Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
1998-01-30
2001-01-30
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S183000, C435S195000, C435S252300, C435S262500, C435S252340
Reexamination Certificate
active
06180381
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to the degradation of polyurethane and more specifically to the enzymatic degradation of polyurethane.
2. Description of the Related Art
Polyurethanes are a diverse group of man-made polymers of considerable economic importance that have a wide range of chemical and physical properties. Polyurethane-based coatings, such as polyurethane paint, are used on many structures such as buildings, vehicles, boats, and aircraft. One of the main benefits of using polyurethane-based coatings is the environmental resistance of polyurethane. However, the environmental resistance also makes it difficult to remove.
Currently, polyurethane-based coatings are removed, for repair and repainting, using organic solvents such as methlyene chloride. However the use of methylene chloride creates large amounts of toxic waste. In fact, some uses of methylene chloride are banned in some States. Alternatively, polyurethane-based coatings are removed using blast cleaning with plastic beads. However, the blasting can damage some types of composite surfaces being cleaned. Moreover, the spent plastic beads create new waste disposal problems. Also, the polyurethane particles removed are not degraded to harmless components.
As an alternative, biological degradation of polyurethane has been proposed. Biological degradation of naturally-occurring polymers, such as chitin and cellulose, involves induction of hydrolytic enzymes by soluble oligomers released form the polymer surface. Through the oligomers, the hydrolytic enzymes break down the polymer into biologically digestible components. However, induction of enzymes for degrading a synthetic polyurethane-coated surface is more difficult because of the lack of soluble material (oligomers) released from weathered, painted surfaces. Accordingly, the direct use of microbes to degrade polyurethane coatings on surfaces is impractical.
SUMMARY OF THE INVENTION
Accordingly, it is an object of this invention to remove a polyurethane coating from a surface without the use of organic solvents or abrasives.
It is a another object of the present invention to remove a polyurethane coating from a surface in an environmental acceptable manner.
It is a further object of the present invention to remove a polyurethane coating from a surface without damaging the surface being cleaned.
These and other objects are achieved by a enzymatic preparation obtained from the culture of a newly developed, man-made mutant strain of
Pseudomonas chlororaphis
that has an enhanced ability to degrade polyurethane. Additionally, a system for the isolation and generation of mutant strains of bacteria having an enhanced ability to degrade polyurethane is disclosed.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
Despite their synthetic polymeric origins, some polyurethanes are susceptible to microbial degradation. Though the specific biological mechanisms are responsible for degradation have not been well-characterized, polyurethanes contain several chemical linkages (bonds) that could be enzymatically hydrolyzed including ester, amide, urethane, urea, and biuret bonds. It has been possible to isolate microorganisms from polyurethane-coated surfaces in the environment.
One microorganism used in the present invention is a mutant strain of
Pseudomonas chlororaphis
designated BC2-12. A subculture of the microorganism may be obtained from the permanent collection of the American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD 20852, where it was deposited on Dec. 14, 1995 and received the number ATCC 55729. The original wild-type microorganism was isolated from a nutrient enrichment culture using weathered paint chips as the inoculum. The wild-type microorganism was screened for production of polyurethanase on culture plates containing colloidal polyurethane and tentatively identified as
Pseudomonas chlororaphis
. This specific strain was designated as
Pseudomonas chlororaphis
BC2. The polyurethanase activity of
Pseudomonas chlororaphis
BC2, however, may be too low to permit practical production of preparations having a high polyurethanase activity.
The production of a genetically altered bacteria that produces large amounts of polyurethane starts with the collection and screening of wild-type bacteria for strains that exhibit the ability to degrade polyurethane. Appropriate wild-type bacteria may be collected, for example, by isolating bacteria strains from polyurethane-based paint fragments that have been exposed to the elements.
These isolated strains are then screened for their ability to degrade polyurethane. The ability to degrade polyurethane can be observed, for example in an appropriate culture medium that contains colloidal polyurethane, which serves as a soluble inducer for the production of polyurethanases. The original polyurethane-Docket containing culture plate is opaque. Conversion of the semi-solid culture medium to a translucent state after inoculation with bacteria and culturing evidences that the wild-type strain produces extracellular polyurethanases.
An suitable vector is then used to introduce a transposon, along with a flanking gene for resistance to a specific antibiotic, into isolates of bacterial strains producing extracellular polyurethanases. The transposon inserts into a random locus within the genome of the bacteria. Chance insertion of the transposon into an appropriate locus provides resistance to the antibiotic and may also destroy the mechanism that normally inhibits the production and/or activity of extracellular polyurethane-degrading enzyme released into the culture medium. Then, the bacterial cultures exposed to the transposon are screened for resistance to the specified antibiotic, which, if present, indicates successful transfer of the transposon DNA into the host bacteria. A Southern Blot or similar test may then confirm the random inseration of the transposon into the genome of the host bacterium.
A wild-type polyurethanase-producing bacterial culture and the mutant bacterial cultures exhibiting resistance to the specified antibiotic are then cultured in the presence of colloidal polyurethane. The cultures are then centrifuged and the supernatant isolated. This supernatant is then assayed for the level of polyurethanase activity. Mutant bacterial cultures that produce supernatants having a polyurethanase activity significantly greater than that of the supernatant produced by the wild-type polyurethanase-producing culture are the desired over-producers of polyurethanases. The supernatant is the desired polyurethanase-containing preparation.
Any vector may be used for insertion of the transposon into the wild-type polyurethanase-producing strains. For example, the transposon can be introduced into the strains by parental matings or conjugation with a donor strain of bacteria such as
E. coli
(including, but not limited to,
E. coli
S17-1 (Miller et al.,
J. Bacteriol,
170:2575-2583 (1988), incorporated herein by reference in its entirety for all purposes) and
E. coli
SM-10 (Simon et al.,
Biotechnology
1:784-791 (1983), incorporated herein by reference in its entirety for all purposes) including a plasmid with the desired transposon flanked by a gene for resistance to a specified antibiotic, direct introduction into the recipient strain of a plasmid including the transposon (flanked by a gene for resistance to a specified antibiotic) by transformation, altering the porosity of the recipient strain's cell membrane, and transduction by phage (including, but not limited to P1) that incorporates the transposon flanked by a gene that imparts resistance to a specified antibiotic).
The transposon may be any transposon which flanks a gene that imparts resistance to a first antibiotic to which the wild-type bacterial recipient will not spontaneously acquire resistance. For example, if the recipient bacteria is
P. chlororaphis
, the transposon miniTn5 flanking a gene encoding for tetracycline resistance is a suitable transposon, since
P. chlororaphis
Campbell James R.
Crabbe Joel R.
Montgomery Michael T.
Thompson Laura
Walz Steven E.
Karasek John J.
Prouty Rebecca E.
Rao Manjunath
Ressing Amy L.
The United States of America as represented by the Secretary of
LandOfFree
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