U.S. patent application number 16/258561 was filed with the patent office on 2019-05-23 for thermolysin for easy-cleaning of insect body stains.
The applicant listed for this patent is Toyota Motor Corporation, Toyota Motor Engineering & Manufacturing North America, Inc.. Invention is credited to Masahiko Ishii, Hongfei Jia, Songtao Wu, Minjuan Zhang.
Application Number | 20190153360 16/258561 |
Document ID | / |
Family ID | 62065507 |
Filed Date | 2019-05-23 |
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United States Patent
Application |
20190153360 |
Kind Code |
A1 |
Wu; Songtao ; et
al. |
May 23, 2019 |
Thermolysin For Easy-Cleaning Of Insect Body Stains
Abstract
A liquid coating material and processes of its use are provided
that includes an enzyme with enzymatic activity toward a component
of a biological stain and a polymeric material. Also provided are
processes for facilitating the removal of a biological stain
wherein an inventive liquid coating material including an enzyme is
capable of enzymatically degrading of one or more components of the
biological stain to facilitate biological stain removal from a
substrate or coating upon which the biological stain is
contacting.
Inventors: |
Wu; Songtao; (Ann Arbor,
MI) ; Jia; Hongfei; (Ann Arbor, MI) ; Ishii;
Masahiko; (Okazaki City Aichi, JP) ; Zhang;
Minjuan; (Ann Arbor, MI) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Toyota Motor Engineering & Manufacturing North America,
Inc.
Toyota Motor Corporation |
Plano
Toyota-shi |
TX |
US
JP |
|
|
Family ID: |
62065507 |
Appl. No.: |
16/258561 |
Filed: |
January 26, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15810700 |
Nov 13, 2017 |
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16258561 |
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15193242 |
Jun 27, 2016 |
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15810700 |
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13567341 |
Aug 6, 2012 |
9388370 |
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15193242 |
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12820101 |
Jun 21, 2010 |
8796009 |
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13567341 |
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14812087 |
Jul 29, 2015 |
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15810700 |
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13229277 |
Sep 9, 2011 |
9121016 |
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14812087 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C11D 3/38627 20130101;
C11D 3/044 20130101; C11D 7/261 20130101; C11D 3/2003 20130101;
C08G 18/6225 20130101; C11D 11/0035 20130101; C08G 18/792 20130101;
C11D 3/38618 20130101; C11D 7/04 20130101; C11D 3/04 20130101; C11D
3/37 20130101; C09D 175/04 20130101; C11D 3/386 20130101; C09D
175/04 20130101; C08L 89/00 20130101 |
International
Class: |
C11D 3/386 20060101
C11D003/386; C11D 3/04 20060101 C11D003/04; C11D 11/00 20060101
C11D011/00; C11D 7/26 20060101 C11D007/26; C11D 3/20 20060101
C11D003/20; C11D 3/37 20060101 C11D003/37; C11D 7/04 20060101
C11D007/04 |
Claims
1. A method of facilitating the removal of a biological stain on a
substrate or a coating comprising: providing a liquid coating
material; incorporating a polymer within the liquid coating
material; and incorporating an enzyme into said liquid coating
material to form a liquid bioactive coating material such that said
enzyme is associable with the polymer and is capable of
enzymatically degrading a component of a biological stain.
2. The method of claim 1 wherein the enzyme is a hydrolase.
3. The method of claim 1 wherein the enzyme is a lipase, a
protease, or an amylase.
4. The method of claim 1 wherein the enzyme is a protease with a
specific activity in excess of 20,000 U/g.
5. The method of claim 1 wherein the pH of the liquid coating
material is greater than 8.0.
6. The method of claim 1 wherein said enzyme is in solution in said
liquid coating material.
7. The method of claim 1 wherein said liquid coating material
comprises an alcohol, said alcohol present at less than 0.8% by
weight.
8. The method of claim 1 wherein said liquid coating material
comprises ammonia, or a derivative of ammonia.
9. The method of claim 1 further comprising applying said bioactive
coating material to a biological stain, thereby promoting removal
of said stain.
10. The method of claim 9 wherein said biological stain is a food
stain or an insect stain.
11. The method of claim 9 wherein said biological stain is present
on a substrate.
12. The method of claim 1 wherein the polymer is one or more of
aminoplasts, melamine formaldehydes, carbamates, polyurethanes,
polyacrylates, epoxies, polycarbonates, alkyds, vinyls, polyamides,
polyolefins, phenolic resins, polyesters, or polysiloxanes.
13. The method of claim 1 wherein the polymer includes a functional
group of acetoacetate, acid, amine, carboxyl, epoxy, hydroxyl,
isocyanate, silane, vinyl, or combinations thereof.
14. The method of claim 1 wherein the enzyme is non-covalently
attached to the polymer.
15. A liquid composition for facilitating biological stain removal
comprising: a liquid coating material; a polymer incorporated into
the liquid coating material; and an enzyme incorporated into the
liquid coating material and not covalently bound to the
polymer.
16. The method of claim 15 wherein the enzyme is a lipase, a
protease, or an amylase.
17. The method of claim 15 wherein the enzyme is a protease with a
specific activity in excess of 20,000 U/g.
18. The composition of claim 15 wherein the pH of the sprayable
composition material is greater than 5.0.
19. The composition of claim 15 wherein said enzyme is in solution
in said liquid bioactive coating material.
20. The composition of claim 15 wherein said liquid bioactive
coating material comprises an alcohol, said alcohol present at less
than 0.8% by weight.
21. The composition of claim 15 wherein said liquid bioactive
coating material is predominantly ammonia, or a derivative of
ammonia.
22. The composition of claim 15 wherein the polymer is one or more
of aminoplasts, melamine formaldehydes, carbamates, polyurethanes,
polyacrylates, epoxies, polycarbonates, alkyds, vinyls, polyamides,
polyolefins, phenolic resins, polyesters, or polysiloxanes.
23. The composition of claim 15 wherein the polymer includes a
functional group of acetoacetate, acid, amine, carboxyl, epoxy,
hydroxyl, isocyanate, silane, vinyl, or combinations thereof.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 15/810,700 filed Nov. 13, 2017, which is a continuation-in-part
of U.S. patent application Ser. No. 14/812,087 filed on Jul. 29,
2015, which is a continuation of U.S. patent application Ser. No.
13/229,277 filed Sep. 9, 2011 (now U.S. Pat. No. 9,121,016), and
U.S. application Ser. No. 15/810,700 is also a continuation-in-part
of U.S. patent application Ser. No. 15/193,242 filed on Jun. 27,
2016, which is a continuation of U.S. patent application Ser. No.
13/567,341 filed Aug. 6, 2012 (now U.S. Pat. No. 9,388,370), which
in turn is a continuation-in-part of U.S. patent application Ser.
No. 12/820,101, filed Jun. 21, 2010 (now U.S. Pat. No. 8,796,009),
the entire contents of each of which are incorporated herein by
reference.
FIELD
[0002] The present invention relates generally to coating
compositions including bioactive substances and methods of their
use to facilitate removal of organic stains.
BACKGROUND
[0003] Many outdoor surfaces are subject to stain or insult from
natural sources such as bird droppings, resins, and insect bodies.
As a result, the resulting stain often leaves unpleasant marks on
the surface deteriorating the appearance of the products.
[0004] Traditional self-cleaning coatings and surface are typically
based on water rolling or sheeting to carry away inorganic
materials. These show some level of effectiveness for removal of
inorganic dirt, but are less effective for cleaning stains from
biological sources, which consist of various types of organic
polymers, fats, oils, and proteins each of which can deeply diffuse
into the subsurface of coatings. Prior art approaches aim to reduce
the deposition of stains on a surface and facilitate its removal
capitalize on the "lotus-effect" where hydrophobic, oleophobic and
super-amphiphobic properties are conferred to the surface by
polymeric coatings containing appropriate nanocomposites. An
exemplary coating contains fluorine and silicon nanocomposites with
good roll off properties and very high water and oil contact
angles. When used on rough surfaces like sandblasted glass,
nanocoatings may act as a filler to provide stain resistance. A
drawback of these "passive" technologies is that they are not
optimal for use in high gloss surfaces because the lotus-effect is
based on surface roughness.
[0005] Photocatalytic coatings are promising for promoting
self-cleaning of organic stains. Upon the irradiation of sun light,
a photocatalyst such as TiO.sub.2 chemically breaks down organic
dirt that is then washed away by the water sheet formed on the
super hydrophilic surface. As an example, the photocatalyst
TiO.sub.2 was used to promote active fingerprint decomposition of
fingerprint stains in U.S. Pat. Appl. Publ. 2009/104086. A major
drawback to this technology is its limitation to use on inorganic
surfaces due to the oxidative impairment of the polymer coating by
TiO.sub.2. Also, this technology is less than optimal for
automotive coatings due to a compatibility issue: TiO.sub.2 not
only decomposes dirt, but also oxidizes polymer resins in the
paint.
[0006] Therefore, there is a need for new materials or coatings
that can actively promote the removal of biological stains on
surfaces or in coatings and minimize the requirement for
maintenance cleaning.
SUMMARY
[0007] The following summary of the invention is provided to
facilitate an understanding of some of the innovative features
unique to the present invention and is not intended to be a full
description. A full appreciation of the various aspects of the
invention can be gained by taking the entire specification, claims,
drawings, and abstract as a whole.
[0008] A process of facilitating the removal of biological stains
is provided including providing a liquid bioactive coating with a
polymer and an enzyme such that the enzyme is capable of
enzymatically degrading a component of a biological stain. An
enzyme is optionally a hydrolase, optionally a protease, lipase or
amylase. In some aspects, a proteins is a thermolysin-like protease
is optionally a member of the M4 thermolysin-like proteases which
include thermolysin or analogues thereof. Optionally, a protease is
a bacterial neutral thermolysin-like-protease from Bacillus
stearothermophilus or an analogue thereof.
[0009] Also provided is a composition for facilitating biological
stain removal including a liquid coating material, a polymer in the
liquid coating material, and an enzyme in the liquid coating
material such that the enzyme is capable of degrading a biological
stain component.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] FIG. 1 represents a schematic of a spray-down application
process of one embodiment of a coating;
[0011] FIG. 2 represents a schematic of stain application and
protease mediated stain removal on a coated substrate;
[0012] FIG. 3 illustrates improved rinsing of insect stains using
the active extracellular fragment of enzyme G. stearothermophilus
TLP (THERMOASE C160) based coating relative to an enzyme free
control;
[0013] FIG. 4 illustrates improved rinsing of insect stains using
the active extracellular fragment of enzyme G. stearothermophilus
TLP (THERMOASE C160) based coating relative to a Lipase PS based
coating;
[0014] FIG. 5 illustrates improved rinsing of insect stains using
the active extracellular fragment of enzyme G. stearothermophilus
TLP (Thermoase C160) based coating relative to an .alpha.-Amylase
based coating;
[0015] FIG. 6 illustrates affects on 100.degree. C. baking for 10
days on surface enzyme activity (A) and stain cleaning time
(B);
[0016] FIG. 7 illustrates increased loading of protease increases
self-cleaning performance with relative enzyme loading
concentrations of 0.2% (A), 2.0% (B), 4.0% (C), 6.0% (D), and 8.0%
(E);
[0017] FIG. 8 illustrates rinsing of insect stains by a (A)
Protease N based SB coating, (B) Protin SD AY-10 based SB coating,
(C) Protease A based SB coating, (D) THERMOASE GL30 based SB
coating (<0.0075 units/cm.sup.2 surface B. stearothermophilus
TLP), or (E) active extracellular fragment of G. stearothermophilus
TLP (THERMOASE C160) based SB coating;
[0018] FIG. 9 illustrates a schematic of a road test protocol for
active stain removal by an embodiment of a coating;
[0019] FIG. 10 illustrates rain or water bath rinsing of enzyme
containing or control coatings after depositing insect bodies
during road driving;
[0020] FIG. 11 illustrates rain or water bath rinsing of enzyme
containing or control coatings after depositing insect bodies
during road driving, WBS represents enzyme coatings; WBB represents
control coatings; (A) panes are before rinsing, (B) 2 hours of
rinsing; diamonds represent control coatings and X represents
bioactive coating;
[0021] FIG. 12 illustrates average road obtained insect stain
removal from panels coated with an enzyme containing coating or a
control coating;
[0022] FIG. 13 illustrates average road obtained insect stain
removal from panels coated with an enzyme containing coating or a
control coating whereby the enzyme containing coatings are prepared
at different buffer pH levels;
[0023] FIG. 14 illustrates the ability of a coating of windshield
washer fluid alone (A) or a coating of windshield washer fluid
containing the active extracellular fragment of enzyme G.
stearothermophilus TLP and its ability to remove insect material
from a glass substrate;
[0024] FIG. 15 illustrates that the presence of the active
extracellular fragment of enzyme G. stearothermophilus TLP
effectively promotes insect stain removal relative to traditional
water and commercial windshield washer fluids;
[0025] FIG. 16 illustrates that the active extracellular fragment
of enzyme G. stearothermophilus TLP surprisingly and specifically
is far superior to other expected enzymes in a coating material at
removing insect stains; and
[0026] FIG. 17 illustrates the specific activity and stability of
the active extracellular fragment of enzyme G. stearothermophilus
TLP in various cleaning fluids.
DETAILED DESCRIPTION
[0027] The following description of particular embodiment(s) is
merely exemplary in nature and is in no way intended to limit the
scope of the invention, its application, or uses, which may, of
course, vary. The invention is described with relation to the
non-limiting definitions and terminology included herein. These
definitions and terminology are not designed to function as a
limitation on the scope or practice of the invention but are
presented for illustrative and descriptive purposes only. While
processes are described as an order of individual steps or using
specific materials, it is appreciated that described steps or
materials may be interchangeable such that the description of the
invention includes multiple parts or steps arranged in many ways as
is readily appreciated by one of skill in the art.
[0028] The present disclosure is based on the catalytic activity of
an enzyme to selectively degrade components of organic stains thus,
promoting active stain removal. Organic stains typically include
organic polymers, fats, oils, and proteins. It was traditionally
difficult to identify an enzyme that was simultaneously
incorporatable into or on a coating or substrate with remaining
activity and successfully promote active breakdown and subsequent
removal of biological stains, particularly stains from insect
sources. Among other discoveries, the inventors unexpectedly
discovered that a particular family of hydrolases, the bacterial
thermolysins (EC 3.4.24.27), particularly the active extracellular
fragment of enzyme G. stearothermophilus TLP (extracellular
Sterolysin) at an activity in excess of 20,000 U/g when in a
bioactive coating material successfully promoted biological stain
removal whereas similar proteases, even other closely related
metalloproteases, were unsuccessful.
[0029] The enzyme is either immobilized into or on coatings or
substrates, or is a component of a fluid (forming a bioactive
liquid coating) used to temporarily contact and coat a surface, and
catalyzes the degradation of biological stain components into
smaller molecules. The small product molecules are less strongly
adherent to a surface or coating incorporating an enzyme, or is
more easily removed with a liquid coating including an enzyme, such
that gentle rinsing, optionally with water, air, or other fluid,
promotes removal of the biological material from the surface or
coating. Thus, the invention has utility as a composition and
method for the active removal of biological stains from
surfaces.
[0030] It is appreciated that the while the description herein is
directed to coatings, the materials described herein may also be
substrates or articles that do not require a coating thereon for
promotion of functional biological stain removal. As such, the word
"coating" as used herein means a material that is operable for
layering on a surface of one or more substrates, or may comprise
the substrate material itself. In some embodiments a coating is a
temporary coating or is otherwise a material designed to be applied
as a rinsing or cleaning agent such as a windshield washer fluid.
As such, the methods and compositions disclosed herein are
generally referred to as an enzyme associated with a coating for
exemplary purposes only. One of ordinary skill in the art
appreciates that the description is equally applicable to
substrates themselves.
[0031] A method as provided herein includes providing a coating
with an enzyme such that the enzyme is enzymatically active and
capable for degrading one or more components of a biological stain
that is applied prior to or after the coating is associated with a
substrate. In particular embodiments a biological stain is based on
bioorganic matter such as that derived from an insect, optionally
an insect body.
[0032] A biological stain as defined herein is a bioorganic stain,
mark, or residue left behind after an organism contacts a substrate
or coating. A biological stain is not limited to marks or residue
left behind after a coating is contacted by an insect body. Other
sources of bioorganic stains are illustratively: insect wings,
legs, or other appendages; bird droppings; fingerprints or residue
left behind after a coating is contacted by an organism; or other
sources of biological stains.
[0033] Enzymes are generally described according to standardized
nomenclature as Enzyme Commission (EC) numbers. Examples of enzymes
operable herein include: EC1, oxidoreductases; EC2, transferases;
EC3, hydrolases; EC4, lyases; EC5, isomerases; or EC6, ligases.
Enzymes in any of these categories can be included in a composition
according to embodiments of the present disclosure.
[0034] In some embodiments, an included enzyme is a hydrolase such
as a glucosidase, a protease, or a lipase. Non-limiting examples of
glucosidases include amylases, chitinase, and lysozyme.
Non-limiting examples of proteases include trypsin, chymotrypsin,
thermolysin, subtilisin, papain, elastase, and plasminogen.
Non-limiting examples of lipases include pancreatic lipase,
lipoprotein lipase, and lipase PS. A lipase can include peptides
having between 2 and about 1000 amino acids or having a molecular
weight in the range of about 150 350,000 Daltons. Illustrative
examples of proteins that function as enzymes are included in U.S.
Patent Application Publication No: 2010/0210745.
[0035] Amylase is an enzyme present in some embodiments of a
coating composition. Amylases have activity that break down starch.
Several types of amylases are operable herein illustratively
including .alpha.-amylase (EC 3.2.1.1) responsible for
endohydrolysis of (1.fwdarw.4)-alpha-D-glucosidic linkages in
oligosaccharides and polysaccharides. .alpha.-Amylase is
illustratively derived from Bacillus subtilis and has the sequence
found at Genbank Accession No: ACM91731 (SEQ ID NO: 1), or an
analogue thereof and encoded by the nucleotide sequence of SEQ ID
NO: 2. A specific example is .alpha.-Amylase from Bacillus subtilis
available from Sigma-Aldrich Co., St. Louis, Mo. Additional
.alpha.-Amylases include those derived from Geobacillus
stearothermophilus (Accession No: AAA22227), Aspergillus oryzae
(Accession No: CAA31220), Homo sapiens (Accession No: BAA14130),
Bacillus amyloliquefaciens (Accession No: ADE44086), Bacillus
lichemformis (Accession No: CAA01355), or other organism, or
analogues thereof. It is appreciated that (.beta.-amylases,
.gamma.-amylases, or analogues thereof from a variety of organisms
are similarly operable in a bioactive coating composition.
[0036] Specific examples of amylase enzymes illustratively have
1000 U/g protease activity or more wherein one (1) U (unit) is
defined as the amount of enzyme that will liberate the non-protein
digestion product form potato starch of Zulkowsky (e.g. starch,
treated with glycerol at 190.degree. C.; Ber. Deutsch. Chem. Ges,
1880; 13:1395). Illustratively, the amylase has activity anywhere
at or between 1,000 U/g to 500,000 U/g, or greater. It is
appreciated that lower activities are operable.
[0037] A protein is optionally a lipase. A wild-type lipase is a
lipase that has an amino acid sequence identical to that found in
an organism in nature. An illustrative example of a wild-type
lipase is that found at GenBank Accession No. ACL68189 and SEQ ID
NO: 5. An exemplary nucleotide sequence encoding a wild-type lipase
is found at Accession No. FJ536288 and SEQ ID NO: 6.
[0038] Lipase activity is illustratively defined in Units/gram. 1
Unit illustratively corresponds to the amount of enzyme that
hydrolyzes 1 .mu.mol acetic acid per minute at pH 7.4 and
40.degree. C. using the substrate triacetin (Sigma-Aldrich, St.
Louis, Mo., Product No. 90240). The lipase of SEQ ID NO: 5 may have
an activity of 200 Units/gram.
[0039] Methods of screening for lipase activity are known and
standard in the art. Illustratively, screening for lipase activity
in a lipase protein or analogue thereof illustratively includes
contacting a lipase or analogue thereof with a natural or synthetic
substrate of a lipase and measuring the enzymatic cleavage of the
substrate. Illustrative substrates for this purpose include
tributyrin and triacetin both of which are cleaved by a
triacylglycerol lipase to liberate butyric acid or acetic acid,
respectively, that is readily measured by techniques known in the
art.
[0040] A protease is optionally a bacterial metalloprotease such as
a member of the M4 family of bacterial thermolysin-like proteases
of which thermolysin is the prototype protease (EC 3.4.24.27) or
analogues thereof. A protease is optionally the bacterial neutral
thermolysin-like-protease (TLP) derived from Geobacillus
stearothermophilus (Bacillus thermoproteolyticus Var. Rokko)
(illustratively sold under the trade name "Thermoase C160"
available from Amano Enzyme U.S.A., Co. (Elgin, Ill.)), with a
sequence of residues 230-548 of SEQ ID NO: 3, or analogues thereof.
A protease is optionally any protease presented in de Kreig, et
al., J Biol Chem, 2000; 275(40):31115-20, or Takagi, M, et al., J
Bacteriol, 1985; 163:824-831, the contents of each of which are
incorporated herein by reference. Illustrative examples of a
protease include the thermolysin-like-proteases from Bacillis
cereus (Accession No. P05806), Lactobacillis sp. (Accession No.
Q48857), Bacillis megaterium (Accession No. Q00891), Bacillis sp.
(Accession No. Q59223), Alicyclobacillis acidocaldarious (Accession
No. Q43880), Bacillis caldolyticus (Accession NO. P23384), Bacillis
thermoproteolyticus (Accession No. P00800), Bacillus
stearothermophilus (Accession No. P43133), Geobacillus
stearothermophilus (P06874), Bacillus subtilis (Accession No.
P06142), Bacillus amyloliquefaciens (Accession No. P06832),
Lysteria monocytogenes (Accession No: P34025; P23224), or active
fragments of each, among others known in the art. In particular
embodiments, a TLP is the active fragment of Geobacillus
stearothermophilus Stearolysin (P06874) encompassing residues 230
to 548, or an active analogue thereof. The sequences at each
accession number listed herein are incorporated herein by
reference.
[0041] Specific examples of proteases illustratively have 10,000
U/g protease activity or more wherein one (1) U (unit) is defined
as the amount the enzyme that will liberate the non-proteinous
digestion product from milk casein (final concentration 0.5%) to
give Folin's color equivalent to 1 .mu.mol of tyrosine per minute
at the reaction initial reaction stage when a reaction is performed
at 37.degree. C. and pH 7.2. Illustratively, the protease activity
is anywhere between 10,000 PU/g to 1,500,000 U/g or any value or
range therebetween, or greater. It is appreciated that lower
protease activities are operable in some embodiments. Protease
activity is optionally in excess of 20,000 U/g. Optionally,
protease activity is between 300,000 U/g and 2,000,000 U/g in
buffer, or any value or range therebetween, or higher.
[0042] Methods of cloning, expressing, and purifying any enzyme
operable herein is achievable by methods ordinarily practiced in
the art illustratively by methods disclosed in Molecular Cloning: A
Laboratory Manual, 2nd ed., vol. 1-3, ed. Sambrook et al., Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989;
Current Protocols in Molecular Biology, ed. Ausubel et al., Greene
Publishing and Wiley-Interscience, New York, 1992 (with periodic
updates); and Short Protocols in Molecular Biology, ed. Ausubel et
al., 52 ed., Wiley-Interscience, New York, 2002, the contents of
each of which are incorporated herein by reference.
[0043] An analogue of an enzyme is optionally a fragment of an
enzyme or includes one or more non-wild-type amino acids in the
peptide sequence. An analogue of an enzyme is a polypeptide that
has some level of activity toward a natural or synthetic substrate
of the enzyme. An analogue optionally has between 0.1% and 200% the
activity of a wild-type enzyme. The term "enzyme" as used herein
includes analogues in some embodiments. In some embodiments, the
term "enzyme" is exclusive of an analogue of a wild-type
enzyme.
[0044] An enzyme is a "peptide," "polypeptide," and "protein"
(terms used herein synonymously) and is intended to mean a natural
or synthetic compound containing two or more amino acids having
some level of activity toward a natural or synthetic substrate of a
wild-type enzyme. A wild-type enzyme is an enzyme that has an amino
acid sequence identical to that found in an organism in nature. An
illustrative example of a wild-type protease is that found at
GenBank Accession No. P06874 and SEQ ID NO: 3.
[0045] A protein optionally functions with one or more cofactor
ions or proteins. A cofactor ion is illustratively a zinc, cobalt,
or calcium.
[0046] Cloning, expressing, and purifying any protein operable
herein is achievable by methods ordinarily practiced in the art
illustratively by methods disclosed in: Molecular Cloning: A
Laboratory Manual, 2nd ed., vol. 1-3, ed. Sambrook et al., Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989;
Current Protocols in Molecular Biology, ed. Ausubel et al., Greene
Publishing and Wiley-Interscience, New York, 1992 (with periodic
updates); and Short Protocols in Molecular Biology, ed. Ausubel et
al., 52 ed., Wiley-Interscience, New York, 2002.
[0047] Naturally derived amino acids present in an enzyme
illustratively include the common amino acids alanine, cysteine,
aspartic acid, glutamic acid, phenylalanine, glycine, histidine,
isoleucine, lysine, leucine, methionine, asparagine, proline,
glutamine, arginine, serine, threonine, valine, tryptophan, and
tyrosine. It is appreciated that less common derivatives of amino
acids that are either found in nature or chemically altered are
optionally present in a protein as well such as alpha-asparagine,
2-aminobutanoic acid or 2-aminobutyric acid, 4-aminobutyric acid,
2-aminocapric acid (2-aminodecanoic acid), 6-aminocaproic acid,
alpha-glutamine, 2-aminoheptanoic acid, 6-aminohexanoic acid,
alpha-aminoisobutyric acid (2-aminoalanine), 3-aminoisobutyric
acid, beta-alanine, allo-hydroxylysine, allo-isoleucine,
4-amino-7-methylheptanoic acid, 4-amino-5-phenylpentanoic acid,
2-aminopimelic acid, gamma-amino-beta-hydroxybenzenepentanoic acid,
2-aminosuberic acid, 2-carboxyazetidine, beta-alanine,
beta-aspartic acid, biphenylalanine, 3,6-diaminohexanoic acid,
butanoic acid, cyclobutyl alanine, cyclohexylalanine,
cyclohexylglycine, N5-aminocarbonylornithine, cyclopentyl alanine,
cyclopropyl alanine, 3-sulfoalanine, 2,4-diaminobutanoic acid,
diaminopropionic acid, 2,4-diaminobutyric acid, diphenyl alanine,
N,N-dimethylglycine, diaminopimelic acid, 2,3-diaminopropanoic
acid, S-ethylthiocysteine, N-ethylasparagine, N-ethylglycine,
4-aza-phenylalanine, 4-fluoro-phenylalanine, gamma-glutamic acid,
gamma-carboxyglutamic acid, hydroxyacetic acid, pyroglutamic acid,
homoarginine, homocysteic acid, homocysteine, homohistidine,
2-hydroxyisovaleric acid, homophenylalanine, homoleucine,
homoproline, homoserine, homoserine, 2-hydroxypentanoic acid,
5-hydroxylysine, 4-hydroxyproline, 2-carboxyoctahydroindole,
3-carboxylsoquinoline, isovaline, 2-hydroxypropanoic acid (lactic
acid), mercaptoacetic acid, mercaptobutanoic acid, sarcosine,
4-methyl-3-hydroxyproline, mercaptopropanoic acid, norleucine,
nipecotic acid, nortyrosine, norvaline, omega-amino acid,
ornithine, penicillamine (3-mercaptovaline), 2-phenylglycine,
2-carboxypiperidine, sarcosine (N-methylglycine),
2-amino-3-(4-sulfophenyl)propionic acid,
1-amino-l-carboxycyclopentane, 3-thienylalanine,
epsilon-N-trimethyllysine, 3-thiazolylalanine, thiazolidine
4-carboxylic acid, alpha-amino-2,4-dioxopyrimidinepropanoic acid;
and 2-naphthylalanine.
[0048] An enzyme is obtained by any of various methods known in the
art illustratively including isolation from a cell or organism,
chemical synthesis, expression of a nucleic acid sequence, and
partial hydrolysis of proteins. Chemical methods of protein
synthesis are known in the art and include solid phase peptide
synthesis and solution phase peptide synthesis or by the method of
Hackeng, T M, et al., Proc Natl Acad Sci USA, 1997; 94(15):7845-50.
An enzyme may be a naturally occurring or non-naturally occurring
protein. The term "naturally occurring" refers to a protein
endogenous to a cell, tissue or organism and includes allelic
variations. A non-naturally occurring protein is synthetic or
produced apart from its naturally associated organism or is
modified and is not found in an unmodified cell, tissue or
organism.
[0049] Modifications and changes can be made in the structure of an
enzyme and still obtain a molecule having similar characteristics
as an active enzyme (e.g., a conservative amino acid substitution).
For example, certain amino acids can be substituted for other amino
acids in a sequence without appreciable loss of activity or
optionally to reduce or increase the activity of an unmodified
protein. Because it is the interactive capacity and nature of a
protein that defines the protein's functional activity, certain
amino acid sequence substitutions can be made in a protein sequence
and nevertheless obtain a protein with like or other desired
properties. Enzymes with an amino acid sequence that is not 100%
identical to that found in nature are termed analogues. An analogue
optionally includes one or more amino acid substitutions,
modifications, deletions, additions, or other change recognized in
the art with the proviso that any such change produces an enzyme
with the same type of activity (e.g. hydrolase) as the wild-type
sequence. In making such changes, the hydropathic index, or the
hydrophilicity of amino acids can be considered. In such changes,
the substitution using amino acids whose hydropathic indices or
hydrophilicity values are within .+-.2, those within .+-.1, and
those within .+-.0.5 are optionally used.
[0050] Amino acid substitutions are optionally based on the
relative similarity of the amino acid side-chain substituents, for
example, their hydrophobicity, hydrophilicity, charge, size, and
the like. Exemplary substitutions that take various of the
foregoing characteristics into consideration are well known to
those of skill in the art and include (original residue: exemplary
substitution): (Ala: Gly, Ser), (Arg: Lys), (Asn: Gln, His), (Asp:
Glu, Cys, Ser), (Gln: Asn), (Glu: Asp), (Gly: Ala), (His: Asn,
Gln), (Ile: Leu, Val), (Leu: Ile, Val), (Lys: Arg), (Met: Leu,
Tyr), (Ser: Thr), (Thr: Ser), (Tip: Tyr), (Tyr: Trp, Phe), and
(Val: Ile, Leu). In particular, embodiments of the proteins can
include analogues having about 50%, 60%, 70%, 80%, 90%, 95%, or 99%
sequence identity to a wild-type protein.
[0051] It is further appreciated that the above characteristics are
optionally taken into account when producing a protein with reduced
or increased enzymatic activity. Illustratively, substitutions in a
substrate binding site, exosite, cofactor binding site, catalytic
site, or other site in a protein may alter the activity of the
enzyme toward a substrate. In considering such substitutions the
sequences of other known naturally occurring or non-naturally
occurring like enzymes may be taken into account. Illustratively, a
corresponding mutation to that of Asp213 in thermolysin is operable
such as that done by Mild, Y, et al., Journal of Molecular
Catalysis B: Enzymatic, 1996; 1:191-199. Optionally, a substitution
in thermolysin of L144 such as to serine alone or along with
substitutions of G8C/N60C/S65P are operable to increase the
catalytic efficiency by 5-10 fold over the wild-type enzyme.
Yasukawa, K, and Inouye, K, Biochimica et Biophysica Acta
(BBA)-Proteins & Proteomics, 2007; 1774:1281-1288. The
mutations in the bacterial neutral protease from Bacillus
stearothermophilus of N116D, Q119R, D150E, and Q225R as well as
other mutations similarly increase catalytic activity. De Kreig, A,
et al., J. Biol. Chem., 2002; 277:15432-15438. De Kreig also teach
several substitutions including multiple substitutions that either
increase or decrease the catalytic activity of the protein. Id. and
De Kreig, Eur J Biochem, 2001; 268(18):4985-4991. Other
substitutions at these or other sites optionally similarly affect
enzymatic activity. It is within the level of skill in the art and
routine practice to undertake site directed mutagenesis and screen
for subsequent protein activity such as by the methods of De Kreig,
Eur J Biochem, 2001; 268(18):4985-4991.
[0052] An enzyme is optionally an analogue of a wild-type enzyme.
An analogue of an enzyme has an amino acid sequence that when
placed in similar conditions to a wild-type protein possess some
level of the activity of a wild-type enzyme toward the same
substrate. An analogue optionally has 500%, 250%, 200%, 150%, 110%,
99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%,
70%, 60%, 50%, 25%, 10%, 5%, or any value or range of values
therebetween, the activity of a wild-type protein. Any modification
to a wild-type protein may be used to generate an analogue.
Illustratively, amino acid substitutions, additions, deletions,
cross-linking, removal or addition of disulfide bonds, or other
modification to the sequence or any member of the sequence may be
used to generate an analogue. An analogue is optionally a fusion
protein that includes the sequences of two or more wild-type
proteins, fragments thereof, or sequence analogues thereof.
[0053] Methods of screening for protein activity are known and
standard in the art. Illustratively, screening for activity of an
enzyme illustratively includes contacting an enzyme with a natural
or synthetic substrate of an enzyme and measuring the enzymatic
cleavage of the substrate. Illustrative substrates for this purpose
include casein, which is cleaved by a protease to liberate
folin-positive amino acids and peptides (calculated as tyrosine)
that are readily measured by techniques known in the art. The
synthetic substrate furylacryloylated tripeptide
3-(2-furylacryloyl)-L-glycyl-L-leucine-L-alanine obtained from
Bachem AG, Bubendorf, Switzerland is similarly operable.
Illustrative substrates of .alpha.-Amylase include long chain
carbohydrates such as amylose or amylopectin that make up starch.
Other methods of screening for .alpha.-Amylase activity include the
colorimetric assay of Fischer and Stein, Biochem. Prep., 1961, 8,
27-33. It is appreciated that one of ordinary skill in the art can
readily envision methods of screening for enzyme activity with the
enzyme present in or on a variety of materials.
[0054] An enzyme is illustratively recombinant. Methods of cloning,
synthesizing or otherwise obtaining nucleic acid sequences encoding
a protein are known and standard in the art. Similarly, methods of
cell transfection and protein expression are similarly known in the
art and are applicable herein. Exemplary cDNA encoding the protein
sequence of SEQ ID NO: 1 is the nucleotide sequence SEQ ID NO: 2.
Exemplary cDNA encoding the protein sequence of SEQ ID NO: 3 is the
nucleotide sequence found at accession number M11446 and SEQ ID NO:
4. Exemplary cDNA encoding the protein sequence of SEQ ID NO: 5 is
the nucleotide sequence SEQ ID NO: 6.
[0055] An enzyme may be coexpressed with associated tags,
modifications, other proteins such as in a fusion protein, or other
modifications or combinations recognized in the art. Illustrative
tags include 6.times.His, FLAG, biotin, ubiquitin, SUMO, or other
tag known in the art. A tag is illustratively cleavable such as by
linking to protein via a target sequence that is cleavable by an
enzyme known in the art illustratively including Factor Xa,
thrombin, SUMOstar protein as obtainable from Lifesensors, Inc.,
Malvern, Pa., or trypsin. It is further appreciated that chemical
cleavage is similarly operable with an appropriate cleavable
linker.
[0056] Protein expression is illustratively accomplished following
transcription of a protein nucleic acid sequence, translation of
RNA transcribed from the protein nucleic acid sequence or analogues
thereof. An analog of a nucleic acid sequence is any sequence that
when translated to protein will produce a wild-type protein or an
analogue of a wild-type protein. Protein expression is optionally
performed in a cell based system such as in E. coli, Hela cells, or
Chinese hamster ovary cells. It is appreciated that cell-free
expression systems are similarly operable.
[0057] It is recognized that numerous analogues of protein are
operable and within the scope of the present invention including
amino acid substitutions, alterations, modifications, or other
amino acid changes that increase, decrease, or not do alter the
function of the protein sequence. Several post-translational
modifications are similarly envisioned as within the scope of the
present disclosure illustratively including incorporation of a
non-naturally occurring amino acid, phosphorylation, glycosylation,
addition of pendent groups such as biotin, avidin, fluorophores,
lumiphores, radioactive groups, antigens, or other molecules.
[0058] It is recognized that numerous analogues of an enzyme are
operable and within the scope of the present disclosure including
amino acid substitutions, alterations, modifications, or other
amino acid changes that increase, decrease, or not alter the
function of the enzyme protein sequence. Several post-translational
modifications are similarly envisioned as within the scope of the
present invention illustratively including incorporation of a
non-naturally occurring amino acid, phosphorylation, glycosylation,
addition of pendent groups such as biotinylation, fluorophores,
lumiphores, radioactive groups, antigens, or other molecules.
[0059] An inventive method uses an inventive composition that is
one or more enzymes incorporated into a substrate itself or into a
coating, optionally for application on a substrate. The enzyme is
optionally non-covalently associated and/or covalently attached to
the substrate or coating material or is otherwise associated
therewith such as by bonding to the surface or by intermixing with
the substrate/coating material during manufacture such as to
produce entrapped enzyme. In some embodiments the enzyme is
covalently attached to the substrate or coating material either by
direct covalent interaction between the enzyme and one or more
components of the substrate or coating material or by association
via a link moiety such as that described in U.S. Pat. App. Publ.
No. 2008/0119381, the contents of which are incorporated herein by
reference. In some embodiments, such as in coatings useful as
cleaning agents, illustratively, windshield washing solutions, an
enzyme is in solution or suspension within the coating
solution.
[0060] There are several ways to associate an enzyme with a
substrate or coating. One of which involves the application of
covalent bonds. Specifically, free amine groups of the enzyme may
be covalently bound to an active group of the substrate. Such
active groups include alcohol, thiol, aldehyde, carboxylic acid,
anhydride, epoxy, ester, or any combination thereof. This method of
incorporating enzyme delivers unique advantages. First, the
covalent bonds tether the enzymes permanently to the substrate and
thus place them as an integral part of the final composition with
much less, if any at all, leakage of the protease. Second, the
covalent bonds provide extended enzyme lifetime. Over time,
proteins typically lose activity because of the unfolding of their
polypeptide chains. Chemical bonding such as covalent bonding
effectively restricts such unfolding, and thus improves the protein
life. The life of a protein is typically determined by comparing
the amount of activity reduction of a protein that is free or being
physically adsorbed with that of a protein covalently-immobilized
over a period of time.
[0061] Enzymes are optionally uniformly dispersed throughout the
substrate or coating network to create a substantially homogenous
protein platform. In so doing, enzymes may be first modified with
polymerizable groups. The modified enzymes may be solubilized into
organic solvents, optionally, in the presence of surfactant, and
thus engage the subsequent polymerization with monomers such as
methyl methacrylate (MMA) or styrene in the organic solution. The
resulting composition optionally includes enzyme molecules
homogeneously dispersed throughout the network.
[0062] Enzymes are optionally attached to surfaces of a substrate.
An attachment of proteases corresponding to approximately 100%
surface coverage was achieved with polystyrene particles with
diameters range from 100 to 1000 nm.
[0063] Chemical methods of protease attachment to materials will
naturally vary depending on the functional groups present in the
enzyme and in the material components. Many such methods exist. For
example, methods of attaching proteins (such as enzymes) to other
substances are described in O'Sullivan et al, Methods in
Enzymology, 1981; 73:147-166 and Erlanger, Methods in Enzymology,
1980; 70:85-104, each of which are herein incorporated herein by
reference.
[0064] Enzymes are optionally present in a coating that is layered
upon a substrate wherein the enzyme is optionally entrapped in the
coating material, admixed therewith, modified and integrated into
the coating material or layered upon a coating similar to the
mechanisms described for interactions between a protease and
substrate material.
[0065] Materials operable for interactions with an enzyme to form
an active substrate or coating illustratively include organic
polymeric materials. The combination of these materials and an
enzyme form a protein-polymer composite material that is used as a
substrate material or a coating.
[0066] Methods of preparing protein-polymer composite materials
illustratively include use of aqueous solutions of enzyme and
non-aqueous organic solvent-borne polymers to produce bioactive
organic solvent-borne protein-polymer composite materials.
[0067] Methods of preparing protein-polymer composite materials are
illustratively characterized by dispersion of enzyme in
solvent-borne resin prior to curing and in the composite materials,
in contrast to forming large aggregates of the bioactive proteins
which diminish the functionality of the proteases and
protein-polymer composite materials. Enzymes are optionally
dispersed in the protein-polymer composite material such that the
enzymes are unassociated with other bioactive proteins and/or form
relatively small particles of associated proteins. Illustratively,
the average particle size of enzyme particles in the
protein-polymer composite material is less than 10 .mu.m (average
diameter) such as in the range of 1 nm to 10 .mu.m, inclusive.
[0068] Curable protein-polymer compositions are optionally
two-component solvent-borne (2K SB) compositions. Optionally, one
component systems (1K) are similarly operable. Illustratively, an
enzyme is entrapped in a coating material such as a latex or enamel
paint, varnish, polyurethane gels, or other coating materials.
Illustrative examples of incorporating enzymes into paints are
presented in U.S. Pat. No. 5,998,200, the contents of which are
incorporated herein by reference.
[0069] In two-component systems the two components are optionally
mixed shortly before use, for instance, application of the curable
protein-polymer composition to a substrate to form an enzyme
containing coating such as a bioactive clear coat. Generally
described, the first component contains a crosslinkable polymer
resin and the second component contains a crosslinker. Thus, the
emulsion is a first component containing a crosslinkable resin and
the crosslinker is a second component, mixed together to produce
the curable protein-polymer composition.
[0070] A polymer resin included in methods and compositions of the
present disclosure can be any film-forming polymer useful in
coating or substrate compositions, illustratively clear coat
compositions. Such polymers illustratively include, aminoplasts,
melamine formaldehydes, carbamates, polyurethanes, polyacrylates,
epoxies, polycarbonates, alkyds, vinyls, polyamides, polyolefins,
phenolic resins, polyesters, polysiloxanes; and combinations of any
of these or other polymers.
[0071] In particular embodiments, a polymer resin is crosslinkable.
Illustratively, a crosslinkable polymer has a functional group
characteristic of a crosslinkable polymer. Examples of such
functional groups illustratively include acetoacetate, acid, amine,
carboxyl, epoxy, hydroxyl, isocyanate, silane, vinyl, other
operable functional groups, and combinations thereof.
[0072] Examples of organic crosslinkable polymer resins includes
aminoplasts, melamine formaldehydes, carbamates, polyurethanes,
polyacrylates, epoxies, polycarbonates, alkyds, vinyls, polyamides,
polyolefins, phenolic resins, polyesters, polysiloxanes, or
combinations thereof.
[0073] A cross linking agent is optionally included in the
composition. The particular crosslinker selected depends on the
particular polymer resin used. Non-limiting examples of
crosslinkers include compounds having functional groups such as
isocyanate functional groups, epoxy functional groups, aldehyde
functional groups, and acid functionality.
[0074] In particular embodiments of protein-polyurethane composite
materials, a polymer resin is a hydroxyl-functional acrylic polymer
and the crosslinker is a polyisocyanate.
[0075] A polyisocyanate, optionally a diisocyanate, is a
crosslinker reacted with the hydroxyl-functional acrylic polymer
according to embodiments of the present invention. Aliphatic
polyisocyanates are preferred polyisocyanates used in processes for
making protein-polymer composite materials for clearcoat
applications such as in automotive clearcoat applications.
Non-limiting examples of aliphatic polyisocyanates include
1,4-butylene diisocyanate, 1,4-cyclohexane diisocyanate,
1,2-diisocyanatopropane, 1,3-diisocyanatopropane, ethylene
diisocyanate, lysine diisocyanate, 1,4-methylene bis (cyclohexyl
isocyanate), diphenylmethane 4,4'-diisocyanate, an isocyanurate of
diphenylmethane 4,4'-diisocyanate,
methylenebis-4,4'-isocyanatocyclohexane, 1,6-hexamethylene
diisocyanate, an isocyanurate of 1,6-hexamethylene diisocyanate,
isophorone diisocyanate, an isocyanurate of isophorone
diisocyanate, p-phenylene diisocyanate, toluene diisocyanate, an
isocyanurate of toluene diisocyanate, triphenylmethane
4,4',4''-triisocyanate, tetramethyl xylene diisocyanate, and
meta-xylene diisocyanate.
[0076] Curing modalities are those typically used for conventional
curable polymer compositions.
[0077] Protease-polymer composite materials used in embodiments of
the present disclosure are optionally thermoset protein-polymer
composite materials. For example, a substrate or coating material
is optionally cured by thermal curing. A thermal polymerization
initiator is optionally included in a curable composition. Thermal
polymerization initiators illustratively include free radical
initiators such as organic peroxides and azo compounds. Examples of
organic peroxide thermal initiators illustratively include benzoyl
peroxide, dicumylperoxide, and lauryl peroxide. An exemplary azo
compound thermal initiator is 2,2'-azobisisobutyronitrile.
[0078] Conventional curing temperatures and curing times can be
used in processes according to embodiments of the present
disclosure. For example, the curing time at specific temperatures,
or under particular curing conditions, is determined by the
criteria that the cross-linker functional groups are reduced to
less than 5% of the total present before curing. Cross-linker
functional groups can be quantitatively characterized by FT-IR or
other suitable method. For example, the curing time at specific
temperatures, or under particular curing conditions, for a
polyurethane protein-polymer composite of the present invention can
be determined by the criteria that the cross-linker functional
group NCO is reduced to less than 5% of the total present before
curing. The NCO group can be quantitatively characterized by FT-IR.
Additional methods for assessing the extent of curing for
particular resins are well-known in the art. Illustratively, curing
may include evaporation of a solvent or by exposure to actinic
radiation, such as ultraviolet, electron beam, microwave, visible,
infrared, or gamma radiation.
[0079] One or more additives are optionally included for modifying
the properties of the protease -polymer composite material and/or
the admixture of organic solvent and polymer resin, the aqueous
lipase solution, the emulsion, and/or the curable composition.
Illustrative examples of such additives include a UV absorbing
agent, a plasticizer, a wetting agent, a preservative, a
surfactant, a lubricant, a pigment, a filler, and an additive to
increase sag resistance.
[0080] A substrate or coating including an enzyme is illustratively
an admixture of a polymer resin, a surfactant and a non-aqueous
organic solvent, mixed to produce an emulsion. The term
"surfactant" refers to a surface active agent that reduces the
surface tension of a liquid in which it is dissolved, or that
reduces interfacial tension between two liquids or between a liquid
and a solid.
[0081] Surfactants used can be of any variety including amphoteric,
silicone-based, fluorosurfactants, anionic, cationic and nonionic
such as described in K. R. Lange, Surfactants: A Practical
Handbook, Hanser Gardner Publications, 1999; and R. M. Hill,
Silicone Surfactants, CRC Press, 1999, incorporated herein by
reference. Examples of anionic surfactants illustratively include
alkyl sulfonates, alkylaryl sulfonates, alkyl sulfates, alkyl and
alkylaryl disulfonates, sulfonated fatty acids, sulfates of
hydroxyalkanols, sulfosuccinic acid esters, sulfates and sulfonates
of polyethoxylated alkanols and alkylphenols. Examples of cationic
surfactants include quaternary surfactants and amineoxides.
Examples of nonionic surfactants include alkoxylates,
alkanolamides, fatty acid esters of sorbitol or manitol, and alkyl
glucamides. Examples of silicone-based surfactants include siloxane
polyoxyalkylene copolymers.
[0082] In some embodiments, a coating is formed of materials that
produce a liquid bioactive coating material suitable for use as a
cleaning fluid, illustratively a windshield washer fluid. As one
example, the inventors surprisingly discovered that inclusion of
the active extracellular fragment of enzyme G. stearothermophilus
TLP at an activity in excess of 20,000 U/g in a coating material
provides unexpectedly superior insect biological stain removal
relative to other enzymes, and particularly other proteases. A
coating material is optionally a cleaning fluid. Illustrative
examples of a cleaning fluid include those described in: U.S. Pat.
Nos. 6,881,711; and 6,635,188; the contents of which are
incorporated herein by reference. Illustrative examples of fluids
for use as a coating material include those sold as BUGWASH by
Prestone Products, Corp., XTREME BLUE by Camco Mfg, Inc.,
Greenboro, NC, and RAIN X Delcer, from ITW Global Brands, Houston,
Tex.
[0083] While any commercially available windshield washer fluid can
be used as a cleaning fluid for addition of an enzyme, the
inventors surprisingly discovered that coating materials that are
low in alcohol content (e.g. less than 0.8%) and have a pH in
excess of 5.0, optionally in excess of 8.0, optionally between 5.0
and 12.0, or any value or range therebetween, possess far superior
protease activity stability. Optionally, a pH is between 10.0 and
11.0. The pH activity and stability data are surprising for the
additional reasons that it is known in the art that the enzyme has
an optimal activity at about pH 7.6 with rapidly decreasing
activity at higher pH levels. For example, the activity in
Britton-Robinson buffer is less than 20% peak activity at a pH at
or above 10.0. Additionally, stability falls of significantly at a
pH above 5. The combined high activity and stability of the active
extracellular fragment of enzyme G. stearothermophilus TLP, for
example, at relatively high pH values such as above 5.0, optionally
above 8.0, particularly above 10.0, more particularly between 10.0
and 11.0, creates an unexpectedly cleaning coating material
relative to coating materials combined with other proteases.
[0084] In some embodiments, a liquid bioactive coating material
includes a surfactant, an ammonia compound, an alcohol, and water.
Water is optionally a predominant. A surfactant in such embodiments
is illustratively a nonionic surfactants, anionic surfactants,
cationic surfactants, zwitterionic surfactants, and mixtures
thereof, with specific examples of surfactants being octylphenol
ethoxylates, alkyl polyglycosides, sodium alkyl sulfates, and
mixtures thereof. A surfactant is optionally present in an amount
at or between 0.001% to about 0.25% (by weight). An ammonia
compound is illustratively ammonium carbamate, ammonium carbonate,
ammonium bicarbonate, ammonium hydroxide, ammonium acetate,
ammonium borate, ammonium phosphate, an alkanolamine having 1 to 6
carbon atoms, and ammonia, or combinations thereof. An ammonia
compound is optionally present at or between 0.005% to about 1.0%
(by weight of NH.sub.3). An alcohol is illustratively one or more:
water miscible alcohols having 1 to 6 carbon atoms, water miscible
glycols and glycol ethers having 2 to 15 carbon atoms and mixtures
thereof. Preferred alcohols include methanol, ethanol, isopropanol,
propanol, butanol, furfuryl alcohol, tetrahydrofurfuryl alcohol
("THFA") and 1-amino-2-propanol. Preferred glycols and glycol
ethers include ethylene glycol, propylene glycol, 2-butoxyethanol
sold as BUTYL CELLOSOLVE.RTM., 2-methoxyethanol,
1-methoxy-2-propanol, ethylene glycol dimethyl ether,
1,2-dimethoxypropane, 2-(2-propoxyethoxy)ethanol,
2-[2-(2-propoxyethoxy)ethoxy]ethanol,
2-(2-isopropoxyethoxy)ethanol, 2-[2-(2
isopropoxyethoxy)ethoxy]ethanol, 2-(2-butoxyethoxy)ethanol,
2-[2-(2-butoxyethoxy)ethoxy]ethanol, 2-(2-isobutoxyethoxy)ethanol,
2-[2-(2 isobutoxyethoxy)ethoxy]ethanol,
2-(2-propoxypropoxy)-propan-1-ol,
2-[2-(2-propoxypropoxy)propoxy]propan-1-ol,
2-(2-isopropoxypropoxy)-propan-1-ol,
2-[2-(2-isopropoxypropoxy)propoxy]propan-1-ol,
2-(2-butoxypropoxy)-propan-1-ol,
2-[2(2-butoxypropoxy)propoxy]propan-1-ol,
2-(2-isobutoxypropoxy)-propan-1-ol and
2[2-(2-isobutoxypropoxy)propoxy]propan-1-ol. Preferably, ethanol,
isopropanol, 2-butoxyethanol or 1-amino-2-propanol methanol,
ethanol, isopropanol, propanol, butanol, furfuryl alcohol,
tetrahydrofurfuryl alcohol, 1-amino-2-propanol, ethylene glycol,
propylene glycol, and 2-butoxyethanol, or combinations thereof are
used.
[0085] A liquid bioactive coating material is optionally formed by
combining a coating material with one or more proteases such that
the protease is in solution or suspension. A protease containing
coating material is optionally mixed such as by vortex mixing until
a solution of protease is achieved. The amount of enzyme is
illustratively 0.1 to 50 mg in .about.4 L coating material.
[0086] When a surface, which is optionally a substrate or a coated
substrate, is contacted with biological material to produce a
biological stain, the enzyme or combinations of enzymes in a
coating material are placed in contact with the stain, or
components thereof. The contacting allows the enzymatic activity of
the enzyme to interact with and enzymatically alter the components
of the stain improving its removal from the substrate or
coating.
[0087] Enzyme containing substrates or coatings have a surface
activity generally expressed in Units/cm.sup.2. Substrates and
coatings optionally have functional surface activities of greater
than 0.0075 Units/cm.sup.2. In some embodiments surface activity is
between 0.0075 Units/cm.sup.2 and 0.05 Units/cm.sup.2 inclusive.
Optionally, surface activity is between 0.0075 Units/cm.sup.2 and
0.1 Units/cm.sup.2 inclusive. Optionally, surface activity is
between 0.01 Units/cm.sup.2 and 0.05 Units/cm.sup.2 inclusive.
[0088] It is appreciated that the inventive methods of facilitating
stain removal will function at any temperature whereby the enzyme
is active. Optionally, the inventive process is performed at
4.degree. C. Optionally, an inventive process is performed at
25.degree. C. Optionally, an inventive process is performed at
ambient temperature. It is appreciated that the inventive process
is optionally performed from 4.degree. C. to 125.degree. C., or any
single temperature or range therein.
[0089] The presence of enzyme combined with the material of a
substrate or a coating, optionally, with water or other fluidic
rinsing agent, breaks down stains for facilitated removal.
[0090] Methods involving conventional biological techniques are
described herein. Such techniques are generally known in the art
and are described in detail in methodology treatises such as
Molecular Cloning: A Laboratory Manual, 3rd ed., vol. 1-3, ed.
Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., 2001; Current Protocols in Molecular Biology, ed.
Ausubel et al., Greene Publishing and Wiley-Interscience, New York,
1992 (with periodic updates); and Short Protocols in Molecular
Biology, ed. Ausubel et al., 52 ed., Wiley-Interscience, New York,
2002.
[0091] Various aspects of the present invention are illustrated by
the following non-limiting examples. The examples are for
illustrative purposes and are not a limitation on any practice of
the present invention. It will be understood that variations and
modifications can be made without departing from the spirit and
scope of the invention.
EXAMPLE 1
[0092] Production of a bacterial neutral protease from Bacillus
stearothermophilus containing material operable for coating a
substrate.
[0093] Materials: Freeze-dried crickets are purchased from
PetSmart. Cricket bodies reportedly contain 58.3% protein. (D.
Wang, et al., Entomologic Sinica, 2004; 11:275-283, incorporated
herein by reference.) .alpha.-Amylase, Lipase PS, Protease N,
Protease A, Protin SD AY-10, active extracellular fragment of G.
stearothermophilus TLP (THERMOASE C160), and THERMOASE GL30 (low
activity preparation of B. stearothermophilus TLP) are obtained
from AMANO Enzyme Inc. (Nagoya, JAPAN). Polyacrylate resin
Desmophen A870 BA, and the hexamethylene diisocyanate (HDI) based
polyfunctional aliphatic polyisocyanate resin Desmodur N 3600 are
obtained from Bayer Corp. (Pittsburgh, Pa.). The surfactant BYK-333
is obtained from BYK-Chemie (Wallingford, Conn.). 1-butanol and
1-butyl acetate are obtained from Sigma-Aldrich Co. (Missouri,
USA). Aluminum paint testing panels are purchased from Q-Lab Co.
(Cleveland, USA). All other reagents involved in the experiments
are of analytical grade.
[0094] Enzyme based 2K SB PU coatings are prepared by either a
draw-down method or by spray application and used for subsequent
biological stain removal experiments. Each enzyme is dissolved in
DI water to a final enzyme solution concentration of 200 mg/mL for
all water borne (WB) coatings. For solvent borne (SB) enzyme
prepared coatings 50 mg/mL enzyme is used. A solution of 150 ml of
deionized water containing 1.5 g of the active extracellular
fragment of enzyme G. stearothermophilus TLP is first purified by
ultrafiltration (molecular weight cut-off of 30 kDa, all liquids
were kept on ice).
[0095] For the draw-down method or coating preparation, the
surfactant BYK 333 is diluted with 1-butanol to the concentration
of 17% by weight. The resin part of the 2K SB PU coating is
prepared by mixing 2.1 g of Desmophen A 870 with 0.5 mL of 1-butyl
acetate and 0.1 mL surfactant in a 20 mL glass vial. The solution
is mixed using a microspatula for 1 min followed by addition of 0.6
mL of enzyme solution (or DI water for control coating without
enzyme) followed by mixing for another 1 min. This solution is then
poured out into a 20-mL glass vial with 0.8 g of NA 3600 and
stirred for 1 min. This formulation produces an enzyme
concentration of 6% by weight. Pre-cleaned aluminum testing panels
are coated with the enzyme containing coating material using a
draw-down applicator with a wet film thickness of 2 mils. The
coating panels are baked at 80.degree. C. for 30 minutes and then
cured at ambient temperature for 7 days.
[0096] For the spray application method, coating are prepared
essentially as described in FIG. 1.
[0097] A cleaning fluid as a coating material including the active
extracellular fragment of enzyme G. stearothermophilus TLP is
produced by intermixing the TLP at 0.1 mg to 20 mg/liter of the
cleaning fluid in a mixing vessel at room temperature at least one
hour prior to use. The cleaning fluid is formed from
1-amino-2-propanol (0.2% w/w),
4-(1,1,3,3-Tetramethylbutyl)phenyl-polyethylene glycol
t-Octylphenoxypolyethoxyethanol Polyethylene glycol
tert-octylphenyl ether (TRITON X-100) (0.04% w/w), ammonia (0.084%
w/w; from 28% NH.sub.3 in water); with the balance water. All of
the components of the cleaning fluid are obtained commercially as
follows: TRITON X-100 from Union Carbide/Dow Chemical; ammonia and
1-amino-2propanol from Sigma-Aldrich Chemical Company Inc.; and
active extracellular fragment of Geobacillus stearothermophilus TLP
(THERMOASE C160) from Amano Enzyme, Inc. A cleaning fluid is mixed
with the active extracellular fragment G. stearothermophilus TLP by
agitation or by vortex mixing. The resulting G. stearothermophilus
TLP containing cleaning fluid is stored at ambient temperature.
EXAMPLE 2
[0098] Preparation of biological stains and application to a coated
substrate. An exemplary schematic of an experimental procedure is
provided in FIG. 2. 60 g of Freeze-dried crickets are chopped into
powder by a blender (Oster, 600 watt) for 10 min. The stain
solution is prepared by vigorously mixing 2 g of cricket powder
with 6 mL of DI water. A template of uniform spacing is used to
apply the stain on the coating surface. The cricket stains are
dried at 40.degree. C. for 5 min followed by placing the coating
panels into a glass dish and rinsing with 200 mL of DI water under
300 rpm shaking at RT for various times. The time of the stain
removal is recorded. In order to reduce random error, the time of
the first and last drop removed are not included. The average
rinsing time of eight stain spots is averaged for stain removal
time.
EXAMPLE 3
[0099] Drying time affects stain removal time. Stained coated
substrate panels prepared with coatings as in Example 1 and insect
stains as in Example 2 are subjected to drying at 40.degree. C. for
various times. The rinsing time of stain drops strongly depends on
the drying time. The control protease free coating, after being
dried for 5 min, produces firmly adhered stain drops that are not
removed by rinsing for 3 hr (Table 1).
TABLE-US-00001 TABLE 1 Drying Time (min) 3 3.2 5 Average washing
time (min) 2.8 4.9 >180
[0100] For the active extracellular fragment of enzyme G.
stearothermophilus TLP containing coated panes, the rinsing time
increases with longer drying time yet at equivalent drying times
relative to control the protease containing coating promotes
dramatically improved stain removal. (Table 2).
TABLE-US-00002 TABLE 2 Drying Time (min) 5 10 Average washing time
(min) 28.7 79.3
EXAMPLE 4
[0101] Increased rinsing intensity reduces stain removal time. The
panels prepared as in Examples 1 and 2 are subjected to various
rinsing intensities. Reduced rinsing time is achieved by increasing
rinsing intensity for the active extracellular fragment of enzyme
G. stearothermophilus TLP containing coatings on substrates (Table
3).
TABLE-US-00003 TABLE 3 Shaking speed (rpm) 200 250 300 Average
washing time (min) 56.5 44.4 28.7
EXAMPLE 5
[0102] Coatings containing various enzymes are prepared as in
Example 1. Each coating is analyzed for performance by measurement
of average rinsing time using a standard protocol of applying a
cricket stain to a coated substrate, drying for 5 min at 40.degree.
C. and rinsing in water or in protease containing cleaning fluid at
an intensity of 300 RPM. For removal of insect stains by a TLP
containing cleaning fluid, the fluid is prepared as described in
Example 1. Insect material is applied onto a glass substrate dried
on a heating plate at 60.degree. C. for a period. After drying,
drops of 50 .mu.1 enzyme containing cleaning fluids of Example 1
are added onto dry stain spots via a multi-channel pipette,
followed by an incubation for 10 minutes at ambient temperature.
The identical volume of control (protease free) washer fluids are
added onto the control spots on the same substrate. The substrate
is then immersed face-up into a deionized water bath while
horizontally shaking at 100 rpm. The stain spots retained on the
coatings after a desired shaking time are counted during the
washing for quantitative analysis. The control and various protease
containing coatings are also evaluated for roughness, contact
angle, and gloss. The results are depicted in Table 4.
TABLE-US-00004 TABLE 4 Average Roughness Contact Gloss Coatings
Washing time (.mu.m) Angle (60.degree.) SB control coating >3 hr
0.053 76.2 163.0 Lipase PS based SB coating >3 hr 0.063 88.0
147.9 .alpha.-Amylase based >3 hr 0.078 80.5 148.4 SB coating
Thermoase C160 based 28 min 0.078 86.4 148.4 SB coating
[0103] The active extracellular fragment of enzyme G.
stearothermophilus TLP based coatings as well as other TLP
containing cleaning fluids have an improved self-cleaning function
against insect body stains compared with other coating materials
containing either no enzyme or alternative enzymes (no enzyme,
Lipase PS, and .alpha.-Amylase). In addition, the coating surface
properties are insignificant different between the active
extracellular fragment of enzyme G. stearothermophilus TLP based
coating and the control, Lipase PS, or .alpha.-Amylase based
coatings. These results indicate that physical characteristics of
the coatings are not differentially affecting the coating
performance.
[0104] The rinsing times of each enzyme containing coating is
compared. FIG. 3 demonstrates comparison of a control SB coating
(enzyme free, left panel) with an active extracellular fragment of
enzyme G. stearothermophilus TLP based coating (right panel). After
30 minutes of rinsing the active extracellular fragment of enzyme
G. stearothermophilus TLP based coating shows significant stain
removal. The control shows no significant stain removal out to 3
hours of rinsing.
[0105] Similar results are observed comparing an active
extracellular fragment of enzyme G. stearothermophilus TLP based
coating with a lipase and .alpha.-Amylase based coating. In FIGS. 4
and 5 respectively, lipase and .alpha.-Amylase (left panels) show
significant adherence of insect stains remaining for the entire
three hour rinsing period. In contrast the active extracellular
fragment of enzyme G. stearothermophilus TLP based coatings show
dramatic stain removal after an initial 30 min rinsing period with
essentially complete stain removal by three hours.
[0106] Similarly excellent results are observed for cleaning fluids
containing active extracellular fragment of enzyme G.
stearothermophilus TLP. FIG. 14 demonstrates a comparison of the
cleaning fluid of Example 1 with or without the active
extracellular fragment of enzyme G. stearothermophilus TLP with (A)
representing glass substrate with insect stains before rinsing, and
(B) after rinsing. The cleaning fluid including the active
extracellular fragment of enzyme G. stearothermophilus TLP is far
superior in promoting insect stain removal from a glass substrate
than the same fluid in the absence of the enzyme.
EXAMPLE 6
[0107] Affect of surface heating on protease function. Panels
coated with the active extracellular fragment of enzyme G.
stearothermophilus TLP based coatings as in Example 1 are subjected
to baking temperatures of 100.degree. C. for 10 days followed by
determination of change in surface enzyme activity. Proteolytic
surface activity of protease containing coatings is determined
following the method of Folin and Ciocalteau, J. Biol. Chem., 1927;
73: 627-50, incorporated herein by reference. Briefly, 1 mL of 2%
(w/v) casein in sodium phosphate (0.05 M; pH 7.5) buffer solution
is used as substrate together with 200 .mu.l of sodium acetate, 5
mM calcium acetate (10 mM; pH 7.5). The substrate solution is
pre-incubated in a water bath for 3 min to reach 37.degree. C. The
reaction is started by adding one piece of sample plate coated with
the active extracellular fragment of enzyme G. stearothermophilus
TLP based coating (1.2.times.1.9 cm.sup.2) followed by shaking for
10 min at 200 rpm at which time the reaction is stopped by adding 1
ml of 110 mM tricholoroacetic acid (TCA) solution. The mixture is
incubated for 30 min at 37.degree. C. prior to centrifugation. The
equivalent of tyrosine in 400 .mu.L of the TCA-soluble fraction is
determined at 660 nm using 200 .mu.L of 25% (v/v) Folin-Ciocalteau
reagent and 1 mL 0.5 M sodium carbonate. One unit of activity is
defined as the amount of enzyme hydrolyzing casein to produce
absorbance equivalent to 1.0 .mu.mol of tyrosine per minute at
37.degree. C. This result is converted to Units/cm.sup.2. FIG. 6
illustrates that the active extracellular fragment of enzyme G.
stearothermophilus TLP surface activity is reduced by approximately
50% after long term high temperature baking (FIG. 6A).
Coincidently, the time of stain cleaning is increased (FIG.
6B).
EXAMPLE 7
[0108] Enzyme loading is titered in coatings prepared and coated
onto substrate panels as in Example 1 and with insect stains
applied as in Example 2 at loading concentrations of enzyme of 0.2%
(A), 2.0% (B), 4.0% (C), 6.0% (D), and 8.0% (E) active
extracellular fragment of enzyme G. stearothermophilus TLP, and the
thermolysin-like-proteins from Bacillis cereus, Lactobacillis sp.,
Bacillis megaterium, Alicyclobacillis acidocaldarious, Bacillis
caldolyticus, Bacillis thermoproteolyticus, Bacillus
stearothermophilus, Bacillus subtilis, Bacillus amyloliquefaciens),
and Lysteria monocytogenes. The panels are baked for 5 min at
40.degree. C. and washed at 300 RPM for three hours. Increased
protease loading correlates with increased rinsing performance
(FIG. 7A-E depicting results for the active extracellular fragment
of enzyme G. stearothermophilus TLP).
EXAMPLE 8
[0109] Comparison of various protease types on insect stain
removal. Coatings are prepared as in Example 1 using protease N
(bacillolysin) as a putative cysteine protease, Protin SD AY10
(subtilisin from Bacillus licheniformis) as a putative serine
protease, protease A as an exemplary metalloprotease, and the
active extracellular fragment of enzyme G. stearothermophilus TLP
and thermolysin-like-proteins of Example 7, and coated onto
substrates as in Example 1 with insect staining as in Example 2.
The different enzyme containing coatings are compared after baking
for 5 min at 40.degree. C. and rinsing at 300 RPM for 3 hours.
Surprisingly, only the active extracellular fragment of enzyme G.
stearothermophilus TLP based coatings show the dramatically
improved self-cleaning function which is not observed by coatings
including, a serine protease, a cysteine protease, or another
metalloprotease. (Table 5 and FIG. 8.)
TABLE-US-00005 TABLE 5 Washing Activity Protease in Coatings
Protease Group time (KU/g) Bacillolysin Cysterine protease >3 hr
150 Subtilisin Serine protease >3 hr 90 Oryzin Metalloprotease
>3 hr 20 TLP Metalloprotease >3 hr 300 Sterolysin
Metalloprotease 28 min 1600
EXAMPLE 9
[0110] Test panels are prepared as in Example 1 and are mounted
onto the front bumpers of test vehicles. A schematic of a road-test
protocol is illustrated in FIG. 9. Real-life insects are collected
from the road by driving. The vehicle is driven during summer
evenings for .about.500 miles to collect insect bodies. The average
speed of driving is 65 mph.
[0111] Within three days of insect collection the panels are rinsed
either in natural rain (driving condition) or in lab on a water
bath at a rate of shaking rate of 200 rpm. Photos are taken prior
to and after the rinsing procedure. Panels are visually checked and
counted prior to, during, and after rinsing to identify differences
in stain removal from test and control panels.
[0112] A clear increase in stain-removal effectiveness under mild
rinsing is observed on enzyme-containing coatings relative to
control coatings without enzyme as is illustrated in FIGS. 10 and
11. The road test is repeated three times and the average percent
remaining insect stains in enzyme containing and control coatings
after rinsing for various times are plotted in FIG. 12. The enzyme
containing coatings promote active insect stain removal using
environmentally obtained insects under normal road driving
conditions.
EXAMPLE 10
[0113] Enzyme containing coatings are prepared as in Example 1
using buffers of pH 6.4 and pH 11. Coated aluminum plates are
subjected to insect staining as in Example 9. Enzyme containing
coatings prepared at both pH levels are superior to control (FIG.
13).
EXAMPLE 11
[0114] Various cleaning fluids are analyzed for performance by
measurement of average rinsing time using a standard protocol. For
removal of insect stains by an active extracellular fragment of
active extracellular fragment of enzyme G. stearothermophilus TLP
containing cleaning fluid, the fluid is prepared as described in
Example 1. Control cleaning fluids of Rain-X Bug Pre-wash Gel
(Commercial Detergent 1), Rain-X Foaming Car Wash (Commercial
Detergent 2), a sodium chloride solution, and water alone are
compared. Insect material is applied onto a glass substrate dried
on a heating plate at 60.degree. C. for a period. After drying,
drops of 50 .mu.1 enzyme containing cleaning fluids of Example 1
are added onto dry stain spots via a multi-channel pipette,
followed by an incubation for 10 minutes at ambient temperature.
The identical volume of control (protease free) washer fluids are
added onto the control spots on the same substrate. The substrate
is then immersed face-up into a deionized water bath while
horizontally shaking at 100 rpm. The substrate is agitated for 1
hour and the time of each spot removal is recorded. FIG. 15
illustrates that the water, NaCl, and both commercial detergents
leave insect material on the glass substrate for greater than one
hour, whereas the active extracellular fragment of enzyme G.
stearothermophilus TLP containing cleaning fluid removes the insect
stain much more quickly.
[0115] Similar experiments are performed using cleaning fluids
prepared as in Example 1 with substitution of lipase (LIPASE PS),
.alpha.-Amylase, or active extracellular fragment of G.
stearothermophilus TLP (THERMOASE C160), or no enzyme. The
experiments above are repeated with the exception that shaking in
water is continued out to three hours. As illustrated in FIG. 16,
the active extracellular fragment of enzyme G. stearothermophilus
TLP containing cleaning fluid was the only enzyme tested with the
ability to remove the insect stain from the glass substrate in the
test period indicating that this protease is unique in its ability
to promote removal of insect material from a surface.
EXAMPLE 12
[0116] A cleaning fluid containing the active extracellular
fragment of enzyme G. stearothermophilus TLP with high pH
demonstrates increased specific activity and excellent stability
relative to the active extracellular fragment of enzyme G.
stearothermophilus TLP containing fluids with lower pH. Various
commercial enzyme free cleaning fluids (RAIN-X De Icer; PRESTONE
BUG WASH; and EXTRME BLUE) and water are used as a base for the
addition of the active extracellular fragment of enzyme G.
stearothermophilus TLP using the procedure of Example 1. The
protease containing cleaning fluids are stored at ambient
temperature and assayed at various timepoints using the procedure
of Example 11 for their ability to promote insect stain removal. As
is illustrated in FIG. 17, the high pH material shows both
surprisingly higher specific activity at all time points and
excellent stability.
[0117] Various modifications of the present invention, in addition
to those shown and described herein, will be apparent to those
skilled in the art of the above description. Such modifications are
also intended to fall within the scope of the appended claims.
[0118] It is appreciated that all reagents are obtainable by
sources known in the art unless otherwise specified or synthesized
by one of ordinary skill in the art without undue experimentation.
Methods of nucleotide amplification, cell transfection, and protein
expression and purification are similarly within the level of skill
in the art.
[0119] Patents and publications mentioned in the specification are
indicative of the levels of those skilled in the art to which the
invention pertains. These patents and publications are incorporated
herein by reference to the same extent as if each individual
application or publication was specifically and individually
incorporated herein by reference.
[0120] The foregoing description is illustrative of particular
embodiments of the invention, but is not meant to be a limitation
upon the practice thereof. The following claims, including all
equivalents thereof, are intended to define the scope of the
invention.
Sequence CWU 1
1
61659PRTBacillus subtilis 1Met Phe Ala Lys Arg Phe Lys Thr Ser Leu
Leu Pro Leu Phe Ala Gly1 5 10 15Phe Leu Leu Leu Phe His Leu Val Leu
Ala Gly Pro Ala Ala Ala Ser 20 25 30Ala Glu Thr Ala Asn Lys Ser Asn
Glu Leu Thr Ala Pro Ser Ile Lys 35 40 45Ser Gly Thr Ile Leu His Ala
Trp Asn Trp Ser Phe Asn Thr Leu Lys 50 55 60His Asn Met Lys Asp Ile
His Asp Ala Gly Tyr Thr Ala Ile Gln Thr65 70 75 80Ser Pro Ile Asn
Gln Val Lys Glu Gly Asn Gln Gly Asp Lys Ser Met 85 90 95Ser Asn Trp
Tyr Trp Leu Tyr Gln Pro Thr Ser Tyr Gln Ile Gly Asn 100 105 110Arg
Tyr Leu Gly Thr Glu Gln Glu Phe Lys Glu Met Cys Ala Ala Ala 115 120
125Glu Glu Tyr Gly Ile Lys Val Ile Val Asp Ala Val Ile Asn His Thr
130 135 140Thr Ser Asp Tyr Ala Ala Ile Ser Asn Glu Val Lys Ser Ile
Pro Asn145 150 155 160Trp Thr His Gly Asn Thr Gln Ile Lys Asn Trp
Ser Asp Arg Trp Asp 165 170 175Val Thr Gln Asn Ser Leu Leu Gly Leu
Tyr Asp Trp Asn Thr Gln Asn 180 185 190Thr Gln Val Gln Ser Tyr Leu
Lys Arg Phe Leu Asp Arg Ala Leu Asn 195 200 205Asp Gly Ala Asp Gly
Phe Arg Phe Asp Ala Ala Lys His Ile Glu Leu 210 215 220Pro Asp Asp
Gly Ser Tyr Gly Ser Gln Phe Trp Pro Asn Ile Thr Asn225 230 235
240Thr Ser Ala Glu Phe Gln Tyr Gly Glu Ile Leu Gln Asp Ser Ala Ser
245 250 255Arg Asp Ala Ala Tyr Ala Asn Tyr Met Asp Val Thr Ala Ser
Asn Tyr 260 265 270Gly His Ser Ile Arg Ser Ala Leu Lys Asn Arg Asn
Leu Gly Val Ser 275 280 285Asn Ile Ser His Tyr Ala Ser Asp Val Ser
Ala Asp Lys Leu Val Thr 290 295 300Trp Val Glu Ser His Asp Thr Tyr
Ala Asn Asp Asp Glu Glu Ser Thr305 310 315 320Trp Met Ser Asp Asp
Asp Ile Arg Leu Gly Trp Ala Val Ile Ala Ser 325 330 335Arg Ser Gly
Ser Thr Pro Leu Phe Phe Ser Arg Pro Glu Gly Gly Gly 340 345 350Asn
Gly Val Arg Phe Pro Gly Lys Ser Gln Ile Gly Asp Arg Gly Ser 355 360
365Ala Leu Phe Glu Asp Gln Ala Ile Thr Ala Val Asn Arg Phe His Asn
370 375 380Val Met Ala Gly Gln Pro Glu Glu Leu Ser Asn Pro Asn Gly
Asn Asn385 390 395 400Gln Ile Phe Met Asn Gln Arg Gly Ser His Gly
Val Val Leu Ala Asn 405 410 415Ala Gly Ser Ser Ser Val Ser Ile Asn
Thr Ala Thr Lys Leu Pro Asp 420 425 430Gly Arg Tyr Asp Asn Lys Ala
Gly Ala Gly Ser Phe Gln Val Asn Asp 435 440 445Gly Lys Leu Thr Gly
Thr Ile Asn Ala Arg Ser Val Ala Val Leu Tyr 450 455 460Pro Asp Asp
Ile Ala Lys Ala Pro His Val Phe Leu Glu Asn Tyr Lys465 470 475
480Thr Gly Val Thr His Ser Phe Asn Asp Gln Leu Thr Ile Thr Leu Arg
485 490 495Ala Asp Ala Asn Thr Thr Lys Ala Val Tyr Gln Ile Asn Asn
Gly Pro 500 505 510Glu Thr Ala Phe Lys Asp Gly Asp Gln Phe Thr Ile
Gly Lys Gly Asp 515 520 525Pro Phe Gly Lys Thr Tyr Thr Ile Met Leu
Lys Gly Thr Asn Ser Asp 530 535 540Gly Val Thr Arg Thr Glu Lys Tyr
Ser Phe Val Lys Arg Asp Pro Ala545 550 555 560Ser Ala Lys Thr Ile
Gly Tyr Gln Asn Pro Asn His Trp Ser Gln Val 565 570 575Asn Ala Tyr
Ile Tyr Lys His Asp Gly Ser Arg Val Ile Glu Leu Thr 580 585 590Gly
Ser Trp Pro Gly Lys Pro Met Thr Lys Asn Ala Asp Gly Ile Tyr 595 600
605Thr Leu Thr Leu Pro Ala Asp Thr Asp Thr Thr Asn Ala Lys Val Ile
610 615 620Phe Asn Asn Gly Ser Ala Gln Val Pro Gly Gln Asn Gln Pro
Gly Phe625 630 635 640Asp Tyr Val Leu Asn Gly Leu Tyr Asn Asp Ser
Gly Leu Ser Gly Ser 645 650 655Leu Pro His21980DNABacillus subtilis
2atgtttgcaa aacgattcaa aacctcttta ctgccgttat tcgctggatt tttattgctg
60tttcatttgg ttctggcagg accggcggct gcgagtgctg aaacggcgaa caaatcgaat
120gagcttacag caccgtcgat caaaagcgga accattcttc atgcatggaa
ttggtcgttc 180aatacgttaa aacacaatat gaaggatatt catgatgcag
gatatacagc cattcagaca 240tctccgatta accaagtaaa ggaagggaat
caaggagata aaagcatgtc gaactggtac 300tggctgtatc agccgacatc
gtatcaaatt ggcaaccgtt acttaggtac tgaacaagaa 360tttaaagaaa
tgtgtgcagc cgctgaagaa tatggcataa aggtcattgt tgacgcggtc
420atcaatcata ccaccagtga ttatgccgcg atttccaatg aggttaagag
tattccaaac 480tggacacatg gaaacacaca aattaaaaac tggtctgatc
gatgggatgt cacgcagaat 540tcattgctcg ggctgtatga ctggaataca
caaaatacac aagtacagtc ctatctgaaa 600cggttcttag acagggcatt
gaatgacggg gcagacggtt ttcgatttga tgccgccaaa 660catatagagc
ttccagatga tggcagttac ggcagtcaat tttggccgaa tatcacaaat
720acatctgcag agttccaata cggagaaatc ctgcaggata gtgcctccag
agatgctgca 780tatgcgaatt atatggatgt gacagcgtct aactatgggc
attccataag gtccgcttta 840aagaatcgta atctgggcgt gtcgaatatc
tcccactatg catctgatgt gtctgcggac 900aagctagtga catgggtaga
gtcgcatgat acgtatgcca atgatgatga agagtcgaca 960tggatgagcg
atgatgatat ccgtttaggc tgggcggtga tagcttctcg ttcaggcagt
1020acgcctcttt tcttttccag acctgaggga ggcggaaatg gtgtgaggtt
cccggggaaa 1080agccaaatag gcgatcgcgg gagtgcttta tttgaagatc
aggctatcac tgcggtcaat 1140agatttcaca atgtgatggc tggacagcct
gaggaactct cgaacccgaa tggaaacaac 1200cagatattta tgaatcagcg
cggctcacat ggcgttgtgc tggcaaatgc aggttcatcc 1260tctgtctcta
tcaatacggc aacaaaattg cctgatggca ggtatgacaa taaagctgga
1320gcgggttcat ttcaagtgaa cgatggtaaa ctgacaggca cgatcaatgc
caggtctgta 1380gctgtgcttt atcctgatga tattgcaaaa gcgcctcatg
ttttccttga gaattacaaa 1440acaggtgtaa cacattcttt caatgatcaa
ctgacgatta ccttgcgtgc agatgcgaat 1500acaacaaaag ccgtttatca
aatcaataat ggaccagaga cggcgtttaa ggatggagat 1560caattcacaa
tcggaaaagg agatccattt ggcaaaacat acaccatcat gttaaaagga
1620acgaacagtg atggtgtaac gaggaccgag aaatacagtt ttgttaaaag
agatccagcg 1680tcggccaaaa ccatcggcta tcaaaatccg aatcattgga
gccaggtaaa tgcttatatc 1740tataaacatg atgggagccg agtaattgaa
ttgaccggat cttggcctgg aaaaccaatg 1800actaaaaatg cagacggaat
ttacacgctg acgctgcctg cggacacgga tacaaccaac 1860gcaaaagtga
tttttaataa tggcagcgcc caagtgcccg gtcagaatca gcctggcttt
1920gattacgtgc taaatggttt atataatgac tcgggcttaa gcggttctct
tccccattga 19803548PRTGeobacillus stearothermophilus 3Met Asn Lys
Arg Ala Met Leu Gly Ala Ile Gly Leu Ala Phe Gly Leu1 5 10 15Leu Ala
Ala Pro Ile Gly Ala Ser Ala Lys Gly Glu Ser Ile Val Trp 20 25 30Asn
Glu Gln Trp Lys Thr Pro Ser Phe Val Ser Gly Ser Leu Leu Asn 35 40
45Gly Gly Glu Gln Ala Leu Glu Glu Leu Val Tyr Gln Tyr Val Asp Arg
50 55 60Glu Asn Gly Thr Phe Arg Leu Gly Gly Arg Ala Arg Asp Arg Leu
Ala65 70 75 80Leu Ile Gly Lys Gln Thr Asp Glu Leu Gly His Thr Val
Met Arg Phe 85 90 95Glu Gln Arg His His Gly Ile Pro Val Tyr Gly Thr
Met Leu Ala Ala 100 105 110His Val Lys Asp Gly Glu Leu Ile Ala Leu
Ser Gly Ser Leu Ile Pro 115 120 125Asn Leu Asp Gly Gln Pro Arg Leu
Lys Lys Ala Lys Thr Val Thr Val 130 135 140Gln Gln Ala Glu Ala Ile
Ala Glu Gln Asp Val Thr Glu Thr Val Thr145 150 155 160Lys Glu Arg
Pro Thr Thr Glu Asn Gly Glu Arg Thr Arg Leu Val Ile 165 170 175Tyr
Pro Thr Asp Gly Thr Ala Arg Leu Ala Tyr Glu Val Asn Val Arg 180 185
190Phe Leu Thr Pro Val Pro Gly Asn Trp Val Tyr Ile Ile Asp Ala Thr
195 200 205Asp Gly Ala Ile Leu Asn Lys Phe Asn Gln Ile Asp Ser Arg
Gln Pro 210 215 220Gly Gly Gly Gln Pro Val Ala Gly Ala Ser Thr Val
Gly Val Gly Arg225 230 235 240Gly Val Leu Gly Asp Gln Lys Tyr Ile
Asn Thr Thr Tyr Ser Ser Tyr 245 250 255Tyr Gly Tyr Tyr Tyr Leu Gln
Asp Asn Thr Arg Gly Ser Gly Ile Phe 260 265 270Thr Tyr Asp Gly Arg
Asn Arg Thr Val Leu Pro Gly Ser Leu Trp Thr 275 280 285Asp Gly Asp
Asn Gln Phe Thr Ala Ser Tyr Asp Ala Ala Ala Val Asp 290 295 300Ala
His Tyr Tyr Ala Gly Val Val Tyr Asp Tyr Tyr Lys Asn Val His305 310
315 320Gly Arg Leu Ser Tyr Asp Gly Ser Asn Ala Ala Ile Arg Ser Thr
Val 325 330 335His Tyr Gly Arg Gly Tyr Asn Asn Ala Phe Trp Asn Gly
Ser Gln Met 340 345 350Val Tyr Gly Asp Gly Asp Gly Gln Thr Phe Leu
Pro Phe Ser Gly Gly 355 360 365Ile Asp Val Val Gly His Glu Leu Thr
His Ala Val Thr Asp Tyr Thr 370 375 380Ala Gly Leu Val Tyr Gln Asn
Glu Ser Gly Ala Ile Asn Glu Ala Met385 390 395 400Ser Asp Ile Phe
Gly Thr Leu Val Glu Phe Tyr Ala Asn Arg Asn Pro 405 410 415Asp Trp
Glu Ile Gly Glu Asp Ile Tyr Thr Pro Gly Val Ala Gly Asp 420 425
430Ala Leu Arg Ser Met Ser Asp Pro Ala Lys Tyr Gly Asp Pro Asp His
435 440 445Tyr Ser Lys Arg Tyr Thr Gly Thr Gln Asp Asn Gly Gly Val
His Thr 450 455 460Asn Ser Gly Ile Ile Asn Lys Ala Ala Tyr Leu Leu
Ser Gln Gly Gly465 470 475 480Val His Tyr Gly Val Ser Val Asn Gly
Ile Gly Arg Asp Lys Met Gly 485 490 495Lys Ile Phe Tyr Arg Ala Leu
Val Tyr Tyr Leu Thr Pro Thr Ser Asn 500 505 510Phe Ser Gln Leu Arg
Ala Ala Cys Val Gln Ala Ala Ala Asp Leu Tyr 515 520 525Gly Ser Thr
Ser Gln Glu Val Asn Ser Val Lys Gln Ala Phe Asn Ala 530 535 540Val
Gly Val Tyr54541881DNAGeobacillus stearothermophilus 4gatcaggaag
cattgcgcta tggacgaagt gagcctcctt tcgttctcgg gatatagccg 60aaaagaacca
ggggaggaaa aacgaaagtc cgggccgtgc acggagggcg tgtcattgcc
120gttcattttc ccaatacaat aaggatgact attttggtaa aattcagaat
gtgaggaatc 180atcaaataca tattcaagaa aggggaagag gagaatgaac
aaacgggcga tgctcggggc 240gatcgggctg gcgttcggcc tgctggcggc
gccgatcggc gcttcggcga agggggaatc 300gatcgtctgg aacgaacaat
ggaagacgcc gtcattcgtg tccggttcgt tgctaaacgg 360aggggaacaa
gcgctggaag agctcgttta tcaatacgtc gatcgggaaa acggcacatt
420ccgcctcggc ggacgcgccc gcgaccgttt ggcgctgatc ggcaaacaga
ctgacgaact 480tggccatacc gtgatgcggt ttgaacagcg gcatcacggt
ataccggttt acggcaccat 540gctggctgcc catgtgaaag atggcgagct
gatcgcgctg tcggggtctt taattcccaa 600tttagacggc cagccgcggt
tgaaaaaggc gaaaacggtc accgtccaac aggcggaagc 660tattgccgag
caagacgtaa cggagacagt gacgaaggag cggccgacaa ccgaaaacgg
720cgagcggacg cggctcgtca tttacccgac tgatggcacg gcccgcctcg
cttatgaagt 780gaacgtccgc tttttaacac cggttcccgg caactgggtg
tatatcattg atgcaaccga 840tggggccatt ttgaataagt tcaaccaaat
cgacagccgc cagcccggcg gcgggcagcc 900ggtcgccggc gcgtcgacgg
tcggcgtggg ccggggtgtg ttgggggatc agaaatatat 960caatacgacg
tattcctcgt attacggcta ctactatttg caagacaata cgcgcggcag
1020cggcattttt acgtatgacg gacgaaaccg caccgttttg cccggcagct
tgtggaccga 1080tggcgacaac caatttaccg ccagctatga cgcggcggcc
gtggacgccc attattacgc 1140cggcgtcgtg tatgattact acaaaaatgt
gcacggccgg ctgagctatg acggcagcaa 1200cgccgccatc cgttcgaccg
tccattatgg ccgcggctac aacaacgcgt tttggaacgg 1260ttcgcaaatg
gtgtacggcg atggcgacgg acagacgttt ttgccgtttt ccggcggcat
1320tgacgtcgtg gggcatgagt tgacccatgc ggtgacggat tatacggccg
ggcttgttta 1380ccaaaacgaa tctggcgcca tcaatgaagc gatgtccgat
attttcggca cgctcgtgga 1440gttctacgcc aaccgcaacc cggactggga
gattggcgaa gacatttaca cgcctggggt 1500cgccggcgat gcgctccgct
cgatgtccga cccggcgaaa tacggcgatc cggatcatta 1560ttccaaacgg
tacaccggca cgcaagacaa cggcggcgtc catacaaaca gcggcatcat
1620caataaagcg gcgtacttgc tcagccaagg cggcgtccat tatggcgtga
gcgtcaacgg 1680catcggccgc gacaaaatgg ggaaaatttt ctaccgggcg
cttgtctact atttgacgcc 1740gacgtcgaac ttcagccagc tgcgtgccgc
ctgcgtgcaa gcggccgctg atttgtacgg 1800gtcgacaagc caagaagtca
actcggtgaa acaggcgttc aatgcggttg gagtgtatta 1860agacgatgag
gtcgtacgcg t 18815298PRTAspergillis niger 5Met Phe Leu Arg Arg Glu
Phe Gly Ala Val Ala Ala Leu Ser Val Leu1 5 10 15Ala His Ala Ala Pro
Ala Pro Ala Pro Met Gln Arg Arg Asp Ile Ser 20 25 30Ser Thr Val Leu
Asp Asn Ile Asp Leu Phe Ala Gln Tyr Ser Ala Ala 35 40 45Ala Tyr Cys
Ser Ser Asn Ile Glu Ser Thr Gly Thr Thr Leu Thr Cys 50 55 60Asp Val
Gly Asn Cys Pro Leu Val Glu Ala Ala Gly Ala Thr Thr Ile65 70 75
80Asp Glu Phe Asp Asp Ser Ser Ser Tyr Gly Asp Pro Thr Gly Phe Ile
85 90 95Ala Val Asp Pro Thr Asn Glu Leu Ile Val Leu Ser Phe Arg Gly
Ser 100 105 110Ser Asp Leu Ser Asn Trp Ile Ala Asp Leu Asp Phe Gly
Leu Thr Ser 115 120 125Val Ser Ser Ile Cys Asp Gly Cys Glu Met His
Lys Gly Phe Tyr Glu 130 135 140Ala Trp Glu Val Ile Ala Asp Thr Ile
Thr Ser Lys Val Glu Ala Ala145 150 155 160Val Ser Ser Tyr Pro Asp
Tyr Thr Leu Val Phe Thr Gly His Ser Tyr 165 170 175Gly Ala Ala Leu
Ala Ala Val Ala Ala Thr Val Leu Arg Asn Ala Gly 180 185 190Tyr Thr
Leu Asp Leu Tyr Asn Phe Gly Gln Pro Arg Ile Gly Asn Leu 195 200
205Ala Leu Ala Asp Tyr Ile Thr Asp Gln Asn Met Gly Ser Asn Tyr Arg
210 215 220Val Thr His Thr Asp Asp Ile Val Pro Lys Leu Pro Pro Glu
Leu Leu225 230 235 240Gly Tyr His His Phe Ser Pro Glu Tyr Trp Ile
Thr Ser Gly Asn Asp 245 250 255Val Thr Val Thr Thr Ser Asp Val Thr
Glu Val Val Gly Val Asp Ser 260 265 270Thr Asp Gly Asn Asp Gly Thr
Leu Leu Asp Ser Thr Thr Ala His Arg 275 280 285Trp Tyr Thr Ile Tyr
Ile Ser Glu Cys Ser 290 2956897DNAAspergillis niger 6atgtttctcc
gcagggaatt tggggctgtt gcagccctat ctgtgctggc ccatgctgct 60cccgcacctg
ctccgatgca gcgtagagac atctcctcta ccgtcttgga caatatcgac
120ctcttcgccc aatacagtgc agcagcttac tgctcctcca acatcgagtc
caccggcacg 180actctgacct gcgacgtagg caattgccct ctcgtcgagg
cagccggtgc cacgaccatc 240gatgagtttg acgacagcag cagctacggc
gacccgacgg ggttcatcgc cgttgacccg 300acgaacgagt tgatcgttct
gtctttccgg ggtagttccg acctctcgaa ctggattgcc 360gacctagact
tcggcctcac ctccgtaagc agcatctgtg atggctgtga gatgcacaag
420ggcttctatg aggcctggga agtcattgcc gacaccatca catccaaggt
ggaggccgct 480gtctccagct atccggacta caccctcgtg ttcaccggac
acagctacgg cgctgcattg 540gcggctgtcg cggccaccgt actccgcaac
gccggataca ctcttgacct gtacaacttc 600ggccagcccc gtatcggcaa
ccttgcttta gctgactaca tcaccgacca aaacatgggc 660agcaactacc
gcgtcacgca caccgacgac atcgtgccta agctgcctcc ggagctgctg
720ggctaccacc acttcagtcc ggagtactgg atcaccagcg gtaatgatgt
gacggtgact 780acgtcggacg tgaccgaggt tgtgggggtg gattcgacgg
atgggaatga cggcacgctg 840cttgacagta cgactgccca tcggtggtac
acgatctaca ttagtgaatg ctcgtag 897
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