JPH03280876A - Lipase gene and use thereof - Google Patents

Abstract

NEW MATERIAL: A pseudomonas cell containing a lipase gene of a chromosome and at least one multi-copy vector containing a lipase gene expressing lipase in pseudomonas cell, wherein said lipase is derived from a cell strain ATCC21808.

USE: A component of a cleaning product, etc.

PROCESS: A cell of a pseudomonas strain composed of multiple copies of a pseudomonas lipase gene encoding a lipase derived from a microbial strain ATCC21808 and a lipase gene of a genom is grown for a period under a condition sufficient for expressing the lipase.

COPYRIGHT: (C)1991,JPO

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to the preparation of lipase in Durham negative hosts and the use of lipases with said hosts.

Prior Art Microorganisms produce a variety of products of commercial interest. Natural product manufacturing methods suffer from low yields, difficulty in isolating the product,
Due to high manufacturing costs etc., it is often useless. In some cases, a particular host may not tolerate transformation, efficient production, or scale-up to commercially useful production levels.

In other cases, the yield may be temporary or variable; the plasmid may be unstable, especially if the gene expressing the protein of interest is plasmid-based. It is therefore of interest to be able to modify organisms and consider efficient production of naturally occurring products of interest.

Recombinant DNA technology provides the opportunity to modify cells. Such techniques include the use of chains that encode peptide products, and the use of open reading frames to express replication initiation regions associated with a gene other than natural peptides.
Examples include creating mixed chromosomal genes by linking to a specific gene (frame), preparing for rough outgrowth of specific genes, and selecting hosts that provide improved phenotypes. These steps are labor intensive and require extensive experimentation to isolate and identify the linkage, modify the structure to the appropriate one, transform it into the host, and demonstrate product formation. I need. Each of these means has a wide range of operations,
Includes proof that the desired operation has been performed and that no sudden displacements have occurred. Each construct has a novel NA on the host cell.
There is uncertainty regarding the effects of linkage and to what extent host cells will adapt to the new linkage and prepare for expression. Finally, there is the nature of lipase, its properties, and whether it has commercial applications.

Related Literature There is a large body of literature on the cloning and identification of microbial lipases. For example, Japan Patent Application Ring No. 59-43899,
Publication No. 6O-188072 (published September 25, 1985); Odera et al., J.

Ferment-Technol, (1986), 6
4.363-371; Kujimiya et al., Biochem, B 1op189; Haas (H
aas), J, A, O, C, S (1987), l.
Abstract No. l 42; Gotsutsu (Gotsutsu)
tz) et al., Nucleic Ac1d Res, (19
85), above 3. 5895-5906 and WO361
See 06743.

A large body of literature has investigated the characteristics of lipases from various microorganisms. For example, Lee and Iandolo, J., Bacteriol.
ogy (1985), Vol. 1, 288-293; (19
86), No. 15385-391; Seitz
, J.A., O.C.

s, (1974), l 2-16; Kosugi (Kosugi)
sugi) and Kamibayashi
), J. Ferment.

Technol, (1971), Sat 9, 968-980
;Lu and Liska, Appl
, Micro.

(1969), Yu, 108-113;
ino) et al., Agric, Biol, C:hem, (1
987), l, 181-183; A mma
r) and McDaniel, Mik.
robiol, (1984), supra 39.61-68, and Kokusho x-(Kokusho) 159-116
4; (1982) 46, 743 1750.

Literature related to wide host range plasmids: 351;
I toh et al., Plasmid (1984), 11.206-220; I toh and Bar 299-308;
r 1an) et al., Gene (1981), 16.237
-247; and Friedman
) et al., Gene (1982), supra 8, 289-296.

Interesting patents related to lipase include JP (Japanese Patent) 86154508 (JP622/I 0981);
JP86154509 (JP622/10986); J
P86154510 (JP622/10987); EP
(European patent) 86/72307 (JP622/2
8279);EPO238023;EPO243338
can be mentioned.

SUMMARY OF THE INVENTION Expression constructs, vectors, transformed hosts, and methods of using the hosts are provided for the efficient production of specific Pseudomonas lipase enzymes with desired properties. Organisms and products are
It can be used as a component in detergents and lipid stain removal barrel cleaning products, as a component in the hydrolysis and production of esters, i.e. monoglycerides, decomposition of ester stereoisomers, and lactone formation by cyclization of hydroxy-substituted dicarboxylic acids. They are precursors for the synthesis of aroma molecules and the production of peracids.

DETAILED DESCRIPTION OF THE INVENTION DNA constructs containing the pseudomonas lipase gene are provided. The construct is transformed into a suitable host for expression of the lipase, especially as a stable episomal element.

Lipases have been isolated and have a variety of commercial applications.

The lipase gene used is the secretable Pseudomonas lipase present in Pseudomonas sp. (sp.) ATCC 21808. The gene and its constituents are 7 fragments (fr) from the indicated strains.
It may be obtained by transforming a Lip-host (a host lacking lipase activity) with a vector containing the lipase activity. Various Pseudomonas Lip-strains, such as Pseudomonas oleoporans (P, Ol5ovora
ns) There is a strain of ATCC8062Rif'. Lip+ transformant (
The presence of a host with lipase activity can be detected by its ability to hydrolyze lipid esters such as castor oil.

Once the 7 fragment containing the gene is identified, the fragment can be manipulated in a variety of ways.

In particular, if the initial library or gene bank contains fragments of 10 kbp or more,
Fragments may be subjected to further restriction using different endonucleases to prepare fragments in the range of about 2 to 1 okbp. The small 7-ragment is
It has many advantages such as reduced number of available restriction sites, simple manipulation, and avoidance of deleterious linkages.

In addition, an open reading frame (openre
One may wish to identify the 5″-untranslated region that serves as a regulatory region for transcription and translation initiation (adding frame) and initiation of transcription and translation. Regulatory regions can be created in a variety of ways, for example by replacing the transcription initiation region with a different region. ,
Alternatively, regions that provide for induction regulation may be combined and denatured by changing the temperature or by removing or removing metabolites. If desired, promoter regions may be added or replaced so that two or more translation initiation regions are arranged in tandem.

Translation initiation regions that may be used in Pseudomonas include β-galactosidase, α-amylase, glycerol-6-7 phosphate, dehydrogenase, alkaline phosphatase, protease, toluene decomposition, etc.
This is a region for promoters, 9-promoter promoters, etc. The region may be endogenous to the Pseudomonas species host and may be endogenous to the Pseudomonas species host and
.

It may be exogenous to Pseudomonas, such as regions that are functional in Pseudomonas from E. coli, Bacillus).

Restriction fragments may be selected to contain the complete gene, including the regulatory regions necessary for expression.

Generally, natural fragments are about 0.5 kbp or more, more commonly 3 kbp or more, and about 10 kbp.
p, more usually no more than 8 kbp.

In some cases, it is desirable to engineer linkage by replacing the 5°-untranslated region with a different regulatory region.

The 5'-untranslated region may be removed by restriction, excision, blazer repair, in vitro alteration, and the like. All or part of the structural gene encoding the lipase may be synthesized. This is particularly the case when one or more sudden mutations are introduced into the lipase by denaturing, inserting or deleting base pairs of the structural gene.

A cloning vector may also serve as an expression vector, in which case the cloning vector
It has a replication system that is functional in the host. In particular, if the fragment is identified by lipase expression in a negative background, the vector is isolated and
It may then be transformed into an expression host for production of the desired lipase. If desired, the gene may be isolated, manipulated, and inserted into different vectors for transformation and expression of the lipase gene.

Vectors are characterized by having one or more replication systems. For example, if the replication system is a cloning host, I;
For example, a shuttle vector may be used if present for E. coli or an expression host, such as Pseudomonas sp. Additionally, there is usually one or more selectable markers for host cells transformed with the vector. The marker may be biocidal resistant, such as antibiotic resistant, prototropic to an auxotrophic host, and the like. Other functions that may exist include mobility.
11. A copy system with no X transmission (no X transmission), high copy number, and no tome. In particular, it is a polylinker that is sensitive to temperature.

Vectors can be prepared using conventional methods such as transfection, conjugation, electroporation, etc.
(oration), injection, fusion, transduction, etc.; all of these methods involve
into a cellular host and prepare for the replication of such NA.

In some cases, it is desirable to have many multicopy plasmids in the same host to promote the copy number of the lipase gene. This refers to incompatibility groups with different replication systems,
For example, different P, incompatibility groups (P, im
compatibility group).

Although expression of the lipase may be carried out in any conventional host, particularly a prokaryotic host, it is desirable that the host to be controlled be able to be transformed into a Pseudomonas host, particularly a Pseudomonas host from which the gene can be isolated. By choosing a Pseudomonas host, which is already a relatively high reverse producer (positive background selected for high production), improved results can be obtained.

Recombinant expression vectors should be multicopy, providing 5 or more copies, usually 10 or more, preferably about 15 or more copies, and may not have more than 100, or usually more than about 20 copies. The transformed host has at least 10 times the amount of lipase naturally produced by the untransformed host, preferably about 15 times or more, and more preferably about 20 times or more. Untransformed hosts are typically incubated at a concentration of 520 U/mff under the conditions described in the experimental section for approximately 24-96 hours.

Lipases of interest are temperature-stable and have a wide pH range.
active in the range and characterized by having an optimum value on the alkaline side. Additionally, lipases are commonly secreted.

Particular replication systems include P-1 or P-4 incompatibility group plasmids, such as pKT231 (Padosarian (B
Adsar 1an) et al., Gene (1981), 16
．． 237-247), RP4, RK2, pVsl (I toh and Hass, Gene
(1985), 36, 27-36), pRK290, etc. A particular host of interest is Pseudomonas oleovorans (oleov
orans), Pseudomonas-1 subi, ATCC2
1808, Pseudomonas putida,
(Bacillus 5ubtilus),
and Bacillus superioris niformis (Iichenifo
rmis).

The transformant is grown under conditions that maintain viability and promote stable structure of the lipase. The cell concentration is generally about 10'~
It is in the range of 1011 cells/mQ. , the temperature is generally 28
It is in the range of °C to 39 °C. The nutrient medium that can be used is PY
EO (see Example 5 below).

Other usage conditions include normal rotary vibration (250r
pm) and baffles (baf f Ie).

Lipase may be isolated in conventional manner by cell separation, concentration of surface float or filtration, precipitation with ethanol, redispersion and reprecipitation if appropriate. The enzyme was further subjected to reverse phase chromatography and pre-isoelectric focusing (pre-isoelectric focusing).
parative isoelectuic focus
ing).

Of particular interest are fully mature secreted lipase enzymes having an amine terminus with substantially the following amino acids and DNA linkages at the N-terminus: Ala Asp Asn Tyr Ala Ala T
hr Arg Tyr Pro Ile 5' GCC
GACAACTACGCG GCG ACG CGT
TAT CCGTC Estimated active position amino acid and encoding DNA
The chain is 1Leu Val Gly
His Ser Gln Gly Gly 5'CT
CGTCGGCCACAGCCAGGGCGGG where the underlined amino acids are those that are conserved in lipases from many sources.

The predominant composition is applicable to the hydrolysis of esters, in particular the hydrolysis of fats to produce fatty acids and monoglycerides. The process may be reversed if desired, and lipases may be used to esterify polyols such as glycerol or sugar. Furthermore, it has been used for the enantioselective hydrolysis of esters and enantiomerically enriched synthons.
thon) may be prepared.

Therefore, esters containing stereoisomeric fatty acids and/or esters may be used to obtain products enriched in specific enantiomers.

The lipases of the present invention can be usefully used as lipolytic additives to liquid and granular detergents. Detergent compositions generally contain the essential ingredients surfactants, detergent builders, bleaching agents, fluorescent whitening agents and adjuvants.

Adjuvants may be enzymes such as proteolytic and lipolytic enzymes. Lipases should be stable in detergent solutions in the presence of commonly used detergent proteases.

Additionally, the lipases described herein provide improved detergency and better stability, especially for enzyme-sensitive soils (fats, etc.). Furthermore, this lipase
It may also be used in an activation system for in situ generation of bleaching peracids.

Detergent compositions using lipases are exemplified in the literature. For example, EPA258008 (this document is hereby cited as part of the specification, especially page 4, line 22~
g5, line 24).

The present invention will be explained below using Examples, but the present invention is not limited to the present invention in any way.

Experimental Example I Part I: B, SP, (P, sp,) AT
CC21808 (American Type Culture Collection (A+nerican Type Cult)
ureCo11ection), Rock Billet (Rock
Genomic DNA from M.D. (MD))
The isolated genomic DNA of Pseudomonas sp.
Isolated from CC21808. It secretes the lipase enzyme, and Amano Pharmaceuti
It was first isolated from soil by scientists at Cal) Co., Ltd. (Nagoya, Japan). Its microbial characteristics, i.e. its morphology, culture properties, and other microbiological characteristics, are described in US Pat.

B, SP, (P, sp,) ATCC21808
The bacteria are Durum-negative non-spores that form polar flagellated aerobic bacilli.

The Beef Sp.
g (Difco Laboratories)
rations), Detroit, Ml.
) and 5 g of Pegton (Difco Laboratories,
(Detroit, MI) overnight at 30°C.

Bacteria were harvested by centrifugation at 2950 xg for 20 min at 40°C and incubated with 0.05 M Tris (Sigma Chemical Co., Ltd., St. Louis, MO). )) and resuspended in 0.05 M ethylenediaminetetraacetic acid (EDTAX Sigma Chemical Co., St. Louis, MO) at pH 8. Next, before lysing the cells, lysozyme (I m
y/m12) (Sigma Chemical Co., St. Louis, MO). cells at 0.5% (w/V
) Sodium dodecyl sulfate (SDS) (Pierce Chemical Co., Ltd., Rockford, I.L.)
IL)), 0.05M Tris pH 7.5, and 0.4
Dissolved in 1/4 volume of detergent solution containing M EDTA. Cell lysis was completed by culturing the disrupted cells at 50°C for 1 hour. Additionally, proteins in the cell lysate were extracted with protease K (Sigma) during the heating step.

Chemical Co., St. Louis, M.O.
1 g/an).

Nucleic acids (DNA and RNA) were purified using unbuffered phenol/chloroform (1:l, v/v
Biotechnologies, Inc., New York, CT (CT) twice and with chloroform/isoamyl alcohol (Sigma Chemical Co., St. Louis, MA
It was separated from other cellular components by repeated extraction. Nucleic acids were purified with 7.5M ammonium acetate (International Biotechnology Seeds Co., Ltd., New Burpen,
2 volumes of cold 100% ethanol (International Biotechnology Seeds Ltd., Neubergen, CT) were added to precipitate. Nucleic acid was dispensed onto a sterile glass Pasteur pipette. The nucleic acid was dissolved in 0.05M Tris pH 7.

Redissolved in a solution containing 5.1 mM EDTA, and 200 pg/ml RNase A (Sigma Chemical Co., MA, St. Louis, MA). Arnase A hydrolyzed contaminant RNA.

The DNA was re-extracted with chloroform/isoamyl alcohol, reprecipitated as described above, spun up and 0.5%.
Redissolved in 05M Tris pH 7.5 and 1mM EDTA. Approximately 500 μg of DNA was transferred to B, SP, AT
CC21808 cells were isolated from a 50ml culture.

Part 2 Isolation of Niblasmid Vector pcP13 DNA Plasmid FpCP13 (F, Au5
ubel'), Department of Molecular
Biology (Dept, of Molecular
Biology), Massachusetts General
Hospital (Massachusetts Gener
al Ho5pital), Boston, M.N.
A)) is a broad host range cosmid vector derived from plasmid pLAFR1 and described by Friedman et al., Gen.
e (198, 2), supra 8, 289-296. Vector pcP13 has a size of 23 kilobases (
kilobasesX K b) to encode tetracytalin (Tc) and kanamycin (Km) resistance;
Although it contains many unique endonuclease restriction sites and is mobile (Mob),
Self-not transmissible (Tra-).

Plasmid pcP13 was produced by I toh et al., P
Iasmid (1984), 1, supra, 206-220, was slightly modified to obtain Escherichia coli strain HB1.
01 cells. E, Corey (E, c
oli-) HB 101 (pCP 13 ) cells,
Grow in Luria liquid culture medium overnight on a rotary shaker (175 rpm) at 32° C. with aeration.

Luria liquid culture medium contains 10 g (grams/liter) of tryptone (Difco Laboratories, Detroit).
MI), 5 g of yeast extract (Day 7 Co Laboratories, Detroit MI), and 10 g of sodium chloride (NaCQ) (Sigma Chemical Co., MA, St. Louis, MI). This medium is 2
3 μg/mQ tetracycline (Tc) (Calbiochem), La Jol
la), Seanie (CA)).

E. coli cells were harvested by centrifugation at 5000×g for 10 minutes at 4°C. The cell was washed with a detergent solution containing 20 cetyl ether (Brij-58 (Brij-58X Sigma Chemical Co., St. Louis, MO) and sodium deoxycholate (Sigma Chemical Co., St. Louis, MO). Dissolved.

After dissolving (10-20 minutes), the clear lysate was
40°C for 0 minutes! , by centrifugation at 39,000×g. After decanting the supernatant layer, PEG60
00 (polyethylene glycol) (Sigma Chemical Co., St. Louis, MO) and 3% (w/v) sodium chloride were added. The lysate was incubated overnight at 40°C to complete precipitation of plasmid DNA.

The precipitated plasmid DNA was incubated at 4°C for 10 minutes at 1400 °C.
Collected by centrifugation at 0×g.

DNA pellet (0.05M), pH 8, 5mM
Dissolved in EDTA, and 0.05M sodium chloride,
and then 9g of cesium chloride (International
Purified in a cesium chloride gradient using 0.75 m<1101/ethidium bromide .

After incubating the DNA solution at 4°C for 60 minutes, the polyethylene glycol precipitate that had become a linear mass was incubated at 4°C for 45 minutes.
Lysed by centrifugation at 12,000xg for minutes. isolated from plasmid DNA. plasmid DNA
was separated from chromosomal DNA by centrifugation in a Beckman Ti70.10-tar at 4500 Orpm at 18°C for 45 hours. A lower band of plasmid pcP13 DNA
was collected with a syringe. Ethidium bromide equilibrated against a saturated aqueous solution of cesium chloride in n-butanol (Sigma Chemical Co., MA, St. Louis, MA).
was removed by extraction three times. 0 cesium chloride
．． 01M Tris, 1mM EDT A pH 8,0 (T
E) Dialysis was performed using three changes of buffer. Approximately 400μg pC
P13 DNA was isolated from 1 liter of culture fluid.

Part 3: BSP ATCC21808DNA
Isolation of 7 fragments of 15-3 OKb size of total DNA from B.S.P. ATCC21808 (
550 μg) and restriction enzyme 5hoI from New England Bio.
Labs, Beverly, MA 31.25-L = Knit Unit
0.01M magnesium chloride (MgC1z), 100
μg/mR bovine serum albumin (Bovine Se
rum albumin (BSA)), and the above solution was incubated at 37° C. for 1 hour. One unit of restriction enzyme is 0.05 mO, (7) f.

It is defined as the amount of enzyme required to completely digest 1pg of lambda DNA in 37°0160 minutes. Next, the restriction enzyme was added to unbuffered phenol/chloroform (
It was removed from the digestion mixture by extraction twice with 1:Lv/l and twice with chloroform/isoamyl alcohol (24:1, v/v). Digested DNA was reprecipitated by adding 0.5 volumes of 7.5M ammonium acetate and 2 volumes of cold 100% ethanol.

After incubation at −70°C for 30 min, the precipitated DNA was collected in an Eppendorf centrifuge (54
15;7' Brinkman Instruments (Brink
man Instruments Inc., Westbury, NY) by centrifugation at 13,000×g for 30 minutes at 4°C. After washing the DNA pellet once with 70% cold ethanol, the DNA was soaked in 1 m TE buffer pH 8.
Redissolved in Q. Small aliquots of partially digested DNA were analyzed by agarose gel electrophoresis. According to this analysis, the majority of the DNA was longer than 1 OKb.

Fragments of 15-30 Kb size were separated by sucrose density centrifugation. Polyaromer (poly
al Iomer) ultracentrifuge tube (Beckman
Instruments Co., Ltd., Palo Aldo
Alto), Sheeny (CA)) 11.4m (l of 2
5% (W/V) saccharose (International Biotechnology Co., Ltd., New York, CT) solution (0.02M 1-Lys pH 8, 5mM EDTA)
, and prepared in 1M NaCl),
Continuous (
A 10-40%; w/v) sucrose gradient was assembled. After gradually dissolving the saccharose solution over 4 hours at 4°C, the partially digested DNA solution was dissolved in IM NaCQ,
Readjusted to 0.02M Tris, pH 8, and 5mM EDTA and applied to the top of a continuous sucrose gradient.

Ultracentrifugation was performed in a Beckman 5W40Ti rotor for 26 min.
The test was carried out at 0Qrpm and 18°C for 24 hours. Fractions were collected and analyzed for DNA content by agarose gel electrophoresis. Fractions containing DNA of 15-30 Kb size were combined.

Next, the saccharose in the DNA solution was removed and the solution was mixed with Centricon-30 (Centricon-'30).
) Mymaro concentrator (Amicon Co., Ltd., Danvers, M.A.)
)).

Part 4: Plasmid Cloning of P.S.P. ATCC21808 DNA Recombinant with Vector pcP13 60 micrograms of plasmid pcP13 DNA and 180 U of restriction enzyme XhoI were combined in 250 μQ of the same digestion buffer solution described in the previous section and incubated at 37°C.
The cells were cultured for 18 hours. D by agarose gel electrophoresis
According to NA analysis, digestion was complete. Proteins and other contaminants were removed from the digestion mixture by extraction twice with unbuffered phenol/chloroform (1:1, v/v) and once with chloroform/isoamyl urea (24:1, v/v). Removed. Plasmid DNA O15
Precipitated with 7.5M ammonium acetate and 2 volumes of cold 100% ethanol.

After incubation for 20 minutes at 70°C, the precipitated DNA was incubated at 4°C in an Eppendorf centrifuge.
The cells were harvested by centrifugation at 13,000×g for 0 min. After washing the plasmid DNA pellet three times with 70% cold ethanol, the DNA was redissolved in 120 μQ of TE buffer.

The XhoI-digested pcP13 DNA is dephosphorylated, and the plasmid is transferred in the subsequent ligation reaction (recirc
curarization). This was accomplished as follows. Digested pcP13 DNA and 22 U of calf intestine alkaline 7 male 7 atase (
Whalinger Mannheim Piochemicals (Bo
ehringer Mannheim Bioche
micals), Indiana
apol is), IN) in a 200 μQ solution containing 0.05 M l-Lis pH 9.5; and 25 μg/mQ spermine (Sigma Chemical Co., St. Louis, MA). and cultured at 37°C for 60 minutes. One unit of alkaline phosphatase is 1M jetanolamine, 0.01M 4-nitrophenyl phosphate, 0.0
3 in a buffer solution consisting of 25M MgCQ, pH 9.8.
1 μ of 4-nitrophenyl phosphate for 1 min at 7°C.
The mole was defined as the amount of enzyme that hydrolyzes.

Phosphatases were inactivated by incubation at 68° C. for 10 minutes, and contaminating proteins were removed in the same manner as the 7enol/chloroform and chloroform/isoamyl alcohol extractions as described in the previous puff graph. Furthermore, dephosphorylated pCP 13 DNA is precipitated,
Incubate overnight at −20 °C and 70%
Washed with ethanol. Speedback Concentrator (Spe
ed-VacConcentrator) (Savant Instruments)
ents) Co., Ltd., Farming Dale (F armi
After drying the plasmid DNA pellet for 10 minutes in a plasmid (Ngdale, NY), the plasmid DNA pellet was
was redissolved in TE buffer solution.

Next, 0.5 μg of digested and size-selected bees;
SP ATCC21808 DNA, 4.5g digested p
CP13 DNA and 20 U of T4 DNA ligase were added to New England Biolabs.
nd Biolabs), Beverly
, M.A.), 0.025M Tris-H
(12 (pH 7,8), O-IM MgC (h, 0.0
The cells were mixed in 20μα of a solution containing 4M β-mercaptoethanol and 4mM adenosine 5'-triphosphate (ATP), and cultured at 4°C for 16 hours.

One unit of ligase is a 20μQ solution (0.05M
1-Lis-HCQ pH7,8,0,01M MgCQ
2.0.02M dithiothreitonodindiLhioth
reitol), 1mM ATP, 50μg/mQBS
Hi of lambda DNA for 30 minutes at 16°C in
ndI[[50% ligation of fragment and 0.12μ
M (defined as the amount required to give a 5' DNA end concentration of 300 μg/m(2). l O: l
Using a vector to insert the ratio of (w/w) length 11
1. A preferred substrate for promoting the formation of DNA strands and for in vitro packaging of DNA molecules into lambda virus particles. be.

Part 5 BSP containing Niblasmid, ATCC2
In vitro packaging of 1808 DNA into lambda particles In vitro packaging of 1808 DNA into recombinant cosmids (c
osmids) into the host cell. Packagene extract (
Promega Biote
c), Madison, Double Eye (
Melt WI)) on ice, BSP, ATC
Half of the C21808 DNA/pcP13 ligation mixture was added and the mixture was incubated at 22°C for 2 hours. The packaging mixture was then added to Q, 5 m12 of 0.1 M NaCff
, 0.01 M Tris pH 7.9, and 0.01 M magnesium sulfate (MgSO4X Sigma Chemical Co., MA, St. Louis, MA). This lambda particle (recombinant pcP13/PSP, ATC
(containing C21808 DNA molecules) were stored at 4°C over chloroform.

Part 6: BSP. ATCC21808DNA
and transduction of plasmids containing markers of antibiotic resistance into E. coli strain HB.
The IOI was mixed with tryptone broth (TB) medium (the medium contains 10 g of tryptone (grams per gram)).
liter), NaCQ 5g, maltose 2g (Sigma・
Chemical Co., St. Louis, MA) and 2.46 g of Mg5O6) at 175 rpm on a rotary shaker at 30°C overnight in flasks. 100 μa resuspended HB 101 bacteria (0,
01M MgSO4) and 50 μQ of the packaging mixture (containing lambda particles) were mixed together in a tube and incubated at 37° C. for 20 minutes. Next, 1 ml of preheated Luria liquid culture medium was added and the tubes were placed in a rotary shaker at 150 rpm for 60 min.
Cultured at 7°C. Transduced HBIOI cells were placed in an Eppendorf centrifuge at 13,000×g.

Centrifuged for 5 minutes at room temperature, harvested, and subsequently resuspended in a small volume of Luria liquid culture medium. HBIOI cells were grown on Luria agar plates containing 15 μg/mQTc.

This selective medium allows the growth of only E. coli HBIOI cells containing plasmid pcP13 (containing the tetracycline resistance gene). To determine the number of cells containing recombinant DNA, Tc-resistant cells were screened for resistance to Km (Sigma Chemical Co., MA, St. Louis, MA).
en) L/ta. Unique pcP of 7 fragments of DNA
Insertion of 13 into the Xho111@ site inactivates the plasmid gene encoding Km resistance, thus
The frequency of m sensitivity is the frequency of recombinant pcP in the total number of cells.
The number of E9 Corey cells containing 13 plasmids is given. Each Tc resistance cell that also has Km sensitivity is
pcP containing ATCC21808 DNA
It was shown that it had 13 plasmids. Twelve hundred H8101 recombinant strains were individually isolated.

Example 2 Part 1: Preparation of a Pool of E., Corey HBIOI Recombinant Strains 1200 Individually Isolated Tc'Km'HB I Ol
The strain is a recombinant strain (BSP, ATCC2
1808/pCP l3) DNA plasmids were spotted on the surface of Luria agar plates (50 clones per l plate) supplemented with 15 μg/mff Tc. After incubating the agar plates for 10 hours at 37°C, 3-Lulia liquid medium containing 15 μg/mQTc was added to each plate to break up the bacteria on each plate together. 2 plates (100HBI
The resuspended cells from the HBIOI clones were combined, thereby forming 12 pools of HBIOI clones.

Part 2: BSP, ATCC21808DNA
Transfer donor and recipient endocells from E. coli to B. oleoporans by conjugation of recombinant plasmids containing B.
J. of Bacteriology (1984),
No. 159.335-340 were prepared for bonding using a slightly modified method.

Tubes containing 3- of Luria liquid culture medium were inoculated with E. coli (donor) pool from Luria agar (+15 μg/m + 2 Tc) stock plates and roller drums (2-2 Brunswick Scientist (7)
-/ri(New Brunswick 5cienti
fic) Co., Ltd., New Brunswick, N.
The cells were cultured overnight at 30°C in New York (NJ). Using these late cultures, A4m of 0.15. (420n
The same medium was inoculated with lomQ (optical scattering at the wavelength of 100 nm). These cultured bacteria were incubated at 37°C for 3 hours at 200 rpm.
Cultured on a rotary shaker at m.

Luria liquid culture medium 3rn containing 15μg/mQKm
In the tube containing Q.Ecoli HBI containing plasmid pRK2103 from the stock plate
Inoculated with OI 1 and cultured in roller drums as described above. - Using a late culture, 0.1 A42° was inoculated with 620+++Q of the same medium. This cultured bacteria is also 3
The cells were cultured at 20 Orpm for 3 hours at 7°C. plasmid p
RK2013 is a kanamycin-resistance helper plasmid consisting of the RK2 transgene cloned onto the ColEl replicon. (Q, pitta), Dept. of Biology (D.
ept, of Biology, University of California, San Diego, La Jolla
Jolla), Cx-(CA)). It is pcP1
3 provides the proteins necessary for efficient mobilization (transfer) of recombinant plasmids, as described by Ditta et al., Pro.
c, Natl, Acad, Sci, USA (198
0), 77.7343-7351.

B. oleoporans A from a stock plate containing the same medium containing 10 μg/mQ of nutrient liquid culture medium containing 7 ampsin (Sigma Chemical Co., St. Louis, MA).
TCC8062 (American Type Culture Collection, Rockville, MD-) Rif' recipe end cells were inoculated and incubated at 30°C on a rotary shaker at 20°C.
Cultured overnight in Orpm. - Using late culture bacteria, 0.3
of AHo were inoculated with 601 of the same medium. The cultured bacteria were also cultured in the same manner as above. Bee, oleoporans A
TCc8062 J, Bacteriology
(1941), 41373-386.

Incubate donor and recipient endocells at 440°C for 10 min.
Centrifuged and harvested at 0×g. Donor and recipient endocells were washed once each with Luria liquid medium and nutrient liquid medium to remove antibiotics. lastly,
Culture the bacteria with a nutrient liquid culture medium in a volume of 0.25
resuspended in

HB 101 clone pool, HBIOI (pRK20
13) Mix helper cells and B. oleoporans recipients at a ratio of 5 donor and 5 helper plasmid cells per recipient endocell,
ricel) membrane filter (diameter 25mm, 0
．． Pores with a size of 45 μm, Ge12man Instrument Co., Ltd., Ann Arbor, MI (
M I )) and cultured on nutrient agar medium for 30°02 days. The cells were then resuspended in nutrient liquid culture medium, followed by dilution, and 50 μg aliquots were added to 50 μQ/mQ rifampsin, 25 i
t g/mffT c and 0.05% (v/v) castor oil (Sigma Chemical Co., St. Louis, MO) supplemented with nutrient agar plates. These plates were heated to 30℃1
Cultured for 0 days. Castor oil was emulsified into the preparation to form a translucent agar medium.

This agar medium is used to detect B. oleoporans that have received a lipase gene that can be expressed on a recombinant plasmid. These lipase-positive colonies hydrolyze castor oil triglycerides, thereby forming a clear zone around the colony. The number of recipe ends upon plating onto selective media is determined by plating aliquots of 100 μg serial dilutions onto nutrient agar plates supplemented with 50 μg/mQ Rifampsin.

transconjugant
Recipient endocells that received the recombinant pCP13 plasmid had between 2X10-' and 2X1 per lucipient.
Appears with a frequency of 04. Approximately 3 cells containing recombinant plasmids from each of the E. coli clone pools
2500 transconjugants, or approximately 2500 transconjugants per experiment, are grown on the selection plate.

Of the 2375 transconjugants from pool number 13 that were mated, 4 colonies produced a clear zone indicating the presence of lipase activity. Similarly, 35 of the 2850 transzygotes from pool number 23 mated produced clear zones. When transconjugants exhibiting lipase activity were linearly cultured on the same agar plates containing castor oil, a clear zone (lipase) was again formed in each strain.

Part 1: Isolation of plasmids containing lipase genes from B. oleoporans Three different B. oleoporans strains containing lipase clones were isolated at 10 μg/mQRif and 5 μg/m
The cells were grown in tubes containing 5-nutrient liquid culture medium supplemented with QTc. The tubes were incubated overnight at 30°C in a roller drum. Aliquot 1.5 m of cells by centrifugation in a 1.5 mff volume microcentrifuge tube for 1 min at 13,000 x g at room temperature.
Incorporated one. The cells were treated with 0.05M glucose (
Sigma Chemical Co., St. Louis, MA), 0.01M EDTA, 0.025M Tris pH
8, and sterile Dosevic Qoothpick in 200 μg of cold solution containing 4 mg/raQ lysozyme.
) and incubated for 5 minutes at room temperature. 400μ of freshly made 0°2N sodium hydroxide (NaOHX Nguma Chemical Co., MA, St. Louis)
Cell lysis was completed by adding Q and U1% (v/v) SDS, and the cells were incubated for 5 minutes at 4°C. Addition of 300 μg of cold 3M sodium acetate (Sigma Chemical Co., MA, St. Louis, MA) pH 4.8, incubation at 4°C for 5 minutes,
Proteins and other cell debris were precipitated by centrifugation at 3000 Xg for 10 min at 4°C. Transfer 700 μQ of the supernatant to a new 1.5 m 12 volume microcentrifuge tube and add the same volume of unbuffered (1:Lv/
v) Extraction with phenol/chloroform to remove remaining contaminating proteins.

Nucleic acids in 500 μA of the extracted aqueous layer were added to 2 volumes of cold 1
00% ethanol was added for precipitation.

= After incubating at 70°C for 25 minutes, the precipitated DNA was
It was centrifuged at 0xg for 4°020 minutes and collected. The nucleic acid pellet was washed three times with 70% ethanol to remove remaining salts and phenol/chloroform. The pellet is then placed in a Speed-Vac concentrator (Speed-Vac concentrator).
Cancentrator) for 7 minutes. Nucleic acids were resuspended in 50μα of TE pH8 buffer.

Part 2: Preparation of Reactive E, Corey Components for Format Conversion Prepare tubes containing 5 mQ of Luria liquid culture medium.
The cells were inoculated with coli~HBIOI bacteria and grown overnight at 30°C in a roller drum. Next, add a flask containing 400 ml of Luria liquid culture medium to 0.02 A. .

was inoculated with HBOI on a rotary vibrator for 20 min.
Cells were incubated for 20 minutes at 64°C, followed by centrifugation at 2000×g for 15 minutes at 14°C. The introduced E. coley cell was used for transformation by Silhav.
Ira, gene fusion experiments (Experiments wi
th Gene Fusion), Cold Spring Harbor Laboratory (Cold Spring
Harbor Laboratory), N.Y. (
(1984), p. 169, using the calcium chloride method.

Part 3 Transformation of Niblasmids into E. coli Ten microliters of the partially purified DNA from Part 1 was transferred to Silverby et al., Gene Fusion Experiments, Cold Spring Harbor Laboratory, N.W.
984), p. 170, into E. coley cells. The cells were incubated at 37°C overnight with 15 μg
/mQTc on Syrian agar plates. This choice of medium allows the growth of only cells containing the recombinant pcP13 plasmid with the lipase gene and TC resistance marker.

Example 4 Physical Analysis of P.S. ATCC 21808 Lipase Recombinant pcp13 plasmids were partially purified from cultures of three different Lip+ B. oleoporans strains as described in Example 3, Bartl. half (2
5 μa) of partially purified plasmid DNA was
0μQ solution (0,025M Tris-HCQ pH 7,8,
0.05M NaCl2, 0゜01M MgCQ, 1
The restriction enzyme
The cells were mixed with 7.5 U of I and incubated overnight at 37°C.

Digested plasmid DNA was analyzed by 0.7% agarose gel electrophoresis. Gel and reservoir buffer
ir buffer) was 0.089M tris-borate and 0.089M boric acid (Mallinckrodt).
linkrodt) Co., Ltd., Paris
It was KY-U. Gel (DNA) at 0.
Staining with 5 μg/m+2 ethidium bromide for 25 minutes at room temperature and washing with 1 mM MgSO, 30 minutes at room temperature to remove non-DNA-bound ethidium bromide.

The DNA fragment patterns and sizes are shown in Table 1.

Table 1 Size analysis of lipase-positive recombinant plasmid Fragment No. pHSH1pHSH2pHSH7
19,4 27,4 54,9 63,13,1 71,2 Total Kb 23.7 21.2 23.8 Each of the three recombinant plasmids had just over 20 Kb of Pseudomonas DNA . 3 normal 7 fragments, a total of 13.2 liper genes
Kb is underlined as shown in Table 1 above.

Example 5 Active peptone/yeast extraction (PYE) medium (pH 7°5)
(Peptone 8g (grams/liter), yeast extract 2
g, and 25 g of NaCl) at 5 μg/mQ
7 ampsin and 15 μg/mQTc and containing a lipase clone.
The cells were inoculated with the TCC8062Rif' strain. In addition, Bee 6 oleoporans ATC, which does not contain recombinant clones,
C8062 and PSP ATCC21808R
if' served as a control (positive and negative, respectively) and was grown in the medium described above, but without Tc. Tubes were incubated overnight at 30°C in a roller drum. This was followed by a 500 IIIQ volume "baffled" cell containing 50 mff of the above medium supplemented with 0.5% (w/v) olive oil (Sigma Chemical Co., St. Louis, MO). A Renmeyer flask (three wells in the bottom of the flask) was inoculated at 0.08 A, 1° and incubated for 3 days at 30° C. in a rotary shaker.

Culture aliquots (IIIQ) were centrifuged in an Eppendorf centrifuge at 13,000×g for 1 minute. The lipase activity of the culture supernatant was measured by spectrophotometer analysis using p-nitrophenyl palmitate (PNPP) as an artificial culture medium. The reaction volume was 1 mg, and the reaction mixture was 0. IMF Squirrel pH7.

5.0.5% (v/v) Triton X
-100 (Sigma Chemical Co., St. Louis, MO) and 0,2 wa9/rnQ p-nitrophenyl palmitate (Sigma Chemical Co., St. Louis, MO). Lipase assays were initiated by adding an aliquot of culture supernatant into the reaction mixture and increasing absorbance at 400 nm for 1 minute. Lipase mediated the hydrolysis of PNPP releasing the p-nitrophenyl moiety producing a yellow color. Lipase activity is shown in Table 2.

Table 2 Heat, 7i and 7) -Pon XATCC8062Rif
': (No plasmid > 0.0
0 (Lipase clone 1) 0.017 (
Lipase clone 2) 0-017 (Lipase clone 3) 0.010hi, xZ
The ATCC21808Rifr18.5 control P. oleoporans strains did not show any lipase activity. On the other hand, B, SP, ATCC2180
No. 8 had high lipase activity. Lipase activity 1
The unit was heated with PNPs for 1 minute at 30°C under the analysis conditions described above.
P is defined as the amount of lipase enzyme required to hydrolyze 1 μmole. The B. oleovorans strain containing the recombinant plasmid with the lipase gene had a level of lipase activity of approximately 0.1% compared to B. sp. ATCC21808. This low but important lipase activity probably lies in the inability of the transcription and/or translation machinery of B. olevobalance and the regulation (initiation) of B. sp. ATCC21808.
Recognize the chain.

Example 6 Part 1: Detection of smaller common DNA fragments containing lipase genes As described in Example 4, BSP ATCC21808DN containing three types of lipase genes.
The A insert has 13.2 Kb of conventional DNA. 3
Two types of Lip+ plasmid DNA were isolated as described in Example 1, Part 2. 0.5 μy of each of the three types of recombinant plasmids, restriction enzyme H4nd
An IOU of ln was mixed in a 20 μQ solution containing the same components as the digestion buffer in Example 4 and incubated at 37°C for 1 hour. Digested plasmid DNA was analyzed by agarose gel electrophoresis. The three unique NA fragment patterns likely represent a single 8. A common DNA fragment of OKb is shown.

Part 2: 8. Plasmid pHSHI DNA 100 μΩ in a solution containing the same components as the digestion buffer in Isolation Example 4 of Hindn, which produced the common 7 fragments of OKb.
A mixed solution of 300 U of μy and Hindl[I was added to the
The cells were cultured at 7°C for 1 hour. The DNA digested fragments were then separated by vertical agarose gel electrophoresis. -Agarose gel concentration was 0.9%. Gel and holding buffers were 0.04M Tris-acetate and 2mM
It was DTA.

Gels were stained and destained as described in Example 4. 8
．． The gel containing the OKb DNA band was excised with a razor blade, passed through an 18 gauge needle, and soaked in 15+ml of distilled water H,O.
12 was extruded into a tube containing 12. 4 tubes
Rocked back and forth overnight at °C.

The DNA solution containing the 8.0 Kb7 fragment was centrifuged and concentrated several times to remove the agarose, and the DNA solution was centrifuged several times to remove the agarose.
H,0 was removed by extraction with butanol.

Finally, the DNA was precipitated and resuspended in TE pHg as described in Example 1.

Part 3: Preparation of plasmid pcP13 for cloning A mixture of 360 μl of plasmid pCP1 and 180 U of the restriction enzyme dI in a 250 μC solution containing the same components as the digestion buffer as described in Example 4 was added.
Cultured overnight at 7°C. Restriction enzymes and other contaminating proteins were removed by organic solvent extraction as described in Example 1 Part 3. The linearized pcP13 DNA was also taken up by ethanol precipitation, washed three times with 70% ethanol, and resuspended as described in Example 11 Part 3. Analysis of the plasmid DNA by agarose gel electrophoresis indicated that the digestion was complete.

HindI [[Digested pcP13 DNA was prepared using Example 1
Dephosphorylation was performed as described in Part 1, Part 4 to prevent plasmid transport in the subsequent ligation reaction.

Additionally, contaminating proteins were removed as described in Example 1, Part 3, and the dephosphorylated DNA was precipitated, incorporated, and washed and resuspended in TE pHB.

Part 4: Recombining the F2 fragment with plasmid pCP13 Equal amounts (0.5 μg) of F2 and pcP13 DNA
and 20 U of T4 DNA ligase were combined in a 20 μg solution (containing the same ligation buffer as described in Example ↓, Part 4) and incubated overnight at 14°C. Analysis of the DNA ligation mixture by agarose gel electrophoresis revealed that
The ligation was complete.

Part 5: Isolation of E.Coley Cells Containing the F2/pCP13 Recombinant Plasmid Competent E.Coley HBIOI cells were prepared as described in Example 3, Part 2.

HBIO 1/2 of the ligated DNA prepared in the previous part
I cells were transformed, and the format transformant was transformed into Example 3 Part 3.
Selected as described in.

The plasmid content of 22Tc'HB l 01 transformants was analyzed as described in Example 4. In this case, partially purified plasmid DNA is treated with the restriction enzyme Hindl.
Digested with lI. Agarose gel electrophoresis"C089%
All 22 transformants contained a recombinant pcP13 plasmid with an 8.0 Kb F27 fragment, as analyzed by agarose concentration).

Part 6: Determination that the F2 fragment contains the complete lipase gene F2/pcP13 recombinant plasmid (pHSH17
) from E. Corey HBOI to P. oleoporans ATC as described in Example 2 Part 2.
Transferred to C8062Rif' by conjugation. All P. oleoporans Tc' transconjugant colonies formed clear zones on agar plates containing semi-transparent castor oil emulsion. Four Peas, Oleoporans Tc'
The plasmid content of the transconjugants was analyzed as described in the previous part. According to agarose gel electrophoresis analysis,
All four transconjugants contained a recombinant pcP13 plasmid with an 8.0 Kb F27 fragment. This result shows that the F2 fragment is a complete P.
It is shown that it contains the sp. ATCC21808 lipase gene.

Example 7 Conjugation of Plasmids Containing Lipase Genes and Antibiotic Resistance Markers into Pseudomonas aeruginosa and Pseudomonas fluorescens Recombinant plasmid pHSH17, assembled as described in Example 6, was transformed into a D.
It was used as a source of NA7 fragment (F2). Plasmid pHSH17 was conjugated as described in Example 2 Part 2 from Corey HB 101 to Ayurugil 2 PAO 2003 (P, Holloway (E)).

Hof loway), Dept of Genetics, Monash University
ity), C1ayton), Victoria (V1ctoria), Australia) Rif'
and P, Fluorescence ATCC948Rif'
It was transferred to. P, Aeruginosa PA 0200
Because 3Rif'' has a low intrinsic lipase activity, the transconjugant formed a slightly wider clear zone on castor oil agar plates (see Example 2 Bart 2).

Additionally, the P.fluorescence transition conjugate formed a clear zone. The plasmid content of several P. aeruginosa and P. fluorescens Tc' transconjugants was analyzed as described in Example 6 Part 5.

According to agarose gel electrophoresis analysis, all transconjugants of both species contained plasmid pHSH17.

The lipase activities of P. aeruginosa, P. fluorescens, and P. oleoporans strains containing pHSH17 were determined as described in Example 5. As in Example 5, a strain not containing pHSHI7 served as a control and was grown as described. Lipase activity is shown in Table 3. The control P. oleoporans and P. fluorescens strains had no lipase activity, whereas P. aeruginosa had low lipase activity. P. fluorescens (pHSH17) recombinant strain had low lipase activity, and its activity was slightly higher than that of P. oleoporans (pHSH17). On the other hand, P. aeruginosa (pHSHI
7) is P, fluoresce>7. It had a lipase activity that was approximately 5 times that of the strain (after subtracting the background).
.

Table 3 Lipase activity of different Pseudomonas strains (without plasmid) (pf(SnI2) Hi-, 7x Luki/-fPA 02003 Rifr(
No plasmid) (+)H5H17) He, Fluorescence ATCC948R1fr (No plasmid) /-ucL117'+ 0.00 0.03 0.19 0.40 0.00 Example 8 Plasmid pME 290 (Cb Km Mob-;
DeHaas (D, ) (aas), Microbiologisches Institutenito (Mikrobiol)
ogisches I n5titute)
Enossische TechnischeHoc
P. aeruginosa plasmid pV S 1 (H
gSuMob IncP (Uncategorized)
h) et al., P lasmid (1984), 11, 206
-220), but the size is 6.8K
b, carrying the gene encoding carbenicillin (Cb) and Km resistance, insertion inactive (insertiona
It has several sites for cloning DNA with different properties through activation (Ito and Haas, Gene (1985), Dan.
27-36), in addition, non-mobile plasmid pME29
0 can be introduced by transformation and maintained in various species of Pseudomonas at levels of between 7 and 10 copies per cell.

HindI [+ is 8,0 Kb containing the lipase gene
F2 DNA fragment (isolated as described in Example 6 Part 2) was generated.

Plasmid pME290 was transformed into P. aeruginosa PA02003 R by the method described in Example 11 Part 2.
If' (pME 290) was purified from cells. Bacteria with 5g/Q yeast extract and 100μg/w
The cells were grown in nutrient liquid culture medium supplemented with rQ carbenicillin (Sigma Chemical Co., St. Louis, MO).

17.5μ9 plasmid pME290DNA and 5
2.5 U of the restriction enzyme HindDI was combined in a 250 μQ solution containing the same components as the digestion buffer used in Example 4 and incubated overnight at 37°C. Restriction enzymes and other contaminating proteins were removed and the cultured plasmid DNA was prepared in Example 1.
Harvested and resuspended as described in Part 3. Next, the digested pME290 DNA was added to Example 1 Part 4.
The dephosphorylation reaction was performed as described in . Additionally, contaminating proteins were removed as described in Example 1 Part 3, and the dephosphorylated DNA was precipitated, harvested, washed, and resuspended in TE pH 8 buffer.

Equal amounts (0.5 μg) of DNA fragments F2 and p
ME290 DNA was ligated together as described in Example 6 Part 4. The DNA ligation mixture was analyzed by agarose gel electrophoresis and the ligation was complete.

P. aeruginosa PA020 for effective transformation
03Rif' cell at 175 rpm before MgCQ1 treatment.
Lederber cells were grown in Luria liquid culture medium at 37°C on a rotary shaker for 3-h.
g) and Cohen, J., Bact.
er corner package ■ (1974), 1, 9. IQ72-1074
It was prepared by the method of

To transform the plasmid into P. aeruginosa, half of the DNA ligation mixture prepared above was transformed into Lederberg and Cohen, J.

Bacteriology (1974), 1, 9, 10
P. aeruginosa PA by the method of 72-1074
02003Rif' into component cells.

The cells were cultured overnight at 37°C on nutrient agar plates containing 500 μg/mQ carbenicillin. This selective culture medium allows pME2 with cb resistance markers to be
Only cells containing 90 plates can be grown.

To determine the number of cells containing recombinant DNA,
cb' cells were blocked for sensitivity to kanamycin. 7 fragments of DNA as the only Hi of pME290
By inserting into the ndI [[ restriction site, we inactivate the plasmid encoding Km resistance, so the frequency of Km sensitivity makes it possible to reduce You can see the number of Aeruginosa cells. 2°30% of the Cb' resistance cell is
pME2, which is also Km' and contains fragment F2
90 plasmid is shown (referred to as pHES3).

Lipase activity of P. aeruginosa (pHES3) strain was determined as described in Example 5.

Plasmid pME290 and P.S.P. ATCC
P. aeruginosa strains containing 21808Rif' were used as negative and positive controls (respectively).
and was grown as described in Example 5. Lipase activity is shown in Table 4. The control pea, Aeruginosa (pME290), had low intrinsic lipase activity. The P. aeruginosa (pHES3) recombinant strain had twice the lipase activity of P. aeruginosa (pME 290) (after background subtraction). In addition, P. aeruginosa (pHES3
) lipase activity of P, SP, ATCC218
It is about 5% of the activity of 08Rif'.

(The following is the margin) Table 4 PAO2003Rifr (pME290) (pHES3) B, SP, ATCC 0,193 0,661 Hindnl contains lipase gene 8, Ok
Generate bF2 DNA fragment (isolated as described in Example 6 Part 6);

pME 285 (HgKmMob”; Day-1)\
-S, Microbiology Instatenito, Eidogenesie Technissie Hochschule,
Zurich, Switzerland) was chosen as the cloning vector. Because it is the same bee as pME290,
It is derived from P. aeruginosa plasmid (pVSl) and has a selectable mercury resistance (Hg) gene.

Plasmid pME285 (I- and /X-S, Ge
ne (1985), 36.27-36) in Example 8, Aeruginosa PAO2003R
if' (pME 285) was purified from cells.

However, the bacterial growth medium is 2.5μg/mff
Mercury chloride (Hg CLX Sigma Chemical Co., Ltd.)
St. Louis, M.O.) and 100 μg/m12K
It is supplemented by m. lo μg of plasmid pME285 DNA and 30 U of the restriction enzyme HindI [I] were added to 12 μg of the digestion buffer containing the same components as the digestion buffer used in Example 4.
Combined in 5μQ solution.

After incubating the reaction overnight at 37°C, the digested pME28
5 plasmids were dephosphorylated as described in Example 1 Part 4. However, the final volume was 216 μg. Contaminating proteins were further removed as described in Example 1 Part 3, and the dephosphorylated DNA was precipitated, harvested, washed, and resuspended in TE pH 8.

Fragment F2 was ligated into plasmid pME285 as described in Example 8. One-half of the DNA ligation mixture was transformed into reactive cells of P. aeruginosa PAE 2003 Rif' as described in Example 8. In this case, the transformed cells were treated at 50 pg/
Cultured for 2 days at 30°C on nutrient agar plates containing mQRif and 30 μg/mQ HgC (k). With this selective culture medium, pME2 containing the Hg resistance gene
Only cells containing the 85 plasmid are allowed to grow. To determine the number of cells containing recombinant DNA, Hg' cells were blocked for sensitivity to Km.

7 fragments of DNA into the only Hind of pME285
Since insertion into the I[[control@ site inactivates the plasmid gene encoding K+n', the Km sensitivity is
A P. aeruginosa cell containing a recombinant plasmid is shown. All Hg' recombinants are Km' and they are pME28 containing F2 (pHSH43).
5 plasmids.

Since B.S.P. ATCC21808Rif' is naturally very sensitive to mercury, the recombinant plasmid pHSH43 (F2/pME285) was chosen as the source of DNA containing the lipase gene for transformation. Plasmid pHSH43 was isolated from P. aeruginosa PAO20 as described in Example 3 Bart 1.
Partially purified from 03Rif' (pHSH43) cells.

The bacterial growth medium was a nutrient liquid culture medium supplemented with 5 μg/ml 2Rif and 2°5 μg/m + 2Hgc (2. ATCC21808Rif' reactive cells were transformed into MgCQ, prior to treatment, in nutrient liquid culture medium supplemented with 57zg/mffRif on a rotary shaker at 20 Orpm for 3 hours.
Grown at 0°C. A P.S.P.+ ATCC21808Rif' recombinant containing the pHSH43 plasmid was selected and verified as described in Example 9.

Example 11 PSP ATCC21808Rif' (pHS
H43) was grown as described in Example 5, except that the growth medium was supplemented with 5 μg/mQHgcQx. In addition, plasmid-free P, SP, ATCC
21808Rif' strain and plasmid pME285
The strain containing . Plasmid-free strains and pME285-containing strains at 5 μg/maRif only and 5 p g/
m(IRif+ 2.5 p g/m(lHgc(lx
+ 100/'g/rnQ Km was grown in medium with solenoid.

Quantitative lipase activity determinations were performed as described in Example 5. Lipase activity is shown in Table 5.

P, SP, ATCC21808Rif' (pH
SH43) is a plasmid-containing strain and pME2
It had the highest lipase activity, which was 17-19 times higher than the 85-containing strain.

Table 5 Lipase activity of P.S.P. 21808Rif' strain (U/m (2 supernatants) 24 hours 48 hours 72 hours 21808 6.11 7.88 6.642
1808 (pME285) 4.86 8.78 8.
9421808 (pHSH43) 129 152 14
1 Example 12 Part 1: pHSH43B, SP, ATCC21
Construction of a recombinant plasmid that can be maintained in 808 p.i. with two compatible plasmids, each containing a lipase gene. SP, ATCC2180
8Rif' was constructed to increase lipase production.

The DNA insert containing the lipase gene is F2 6.5
Kb subfragment. F2DNA (4,25
μg) and 12.75 U of the restriction enzyme BamHI (2N England Biorapoz, B) (Lee, MNY) were mixed together in a 50 μa solution and the solution was prepared for the same digestion as described in Example 1 Part 3. contained a buffer (but 0°1M NacI2 was used),
Cultured overnight at 37°C. Proteins were then removed and the digested DNA was taken up, washed, and resuspended in TE pH 8 as described in Example 1 Part 3.

Plasmid pcP13 was chosen as the cloning vector. This is because it is derived from a plasmid with a different incompatibility group than that of pME285. Plasmid pcP13 DNA (2
0 μg) and 60 U of the restriction enzyme HindI [[12
5/712 solution (containing the same digestion buffer described in Example 1 Part 3) and incubated for 1 hour at 37°C. After adjusting the solution to 0.1M NaCl2,
Add 60 U of restriction enzyme BamHI and incubate the digestion at 37°C.
Cultured late.

Then digest pcP13D as described in Example 9.
The NA was dephosphorylated to remove contaminating proteins, and the plasmid DNA was precipitated, harvested, washed, and resuspended in TE pH 8.

The F2 subfragment was ligated into plasmid pcP13 as described in Example 8. The DNA ligation mixture was transformed into E. Coley, HBLol reactive cells as described in Example 3 Parts 2 and 3. The transformed cells were cultured on Syriac agar plates containing 15 μg/m12Tc and incubated overnight at 37°C. The selective medium allows growth of only cells containing the pcP13 plasmid containing the Tc resistance gene. To determine the number of cells containing recombinant DNA, Tc
'Cells were blocked for sensitivity to Km.

The DNA fragment was converted into asymmetric HindlI of pcP13.
Km" indicates E. coli cells containing recombinant plasmids, since insertion into the [/BamH1 site forms a plasmid lacking the kanamycin resistance gene. All Tc' recombinants are Km' Met.

The plasmid content of the 12Tc'Km' format transformants was analyzed by agarose gel electrophoresis as described in Example 4. In this case, partially purified plasmid DNA was added to the 2-step single transformant as described above for 6
．． It contained a recombinant pcP13 plasmid with a 5 Kb fragment (referred to as pHSH31). Plasmid pHS H31 was transferred to oleoporans as described in Example 3 Part 2. B. oleoporans (pHSH31) produced a clear zone on the selection plate.

Since B.S.P., ATCC 21808 is naturally high Tc resistant, selectable antibiotic resistance markers were selected from Ruvkun and Ausubel.
), Nature (1981), 289, 85-88, with slight modifications, was introduced into pHSH31 via insertion of transposon Tn5 (which encodes Km'). A defective lambda virus containing Tn5 was developed by R. Maier, Dept. of Biology, The Johns Hopkins University.
pkins Univ, ), Baltimore (B a I
It was a gift from M.D. (MD). E. coley, HBLol containing plasmid pHSH31 was grown in tryptone liquid culture as described in Example 1 Bart 6. Multiplicity of infection (
multiplicity of 1 infection
) was 0.5.

Tn5 translocation into the genome was 15 μg/mQTC and 4
Selected by bacterial culture on Syrian agar plates containing 0 μg/m (2 Km.
Tn5 transfer into H31 is identified by isolating plasmid DNA from several thousand Tc'Km' colonies (as described in Example 1 Part 2). The plasmid DNA was then transformed into HBLol as described in Example 3 Parts 2 and 3. The transformed cells were incubated with l 5 pg/ynQTc and 40 pg/ynQTc.
on Syrian agar plates containing g/mn Km and incubated overnight at 37°C. This selective medium allows T
pHSH31 plasmid with n5 insert (pHSH
31::Tn5) can be grown.

Since Tn5 is 5.7 Kb and does not contain an EcoRI site, the Tn5 insertion into the pcP13 portion of pHSH31 is confirmed by the appearance of a 5.7 kb broad linear pcP13 plasmid band (as described in Example 4). ) Identified by agarose gel electrophoresis. 23 Tc
Tn5 with 18 of 'Km' inserted into pcP13
It contained pHSH31 with . One transformant was selected for this study (pHSH31::Tn5
-6).

Part 2: Conjugation of pHSH31::Tn5-6 into BSP ATCC 21808 (pHSH43) Plasmid pHSH31::Tn5-6 was transformed into Echoli-
HBIO1 to BSP ATCC21808Ri
f' (pHSH43). donor cell, 1
Cultured in Luria liquid culture medium containing 5 μg/m12Tc and 40 μg/mQ Km. B.S.P.
ATCC21808Rif'Hg' transconjugant, 50
μg/maRir, 30 pg/mffHgcQ2
, and selected on nutrient agar plates supplemented with 500 pg/rnD-Km. Plasmid analysis by agarose gel electrophoresis confirmed the presence of both plasmids. This strain composition is from B.S.P., 21808.
shows that it is possible to simultaneously carry two different chimeric plasmids containing the lipase gene.

Part 3 Effect of Olive Oil Concentration on Lipase Activity Additionally, higher olive oil concentrations promote the expression of higher lipase activity. Cultures were grown as described above by shaking the flask. However, olive oil 2
50 C2 (0.05% v/v) was added to each culture after 24 hours of culture. As described below and shown in Table 2,
Although strain 21808 (pHSH43) has a higher maximum lipase activity (19 times that of 21808pME285 and 218
strain 21808 (pHSH43) was 24 times higher than in the previous experiment (359
U/mQ) had the maximum lipase activity.

Table 6 Pea, SP ATCC21 supplemented with olive oil
Lipase activity of 808 strain 21808 2.27 2.92 13.0
21.221808 (pME285) 1.99
8.89 8.1517.021808 (pHSH4
3) 61.7 46.3 72-7 29422.
3 18.5 59 This higher lipase activity is not due to increased cell number. This is because the cell concentration that can be grown is the same in both experiments.

Example 13 A DNA insert containing the lipase gene was prepared from F2 2.
5 was a subfragment. F2DNA (4-25
μg) and IOU of restriction enzyme BgQI
37
．． 5μQ solution (containing the same digestion buffer as described in Example 1 Part 3) and incubated overnight at 37°C. After adjusting the solution to 0.1M Na (j2), 10U of the restriction enzyme EcoRI was added and incubated for 1 hour at 37°C. Proteins were removed as described in Example 1 Part 3, and the digested DNA was taken, washed and resuspended in TE pH8.

Plasmid pKT231 (K, Tim
m1s), Department of Medicine, Biochemistry (Department of Medi)
calB iochemistry), University of Geneva
(Geneva, Switzerland) was chosen as the cloning vector.

Because it has a selectable kanamycin resistance (Kin) gene with a copy number of 15 to 20 in a variety of Durham-negative bacteria with a wide host range, E., Co., IJ, and is mobile-positive. [Bagasarian (B)
agdelasarian) et al., Gene, 16
．． 237-247 (1981)).

Plasmid pKT231 was purified from Enicoley cells containing pKT231 as described in Example Bart 2. However, the bacterial growth medium is 25μg/
mffKm and 25 pg/mQ streptomycin (SmX Sigma Chemical Co., St. Louis;
Supplemented with M.O.). Plasmid pKT231D
Example 4: 3 μg of NA and restriction enzyme BglI [1OU
were combined in 50 μα of digestion buffer as described in .

After incubating the reaction overnight at 37°C, the solution was diluted with O, 1M NaC.
12, add IOU of restriction enzyme EcoRI,
The digestion reaction was then incubated for an additional hour at 37°C. The double-digested pKT231 DNA was then dephosphorylated as described in Example 9 to remove contaminating proteins.
The plasmid DNA was then precipitated, harvested, washed, and resuspended in TEpHg.

A subfragment of F2 DNA was ligated into plasmid pKT231 as described in Example 8. The DNA ligation mixture was transformed into E. coli HBIOI component cells as described in Example 3 Parts 2 and 3. The transformed cells were cultured on Syrian agar plates containing 50 μg/IIIQKI11 and 50 pg/rnQ Sm and incubated overnight at 37°C. This selective medium allows the growth of only cells containing the pKT231 plasmid, which contains both the Km and 5I11 resistance genes. To identify strains containing the desired 2.5 Kb DNA insert, the plasmid content of Ktn'Sm' transformants was analyzed by agarose gel electrophoresis as described in Example 4. In this case, partially purified plasmid DNA was digested with the restriction enzymes Dang121r and IjikuRI in a two-step digestion reaction as described above. 21
Ten of the format transformants contained the recombinant pKT231 plasmid with a 2.5 Kb fragment (p
HES8 [According to the Budapest Treaty <Deposit number with ATCC (hereinafter referred to as the "International Deposit Number") 68160
).

Plasmid pHES8 (International Deposit No. 68160) was transferred to B.S.P. ATCC 21808 by transformation as described in Example 1O. Plasmid pHES8 (International Deposit No. 68160) was purchased from Ecoli HB to 1 [p
HES8 (International Deposit No. 68160)]. Ekoly HB 101 [pHES
8 (International Deposit No. 68160)] The medium used to culture the cells was 50 μg/m12Km and 50 pg/m
Luria liquid culture medium supplemented with QSm. 192 μg of partially purified pHES8 (International Deposit No. 68160) plasmid DNA was transformed into BSP ATCC 21808 Rif' reactive cells as described in Example 10. Transformed. Transformed cells at 500 μg/mffKm and 50 μg/m
Cultured on nutrient agar plates containing ff5m.

This selective medium allows growth of only cells containing the pHE58 recombinant plasmid. pHES8(
ATCC 21808 Rif'Km'Sm' transformants containing the plasmid (International Deposit No. 68160) were selected and verified as described above.

e2, SP-21808Rif'CPHE S 8
(International Deposit No. 68160)] was determined as described in Example 5. However, the growth medium is 50
Supplemented at μg/mffiKm. In addition, the SP ATCC21808Rif' which does not contain plasmids
strain and the strain containing plasmid pKT231 served as a negative control.

Plasmid-free strains and pKT231-containing strains were tested at 5 μg/mQRif alone and at 5 μg/mQRif.
/m (2Rif+50 /jg/mff Km, respectively).

Quantitative lipase activity was determined as described in Example 5. Lipase activity is shown in Table 7. BSP ATCC
21808Rif' [pHE S 8 (International Deposit Number 6)
8160)] are puplast-free strains and puplast-free strains.
It had 42 and 62 times higher lipase activity than the KT231-containing strain.

table Bee.

S.P.

21808RIF' Strain Nori Strain Lipase Activity (U/+++Q of Cultured Bacteria) It is clear from the above results that lipase production at a high concentration can be achieved by using the present invention. Therefore,
Commercially desired concentrations of lipase can be secreted into the supernatant and continuously produced and isolated. Concentrations can be increased by combining further plasmids, especially high copy number plasmids. In that case,
The two plasmids may be compatiblely maintained in the same host using strong promoters, gene integration, expansion, etc. Of particular interest are pseudomonads harboring one or more plasmids.
) and the lipase gene is expressed and secreted.

All publications and patent applications mentioned in the specification are indicative of the level of those skilled in the art to which this invention pertains. Although each respective publication or patent application is as if specifically and individually indicated to be incorporated by reference, all publications and patent applications are herein incorporated by reference to the same extent.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the claims.

Example 14 Muvertl: Construction of a Recombinant Plasmid Containing the Complete Lipase Gene The DNA insert containing the lipase gene was
, a 5 kb subfragment. F2-containing plasmid pHSH1 7 DNA (20 μg) and 20 U of the restriction enzyme HindI[I were mixed together in a 20 μa solution (containing the same digestion buffer as described in Example 1 Part 3) and incubated for 1 hour at 37°C. Add the solution to 0.1M Na
The mixture was adjusted to Cl2, 40 U of the restriction enzyme coRI was added, and cultured at 37°C for 1 hour.

Proteins were removed, digested DNA was taken up, washed, and purified with TEpH as described in Example 1 Bart 3.
resuspended in a.

Cloning vector, plasmid pBR322DNA
(20 μg) of the restriction enzymes HindI [[ and Ec] in a two-step digestion reaction as described above.

Digested with R1. Proteins were removed as described in Example 1 Bart 3, and the digested DNA was harvested, washed, and resuspended.

The subfragment of F2 DNA resulting from digestion of plasmid pHSH17 was ligated into plasmid pBR322 as described in Example 6 Part 4. The DNA ligation mixture was prepared as described in Example 3, parts 28 and 3.
The cells were transformed into Corey HBIOI reactive cells. Transformed cells were cultured on Luria agar plates containing 50 μg/rnQ amvicillin and incubated overnight at 37°C. This selective medium allows the growth of only cells containing the pBR322 plasmid containing the amvicillin resistance gene. To identify strains containing the desired 5.5 kb DNA insert, the plasmid content of Tc' transformants was analyzed by agarose gel electrophoresis as described in Example 4. In this case, the partially purified plasmid DNA was digested with the restriction enzymes HindI [[ and Ec
Digested with oRI. One of the 18 transformants was 5.5K
It contained a recombinant pBR322 plasmid with the b fragment (referred to as pHRA 1).

Part 2: Deletion of the Lipase Gene Promoter Plasmid pHRAI, which has a unique DungQn restriction site, was located near the lipase gene promoter region. The combination of deletions in the lipase promoter region is unique to the restriction site of the exonuclease Ba1231 (International Biotechnology Seeds Co., Ltd., Two Barben, CT).
) was assembled by proceeding with the digestion of plasmid pHRA1.

Ba(+31) is an exonuclease that deletes nucleotides from both the 3' and 5' ends of linear DNA fragments. Plasmid pHRAI DNA (80 μg) and 128 U of restriction enzyme 1 were added to a 48 μα solution (described in Example 1). The proteins were removed and the digested DNA was taken up and washed as described in Example 1 Part 3) and incubated overnight at 37°C. , and resuspended in TEpHB.

10 μg of digested pHRAI DNA and af.
Eight aliquots containing 100 U of 31 Fast were added to a 100 μQ solution (600 mM NaCL 12°5
mM Ca(j+2.12, 5 mM Mg(122
, 20mM Tris-MCI2ph3 and 1mMMEDT
A) and cultured for 45 minutes and 30 minutes. Remove protein as described in Example 1 Part 3;
Digested DNA was harvested, washed, and resuspended in TEpHa.

Part 3: Preparation of a DNA fragment containing a deletion of the lipase gene promoter for fusion to the tac promoter Ba1231-treated pHRAI DNA was added to a 260 μQ solution (25 mM) of Lis-HCQ pH 7,8,50 + oMN.
aCQ, 10mM MgCQz, 100 pg/mQ bovine serum albumin, 0.1mM dATP, dTTP, dC
T.P.

and dGTP, and 1 mM dithiothreitol (d
E., Corey DNA polymerase (Klenow 7 fragment (Klenow 7 fragment))
The cells were made blunt-ended by adding 5 U of LenowFragment; New England Biolabs, Beverly, M.N., and cultured at 37° C. for 1.5 hours. Proteins were removed and digested DNA was taken up, washed, and resuspended in TEpHB as described in Example 1 Part 3.

Multiple EcoRI linker G G A
AT T CC; Stratagene
), La J.

11a, Sheeny (CA)) T, DNA ligase 2
000U was added to 200C2 solution (25mM l-Lith-H (1
pH 7, 8, 10mM MgC (1,,4mM β-mercaptoethanol, 0.4mM ATP and 5 ng/
mQEcoRI linker) was added to the ends of pure pHRAI DNA and incubated overnight at room temperature.

EcoRI linked plasmid pHRAI DNA and restriction enzyme EcoRI
100 U of 100 U were mixed together in a 300 μg solution (containing the same digestion buffer as described in Example 13) and incubated at 37° C. for 6 hours. As described in Example 1 Part 3,
Proteins were removed and digested DNA was taken up, washed, and resuspended in TEphS. DN containing the lipase gene with possible deletions in the promoter region
The A fragment was separated from the pBR322 plasmid fragment by vertical gel electrophoresis as described in Example 6 Part 2.

Part 4: Construction of recombinant plasmid containing taC promoter fused to lipase gene Plasmid pM
MB22 (M, Bagdasa)
rian), Michigan Biotech Institute
ologyI n5tituta), Lang Lang (La
nsing) and MI (Ml) were selected as cloning vectors.

because it has a tac promoter, a variable host range of Durham-negative bacteria, a selectable amvicillin resistance (Ap) gene, and a mobile gene [Badassarian et al., Gene, 26.273-282
(1983)]. Additionally, this plasmid has been successfully used to overexpress the B. aeruginosa phosphomannose Φ isomerase gene in B. ayruginosa.

Plasmid pMMB22 was purified from E. coley cells containing pMMB22 as described in Example 1 Part 2. However, the bacterial growth medium is 25μg/
Supplemented with m (lAp (Sigma Chemical Co., St. Louis, MA). Plasmid pMMB
22 44 μ9 of DNA and 40 U of restriction enzyme EcoRI were added to 1 ml of digestion buffer solution as described in Example 13.
Combined in 25 μQ solution and incubated overnight at 37°C.

EcoRI-digested pMMB22 DNA was dephosphorylated as described in Example 1 Part 4 to prevent plasmid transfer in subsequent ligation reactions. Example 1 Part 3
Contaminating proteins were removed and the dephosphorylated DNA was precipitated, harvested, washed, and resuspended in TEpHB as described in . The purified DNA fragment containing the lipase gene with possible deletions in the promoter region was ligated into plasmid pMMB22 as described in Part 3 above.

Transformation of the Virt 5:' Plasmid into E. coli The DNA ligation mixture was transformed into E. coli DH5 reactive cells as described in Example 3, Parts 2 and 3. Transformed cells at 50 μg/rnQA
The cells were cultured on Syrian agar plates containing p.p., and incubated overnight at 37°C. This selection of selective media allows the growth of only cells containing the pMMB22 plasmid containing the Ap resistance gene. Desired DH with promoterless lipase gene fused to tac promoter
To identify strains containing the 5 insert, Ap' transformants were incubated with 50 pg/rnQAp, 0.05% castor oil and 1 mM isopropyl-thiogalactozide (■
The cells were blocked on Luria agar plates supplemented with PTG, International Biotechnology Seeds, Inc., New Burpen, CT) (sometimes without). One DH5A transformant produced a wide clear zone only when lPTG was present. Controlled by the tac promoter I; the gene is fully induced by non-metabolizable l PTG molecules. This DH strain contained a recombinant 9MMB22 plasmid with the tac promoter fused to the lipase gene [p
HES12 (International Deposit Number 68161)].

Bart 6: Plasmid pHES12 (International Deposit No. 68
Plasmid pHES12 (International Deposit No. 68161) was transferred into B.S.P. ATCC 2180g by conjugation as described in Example 2, Part 2. P, SP ATCC21808 metazygote 50119/rnQ
Selection was made on nutrient agar plates with Rif and 1000 μp/mQAp.

Bart 7: B.S.P. ATCC21808 [p
HES12 (International Deposit No. 68161)] strain of lipase activity Be, Engineering 7 SP ATCC21808Rif' [pH
ES12 (International Deposit No. 68161)] was determined as described in Example 5.

However, the growth medium is a growth medium with 50 μg/rnQ Ap but without 1 mM IPTG.

P.S.P ATCC218 that does not contain plasmids
The 08Rifr strain and the strain containing plasmid pMMB22 served as negative controls. 5 plasmid-free strains and pMMB22-containing strains
tt9/mQ Rif with and without 1mM IPTG and 5 pg/mQ Ri
f+50119/mOAp and l PTG were grown in respective media with and without PTG.
Quantitative lipase activity was performed and determined as described in Example 5. The lipase activity is shown in the table below.

(Hereafter, margin) Bee.

Lipase activity of SP1808 R1fr strain U/rnQ of strain/culture 1mM IPTG Lipase activity 1808 21808
+21808 (pMMB22) 21808 (pMMB22) 121808 [pHES12 (International Deposit Number 68] 61
)] 21808 [pHES12 (International Deposit Number 681
61)] +12.6 12.6 11.7 13.4 26.4 070 1 PSP ATCC grown without PTG
21808Rif' [pHE S 12 (International Accession No. 68161)] had twice the lipase activity of the strain without Puplast or the strain containing the parental plasmid. On the other hand, when grown in the presence of IPTG (7), P,SP ATCC21808Rif' [pHE
S12 (International Deposit No. 68161)] had 85 times the gluepase activity of both control strains.

Example 15 Plasmid pNM185 (K. Timmis).

T 1mm1s, Department of Medicine
Biochemistry, University of Geneva;
Geneva, Switzerland) was chosen as the cloning vector. [Merm□l et al., Journal
of BacterioL 167.447-454
(1986)]. This plasmid has been successfully used to overexpress the xyl E gene in a variety of Gram-negative bacteria.

Plasmids pNM185 and pHES12 were added to pNM185 and pHES12 as described in Example 1 Part 2.
Purified from E. coli cells containing S12.

However, the bacterial growth medium is 50μg/vxQ
It is supplemented with Km. Plasmid pNM185 was digested and dephosphorylated and plasmid pHES12 was digested as described in Example A Part 4. A DNA fragment from pHES12 containing the promoterless lipase gene was ligated into plasmid pNM185 (containing the xyl promoter as described in Example 8).

Bart 2: Transformation of recombinant plasmids into E. coli The DNA conjugate mixture was transformed into E. coli DH5 reactive cells as described in Example 3 Bart 2 and 3. Transformants were selected as described in Example A Part 5. However, it is 50μ9/l112! It
It was used. To identify strains with the desired orientation of the DNA insert relative to the K1 promoter, K1 transformants were incubated at 50 pg/mQ Km.

Blocking was performed on Luria agar plates supplemented with 0.05% castor oil and 5mM m-toluic acid (m-Tolu; Eastman Kodak, Robiesta, S.Y.). Only when m-tor exists,
Some Km' transformants produced large clear zones. These pH5 strains are a recombinant p1 promoter fused to the promoterless lipase gene.
contained the NM185 plasmid [pHES12X
(International Deposit No. 68181)].

Bart 3: pHES12X (International Deposit Number 68181)
Plasmid pHES12X (International Deposit Number 68181)
by joining as described in Example A.
Metastasized to TCC21808.

However, 500 μg/1nQKrn was used.

Bart 4: PSP ATCC 21808 [pH
E S 12X (International Deposit No. 68181)] lipase activity of strain P, S.
The lipase activity of 12X (International Deposit No. 68181) was determined as described in Example A Part 7. However, 50 μg/mQKrnl and 0.1 mM m-Toll were used. Quantitative lipase activity was performed and determined as described in Example 5. The lipase activity of 21808 [pHES12X (International Deposit No. 68181)] is 750
U/mQ, plasmid-free strain or parental strain.
It was about 60 times that of the strain containing the plasmid.

Claims (1)

[Claims] 1. A chromosomal lipase gene and at least one multicopy vector containing a lipase gene capable of expressing lipase in Pseudomonas cells, characterized in that the lipase is derived from strain ATCC21808. and Pseudomonas cell. 2. Vector is pHES3, pHSH43, pHSH3
1, pHES12 (International Deposit No. 68161) or p
The Pseudomonas cell according to claim 1, which is HES12X (International Deposit No. 68181). 3. A plasmid comprising a P-incompatibility replication system, a Pseudomonas lipase gene derived from strain ATCC21808, and a selection marker. 4. Plasmids are pHES3, pHSH43, pHSH
The plasmid according to claim 3, which is pHES12 (International Deposit No. 68161) or pHES12X (International Deposit No. 68181). 5. Cells of a Pseudomonas strain characterized in that the lipase is derived from the strain ATCC21808, consisting of a genome lipase gene and multiple copies of the Pseudomonas lipase gene encoding the lipase, for a period sufficient for the lipase to be expressed. grown under and conditions;
and a method for producing lipase, which consists of removing cells and incorporating lipase. 6. The method of claim 5, wherein the multiple copies are components of at least one vector. 7. Vector is pHES3, pHSH43, pHSH3
1, pHES12 (International Deposit No. 68161) or p
6. The method according to claim 5, which is HES12X (International Deposit No. 68181). 8. The method of claim 5, wherein the cell produces at least 5 times more lipase than in the absence of the multicopy lipase gene. 9. The method of claim 5, wherein the cell consists of two multicopy vectors, each vector consisting of the Pseudomonas lipase gene encoding lipase. 10. A method for producing lipase, which comprises growing a cell of Pseudomonas strain ATCC 21808 comprising plasmids pHSH43 and pHSH31 for a time and under conditions sufficient to express the lipase, and removing the cell and incorporating the lipase. 11. Pseudomonas sp. ATCC218
A composition consisting of a lipase encoded by a lipase gene from 08. 12. Pseudomonas sp. in an aqueous medium. A
A method of hydrolyzing an organic carboxylic ester by combining a lipase encoded by the lipase gene from TCC21808 with a carboxylic ester. 13. Pseudomonas sp. in an aqueous medium. A
By combining the lipase encoded by the lipase gene from TCC21808 and hydroxycarboxylic acid,
A method for preparing lactones from said hydroxycarboxylic acids. 14, Pseudomonas sp. ATCC218
A method for removing lipid stains from fabrics by washing with a detergent formulation consisting of a lipid-removing amount of lipase encoded by the lipase gene from 08. 15, comprising a detergent active composition and a lipase, characterized in that the lipase is produced by a Pseudomonas microorganism comprising a recombinant vector consisting of a gene encoding the lipase, and is expressed in the Pseudomonas microorganism; Detergent composition. 16. The detergent composition of claim 15, wherein the detergent composition is granular and comprises at least one builder, bleach or whitening agent. 17. The detergent composition of claim 15, wherein the detergent composition is liquid and contains at least one bleaching or whitening agent. 18. Lipase has portrait amino acid chain: L-V-
The detergent composition according to claim 15, characterized in that it has G-H-S-Q-G-G. 19. A liquid detergent composition comprising a lipase produced by expression of a gene encoding a lipase, the gene comprising a part of a recombinant vector. 20. A granular detergent composition comprising lipase produced by expressing a gene encoding lipase, the gene comprising a part of a recombinant vector. 21. A detergent composition containing a lipase encoded by a lipase gene from Pseudomonas sp.