WO1996033257A1

WO1996033257A1 - Reusable cleaning solutions containing stabilized enzymes

Info

Publication number: WO1996033257A1
Application number: PCT/JP1996/001053
Authority: WO
Inventors: Kyosuke Owa
Original assignee: Horiuchi Co., Ltd.
Priority date: 1995-04-18
Filing date: 1996-04-18
Publication date: 1996-10-24
Also published as: IL117948A0; AU5346696A

Abstract

Stabilized enzyme-based cleaning solutions and methods of synthesis. The enzyme solutions are stabilized with an enzyme stabilizing agent that stabilizes the enzyme when the cleaning solution is on the shelf and during use. The cleaning solutions are effective and stable over long periods of time thereby allowing repeat usage and reducing environmental waste.

Description

REUSABLE CLEANING SOLUTIONS CONTAINING STABILISED ENZYMES

FIELD OF THE INVENTION The present invention is directed to reusable cleaning solutions comprising stabilized enzymes. More specifically, the present invention is directed to reusable cleaning solutions and methods of preparing reusable cleaning solutions containing enzymes that are stabilized with an enzyme stabilizing agent.

DESCRIPTION OF THE RELATED ART It is well known that enzymes in enzyme-based cleaning solutions are very unstable during both storage and use as a result of the hostile environment of the cleaning solution. Cleaning solutions enzymes may be denatured and inactivated by surfactants or other components of the cleaning composition, the high or low pH of the solution, the high or low temperature of the solution or by the enzymes themselves as a result of proteolytic digestion. A number of methods are known in the art for protecting and stabilizing enzymes in cleaning solutions during storage but little is known about the stabilization of enzymes in cleaning solutions during use.

The use of a combination of a polyol and a boron compound as an enzyme stabilization system for cleaning solutions during storage is known. Canadian Patent No. 1,092,036, for example, teaches enzymatic liquid detergents containing 4-25% polyol and boric acid. U.S. Patent 4,404,115 teaches the combination of alkalimetal sulphite and/or polyol as an enzyme stabilizing system.

U.S. Patent 5,431,842 teaches the use of ortho substituted phenylboronic acids to stabilize proteases in liquid laundry detergents. U.S. Patent No. 4,518,694 teaches the use of carboxylic acids as enzyme stabilizers and U.S. Patent No. 5,073,292 teaches the stabilization of enzymes using proteins containing quaternary nitrogen substitutes.

U.S. Patent No. 4,906,396 teaches coating enzymes in a hydrophobic substance such as silicone oil for enzyme stabilization during storage.

U.S. Patent No. 5,078,802 teaches a method of washing semiconductors with enzymes but does not teach the stabilization of the enzymes.

Many references teach the encapsulation of enzymes which are released from the capsule during cleaning.

U.S. Patent No. 4,908,233 teaches the use of a microcapsule made of a core protein material that is coated with a single water soluble polymer to protect the enzyme. Enzymes are released from the capsule during use as a result of the phase separation of the polymer resulting from the action of an electrolyte in the cleaning solution. The enzyme is no longer stabilized once it is released from the capsule during cleaning.

U.S. Patent 5,281,356 teaches heavy duty liquid detergent compositions containing non-proteolytic enzymes and a capsule containing a proteolytic enzyme wherein the capsule is made of a composite polymer. The capsule described in the '356 patent protects the non-proteolytic enzyme from the proteolytic enzyme during storage but provides no added protection once the proteolytic enzyme is released from the capsule during use .

Although useful during storage, the capsule and chemical-based enzyme stabilization solutions do not provide stability to cleaning enzymes during use. The chemical-based cleaning solutions must first be diluted from the storage concentration in order to function properly; if the chemical inhibitor is not PCI7JP96/01053 diluted, the enzyme is inhibited and the cleaning solution does not function properly. With the capsule-enzyme solutions, once the capsule is broken or dissolved, the encapsulated enzyme is free to digest itself as well as other enzymes in the solution and is subject to inactivation by other components in the cleaning solution.

There is thus a need in the art for a stable enzyme based cleaning solution that is both stable on the shelf and during use. An enzyme solution stabilized during use would permit continued re-use of the cleaning solution saving costs and reducing environmental pollution.

SUMMARY OF THE INVENTION

In order to meet these needs, the present invention is directed to a cleaning solution which includes an enzyme, a surfactant and a mixture of glycerol and ethylene glycol as an enzyme stabilizing agent. Preferably, a mixture of 75% glycerol and 25% ethylene glycol is used. The ratio of the enzyme to the glycerol and ethylene glycol ranges from 1:1 to 1:40 on a volume basis. The enzyme is preferably a lipase or a protease.

Preferably, the surfactant of the cleaning solution is selected from the group comprising anionic, cationic, nonionic, and zwitterionic surfactants. Most preferably, the surfactant is lauric acid diethanolamine or monostearic acid sorbitan.

In one embodiment, the cleaning solution may further contain a solvent, preferably, methylene chloride or water.

In another embodiment the cleaning solution may also contain a pH adjusting agent such as an acid or base. A preferred pH adjusting agent is monoethanolamine . The pH of the solution may be adjusted to pH 9.0 - 14. In yet another embodiment, the cleaning solution may also contain a vapor pressure reducing agent, preferably, liquid paraffin.

The cleaning solution may preferably also contain diethylene glycol monobutyl ester or polyoxyethylene nonyl phenyl ester.

In another embodiment, the cleaning solution may also contain a disinfectant, preferably R(CHC₂H₄)₂N^*HC₂C0(T or R(NHC₂H₄) ₂N^*HCH₂C00^'

The enzyme cleaning solution is preferably prepared by dissolving at least one enzyme in aqueous solution, adding a stabilizing agent to the enzyme solution to form an enzyme and stabilizing solution mixture and allowing the mixture to sit until the mixture becomes clarified.

The enzyme cleaning solution can also be prepared by pressuring the enzyme mixture in a pressure chamber or depressurizing the enzyme mixture in a vacuum chamber.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1-1 is a diagram demonstrating a membrane before water is squeezed out of a membrane. 1-1- (A) represents an area of reduced entropy; 1-1- (B) represents a hydrophobic bond; 1-1- (C) is water with normal structure; 1-1- (D) is structured (iceberg) water.

Figure 1-2- (E) is a diagram demonstrating iceberg oriented water being squeezed out from the hydrophobic core of an enzyme.

Figure 2 is a diagram of a free fixation enzyme. 2- (A) represents a surfactant; 2- (B) represents iceberg (structured) water; 2-(C) represents an area of hydrophobic bonds and 2-(D) represents an enzyme.

FIG. 3 is a diagram of a free fixation enzyme functioning as a cleaning solution. 3- (A) represents a cleaning solution; 3-(B) represents an area of iceber-oriented water; 3-(C) represents an area of enzyme catalysis; 3-(D) is a surfactant and 3-(E) is a cleaning substrate

Figures 4-1 to 4-4 are diagrams demonstrating the recycling of a free fixation enzyme through a single cleaning cycle. 4-1- (A) represents iceberg (structured) water; 4-1- (B) represents enzyme; 4-1- (C) represents an area of an enzyme catalalyzed cleaning reaction; 4-2- (D) represents iceberg (structured) water; 4-3- (E) represents iceberg (structured) water; 4-3- (F) represents an enzyme; 4-

4-(G) represents iceberg (structured) water and 4-4- (H) represents hydrophobic bonds

Figures 5-1- (A) , 5-1- (B) , 5-2- (C) and 5-3- (D) are water clathrate structures.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to enzyme-based cleaning solutions that are both stable on the shelf and during use. These cleaning solutions are comprised of an enzyme, a surfactant and an enzyme stabilizing agent. In order to provide a clear and consistent understanding of the present invention and claims, including the scope given to such terms, the following definitions related to the invention are provided:

Enzymes: Enzymes are biological macromolecules that perform enzymatic reactions. Enzymes useful in the cleaning solution of the invention fall into 6 categories: 1) oxidoreductases, 2) transferases, 3) hydrolases, 4) lyases, 5) isomerases and 6) ligases.

Hydrolase enzymes useful in the invention include proteases, nucleases, glycosidases, upases, phospholipases, phosphatases and sulfatases.

Typical enzymes used in cleaning solutions are disclosed in U.S. Patent 5,431,842 which is hereby incorporated by reference. Enzymes can be manufactured by chemical processes or purified from natural sources such as microorganisms, plants and the like. Enzymes can be readily obtained from companies such as Novo Nordisk, Nagase Enzymes in Japan, Genencor, Sigma, ICN and the like.

Surfactants : Surfactants are molecules that include a hydrophobic moiety and a hydrophilic moiety. The group of surface active agents which are manufactured by chemical processes or purified from natural sources or processes can be anionic, cationic, nonionic, and zwitterionic . Specific surfactants include: lauric acid diethanolamine and monostearic acid sorbitan Such materials are described in Emulsions: Theory and Practice, Paul Becher, Robert E. Krieger Publishing, Malabar, Florida, 1965 which is hereby incorporated by reference.

Coenzyme: Coenzymes are organic molecules associated with a protein that take part in the catalytic reaction, but are only loosely associated with the enzyme in solution. Coenzymes may be regarded as dissociable prosthetic groups. Their protein partners are termed apoenzymes. Neither the apoenzyme nor the coenzyme is, in itself, normally a complete catalyst . In the course of the catalyzed reaction, the coenzyme may be changed chemically in the same way as a substrate and dissociate from the apoenzyme. However, all coenzymes can be regenerated in associated reactions and can complex again with the apoenzyme. Some coenzymes function with many different apoenzymes, and hence play an important role in coupling different biochemical reactions. Coenzymes of importance in central metabolism include the pyridine nucleotides (NAD and NADP) , coenzyme A, flavin adenine dinucleotide (FAD) and pyridoxal phosphate. Cofactor: Cofactors are non-protein compounds that are essential for the catalytic activity of many proteins. An enzyme-cofactor complex is termed a holoenzyme. The protein on its own is termed an apoenzyme. Cofactors may be organic compounds (coenzymes) or metal ions such as magnesium, manganese, calcium, zinc, potassium, etc. Cofactors tightly associated with the protein in the holoenzyme are generally termed prosthetic groups.

Enzyme Stabilizing Agent : A solution when combined with an enzyme which provides a stable environment for the enzyme such that the enzyme is stable during storage and use in cleaning solutions. Examples of enzyme stabilizing agents include mixtures of glycerol and polyethylene glycol . Another compound used to stabilize enzymes in cleaning solutions is polyoxyethylene nonyl phenyl ether. Other enzyme stabilizing agents include: hydrocarbon halides; ether; ketones; fatty acids; nitrogen oxides and organic compounds with more than two functional groups. Other enzyme stabilizing agents include organic compounds with saturated fatty hydrocarbon groups, non-saturated fatty hydrocarbon groups, alicyclic hydrocarbon groups, aromatic hydrocarbon groups, halogen groups, oxygen, sulfur and nitrogen containing groups, ether groups, carboxylic acid and ester groups, acyl groups, heterocyclic groups, and cyclic hydrocarbon groups. pH Adjusting Agents: pH adjusting agents are chemicals useful to adjust the pH of a solution in the range of pH 1-14, typically 9-14. Examples of pH adjusting agents include: monoethanolamine, acids, bases, Trizma buffer, phosphate buffer and the like. pH adjusting agents are available from Sigma Chemical, Aldrich Chemical and other manufacturers.

Vapor Pressure Reducing Agent: Vapor pressure reducing agents are added to solutions to reduce the vapor pressure of the solution thereby reducing the release of fumes from the solution. Examples of vapor pressure reducing agents include liquid paraffin.

Anti-foaming Agents: Anti-foaming agents reduce bubbles in liquid solutions and include such chemicals as propylene glycol.

Oil Dissolving Agents: Oil dissolving agents aid dissolution of substances such as waxes, oils and the like and include such compounds as diethylene glycol monobutyrin ether.

Disinfectants : Disinfectants are often added to cleaning solutions for sterilization purposes and include compounds such as alkyl diamino ethane hydrochloric acid glycine, [R(NHC₂H₄) ₂N^*HCH₂COO^"] and R(CHC₂H₄)₂N^*HC₂COO-

Clarified Solution: A chemical solution that has become clear or transparent to the human eye.

Reusable Cleaning Solution: A cleaning solution that may be used repeatedly (more than once) without significant loss of its cleaning effectiveness.

Iceberg Water: Ordered or structured water associated near hydrophobic residues. Iceberg water can surround enzymes and interact with polar molecules such as surfactants.

Clathrate Water: Ordered or structured water associated near hydrophobic residues. Clathrate water can surround enzymes and interact with polar molecules such as surfactants. Clathrate water is generally produced by pressurizing hydrophobic residues in water.

Free Fixation Enzyme: Clathrate or iceberg structured water surrounding an enzyme that is further surrounded by a surfactant. The structured water and enzyme interact with the surfactant to form a stabilized enzyme.

The invention will be better understood by way of the following examples. EXAMPLE 1

Lipase enzyme obtained from Rhizopus japonicus NR400 (Nagase Enzymes, Japan) at 8,000,000 lipase units/g dry weight is dissolved at a ratio of 1:9 enzyme:water or D₂0 (wt/wt) in a 1000 ml erlenmeyer flask at 25-30°C with mixing. The solution typically clears or becomes transparent in 5 to 10 minutes. The enzyme solution is then added to a mixture of the enzyme stabilizing agent 75% glycerol and 25% ethylene glycol solution at a vol/vol ratio of 10:90 (enzyme/glycerol-glycol) in a 1,000 ml erlenmeyer flask 25-30°C with mixing. Mixing is provided by vigorous shaking of the flask 5-6 times. The mixed solution is then allowed to sit for 2 days at 25- 30°C. During this 2 day period, the solution turns a purple/brown color and changes from a cloudy solution (like coffee) to a clear or transparent solution (like tea) . The enzyme solution is then combined with a surfactant such as monostearic acid solution.

EXAMPLE 2

Protease enzyme obtained from Rhizopus japonicus NR400 (Nagase Enzymes, Japan) at 150,000 protease units/g dry weight is dissolved at a ratio of 1.7:15 enzyme.-water or D₂0 (wt/wt) in a 1000 ml erlenmeyer flask at 25-30°C with mixing. The solution typically clears or becomes transparent in 5 to 10 minutes. The enzyme solution is then added to the enzyme stabilizing agent composed of a mixture of 75% glycerol and 25% ethylene glycol solution at a vol/vol ratio of 16.7:83.3 (enzyme/glycerol-glycol) in a 1,000 ml erlenmeyer flask 25-30°C with mixing. Mixing is provided by vigorous mixing with a mixer for a minute. The mixed solution is then allowed to sit for approximately 2 days at 25-30°C. During this 2 day period, the solution turns a purple/brown color and changes from a cloudy solution (like coffee) to a clear or transparent solution (like tea) . The enzyme solution is then combined with a surfactant such as monostearic acid solution at 6°C.

EXAMPLE 3

The lipase enzyme solution of Example 1 can be combined with other materials to form a cleaning solution useful in cleaning steel, metal, aircraft wings, engine parts and TV tubes. This cleaning solution is also useful for dissolving styrofoam. A preferred lipase cleaning solution composition is:

Components: proportion by wt .

1) Methylene chloride 88.9%

2) Monostearic acid sorbitan (Surfactant) 5%

3) Monoethanolamine (base) 5%

4) Liquid paraffin 1%

5) Lipase enzyme 0.1%

Components 1) and 2) are combined and mixed at room temperature until a clear or transparent solution is formed, generally about 5 minutes. Components 3) , 4) and 5) are then mixed together separately and then added together to the surfactant/methylene chloride mixture. Alternatively, compounds 3) , 4) and 5) are added directly to the surfactant/methylene chloride mixture at room temperature with mixing. The pH of the solution is adjusted to pH 9.8-14, typically 12 with base .

The enzyme cleaning solutions produced by the methods of this example are very stable and have superior cleaning properties. The enzyme cleaning solution showed marked improvement over known fluorine based cleaning solutions in that it cleaned steel, metal, aircraft wings, engine parts and TV tubes significantly faster and could be recycled much easier than known solutions. In addition, the enzyme cleaning solution of this Example is more stable than known enzyme cleaning solutions. See below.

EXAMPLE 4

The protease enzyme solution of Example 2 can be combined with other materials to form a cleaning solution useful in cleaning circuit boards, printing presses, silicon wafers, plastics, lenses and some metals such as drill bits. A preferred protease cleaning solution composition is:

Components: proportion by wt .

1) Laurie acid diethanolamine (surfactant) 2%

2) Water 45.2%

3) Propylene glycol 1.5%

4) Diethylene glycol monobutyrin ether 38%

5 ) [R (NHC₂H₄ ) ₂N^*HCH₂COO-] 0 . 5%

6 ) monoethanolamine (base) 9 . 5%

7 ) Polyoxyethylene nonyl phenyl ether 3%

8 ) Protease 0 . 3 %

Components 1) and 2) are agitated and melted at 70-80°C for about 3 hours or until the solution clears or becomes transparent. The solution is then cooled to room temperature under running water. Components 7) and 8) are then mixed at room temperature in a separate flask to provide an enzyme/polyoxyethylene ether mixture. The mixture of 7) & 8) is then added to the mixture of 1) & 2) . Lastly, components 3), 4), 5), and 6) are mixed together by hand at room temperature prior to adding to the mixture of 1) , 2) , 7) and 8) . The final mixture rolls and boils on its own until the mixing is complete. The mixture is then cooled to room temperature and is ready for use. A single volume of the cleaning solution was used continuously for over 2 months to clean circuit boards of average size 400cm²/piece . It is estimated that a total accumulated area of about 200 m² of circuit boards has been cleaned with the cleaning solution with only about a 15% depletion of the original solution supplied during the testing period. The process has been continued for six hours every day for over two months now and the solution has not lost its original cleansing capability. The enzyme cleaning solution cleans circuit board flux effectively without reacting with any other part on the circuit board.

In addition to being more stable than typical fluorine-based or enzyme-based cleaning solutions, the protease enzyme cleaning solution works significantly faster than cleaning agents of the prior art . It takes approximately two minutes for a typical fluorine-based cleanser to clean circuit board flux whereas it only takes from 30 seconds to one minute for the protease solution to clean an area of similar size.

EXAMPLE 5

Soldering is a major component of electronic circuit board production and produces fluxes that must be removed by washing. The soldering fluxes were analyzed and their composition included Zinc chloride (ZnCl₂) ; ammonium chloride (NH₄C1) ; bicyclic monocyclic (c--C₁₀ H₁₆) { β - C₁₀H₁₆) ; methane monocyclic (p - C₁₀H₁₆) ; abioetic Acid (C₂₀H₃₀O₂) ; α-Pidmaric Acid (a - C₂₀H₃₀O₈) and hydrocarbons.

These compounds are contained mostly in the organic components of fluxes. Furthermore, other foulings such as waxes and paintings are mostly components of the organic compounds containing hydrocarbon series . Previous washing methods have been generally insufficient to efficiently and adequately remove fluxes and other foulings. However, the cleaning solutions of Example 4 effectively removed these materials.

EXAMPLE 6

In addition to the mixture methods disclosed in Examples 1-4, enzyme cleaning solutions can be made by alternative methods. The enzyme mixture can be sprayed with solvent in a jet stream to form a cleaning solution. In addition, the enzyme and solvent can be mixed in a high speed agitator. The enzyme solutions can also be prepared in either a high pressure chamber or in a vacuum chamber. When using a high pressure chamber, common gases such as hydrogen, propane, methane or ethane are used to pressurize the mixtures of Examples 1 and 2.

Example 7

Oxidation enzymes prepared as in Examples 1, 2 or 6, coenzymes and surfactants are placed into a container filled with water and stirred well to prepare a cleaning solution.

Preferred ratios include (weight/weight) :

Oxidation enzymes 39%

Coenzyme 8%

Surfactant 53%

A suitable oxidation enzyme is alcoholoxido reductase (EC.1.1.1.1. ) . A suitable coenzyme is Nicotinic acid amido adeninjicreochidorin acid (C₂₄H₂₈N₇0₁₄P₂) . A suitable surface active agent is Polyooxy tridecyl ether (C.₃H₂₇0(CH₂CH₂0)₂H) .

To prepare a cleaning solution, the oxidation enzyme solution is mixed with a supplementary washing solution in the following ratios: Enzyme Solution 25%

Supplementary washing solution 75%

The supplementary washing solution is available commercially and its components and ratio (weight base) are:

Sodium Hydroxide 16

Sodium Carbonate 26

Trisodium Phosphate 32

Pyrosodium Phosphate 20

Alkyl allyl sulfonic acid sodium 5

Defoaming agent 1

This cleaning solution is strongly alkaline (pH 14) . Oxidation enzymes are normally inactivated under such strong alkali conditions. However, the enzymes prepared as described in Examples 1, 2 and 7 are stable and protected from the alkaline environment of the cleaning solution by the enzyme stabilizing agent. The oxidation enzymes are useful for cleaning electronic circuit boards.

Fibroin membranes can provide similar protection to oxidation enzymes as do the enzyme stabilizing agents. However, the rate of enzyme catalysis is slower when the Fibroin is used compared to the enzymes prepared as discussed above.

Coenzymes may also be included in the cleaning solutions for the purpose of enhancing the activation of oxidation enzymes. The coenzymes are not essential at all times. The use of coenzymes are dependent on the conditions of washing solutions. EXAMPLE 8

Hydrogenation enzymes can be utilized in cleaning solutions. Hydrogenation enzymes were prepared as described in Examples 1,2 and 6 and mixed in the following ratios:

Hydrogenation enzymes 39 %

Coenzymes 8 %

Surfactants 53 %

In this case, the coenzymes and the surfactant were the same as example 7. The hydrogenation solution is mixed with coenzymes and sprayed over electronic printed circuit boards. The hydrogenation enzyme solution can be combined with the supplementary solution of Example 7 to clean circuit boards.

EXAMPLE 9

Enzymes can be replaced with microorganisms in this invention.

EXAMPLE 10

Enzymes are comprised of amino acids linked together in specific sequences to form polypeptide chains. In principle, any enzyme molecule can adopt an enormous number of different shapes or conformations. However, in an aqueous environment, most polypeptide chains fold into only one of these conformations because of the distribution of the polar and nonpolar side chains among the amino acids . Hydrophobic side chains associate together in the interior of enzyme molecules to form a hydrophobic core. In contrast, amino acids with polar side chains arrange themselves near the outside of the enzyme molecule.

The conformational stability of enzymes in aqueous media is dependent on the interactions of amino acid side chains with the surrounding aqueous environment . Water molecules hydrogen bond with polar side groups and with themselves around the hydrophobic core but they cannot hydrogen bond with the hydrophobic core directly.

It is well known that water solvation of nonpolar groups has a destabilizing action on hydrophobic bonds. Proteins. Thomas E. Creighton, W.H. Freeman and Company, New York. 1993. Thus, the hydrophobic core of enzymes is much more stable if water is removed from the center of the enzyme.

Removal of water from the hydrophobic core of enzymes alters the structure of water molecules surrounding the hydrophobic core. The hydrogen bonds existing in water cannot be transferred to the nonpolar hydrophobic core. As a result of this inability to bond to the hydrophobic core, the water around the hydrophobic core becomes ordered when the enzyme is dissolved in water. This ordering of water around the hydrophobic core leads to the formation of hydrogen bonded clathrate-like water structures around the hydrophobic core. This water ordering effect has also been described as iceberg formation. See "Iceberg Formation and Solubility, Kozo Shinoda, Journal of Physical Chemistry 81:1300-1302, 1977. The water ordering effect increases the solubility of the nonpolar molecules in water. Proteins. Thomas E. Creighton, W.H. Freeman and Company, New York. 1993, pp. 159-160.

Water is removed from the hydrophobic core of proteins with the formation of water clathrates or icebergs during protein hydration when dried protein powders are dissolved (hydrated) in aqueous solution. Water removal from the hydrophobic core of enzymes can be accelerated by pressurizing or depressurizing the enzyme solution as described in Example 6.

As discussed above, water removal from the hydrophobic core of membranes coincides with iceberg or clathrate formation. The removal of water can thus be described as "squeezing" iceberg water out from within the hydrophobic core of the enzyme. Once removed from the hydrophobic core, the iceberg water surrounds the hydrophobic core of the enzyme and interacts with the hydrophilic region of the protein. This phenomenon is illustrated in FIG. 1.

Hydrogen gas is preferred for the production of iceberg water with enzymes. However, other gases may be used. It is thought that if the gas used to pressurize the enzymes is varied during the structure of the resulting clathrate or iceberg will vary. Many chemicals are thought to produce clathrates or icebergs, including: Ar, Kr.0₂, N₂.H₂S.CH₄, Xe.H₂Se.PH₄, CH₃F, C0₂, AsH₃.CH₂F₂, N₂OS0₂, CHF₃.CF₄.CH₃C1, Cl₂.SbH₃.CH₂ClF. (CH₂)₂0, C10₂.C₂H₆.CH₃Br, BrCl.C₂H₄. (CH₂)₃.C₂H_SF.CHC1F₂,

COS.C₂H₂.CH₂=CHF.CH₃CHF₂.CH₃SH. (CH₂)₃0, 1, 3-Dioxolane, CH₃.CHBrF₂, SF₆.CBrF₃, Tetrahydrofuran, cyclopentane.CH₂Cl₂,CHCl-F. (CH₃)₂0. Dihydrofuran, C2H5C1.CC12F2.CH3CF2C1.CBrClF2,

C₃H_β.Cyclopentene.C₂F₄.CH₂=CHC1, CH₃CHC1₂.Furan.Acetone, CH₃CH=CH₂.CHCl_j.CCl₃F,

(CH₃)₃CH. (CH₃)₃CF.C₂H₅Br.Propylene.Oxide. Cyclobutanone, and CBr₂F₂.

As a result of hydrogen bonding between water molecules, clathrates or icebergs form a stable shape around solute molecules of the proper size. Possible structures are shown in Figures 5-1- (A) and is 5-2- (A) and (B) . Bromine gas is thought to create a fif een-faced hydrate. Alkylamine groups are thought to create a fourteen-faced hydrate. Fluoro-tetra- iso-amyl-ammonium are thought to create two fifteen- faced and two fourteen faced Iceberg hydrates inside a fused shell. Methyl-ammoniumhydroxide is thought to create a cation coated decahedron hydrate partially open to take in -OH's. Ethanol is also thought to create hydrates similar to that of diagram B-2 below -73.5°C and at proper concentrations. EXAMPLE 11

The formation of clathrates or iceberg structured water around enzymes is thought to increase the interaction of the enzyme with surfactant molecules. The interaction of the iceberg water with a surfactant yields a free-fixation enzyme. Free-fixation enzymes have superior properties to surfactant and enzyme mixtures where the enzyme is not surrounded by iceberg water.

As discussed in Example 10, iceberg structured water is thought to be "squeezed" out from among the hydrophobic core of an enzyme. The phenomenon leaves the enzyme tightly folded and surrounded by the iceberg water. The iceberg water is in turn surrounded by a surfactant forming a free fixation enzyme. See FIG 2.

It is thought that the interaction of the iceberg enzyme and the surfactant provide the superior properties to the free fixation enzyme. Surfactants have both hydrophilic and hydrophobic moieties. The hydrophilic group of the surfactant is thought to associate with the surface of clathrate/iceberg enzyme while the hydrophobic group of the surfactant associates with the external solution. (See FIG. 2.)

The superior qualities of the free fixation enzyme are demonstrated by its use as a cleaning agent. The free fixation enzyme easily reaches organic substrates during cleaning since the free fixation enzyme is easily opened up to expose the enzyme to substrate. Once open, the free fixation enzyme can catalyze substrate as shown in Fig. 3.

Hydrophobic interactions cause the split open free fixation enzyme to close again and return to original shape as shown in Fig. 4. Closure of the free fixation enzyme provides protection for the enzyme from the external environment. (Step 1 through Step 4) This cycle may be repeated many times and it is this aspect of free fixation enzymes that is thought to be responsible for the reusability of the free fixation enzyme cleaning solutions.

Although the invention has been described in some respects with reference to specified preferred embodiments thereof, many variations and modifications will be apparent to those skilled in the art. It is, therefore, the intention that the following claims not be given a restrictive interpretation but should be viewed to encompass such variations and modifications that may be routinely derived from the inventive subject matter disclosed.

Claims

Claims :

1. A reusable cleaning solution comprising: an enzyme; a surfactant, and an enzyme stabilizing agent comprising a mixture of glycerol or ethylene glycol and mixtures thereof .

2. The cleaning solution of claim 1 wherein the ratio of the enzyme to the mixture of glycerol and ethylene glycol ranges from 1:1 to 1:40 on a volume basis .

3. The cleaning solution of claim 1 wherein the surfactant is selected from the group comprising anionic, cationic, nonionic, and zwitterionic surfactants .

4. The cleaning solution of claim 1 further comprising a solvent.

5. The cleaning solution of claim 4 wherein the solvent is methylene chloride.

6. The cleaning solution of claim 1 further comprising a base.

7. The cleaning solution of claim 6 wherein the base is monoethanolamine.

8. The cleaning solution of claim 1 further comprising a vapor pressure reducing agent.

9. The cleaning solution of claim 8 wherein the vapor pressure reducing agent is liquid paraffin,

10. The cleaning solution of claim 3 wherein the surfactant is monostearin acid sorbitan. 11. The cleaning solution of claim 6 wherein the pH of the solution is from 9.0 to 14.

12. The cleaning solution of claim 1 wherein the enzyme is a lipase.

13. The cleaning solution of claim 4 wherein the solvent is water.

14. The cleaning solution of claim 13 further comprising a base.

15. The cleaning solution of claim 14 wherein the base is monoethanolamine.

16. The cleaning solution of claim 15 further comprising diethylene glycol monobutyl ether.

17. The cleaning solution of claim 16 wherein the pH of the solution is from 9 to 14.

18. The cleaning solution of claim 17 wherein the surfactant is lauric acid diethanolamine.

19. The cleaning solution of claim 18 further comprising poloxyethylene nonyl phenyl ether.

20. The cleaning solution of claim 1 wherein the enzyme is a protease.

24. A reusable cleaning solution comprising: methylene chloride; a surfactant; monoethanolamine; liquid paraffin and a lipase.

25. The cleaning solution of claim 24 wherein the surfactant is monostearic acid sorbitan.

26. A reusable cleaning solution comprising: water; a surfactant; propylene glycol ; diethylene glycol monobutryin ether; [R(NHC₂H₄)₂N'HCH₂COO-] ; mononethanolamine; polyoxyethylene nonyl phenyl ether and a protease .

27. The cleaning solution of claim 26 wherein the surfactant is lauric acid diethanolamine.

28. A method of preparing a reusable enzyme containing cleaning solution comprising: dissolving an enzyme in aqueous solution, adding a stabilizing agent to the enzyme solution to form an enzyme and stabilizing agent mixture and allowing the mixture to sit until the mixture becomes clarified.

29. The method of claim 28 wherein the aqueous solution further comprises D₂0.

30. The method of claim 29 wherein the enzyme stabilizing agent comprises a mixture of glycerol and ethylene glycol .

31. The method of claim 29 further comprising the step of combining the mixture with a solvent and a surfactant.

32. The method of claim 31 wherein the solvent is methylene chloride. 33. The method of claim 31 wherein the surfactant is selected from the group comprising lauric acid diethanolamine and monostearic acid sorbitan.

34. A reusable cleaning solution prepared by the steps comprising: dissolving an enzyme in aqueous solution to give an enzyme solution; adding an enzyme stabilizing agent to the enzyme solution to form an enzyme-stabilizing component mixture; and allowing the mixture to sit until a clarified solution is produced.

35. The cleaning solution of claim 34 wherein the aqueous solution further comprises D₂0.

36. The cleaning solution of claim 35 wherein the enzyme stabilizing solution comprises a mixture of glycerol and ethylene glycol .

37. The cleaning solution of claim 36 prepared by the additional step of combining the mixture with a solvent and a surfactant.

38. A reusable cleaning solution prepared by the steps comprising: dissolving an enzyme in an aqueous solution to give an enzyme solution; adding an enzyme-stabilizing agent to the enzyme solution to form an enzyme-stabilizing agent mixture; placing the mixture in a sealed container; pressurizing the mixture by adding gas to the sealed container to form a pressure-treated enzyme; and adding the pressure treated enzyme to a surfactant to form a surfactant-enzyme solution.

39. The cleaning solution of claim 38 wherein the enzyme is selected from the group comprising proteases, lipases, oxido reductases or hydrogenation enzymes an the gas is selected from the group consisting of hydrogen, methane, ethane and propane.

40. A method of cleaning surfaces with a reusable enzyme cleaning solution comprising: dissolving an enzyme in an aqueous solution; adding an enzyme-stabilizing agent to the enzyme solution to form an enzyme-stabilizing agent mixture; placing the mixture in a sealed container; pressurizing the mixture by adding gas to the sealed container to form a pressure-treated enzyme adding the pressure treated enzyme to a surfactant to form a surfactant-enzyme solution; and contacting a surface to be cleaned with the surfactant-enzyme solution.

41. The method of claim 40 wherein the enzyme is selected from the group comprising proteases, lipases, oxido reductases or hydrogenation enzymes an the gas is selected from the group comprising hydrogen, methane ethane and propane.

42. A free fixation enzyme.