:1 1 4.3 94.5 1.2 0 XAD16 2.25:1 3:1 4 19.7 63.6 16.0
0.56 XAD16 2:1 2.67:1 2 23.7 49.9 24.7 0.95 XAD16 2.5:1 3.33:1 2
27.3 41.1 28.9 2.30 XAD16 3:1 3.33:1 1 18.8 71.8 9.40 0.00 XAD16
(5% 2.3:1 3:1 2 91.0 0.15 6.55 2.60 KCl washed) XAD16 (1% 23:1 3:1
2 73.0 0.50 19.2 7.40 KCl washed) XAD1180 2.25:1 3:1 2 7.7 92.4
1.65 0.2 KO.sub.2CCH.sub.3 20% 1 51.3 0.5 38.0 10.1 NOVOZYME .TM.
435 1 8.00 90.7 1.20 0.00
Example 6
(Comparative Example) Transesterification Using NOVOZYM.TM. 435
[0113] The oil XO0355 EE (55 g), together with glycerol (5 g), was
placed in a reaction vessel and NOVOZYME.TM.435 (1.2 g) was added.
A vacuum (<1 mbar) was applied and the temperature was raised to
85.degree. C. The reaction mixture was stirred at this temperature
and pressure for 22 hours, then the product was separated from the
immobilized enzyme and the residual immobilized enzyme was ready to
be used in the next batch.
Example 7
Effect of Varying Reaction Conditions
[0114] Reactivity of ethyl ester fish oil was investigated under
varying reaction conditions (Table 11). The higher ratios of ethyl
ester to glycerol (e.g., 4.5-1) were tested to confirm previous
findings. The reaction was repeated with EE/glycerol ratios of
3.5:1 but only at temperatures around 60.degree. C. with 61% yield
of TGs and 25% EEs remaining in average. A molar ratio: 2.8:1 after
22 hours at 70.degree. C. led to the final mixture consisting of EE
6.3%, TG 78.6%, DG 12.2%, MG 2.8%, at 60-65.degree. C. However, the
yields were considerably lower in two other separate experiments,
when the temperature was lower.
TABLE-US-00013 TABLE 11 (Comparative) Transesterification of ethyl
ester concentrates to the corresponding triglycerides with 2%
NOVOZYME .TM. 435. EE/ Glycerol Components after reaction % molar
Incubation Time (average) ratio temperature (total) TG DG MG EE %
TG* R1- 56-60.degree. C. 22 h 59.1 12.6 1.5 26.8 80.7 3.5:1**
(mostly 56.degree.) R2-3.5:1 65.degree. C. 22 h 67.1 4.9 0.6 27.4
92.4 R4-3.5:1 58-60.degree. C. 22 h 44.1 19.4 3.7 32.8 65.6 (mostly
58.degree.) R5-3.5:1 6-65.degree. C. 22 h 63.2 15.9 1.9 19.1 78.0
(mostly 65.degree.) 2.3:1 62-64.degree. C. 22 h 53.6 34.6 10.4 1.3
54.4 2.8:1 60-65.degree. C. 22 h 70.3 20.5 3.8 5.4 74.3 2.8:1
70.degree. C. 22 h 78.6 12.2 2.8 6.3 84.0 2.8:1 58-60.degree. C. 22
h 37.2 23.3 5.2 34.3 56.1 3.2:1 52-62.degree. C. 22 h 51.0 20.6 2.0
26.4 69.3 (~8-10 h at 52.degree. C.) 3.3:1 60.degree. C. 22 h 35.6
1.3 0 63.2 96.4 3.3:1 60-65.degree. C. 7 h 22.7 9.2 1.0 67.1 69.0
3.5:1 ~65-70.degree. C. 22 h 70.6 7.1 1.9 20.4 88.6 3.8:1
48-63.degree. C. 22 h 59.4 4.7 1.6 34.3 90.4 (mostly 48.degree.)
4.5:1 ~55.degree. C. 22 h 43.5 14.1 2.0 39.9 72.9 4.5:1
58-60.degree. C. 22 h 56.3 8.5 0.9 34.4 85.7 4.5:1 60-62.degree. C.
22 h 65.9 2.5 0.5 31.1 95.6 4.5:1 62-64.degree. C. 22 h 67.7 2.1
0.5 29.7 96.3 4.5:1 60-63.degree. C. 22 h 40.5 13.2 1.6 44.6 73.2
4.5:1 58-60.degree. C. 22 h 1.2 0.6 3.1 95.1 24.5 4.5:1
59-64.degree. C. 20 h 51.7 8.0 0.9 39.4 85.3 (mostly 59.degree.)
4.5:1 65.degree. C. 22 h 69.2 1.5 0.3 29.1 97.5 4.5:1 60-62.degree.
C. 22 h 58.0 27.5 5.1 9.4 64.0 4.5:1 60-65.degree. C. 8 h 43.6 7.3
1.0 48.2 84.0 5.5:1 52-62.degree. C. 22 h 46.4 5.4 0.9 47.3 88.0
(mostly 52.degree.) 6.2:1 60.5.degree. C. 22 h 26.0 13.1 1.2 59.7
64.5 6.7:1 60.degree. C. 5 h 15.2 25.7 7.5 51.6 31.4 7:1
55-60.degree. C. 22 h 1.3 1.1 2.8 94.9 25.0 (mostly 55.degree.)
9.0:1 60-62.degree. C. 22 h 45.4 12.4 2.0 40.2 75.9 ~2.8:1 55-67 22
h 66.3 19.6 4.1 10.1 ~2.8:1 70-75 22 h 69.5 21.5 4.1 4.9 ~2.8:1
70-75 7 h 14.0 25.6 8.1 52.3 ~2.8:1 70-75 7 h 5.9 26.5 12.8 54.8
~2.8:1 50-62 22 h 53.3 22.3 5.6 18.7 ~2.8:1 55-63 22 h 50.7 27.1
7.0 15.2 ~2.8:1 55-65 22 h 47.3 29.8 6.9 16.0 ~2.8:1 55-67 22 h
53.3 27.9 6.4 12.3 ~2.8:1 50-65 22 h 40.1 29.3 9.2 21.4 ~2.8:1
55-67 22 h 59.6 25.8 5.9 8.7 ~2.8:1 55-65 22 h 45.6 29.7 7.2 17.5
~2.8:1 55-65 22 h 42.0 32.6 8.1 17.3 ~2.8:1 55-65 22 h 47.6 29.7
6.5 16.2 ~2.8:1 55-65 22 h 51.8 28.4 5.2 14.6 ~2.8:1 55-65 22 h
54.3 29.1 6.0 10.6 ~2.8:1 55-65 22 h 51.3 28.2 6.3 14.2 ~2.8:1
55-65 22 h 46.1 29.2 6.2 18.5 ~2.8:1 55-65 22 h 46.7 27.5 6.3 19.4
~2.8:1 70-75 3 h 7.7 28.2 7.9 56.2 ~2.8:1 70-75 22 h 79.2 15.8 2.2
2.8 ~2.8:1 70-75 27 h 76.6 18.1 2.4 2.9 ~2.8:1 70-75 44 h 79.2 16.9
1.9 2.1 ~2.8:1 70-75 48 h 79.2 16.7 1.8 2.3 ~2.8:1 70-75 51 h 79.7
16.9 1.6 1.8 ~2.8:1 70-75 3 h 18.9 38.6 9.9 32.6 ~2.8:1 70-75 22 h
70.1 19.4 3.4 7.1 ~2.8:1 70-75 27 h 70.4 19.8 3.3 6.5 ~2.8:1 70-75
44 h 75.4 16.8 3.7 4.1 ~2.8:1 70-75 48 h 74.6 18.8 2.6 4.0 ~2.8:1
70-75 51 h 74.6 19.0 2.5 3.9 ~2.8:1 75-80 22 h 80.5 13.0 2.8 3.7
~2.8:1 75-80 27 h 78.8 14.2 3.9 3.1 ~2.8:1 75-80 45 h 80.7 13.9 3.0
2.5 ~2.8:1 75-80 48 h 79.3 16.6 1.6 2.5 ~3.0:1 70-75 22 h 78.8 12.5
1.0 7.6 ~3.0:1 70-80 22 h 84.1 11.6 0.7 3.6 ~3.0:1 70-80 22 h 74.1
15.9 1.9 8.1 ~3.0:1 70-80 22 h 70.3 18.6 2.4 8.6 ~3.0:1 70-80 22 h
72.5 19.5 2.3 5.7 ~3.0:1 70-80 22 h 81.2 15.7 1.4 1.7 ~3.0:1 75 3 h
29.0 30.9 2.5 35.2 ~3.0:1 75 22 h 74.7 13.9 2.5 8.9 ~3.0:1 75 27 h
75.4 14.8 2.1 7.7 ~3.0:1 75 44 h 82.9 11.5 1.3 4.3 ~3.0:1 75 48 h
83.5 11.7 0.7 4.0 ~3.0:1 75 3 h 46.7 26.8 6.2 20.2 ~3.0:1 75 22 h
69.9 18.9 2.9 8.2 ~3.0:1 75 27 h 71.2 19.4 2.4 7.0 ~3.0:1 75-95 17
h 83.5 11.6 0.9 4.1 ~3.0:1 75-80 17 h 64.5 17.9 2.0 15.5 ~3.0:1
75-80 17 h 60.4 25.1 3.0 11.5 ~3.0:1 75-80 17 h 42.6 30.8 4.3 22.3
~3.0:1 75-80 17 h 58.4 25.3 3.1 13.3 ~3.0:1 80-95 17 h 80.9 12.9
2.1 4.1 ~3.0:1 80-95 22 h 81.8 13.8 0.8 3.6 ~3.0:1 80-85 17 h 75.2
15.2 1.8 7.8 ~3.0:1 80-85 17 h 71.4 20.1 1.9 6.5 ~3.0:1 80-85 17 h
71.3 20.9 1.8 6.0 ~3.0:1 80-85 17 h 62.9 23.2 2.3 11.7 ~3.0:1 80-85
17 h 11.1 0.0 0.0 88.9 ~3.0:1 80-85 17 h 47.3 30.9 3.6 18.1 ~3.0:1
80-85 22 h 74.5 17.7 1.3 6.6 ~3.0:1 90-95 20 h 52.3 28.6 3.8 15.3
~3.0:1 80-95 17 h 73.1 17.1 1.1 8.7 ~3.0:1 80-95 17 h 75.2 21.9 1.7
1.3 ~3.0:1 80-95 17 h 78.4 18.6 1.1 1.9 ~3.0:1 80-90 17 h 65.3 22.1
1.6 11.0 ~3.0:1 80-95 17 h 72.4 28.6 3.8 3.2 ~3.0:1 80-95 17 h 73.9
22.2 1.3 2.6 ~3.0:1 80-112 17 h 73.3 23.2 1.5 2.0 ~3.0:1 75-90 17 h
58.8 27.9 3.0 10.3 ~3.0:1 80-95 17 h 73.5 18.4 0.8 7.3 ~3.0:1
80-104 17 h 49.6 31.5 2.5 16.4 ~3.0:1 80-90 17 h 32.1 40.0 3.1 24.8
~3.0:1 78-90 17 h 12.4 49.9 3.7 34.0 ~3.0:1 80-90 17 h 4.9 56.9 1.8
36.5 (EE40/20) ~3.0:1 80-90 17 h 1.4 32.6 17.9 48.0 (EE40/20)
~3.0:1 80-92 17 h 1.3 19.5 21.9 57.3 (EE40/20) ~3.0:1 80-90 17 h
1.4 5.9 10.3 82.4 (EE40/20)
[0115] Summary of Results
[0116] The recommended reaction temperature is 85.degree. C. The
amount of 2% of the enzyme of the reaction mixture mass provided
good results and is recommended. However, several experiments
indicated that even 1% could be used successfully. Previous
experiments indicated that long reaction times (approximately 22 h)
provided better results, while shorter periods (e.g., 7 h) seemed
to lead to poorer conversions. Conversions at the higher
temperatures may require less time. It was observed that lower
temperatures produced lower degrees of conversion, e.g., when the
temperature dropped during the night to 50.degree. C. (probably due
to the fluctuations in the power grid and overnight lowering room
temperature). Initially, temperatures around 60.degree. C. were
employed. There were temperature fluctuations overnight, and in
many cases the temperature was reduced at least to 55.degree. C.
The conversions were clearly better at temperatures between
70-75.degree. C. It should be born in mind, that relative initial
activity of the enzyme reaches peak around 75.degree. C.
[0117] The immobilized enzyme was reused 5 times when 3.5:1
EE/glycerol ratio was used in the presence of 2% of the enzyme. No
solvent (e.g., hexane) was used to wash the enzyme for each
reaction. Since the enzyme was not washed, there may be a case when
the enzyme gets blocked by a component of the reaction mixture.
[0118] Throughout this application, various publications are
referenced. The disclosures of these publications in their
entireties are hereby incorporated by reference into this
application.
[0119] It will be apparent to those skilled in the art that various
modifications and variations can be made in the present invention
without departing from the scope or spirit of the invention. Other
embodiments of the invention will be apparent to those skilled in
the art from consideration of the specification and practice of the
invention disclosed herein. It is intended that the specification
and examples be considered as exemplary only, with a true scope and
spirit of the invention being indicated by the following
claims.
* * * * *