Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
  • C12N9/18—Carboxylic ester hydrolases (3.1.1)
  • C12N9/20—Triglyceride splitting, e.g. by means of lipase

Definitions

• This invention relates to the preparation, by recombinant DNA technology, of enzymes for the enzymatic degradation of fatty materials, specifically lipolytic enzymes which have characteristics which make them suitable for use as detergent additives.
• a special problem associated with laundry cleaning is the removal of stains of a fatty nature.
• fat-containing dirt is emulsified and removed using a combination of elevated temperature and high alkalinity.
• detergent additives which are effective at the lower washing temperatures, stable in high alkaline detergent solutions and stable under storing conditions in both solid and liquid detergent compositions.
• a group of enzymes which hydrolize triglycerides are lipases (E.C. 3.1.1.3). Lipases are widely distributed, occurring in many different prokaryotic organisms, as well as eukaryotic cells. Depending upon the source of the enzyme, substrate specificity as well as other characteristics including stability under various conditions, varies widely. Lipases have been used in detergent compositions, however those used exhibited low cleaning efficiency under washing conditions and in addition, did not meet the stability requirements for detergent use.
• Lipolytic enzymes which are capable of exhibiting lipase activity under modern washing conditions, i.e., they are stable and effective at high detergent concentrations, at high pH and at low washing temperatures, are produced by certain strains belonging to the species of Pseudomonas pseudoalcaligenes. Pseudomonas stutzeri and Acinetobacter calcoaceticus (see European Patent Application EP-A-0218272). However, these species are potentially pathogenic for plants and animals, and few data are available on the fermentation conditions required for an effective production process for lipases using these microorganisms.
• lipases be secreted by the host organism so that the enzyme may be recovered directly from the extracellular fluid of the fermentation mixture.
• Bacillus species in particular Bacillus subtilis strains have been used with varying degrees of success as host strains for the expression of both foreign and endogenous genes and for the secretion of the encoded protein products.
• Bacillus species in particular Bacillus subtilis strains have been used with varying degrees of success as host strains for the expression of both foreign and endogenous genes and for the secretion of the encoded protein products.
• Sarvas Current Topics in Microbiology and Immunology 125 (1986) 103-125, H.C. Wu and P.C. Tai, eds., Springer Verlag; also see Himeno et al., F.E.M.S. Microbiol. Letters 35 (1986) 1721.
• U.S. Patent No. 3,950,277 and British Patent Specification No. 1,442,418 disclose lipase enzymes combined with an activator and calcium and/or magnesium ions, respectively, which are utilized to pre-soak soiled fabrics and to remove triglyceride stains and soils from polyester or polyester/cotton fabric blends, respectively.
• Suitable microbial lipases disclosed include those derived from Pseudomonas, Aspergillus, Pneumococcus, Staphylococcus, Mycobacterium tuberculosis, Mycotorula lipolytica and Sclerotinia.
• British Patent Specification No. 1,372,034 discloses a detergent composition comprising a bacterial lipase produced by Pseudomonas stutzeri strain ATCC 19154.
• the patent further discloses that the preferred lipolytic enzymes should have a pH optimum between 6 and 10, and should be active in said range, preferably between 7 and 9. (This presumed Pseudomonas stutzeri strain has been reclassified as Pseudomonas aeruginosa).
• European Patent Application (EP-A) 0130064 discloses an enzymatic detergent additive comprising a lipase isolated from Fusarium oxysporum which has a higher lipolytic cleaning efficiency than conventional lipases. Lipolytic detergent additives were also disclosed in, e.g., British Patent Specification No. 1,293,613 and Canadian Patent No. 835,343.
• EP-A-0205208 and EP-A-0206396 disclose use of Pseudomonas and Chromobacter lipases in detergents. For a comprehensive review article on lipases as detergent additives, see Andree et al., J. Appl. Biochem. 2. (1980) 218-229.
• Novel compositions comprising transformed microbial cells, and methods for their preparation, are provided which produce lipolytic enzymes suitable for use in detergent compositions.
• Host microbial cells are transformed using expression cassettes comprising a DNA sequence encoding a lipolytic enzyme which is active at alkaline pH and stable under laundry washing conditions.
• Methods for preparation of the lipolytic enzymes include cloning and expression in microbial systems and screening on the basis of DNA homology.
• DNA sequences comprising a gene encoding a lipolytic enzyme derived from a Pseudomonas pseudoalcaligenes strain and a Pseudomonas aeruginosa strain, respectively.
• lipase genes derived from certain Pseudomonas species.
• Figure 1 Strategy of the lipolytic enzyme gene cloning. For symbols see the legend in Figure 2.
• Figure 2 Restiction map of pAT1. A number of restriction enzyme recognition sites have been determined in plasmid pAT1. pUN121 vector DNA Thai IV 17-1 DNA insert
• - Ap r the pUN121 gene encoding ampicillin resistance
• - Tc r the pUN121 gene encoding tetracycline resistance
• - cl the bacteriophage lambda gene encoding the cl repressor.
• Figure 3 Restriction map of pAT3. The symbols are the same as used in Figure 2.
• Figure 4 Restriction map of pET1. The symbols are the same as used in Figure 2.
• Figure 5 Restriction map of pET3. The symbols are the same as used in Figure 2.
• Figure 6 Restriction map of pAMl. The symbols are the same as used in Figure 2.
• Figure 7 Restriction map of pM6-5. The symbols are the same as used in Figure 2.
• Figure 8 Restiction map of pP5-4. The symbols are the same as used in Figure 2. The position at which partially EcoRI* digested chromosomal DNA of Acinetobacter calcoaceticus GR V-39 (CBS 460.85) was ligated to pUN121 is indicated by EcoRI/EcoRI*.
• Figure 9A after staining with Coomassie Brilliant Blue.
• Figure 9B after staining with ⁇ -naphthyl acetate/Fast Blue BB.
• Lane 1 lysate from E. coli JM101 hsdS recA strain harbour ing pUN121 heated with SDS for 10 min at 95°C.
• Lane 2 lysate from E. coli JMlOl hsdS recA strain harbouring pET3 heated with SDS for 10 min at 95oC.
• Lane 3 culture supernatant from P. stutzeri Vietnamese IV 17-1 strain heated with SDS for 10 min at 95°C.
• Lane 4 the same sample as in lane 2 but heated with SDS for
• Lane 5 the same sample as in lane 3 but heated with SDS for
• Lane 6 the same sample as in lane 2 but incubated with SDS for 10 min at room temperaturet
• Lane 7 the same sample as in lane 3 but incubated with SDS for 10 min at room temperature.
• Lane 8 low molecular weight protein markers from Pharmacia.
• Figure 10 SDS 13% polyacrylamide gel after staining with Coomassie Brilliant Blue.
• Lane 2 low molecular weight protein markers (Pharmacia).
• Figure 11 Restriction map of pTMPv18A.
• pTZ18R vector DNA M-1 DNA insert Symbols used are:
• E. coli origin of replication derived from pBR322 vector
• Figure 12 shows the nucleotide sequence (i.e. the first 942 nucleotides) of the M-1 lipase gene and the derived amino acid sequence of the M-1 lipase.
• the termination codon TGA is indicated by an asterisk.
• the A box represents the active center of the lipase protein.
• the arrow indicates the putative signal peptidase cleavage site.
• the amino terminal sequence of the mature lipase protein is underlined.
• E. coli origin of replication derived from pBR322 vector
• Figure 14 Partial nucleotide sequence of the PAO lipase gene (i.e. from the internal SalI site) and derived amino acid sequence of the PAO lipase.
• the termination codon TAG is indicated by an asterisk.
• the A box represents the active center of the lipase protein.
• Figure 15 The construction of the plasmids pBHAM1 and pBHCM1. Symbols used are:
• Bacillus origin of replication Bacillus origin of replication
• Figure 16 shows the construction of plasmid pBHAMlNl. The symbols are the same as used in Figure 15.
• Figure 17 illustrates the construction of plasmid pTZN1M1. Symbols used are:
• Figure 18 shows the construction of plasmid pMCTM1. Symbols used are:
• Lanes A-D contain periplasmic fractions of E. coli cells harbouring the following constructs: Lane A: pTZ18RN Lane B: pTZN1M1 Lane C: pMCTM1
• Lane D purified M-1 lipase from Pseudomonas pseudoalcaligenes strain M-1.
• the molecular mass of marker proteins (RainbowTM from Amersham) is indicated in kDa at the right.
• Figure 20 The construction of plasmid pBHM1N1. The symbols are the same as used in Figure 15.
• Figure 21 Autoradiograph of 35 S labeled proteins synthesized in vitro. Lanes A-D: Immunoprecipitation of the in vitro translated samples by monoclonal antibodies against M-1 lipase. Lanes E-H: In vitro transcription/translation products of the following plasmids: Lanes A and E: pTZ18RN Lanes B and F: pTMPv18A Lanes C and G: pMCTM1 Lanes D and H: pMCTbliM1
• Figures 22A and 22B Detection of lipase encoding sequences in bacterial DNAs. Five nanogram amounts of plasmid DNA and five microgram amounts of chromosomal DNA were digested with restriction enzyme as indicated, separated on 0.8% agarose gel blotted onto nitrocellulose filters and hybridized with the nick-translated insert of pET3 and pTMPv18A, respectively. Figure 22A shows the autoradiograph after hybridization with the pET3 EcoRI insert.
• Figure 22B shows the autoradiograph after hybridization with the pTMPv18A XhoI-EcoRV insert.
• Lane A Hindlll/SStI digest of plasmid pTMPv18A
• Lane B EcoRI digest of plasmid pET3.
• BRL DNA gel marker MW of 0.5, 1.0, 1.6, 2.0, 3.0, 4.0, 5.0, 6.0, 7 , 8 , 9 , 10, 11, 12 kb
• Lane C SalI digest of P. pseudoalcaligenes M-1 (CBS 473.85)
• Lane D SalI digest of P. pseudoalcaligenes IN II-5 (CBS
• Lane E SalI digest of P. alcaligenes DSM 50342
• Lane F SalI digest of P. aeruqinosa PAC 1R (CBS 136.89)
• Lane G SalI digest of P. aeruginosa PAO2302 (6-1)
• Lane H SalI digest of P. stutzeri Thai IV 17-1 (CBS 461.85)
• Lane I SalI digest of P. stutzeri PG-I-3 (CBS 137.89)
• Lane J SalI digest of P. stutzeri PG-I-4 (CBS 138.89)
• Lane K SalI digest of P. stutzeri PG-II-11.1 (CBS 139.89)
• Lane L SalI digest of P. stutzeri PG-II-11.2 (CBS 140.89)
• Lane N SalI digest of P. gladioli (CBS 176.86)
• Lane O SalI digest of A. calcoaceticus Gr-V-39 (CBS 460.85)
• Lane P SalI digest of S. aureus (ATCC 27661).
• DNA constructs and novel compositions comprising microbial strains producing lipolytic enzymes are provided.
• the lipases of interest in the present invention possess a pH optimum between about 8 and 10.5, exhibit effective lipase activity in an aqueous solution containing a cleaning composition at concentrations of up to about 10 g/l under washing conditions at a temperature of 60°C or below, preferably 30-40°C, and at a pH between about 7 and 11, and preferably between about 9 and 10.5.
• Plasmid constructs comprising a DNA sequence encoding a lipase gene with the desired characteristics are used to transform a host cell which may be either a eukaryotic or prokaryotic cell. The transformed host cell is then grown to express the gene.
• the techniques used in isolating the lipase gene are known in the art, including synthesis, isolation from genomic DNA, preparation from cDNA, or combinations thereof.
• the various techniques for manipulation of the gene are well known, and include restriction, digestion, resection, ligation, in vitro mutagenesis, primer repair, employing linkers and adaptors, and the like. See Maniatis et al., "Molecular Cloning", Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1982.
• the method comprises preparing a genomic library from an organism expressing a lipase with the desired characteristics.
• lipases are those obtainable from Pseudomonas and Acinetobacter, and in particular from strains belonging to the species of Pseudomonas alcaligenes, Pseudomonas pseudoalcaligenes, Pseudomonas aeruginosa, Pseudomonas stutzeri and Acinetobacter calcoaceticus.
• Pseudomonas and Acinetobacter are those obtainable from Pseudomonas and Acinetobacter, and in particular from strains belonging to the species of Pseudomonas alcaligenes, Pseudomonas pseudoalcaligenes, Pseudomonas aeruginosa, Pseudomonas stutzeri and Acinetobacter calcoaceticus.
• EP-A-0218272 which disclosure
• the genome of the donor microorganism is isolated and cleaved by an appropriate restriction enzyme, such as Sau3A.
• the fragments obtained are joined to a vector molecule which has previously been cleaved by a compatible restriction enzyme.
• An example of a suitable vector is plasmid pUN121 which can be cleaved by the restriction endonuclease BclI.
• the amino acid sequence can be used to design a probe to screen a cDNA or a genomic library prepared from mRNA or DNA from cells of interest as donor cells for a lipase gene.
• the lipase DNA or a fragment thereof as a hybridization probe, structurally related genes found in other microorganisms can be easily cloned.
• oligonucleotide probes based on the nucleotide sequence of lipase genes obtainable from the organisms described in EP-A-0218272.
• these oligonucleotides can be derived from the amino acid sequences of lipases of interest.
• Such probes can be considerably shorter than the entire sequence but should be at least 10, preferably at least 14, nucleotides in length. Longer oligonucleotides are also useful, up to the full length of the gene, preferably no more than 500, more preferably no more than 300, nucleotides in length. Both RNA and DNA probes can be used.
• the probes are typically labeled in a detectable manner (e.g., with 32 P, 35 S, 3 H, biotin, or avidin) and are incubated with single-stranded DNA and RNA from the organism in which a gene is being sought.
• Hybridization is detected by means of the label after single-stranded and double-stranded (hybridized) DNA (DNA/RNA) have been separated (typically using nitrocellulose paper).
• Hybridization techniques suitable for use with oligonucleotides are well known to those skilled in the art.
• oligonucleotide probe refers to both labeled and unlabeled forms.
• sequence from the Pseudomonas pseudoalcaligenes lipase is cloned from the strain CBS 473.85 (M-1).
• the cloned lipase of Pseudomonas aeruginosa PAO (ATCC 15692), once sequenced showed a high degree of sequence homology wit the lipase gene sequence of M-1. Even more surprisingly, a similar high degree of sequence homology was found between the lipase gene sequence of the M-1 strain and the chromosomal DNA of a number of Pseudomonas stutzeri isolates [viz.
• PG-I-3 (CBS 137.89), PG-I-4 (CBS 138.89, PG-II-11.1 (CBS 139.89), PG-II-11.2 (CBS 140.89)] and Pseudomonas alcaligenes DSM 50342.
• a "high degree of hybridization" as used in this specification is defined as a continuous sequence stretch of DNA of at least 300 bp wherein at least 67% homology is found.
• the lipase enzymes of P. aeruginosa and P. stutzeri were produced and tested for their cleaning performance in the SLM-test.
• E. coli (Kuhn et al., Gene 44 (1986) 253-263) and for B. subtilis (Gryczan and Dubnau, Gene 20 (1982) 459-469).
• An example of such a positive selection vector for E. coli is pUN121 (Nilsson et al., Nucleic Acids Res. 11 (1983) 8019-8030).
• clones expressing lipolytic enzymes may be identified, for example, using a suitable indicator plate assay, such as agar media containing tributyrin or olive oil in combination with rhodamine B (Kouker and Jaeger, Appl. Env. Microbiol. 53. (1987) 211). Further, replicated colonies may be screened using an adapted soft agar technique based on the procedure described for detecting esterase activity (Hilgerd and Spizizen, J. Bacteriol. 114 (1978) 1184). Alternatively, clones expressing lipolytic enzymes may be identified by using genetic complementation in a suitable lipase-negative recipient strain, such as described by Wohlfarth and Winkler, J. Gen. Microbiol.
• Microbial hosts may include, for example, bacteria, yeasts and fungi, such as E. coli, Kluyveromvces, Aspergillus, Bacillus and Pseudomonas species. Therefore, where the gene is to be expressed in a host which recognizes the wild-type transcriptional and translational regulatory regions of the lipase, the entire gene with its wild-type 5' and 3' regulatory regions may be introduced into an appropriate expression vector.
• Various expression vectors exist employing replication systems from prokaryotic cells.
• an expression cassette which in the 5'-3' direction of transcription has a transcriptional regulatory region and a translational initiation region, which may also include regulatory sequences allowing for the induction of regulation; an open reading frame encoding a lipolytic enzyme, desirably including a secretory leader sequence recognized by the proposed host cell; and translational and transcriptional termination regions.
• the expression cassette may additionally include at least one marker gene.
• the initiation and termination regions are functional in the host cell, and may be either homologous (derived from the original host), or heterologous (derived from the original host), or heterologous (derived from a foreign source or from synthetic DNA sequences).
• the expression cassette thus may be wholly or partially derived from natural sources, and either wholly or partially derived from sources homologous to the host cell, or heterologous to the host cell.
• the various DNA constructs (DNA sequences, vectors, plasmids, expression cassettes) of the invention are isolated and/or purified, or synthesizend and thus are not "naturally occurring".
• RNA messenger RNA
• the amount of mRNA is determined by the copy number of the particular gene, the relative efficiency of its promoter and the factors which regulate the promoter, such as enhancers or repressers. Stability of the mRNA is governed by the susceptibility of the mRNA to ribonuclease enzymes. In general, exonuclease digestion is inhibited by the presence of structural motifs at the ends of the mRNA; palindromic structures, altered nucleotides or specific nucleotide sequences.
• Endonuclease digestion is believed to occur at specific recognition sites within the mRNA and stable mRNAs would lack these sites. There is also some evidence that mRNAs undergoing high levels of translation are also protected from degradation by the presence of ribosomes on the mRNA.
• expression can be regulated by influencing the rate of initiation (ribosome binding to the mRNA), the rate of elongation (translocation of the ribosome across the mRNA), the rate of post-translational modifications and the stability of the gene product.
• the rate of elongation is probably affected by codon usage, in that the use of codons for rare tRNAs may reduce the translation rate.
• Initiation is believed to occur in the region just upstream of the beginning of the coding sequence. In prokaryotes, in most cases this region contains a consensus nucleotide sequence of AGGA, termed the Shine-Dalgarno sequence. While this sequence characterizes the ribosomal binding site, it is evident that sequences both upstream and downstream can influence successful initiation.
• Evidence also points to the presence of nucleotide sequences within the coding region which can affect ribosome binding, possibly by the formation of structural motifs through which the ribosome recognizes the initiation site. Position of the AGGA sequence with respect to the initiating ATG codon can influence expression. It is thus the interaction of all of these factors which determines a particular expression rate. However, the expressed genes have evolved a combination of all of these factors to yield a particular rate of expression. Design of an expression system to yield high levels of gene product must take into consideration not only the particular regions that have been determined to influence expression but also how these regions (and thus their sequences) influence each other.
• Illustrative transcriptional regulatory regions or promoters include, for example, those sequences derived from genes overexpressed in industrial production strains.
• the transcriptional regulatory region may additionally include regulatory sequences which allow expression of the structural gene to be modulated, for example by presence or absence of nutrients or expression products in the growth medium, temperature, etc.
• expression of the structural gene may be regulated by temperature using a regulatory sequence comprising the bacteriophage lambda P L promoter together with the bacteriophage lambda O L operator and a temperature-sensitive repressor. Regulation of the promoter is achieved through interaction between the repressor and the operator.
• expression cassettes capable of expressing a lipolytic enzyme which employ the regulatory sequences of Bacillus amylase and protease genes.
• the structural gene of interest is joined downstream from the ribosomal binding site, so as to be under the regulatory control of the transcriptional regulatory region and the translational initiation region.
• a fused gene may be prepared by providing a 5'-sequence to the structural gene which encodes a secretory leader and a processing signal. If functional in the host cell of choice, the signal sequence of the lipase gene itself may also be employed.
• Illustrative heterologous secretory leaders include the secretory leaders of penicillinase, amylase, protease and yeast alpha-factor.
• the expression cassette may be included within a replication system for episomal maintenance in an appropriate host microorganism or may be provided without a replication system, where it may become integrated into the host genome.
• the manner of transformation of the host microorganism with the various DNA constructs is not critical to this invention.
• the DNA may be introduced into the host in accordance with known techniques, such as transformation, using calcium phosphate-precipitated DNA, conjugation, electroporation, transfection by contacting the cells with a virus, micro-injection of the DNA into cells, or the like.
• the host cells may be whole cells or protoplasts.
• any microorganism may be used which is suitable for production and extraction of a lipolytic enzyme; preferably the host organism is also capable of secreting the enzyme produced whereby the enzyme can be recovered from the cell-free fermentation fluid.
• the host microoganism is also preferably a non-pathogenic organism. Examples of host organisms which fulfill the above criteria include E. coli, Pseudomonas putida and Bacillus strains, especially B. subtilis and B. licheniformis, Streptomyces strains and fungi and yeast strains such as Aspergillus and Kluyveromyces, respectively.
• the host strains may be laboratory strains, or can include industrial strain microorganisms.
• Industrial strains are characterized as being resistant to genetic exchange, such as phage infection or transformation.
• the strains are stable and may or may not be capable of spore formation. They are prototrophic and modified to provide for high yields of endogenous protein products, such as the enzymes alpha-amylase and various proteases.
• the yield of an endogenous protein product obtained in an industrial production process can amount to at least 5 g/l (0.5% w/v).
• Industrial strains also secrete DNases, which result in the degradation of DNA in the medium, providing for protection against genetic exchange.
• the host cell may be grown to express the structural gene. Production levels of lipolytic activity may be comparable to or higher than the original strains from which the genes are derived.
• the host cell may be grown to high density in an appropriate medium to form a nutrient-rich broth. Where the promoter is inducible, permissive conditions will then be employed, for example, temperature change, exhaustion, or excess of a metabolic product or nutrient, or the like.
• the expression product may be isolated from the growth medium by conventional means. Release of the produced lipolytic enzyme may be enhanced by dilute surfactant solutions. Where secretion is not provided for, host cells may be harvested and lysed in accordance with conventional conditions. The desired product is then isolated and purified in accordance with known techniques, such as chromatography, electrophoresis, solvent extraction, phase separation, or the like.
• the subject compositions can be used in a wide variety of ways.
• the transformed host microorganisms can be used for enhanced production of lipase having characteristics which make it useful in detergent compositions.
• the cloned lipase genes may also find use in screening for lipase genes, including identification of a lipolytic gene as a lipase rather than an esterase.
• They may also be used for enzyme engineering using known techniques relating to random or site-directed mutagenesis resulting in lipases with the required altered characteristics.
• the lipolytic enzyme compositions can be used in washing compositions together with a detergent and optionally other ingredients which are commonly used in cleaning compositions.
• these ingredients can include at least one of surfactants, water softeners such as complex phosphates, alkali metal silicates and bicarbonates; fillers such as alkali metal sulfate; other emzymes such as proteases and amylases; bleaching agents; as well as miscellaneous compounds such as perfumes, optical brighteners, etc.
• the lipase activity is preferably in the range of from 1 to 20,000 TLU/g of composition, while the proteolytic enzyme activity is preferably in the range of from 50 to 10,000 Delft Units/g of cleaning composition.
• TLU true lipase unit
• the Delft Units are defined in J. Amer. Oil Chem. Soc. 60 (1983) 1672.
• the cleaning compositions of the invention may be prepared in the usual manner, for example by mixing together the components or by the preparation of an initial premix, which is subsequently finished by mixing with the other ingredients.
• one or more lipase preparations are mixed with one or more of the other compounds to make a concentrate of a predetermined enzymatic activity, which concentrate can then be mixed with the other desired components.
• the lipolytic enzymes of the invention are in the form of an enzymatic detergent additive.
• This additive may also contain one or more other enzymes, for example a protease and/or an amylase, which can be used in modern washing compositions, and one or more other components, which are commonly used in the art, for example a non-ionic, salt, stabilizing agent and/or coating agent.
• the enzymatic detergent additive can comprise in addition to a lipase, a protease and optionally an alphaamylase.
• the proteolytic enzymes are compatible with the lipolytic enzymes in this formulation.
• the enzymatic detergent additives are generally mixed with one or more detergents and other components known in the art to form washing compositions.
• the enzymatic detergent additive is generally used, in the range from 10 2 and 10 7 TLU/g of additive, while the optionally present proteolytic activity is in the range of from 5x10 4 to 10 6 Delft Units/g.
• the enzymatic detergent additives of the invention may be in the form of, for example, granulates or prills, prepared according to methods which are generally known in the art. See, for example, British Patents Nos. 1,324,116 and 1,362,365 and U.S. Patents Nos. 3,519,570, 4,106,991 and 4,242,219.
• the enzymatic detergent additive can be in liquid form with an enzyme stabilizer, for example propylene glycol. They can also be in the form of organic or inorganic slurries, emulsions or encapsulates, immobilized on a soluble or insoluble support or in an aqueous or waterfree solution in the presence of one or more stabilizers. Such additives are preferably used in liquid detergent compositions.
• an enzyme stabilizer for example propylene glycol.
• EP-A-0218272 discloses several strains of bacteria which produce lipases suitable for use in detergents. Of these, Acinetobacter calcoaceticus Gr V-39 (CBS 460.85), Pseudomonas stutzeri Thai IV 17-1 (CBS 461.85), Pseudomonas pseudoalcaligenes IN II-5 (CBS 468.85) and Pseudomonas pseudoalcaligenes M-1 (CBS 473.85) were selected as a source of lipolytic genes.
• Plasmid vector pUN121 (Nilsson et al., Nucleic Acids Research 11 (1983) 8019) which carries an ampicillin resistance gene, a tetracycline resistance gene and the cI repressor gene of bacteriophage lambda was obtained from Dr. M. Uhlen, Royal Institute of Technology, Department of Biochemistry, Teknikringen 10, S-10044 Swiss, Sweden. Transcription of the tetracycline gene is prevented by the cl repressor. Insertion of foreign DNA into the unique restriction sites (BclI, SmaI, HindIII and EcoRI) results in activation of the tetracycline gene. This permits direct (positive) selection of recombinant transformants on Luria broth agar plates containing 8 ⁇ g/ml tetracycline and 50 ⁇ g/ml ampicillin.
• Plasmid and chromosomal DNA was isolated as described by Andreoli, Mol. Gen. Genet. 199 (1985) 372-380. Chromosomal DNA isolated from Thai IV 17-1 and M-1, respectively, was partially Sau3A digested. The DNA was then ligated with T4 DNA ligase to BclI digested pUN121 DNA as described by Maniatis et al. (supra) and transformed into competent cells of E. coli strain JM101 hsdS recA (Dagert and Ehrlich, Gene 6. (1979) 23-28). E. coli JM101 hsdS recA was obtained from the Phabagen Collection (Accession Number PC2495), Utrecht, The Netherlands. Transformants resistant to tetracycline at 8 ⁇ g/ml in Luria broth agar plates were selected for.
• the tributyrin-positive clones were grown overnight in 2TY medium (16 g/l Bacto-tryptone, 10 g/l Bacto-yeast extract, 5 g/l NaCl, pH 7.0) medium and assayed for both the plasmid content (see under 1D) and the ability to convert various ⁇ -naphthyl substrates, an indication of lipolytic activity (see Example 2A).
• the plasmids isolated from the Thai IV 17-1/ pUN121 transformants were designated pAT1 and pAT3. Their structures are presented in Figures 2 and 3, respectively.
• the gene encoding the lipolytic activity was located within a 2.7 kb EcoRI fragment of pAT1 ( Figure 2, dashed line) and within a 3.2 kb EcoRI fragment of pAT3 ( Figure 3, dashed line).
• the two EcoRI fragments were subcloned in appropriate vectors, both for DNA sequencing and for obtaining higher yields of lipolytic activity.
• the plasmid pAMl in E. coli JM101 hsdS recA was deposited with CBS on February 5, 1987, under No. 154.87.
• E. coli DH1 (pM6-5) towards ⁇ -naphthyl esters was determined (Table 1).
• a sample of E. coli DH1 harbouring plasmid pM6-5 was deposited with CBS on February 5, 1987, under No. 153.87.
• Plasmid DNA from one of these clones was isolated and characterized with restriction endonucleases.
• the physical map of this plasmid pP5-4 is shown in Figure 8.
• the hydrolysis of ⁇ -naphthyl esters by crude enzyme preparations from E. coli DH1 (pP5-4) was then determined (Table 1). Plasmid pP5-4 in E. coli DH1 was deposited with CBS on February 5, 1987, under No. 151.87.
• Tributyrin-positive E. coli colonies were inoculated in 100 ml 2TY medium containing ampicillin and tetracycline in a 500 ml conical flask. E. coli cultures were shaken for 40 hrs at 30°C in an orbital shaker at 250 rpm. The Pseudomonas and Acinetobacter strains were grown at 30°C. After 40 hrs the optical density at 575 nm was measured and the broth was centrifuged in a Sorvall RC5B centrifuge in a GSA rotor at 6,000 rpm for 10 min. The supernatant was stored at 4oC until enzyme assay.
• the cells were resuspended in 4 ml lysis buffer (25% sucrose, 50 mM Tris-HCl pH 7.5). Lysozyme was added and after 30 min of incubation at 21°C DNase (20 ⁇ g/ml) was added and the incubation continued for 30 min at 37°C. Triton-X100 (0.1% v/v) was added and the cell suspensions sonicated on ice with a Labsonic 1510 sonifier set (5 strokes for 30 sec at 100 Watts with 1 min intervals). The cell debris was then removed by centrifugation for 15 min. at 12,000 rpm in a Hettich Mikro Rapid/K centrifuge. The supernatant obtained was then assayed for lipolytic activity.
• the assay is based on the hydrolysis of ⁇ - naphthylesters by lipolytic enzymes.
• the ⁇ -naphthyl released reacts with the diazonium salt Fast Blue BB to produce an azo dye absorbing at 540 nm.
• the method is essentially that of McKellar, J. Dairy Res. 53. (1986) 117127, and was performed as follows.
• the reaction tube contained in a final volume of
• Corning centrifuge tubes (15 ml) containing the reaction mixture were incubated at 37oC or specified temperature for 30 min.
• a 0.02 ml 100 mM FB solution (Fast Blue BB salt (Sigma), dissolved in DMSO) was added and the incubation continued for 10 min.
• the reaction was terminated with 0.2 ml 0.72 N TCA (trichloroacetic acid, Riedel-De Haen) and the coloured complex was extracted by vigorous mixing with 2.5 ml 1-butanol (Merck). The layers were separated by centrifugation at 5,000 rpm for 5 min. in a Heraeus Christ minifuge RF.
• the absorbance of the top layer was measured at 540 nm using a LKB ultraspec II spectrophotometer. After subtraction of controls, the readings were converted to TLUs (True Lipase Units) using Candida cylindracea lipase (L1754, Sigma) as a standard.
• TLU is defined as the titratable fatty acids equivalent to the amount of 1 ⁇ mole NaOH/min (see also EP-A-0218272). The results are shown in Table 1 below.
• Sample preparation Four volumes of an appropriate dilution of the enzyme preparations were mixed with one part of sample buffer containing 10% SDS, 10% ⁇ -mercaptoethanol in 0.5 M Tris-HCl, pH 6.8. This solution was divided into three equal parts. One portion received no further treatment but was kept at room temperature until gel electrophoresis took place. The second and third portions were heated for 5 and 10 min at 95°C, respectively, followed by cooling down in ice, then kept at room temperature until subjected to gel electrophoresis. Electrophoresis. The treated samples, in duplicate, were electrophoresed on the Phastgel system at 65 Volts/hour. One gel was stained for protein with Coomassie Brillant Blue according to Pharmacia Development technique file no 200.
• Figure 9A shows the polypeptide patterns after staining with Coomassie Brillant Blue.
• the second gel was washed with 50 mM Tris-HCl pH 7.5, 0.1% Triton X-100 to remove SDS and to reactivate the enzyme activity.
• the presence of lipolytic activity in the washed gel was visualized by a soft agar overlay technique based on the ⁇ -naphthyl acetate/Fast Blue BB salt method described in Example 1C. After incubation for 30 min at 30°C, purple bands became visible against a clear background.
• Figure 9B the lipase from the E. coli pET3 clone and native P.
• Plasmid pLAFR1 is a RK2-derived broad host-range cosmid conferring tetracycline resistance and is mobilizable but not self-transmissable.
• Plasmid pKT248 is a mobilizable R300B-derived broad host range plasmid conferring streptomycin and chloramphenicol resistance. Mobilisation of these vectors from E. coli to Pseudomonas was performed with the aid of the plasmid pRK2013, which contains RK2 transfer functions (Ditta et al., Proc. Natl. Acad. Sci. U.S.A. 77 (1980) 7347-7351) according to the triparental mating procedure of Friedman et al., (Gene 18 (1982) 289-296). A lipase-negative mutant 6-1 of P. aeruginosa strain PAO 2302 (obtained from S. Wohlfarth and U.K. Winkler, Ruhr Universitat, Bochum, FRG) was used as Pseudomonas recipient (J. Gen. Microbiol. 134 (1988) 433440).
• coli JM101 hsdS recA were selected for streptomycin resistance (Sm R ) and contra-selected for chloramphenicol sensitivity (Cm S ). Five thousand Sm R Cm S clones were obtained.
• the two M-1 gene libraries obtained in the E. coli host were replica-plated and screened for lipolytic activity as described in Example 1. None of the 13,000 recombinant transformants examined showed a lipase activity.
• Mobilization of the clone from E. coli to P. aeruginosa PAO 2302 (6-1) was therefore performed as follows. Recombinant plasmids were transferred to Pseudomonas recipients by replica-plating of donor strains to a lawn of the recipient strain (PAO2302/6-1) and helper strain (E. coli MC1061 or DHl harbouring the pRK2013 plasmid). After overnight growth of donor, recipient and helper strain on a Heart Infusion agar plate, exconjugants were selected by replica-plating on minimal agar medium containing 0.2% citrate, methionine (10 ⁇ g/ml) and streptomycin or tetracycline.
• Citrate is not metabolized by E. coli.
• Restoration of the lip phenotype of the lipase-defective mutant 6-1 with recombinant plasmids was tested by replica-plating the P. aeruginosa exconjugants on nutrient broth agar plates containing trioleoylglycerol and the fluorescent dye rhodamine B as described by Kouker and Jaeger, Appl. Env. Microbiol. 53 (1987) 211-213.
• N-terminal sequence analysis was performed after SDS gel-electrophoresis and electro-blotting on Immobilon transfer membrane (Millipore) according to Matsudaira (J. Biol. Chem. 262 (1987) 10035-10038). This analysis yielded the following sequence (using the conventional single letter amino acid code):
• Two synthetic 32-mer oligonucleotides 5'ACC GGC TAC ACC AAG ACC AAG TAC CCC ATC GT-3' and 5'ACC GGC TAC ACC AAG ACC AAG TAC CCG ATC GT-3' were deduced from amino acids 6 to 15 (T G Y T K T K Y P I) of the N-terminal sequence of the mature lipase protein as described above, after taking into consideration the codon bias for Pseudomonaceae and the degeneracy of the genetic code.
• the oligonucleotides were end-labeled using T4 polynucleotide kinase.
• Chromosomal DNA was isolated from Pseudomonas pseudoalcaligenes M-1 strain (CBS 473.85) according to Andreoli (Mol. Gen. Genet. 199 (1985) 372-380), digested with several restriction endonucleases, and separated on an 0.8% agarose gel. Southern blots of these gels, using radiolabeled 32-mer oligonucleotide as a probe revealed the following unique hybridizing DNA bands: 1.8 kb BclI, 2.0 kb PvuII and 1.7 kb SalI.
• M-1 chromosomal DNA was digested separately with these three restriction endonucleases, fractionated by 0.8% agarose gel-electrophoresis and the hybridizing fractions as described above recovered by electro-elution in a bio-trap BT 1000 apparatus from Schleicher and Schull.
• the 1.8 kb BclI digested fraction was ligated into BamHI-cut dephosphorylated vector pTZ18R/19R (purchased from Pharmacia, Woerden, The Netherlands).
• the 2.0 kb PvuII digested fraction was ligated into Smal-cut dephosphorylated vector pTZ18R/19R.
• the 1.7 kb SalI digested fraction was ligated into SalI-cut dephosphorylated vector pZT18R/19R. All three ligations were transformed into competent E.
• plasmid mini-prep was prepared by the alkaline lysis method and digested with the appropriate restriction endonucleases according to the instructions of the supplier.
• Six plasmids contained the expected hybridizing inserts.
• a sample of E. coli JMlOl hsdS recA harbouring plasmid pTMPv18A was deposited with CBS on March 8, 1989, under No. CBS 142.89.
• the E. coli pTZ18R/19R recombinant transformants obtained as described above were replica-plated and screened for lipolytic activity as described in Example 2. None of the 5x10 4 E. coli transformants examined showed lipolytic activity.
• a) the gene expression intiation signals of the M-1 lipase genes were either not recognized by the E. coli transcription/translation system (see, for example, Jeenes et al., Mol. Gen. Genet. 203 (1986) 421-429), b) a regulatory sequence or protein is necessary to switch on this M-1 lipase gene, c) proper folding or secretion of M-1 lipase in E. coli may be inadequate.
• Plasmid pTMPvlSA was digested with a variety of restriction enzymes which have 6 bp recognition sequences. Analysis of the fragment sizes resulting from these experiments allowed the generation of a preliminary restriction endonuclease cleavage map of the 2.0 kb PvuII insert of pTMPv18A. This map is shown in Figure 11.
• the pTZ18R/19R vectors used provide a versatile "all-in-one system" to permit DNA cloning, dideoxy DNA sequencing, in vitro mutagenesis and in vitro transcription (Mead et al., Protein Engineering 1 (1986) 67-74).
• the double-stranded plasmids were converted into single-stranded DNA by superinfection with a helper phage M13K07 obtained from Pharmacia.
• This signal peptide of 24 amino acids is cleaved off during the secretion process after the A E A (-3 to -1) signal peptidase recognition site. It is notable that the region around the cysteine residue of this signal sequence shows close resemblance to a lipoprotein consensus signal peptide (see Von Heyne, ibid.).
• the amino acid sequence predicted from the DNA sequence indicates that mature M-1 lipase consists of a 289 amino acid protein terminating with a TGA stop codon (see Figure 12).
• the predicted molecular weight of this mature protein is 30,323 which is in close agreement with the molecular weight determined for the M-1 lipase (MW 31,500) by SDS-polyacrylamide gel-electrophoresis (see Figure 10).
• the lipase of Pseudomonas aeruginosa is a lipidhydrolyzing enzyme (EC 3.1.1.3), having a MW of about 29,000, which is secreted into the medium during the late exponential growth phase (see Stuer et al., J. Bacteriol.
• the broad host range vector pKT248 (Bagdasarian et al., Gene 16 (1981) 237-247) was used, which is a mobilizable R300B-derived broad host range plasmid conferring streptomycin and chloramphenicol resistance.
• Pseudomonas aeroginosa PAO 1 DNA was partially digested with restriction endonuclease SalI and ligated into the single SalI site of the pKT248 vector.
• the ratio of insert:vector was 5:1 to reduce the possibility of vectorto-vector ligation.
• the ligated PAO/pKT248 DNA was transformed into competent E. coli SK1108 (Donovan and Kushner, Gene 25 (1983) 39-48) as described in Example IB. Transformants of E. coli SK1108 were selected for streptomycin resistance (Sm R ) and contraselected for chloramphemicol sensitivity (Cm S ) .
• the plasmid contained therein was characterized by restriction analysis followed by electrophoresis on agarose gels and was found to contain a SalI insert of 3.1 kb composed of a 1.3 kb, a 0.97 kb and a 0.76 kb SalI subfragment. These inserts were subcloned in appropriate vectors for DNA sequencing and for obtaining expression of the lipase gene.
• Plasmid DNA from a pUC19 derived subclone was isolated and characterized with restriction endonucleases.
• the physical map of this plasmid pSW103 is presented in Figure 13.
• a sample of E. coli JM101 hsdS recA harbouring plasmid pSW103 was deposited with CBS on March 8, 1989 under No. 141.89.
• Sall/PstI and Sall/EcoRI fragments of the 2.3 kb insert of pSW103 were purified and ligated into an appropriate M13mpl8 vector. The entire sequence was established by merging the collection of the obtained pieces of DNA sequence. Most of the DNA sequence has been determined for both strands. This DNA sequence when translated in all possible reading frames revealed that only the 0.97 kb SalI fragment of pSW103 (see Figure 14) encodes for the mature PAO 1 lipase.
• the 1.3 kb SalI fragment of the pSW103 subclone did not encode for, when translated in all possible reading frames, the 24 N-terminal amino acid residues of the PAO 1 lipase protein as determined by direct amino acid sequencing.
• Domain A encodes for an amino acid sequence G-H- S-H-G which was already defined as the active center of both eukaryotic lipases (Wion et al., Science 235 (1987) 1638-1641; Bodmer et al., Biochem. Biophys. Acta 909 (1987) 237-244) and prokaryotic lipases (Kugimiya et al., Biochem. Biophys. Res. Commun. 141 (1986) 185-190).
• the 2.0 kb Kpnl-Hindlll fragment of pTMPv18A carrying the M-1 lipase gene was ligated into Kpnl and Hindlll digested pBHA/C1 vectors.
• the nucleotide sequence of the pBHA1 vector is described in EPA-0275598.
• the pBHC1 vector is identical to the pBHAl vector except for the difference in expression of the following antibiotic resistance genes: the chloramphenicolacetyltransferase gene (Cm) is expressed in E. coli strain WK6 harbouring the pBHC1 vector and the beta-lactamase gene (Ap) is expressed in E. coli strain WK6 harbouring the pBHAl vector.
• the E. coli WK6 strain is described by R. Zell and H. J. Fritz , EMBO J. 6 ( 1987) 1809 .
• the next step was the introduction of a Ndel restriction endonuclease site on the ATG initiation codon of the M-1 preprotein.
• Site-directed mutagenesis on the pBHA/CM-1 plasmids was performed as described by Stanssens et al., in "Protein Engineering and Site-Directed Mutagenesis", 24th Harder Conference (1985), Ed. A.R. Fersht and G. Winter.
• pTZ18RN a pTZ18R derivative which contains a unique Ndel site on the ATG initiation codon of betagalactosidase (see Mead et al., Protein Engineering 1 (1986) 67-74);
• the cells were separated from the culture supernatant by centrifugation and then fractionated into periplasmic and membrane/cytopiasmic components according to Tetsuaki et al.. Appl. Environmental Microbiol. 50 (1985) 298-303. Each fraction was analyzed on SDS-polyacrylamide gels according to Laemmli, Nature 227 (1970) 680-685 consisting of a 13% separation gel and a 5% stacking gel. The gels were run at 60 mA until the Bromophenol Blue (BPB) marker reached the bottom of the gel. Sample preparation and protein blotting procedure were performed as described in EP-A-0253455. The Western blot was analyzed by using polyvalent rabbit antisera against purified lipase from P.
• BPB Bromophenol Blue
• EP-A-0275598 and EP-A-0253455 disclose a method for efficient transfer to Bacillus of a vehicle containing a gene of interest, resulting in Bacillus strains which secrete effectively the desired polypeptide products. This method was used for both the Thai IV 17-1 and the M-1 lipase genes.
• the pBHAM1N1 plasmid (see above) was digested with Ndel and religated. The ligation mixture was transformed into Bacillus licheniformis T9 protoplasts. A number of neomycin resistant tributyrin positive colonies were analyzed and the correct plasmid was obtained.
• the plasmid was called pBHM1N1 (see Figure 20) with the M-1 preprotein behind the Hpall promoter of pUB110 (see Zyprian and Matsubara, DNA 5 (1986) 219-225).
• T9 transformants harbouring the pBHM1N1 plasmid were tested for their ability to hydrolyze ⁇ -naphthyl esters after fermentation in industrial broth according to the method described in Example 2.
• This fragment was ligated into vector pTZ18R cleaved with EcoRI and Kpnl giving rise to plasmid pTZPf31A.
• pTZPf31A and pMCTM1 were cleaved both with BamH1 and Hindlll, ligated and transformed to competent E. coli JM-101 hsdS recA cells.
• the correct plasmid in which the M-1 lipase gene is placed under the control of the Pf3 promoter was obtained and called pTZPf3M1.
• the M-1 expression cassettes present in the plasmids pTMPv18A, pMCTM1 and pTZPf3M1 were inserted into the broad host range vectors pKT231 (Bagdasarion et al., Gene 16 (1981) 237-247), pLAFR3 (Staskawisz et al., J. Bacteriol. 169 (1987 ( 5789-5794) and pJRD215 (Davison et al., Gene 51 (1987) 275-280).
• the lipases produced by these Pseudomonas transformants were analyzed by SDS gel-electrophoresis and migrated identically to the lipase produced from the original P. pseudoalcaligenes strain M-1.
• Plasmid pMCTM1 (see Figure 18) carrying the M-1 lipase gene and the chloroamphenicol resistance gene (Cm). In this construct the lipase promoter is exchanged by a strong tac promoter. B.3. Plasmid pMCTbliM1 carries also the M-1 lipase gene and the chloroamphenicol resistance gene. In this construct the lipase signal sequence was exchanged by the ⁇ -amylase signal sequence (see EP-A-0224294). The promoter was the same as in construct pMCTM1. B.4. Plasmid pTZ18RN (see figure 17) was used as a negative control.
• DNA (0.5 ⁇ g) of the mentioned plasmids was transcribed in vitro. This reaction was carried out by adding 0.5 ⁇ l of 10XTB/10XNTP mix (a mixture of equal volumes of 20xTB and 20xNTP mix; 20xTB contains 800 mM Tris HCl pH 7.5, 120 mM MgCl 2 and 40 mM spermidine; 20xNTP mix contains 10 mM ATP, 10 mM CTP, 10 mM GTP and 10 mM UTP), 0.5 ⁇ l of 0.1M DTT, 0.5 ⁇ l of RNasin (40 u/ ⁇ l, Promega) and 0.5 ⁇ l of T7 RNA polymerase (15 u/ ⁇ l, Promega) or 1 ⁇ l of E. coli RNA polymerase (1 u/ ⁇ l, Boehringer). The reaction mixture was incubated for 1 h at 39.5°C.
• 10XTB/10XNTP mix a mixture of equal volumes of 20xTB
• RNA transcripts In vitro translation of the RNA transcripts was performed according to the instructions supplied by the manufacturer. M-1 lipase was immunoprecipitated as described by Van Mourik (J. Biol. Chem. 260 (1985) 11300-11306) using monoclonal antibodies against M-1 lipase.
• Example 8 we also demonstrate that this hybridization technique enables us to clone homologous lipase genes from other microorganisms.
• chromosomal DNA from the following strains Pseudomonas pseudoalcaligenes M-1 (CBS 473.85), P. pseudoalcaligenes IN II-5 (CBS 468.85), P. alcaligenes (DSM 50342) P. aeruginosa (PAC 1R (CBS 136.85), P. aeruginosa PAO (ATCC 15692) (Wohlfarth and Winkler, 1988, J. Gen. Microbiol. 134, 433-440). P. stutzeri Vietnamese IV 17-1 (CBS 461.85); P. stutzeri PG-I-3 (CBS 137.89), P.
• Filters were prehybridized in 6xSSC, 5xDenhardt, 0.5% SDS and 100 ⁇ g/ml of denatured calf thymus DNA. After two hours of pre-incubation 32P labeled insert of respectively pTMPv18A or pET3 was added and hybridization was carried out at 55oC during 16 hours.
• Insert DNAs were isolated by digestion of pTMPvlSA with Xhol and EcoRV and digestion of pET3 with EcoRI, followed by separation of the fragments by 0.8% agarose gel electrophoresis and recovering of the fragments by the glass milk procedure (Gene CleanTM).
• the insert of pTMPvlSA, a 1 kb XhoI/EcoRV fragment and the insert of pET3, a 3.2 kb EcoRI fragment were labeled in vitro to high, specific activity (2-5 10 8 cpm/ ⁇ g) with ⁇ 32P ATP by nick translation (Feinberg and Vogelstein, Anal. Biochem. 132, 1983, 6-13). It was shown that probe fragments have an average length varying from 300-800 bases.
• Filters were then washed twice for 30' at 55°C in 6xSSC, 0.1% SDS. Filters were dried at room temperature and autoradiographed for 1-3 days by exposure to KODAK X- omat AR films or Cronex 4 NIF 100 X-ray film (Dupont) with intensifying screens at -70°C (Cronex lightning plus GH 220020).
• Tm 81 + 16,6 (log10 Ci) + 0.4 (% G + C) - 600/n -1.5% mismatch (Current protocols in molecular biology 1987-1988, edited by Ausubel et al.)
• n length of the shortest chain of the probe
• Ci ionic strength (M)
• G+C base composition was used to determine the homology which could be detected in our experiments. Assuming a probe length of 300 bases, we were able to detect a homologous gene which shows at least 67% homology within a fragment of 300 bases or more.
• Figure 22A shows the hybridization pattern of the SalI digested chromosomal DNAs, when hybridized with the EcoRI insert of pET3. It can be seen that most of the Pseudomonas strains, contain homologous genes to our pET3 clone. In P. fragi DB 1051 (lane M), A. calcoaceticus Gr-V-39 (lane 0) and S. aureus (lane P) no homologous genes were detected.
• Figure 22B shows the hybridization pattern with the EcoRV/XhoI insert of pTMPv18A. It can be seen that strong hybridization signals were obtained from chromosomal DNAs of P. pseudoalcaligenes, (lane C and D), P. alcaligenes (lane E) and P. stutzeri strains (lane H to lane L). Weaker hybridization was seen in chromosomal DNAs of P. aeruginosa (lane F and G) and no hybridization at all was found with chromosomal DNAs of P. fragi DB1051 (lane M), P. gladioli (lane N), A. calcoaceticus Gr-V-39 (lane O) and S. aureus (lane P).
• a SalI gene library was constructed with the aid of vector pUC19 and transformed into E. coli JM109 (Yanish-Perron et al., Gene 33 (1985) 103-119). Replica filters of the gene library were hybridized with the ⁇ 32P-labeled insert of pTMPv18A using conditions as described in Example 7. Three positive clones, which all carried a 5.0 kb SalI fragment, were isolated from the library: pCH1, pCH2 and pCH3.
• the 5.0 kb SalI fragment of pCH1 was recloned into vector pKT248 (Bagdasarian et al., Gene 16 (1981) 237-247) with the aid of the SalI site, which is located in the chloramphenicol-resistance gene. Streptomycin-resistant chloramphenicol-sensitive transformants were selected in E. coli JM109. One of these transformants, pCH101, which contained the expected 5.0 kbb SalI insert was transformed into Pseudomonas aeruginosa 2302 (6-1) lip- (see Example 5).
• Colonies were grown on NB-calcium triolein agar. After growth, plates were stored at 10oC. After several days a calcium precipitate was visible around the transformed colonies and not around non-transformed P. aeruginosa 2302 (6-1) lip-, which was grown on similar agar plates without antibiotics.
• the cloned 5.0 kb SalI fragment complements the lip- phenotype of P. aeruginosa 2302 (6-1) lip- and therefore encodes an extracellular lipase, which can be produced in a suitable host.
• this lipase shows homology to M-1 lipase and consequently to the other lipases, hybridizing with the pTMPv18A insert, used as a probe.
• the description of the cloning of P. alcaligenes lipase represents a generally applicable method to clone lipase genes that show homology to M-1 lipase.
• M-1 lipase was also compared to other lipases. Overall homology between M-1 lipase and P. aeruginosa PAOl lipase was found to be (78%) and between M-1 lipase and P. fragi lipase to be 48%. Again no homology was found between the amino acid sequence of M-1 lipase and Staphylococcus hyicus lipase. However, close homology does exist between the four lipases in the region of the essential serine 87 of mature M-1 lipase. Kugimiya et al., (supra), postulated that the sequence of this region G-H-S-H-G is the active center of lipase enzymes.
• This example illustrates the performance of the lipase enzymes produced by P. aerugiuosa. P. stutzeri and P. alcaligenes strains which show a high degree of DNA sequence homology with the M-1 gene, in a washing process according to the modified SLM-test where the compatibility of these enzymes in modern laundry cleaning composition was tested.
• the cleaning compositions used were a powder detergent composition (ALL R -base, described in EP-A-0218272) and a liquid detergent formulation (Liquid TIDE R , also described in EP-A-0218272, but without inactivation of the protease).
• the lipase enzymes were prepared by culturing the bacteria according to the following procedure: an inoculum culture was prepared by culturing the bacteria in 100 ml Brain Heart Infusion (BHI) medium in a rotary shaker at 30o-C, for 24 hours. A lab-fermentor (2 litres) containing 1.0 litre of a medium with the composition mentioned below was then inoculated.
• the fermentation was run at 30°C. Sixteen hours after inoculation a feed of soya oil was started at the rate of 1 g/h and continued for the rest of the fermentation. The aeration rate was 60 1/h and the agitation rate 700 rpm. The fermentation was run for a total of 64 hours.
• the detergents tested were a powder detergent composition (ALL R base) and a liquid formulation (TIDE R liquid). The concentration of ALL base washing solution was 4 g/l (pH 9.5).
• the concentration of liquid TIDE was 1.5 g/l (pH 7.5).
• the solutions were buffered with 0.1 M Tris/HCl.
• the washing process was started by adding the enzyme preparation and immediately thereafter the soiled swatch, to the Erlenmeyer flask and shaking for 40 min or longer at 40oC.
• the final lipase concentration was 2 TLU/ml.
• the swatch was rinsed with SHW then dried at 55°C for one hour.
• the dried swatches as such were washed again in a second wash cycle using a fresh detergent and enzyme solution for 40 minutes.
• the swatch was treated with 0.01N HCl for 5 min, rinsed and dried at room temperature overnight.
• the dried swatches were extracted by rotation in a glass tube containing a 5 ml of solvent (n-hexane/isopropylalcohol/ formic acid: 975:25:2.5 (v/v), 1 ml/min).
• the residual triglyceride, diglyceride and the free fatty acid formed were determined by HPLC.
• Integrator SP 4270 (Spectra Physis)
• triolein The retention time of triolein was 1.2 min., that of 1,3 diglycerides was 2.5 min., that of 1,2 diglycerides was 3.6 min. and that of oleic acid was 1.6 min.
• the peak area or peak height was determined as an indication of the recovery of the triglyceride and fatty acid.
• the recovery of triglyceride after extraction from the unwashed swatch was taken as 100%.
• the ratio of the refractive index responses between triolein and oleic acid was found to be 1.0 on the basis of peak height.
• the results of these SLM-tests are shown in the following tables. In these tables the triglycerides recovery and the total lipids recovery are shown.
• Total lipids recovery is triglycerides plus 1,2- and 1,3-diglycerides plus free fatty acids.
• the difference between total lipids recovery and triglycerides recovery is a measure of lipase activity and performance.
• the difference between the total lipid recovery and the control (without lipase enzyme) indicates the removal of oily stain from the fabric and demonstrates that these enzymes are stable and effective under realistic washing conditions, as simulated in the SLM-test.

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