EP0260299A1 - Procede de mutagenese et de criblage et produit obtenu - Google Patents

Procede de mutagenese et de criblage et produit obtenu

Info

Publication number
EP0260299A1
EP0260299A1 EP87901901A EP87901901A EP0260299A1 EP 0260299 A1 EP0260299 A1 EP 0260299A1 EP 87901901 A EP87901901 A EP 87901901A EP 87901901 A EP87901901 A EP 87901901A EP 0260299 A1 EP0260299 A1 EP 0260299A1
Authority
EP
European Patent Office
Prior art keywords
subtilisin
amino acid
mutant
substituted
acid position
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
EP87901901A
Other languages
German (de)
English (en)
Other versions
EP0260299A4 (fr
Inventor
Philip N. Bryan
Michele L. Rollence
Michael W. Pantoliano
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Enzon Labs Inc
Original Assignee
Genex Corp
Enzon Labs Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Genex Corp, Enzon Labs Inc filed Critical Genex Corp
Publication of EP0260299A1 publication Critical patent/EP0260299A1/fr
Publication of EP0260299A4 publication Critical patent/EP0260299A4/fr
Ceased legal-status Critical Current

Links

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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/102—Mutagenizing nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
    • C11D3/16—Organic compounds
    • C11D3/38—Products with no well-defined composition, e.g. natural products
    • C11D3/386—Preparations containing enzymes, e.g. protease or amylase
    • 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)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
    • C12N9/54—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea bacteria being Bacillus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids

Definitions

  • the present invention relates to a method for mutating genetic material and screening mutant genetic material for a desired mutation.
  • the invention further relates to mutant genetic material produced according to the disclosed method, as well as to expression products of such mutant genetic material.
  • the largest class of naturally occurring proteins is made up of enzymes. Each enzyme generally catalyzes a different kind of chemical reaction, and is usually highly specific in its function. An enzyme molecule contains an active site to which a specific substrate is bound during a catalytic cycle. Although there may be slight variations in a distinct type of naturally occurring enzyme within a given species of organism, enzyme molecules of a specific type produced by organisms of the same species generally are substantially identical with respect to substrate specificity, thermal stability, activity levels under various conditions (e.g., temperature and pH), oxidation stability, and the like. Such characteristics of a naturally occurring or "wild-type" enzyme are not necessarily optimized for utilization outside of the natural environment of the enzyme. It may thus be desirable to alter a natural characteristic of an enzyme to optimize a certain property of the enzyme for a specific use, or for use in a specific environment.
  • the amino acid sequence of an enzyme determines the characteristics of the enzyme, and the enzyme's amino acid sequence is specified by the nucleotide sequence of a gene coding for the enzyme.
  • a change of the amino acid sequence of an enzyme may alter the enzyme's properties to varying degrees, or may even inactivate the enzyme, depending on the location, nature and/or magnitude of the change in the amino acid sequence.
  • Naturally occurring bacterial proteases are currently used for many purposes, among these is as an additive to washing preparations. Many stains on clothes are proteinaceous and wide-specificity proteases can substantially improve removal of such stains. Unfortunately, naturally-occurring proteases lose activity when stored in solution with detergents. Typically this decay of activity is geometric in nature, that is, a certain percentage of activity is lost in each time interval.
  • the present invention provides a method to develop novel proteases with enhanced thermal stability and which survive prolonged storage in liquid detergents much longer than naturally-occurring proteases.
  • a method for mutating cloned DNA and thereafter identifying mutants comprises creating a single-stranded target region in a cloned DNA segment, and introducing a mutation into the target region by treating the target region with a chemical mutagenizing agent capable of introducing mutations into single-stranded DNA.
  • the target region then is rendered double-stranded to form mutated double-stranded DNA, and a microorganism is transformed with an expression vector containing the mutated double-stranded DNA.
  • the transformed microorganism is cultivated under conditions wherein the mutated DNA is expressed to form an expression product, and the expression product is screened to identify a desired mutation in the DNA segment.
  • FIG. 1 is a schematic diagram showing the subtilisin gene cloned in plasmid pGX4330 for mutagenesis and screening according to the invention.
  • FIG. 2 is a schematic diagram showing a gapped duplex DNA molecule for mutagenesis according to the invention.
  • FIG. 3 is a portion of the variant subtilisin DNA sequence showing the single base substitution in the subtilisin gene which produces subtilisin of enhanced thermal stability according to the invention.
  • FIG. 4 is a graphic illustration showing thermal inactivation of variant subtilisin according to the invention (7150) and subtilisin wild-type at 65°C in 10 mM CaCl 2 , 50 mM KCl, 50 mM Tris-HCl, pH 8.0.
  • FIG. 5 is a graphic illustration showing thermal inactivation of variant subtilisin according to the invention (7150) and subtilisin wild-type at 45°C in 1.0 mM EDTA, 50 mM KC1, 50 mM Tris-HCl, pH 8.0.
  • FIG. 6 is a graphic illustration showing thermal inactivation of variant subtilisin according to the invention (7150) and subtilisin wild- type at 40°C, in 1.0 mM EDTA, 20 mM 3-(cyclohexylamino) propanesulphonic acid (CAPS), pH 10.5.
  • FIG. 7 is a graphic illustration showing differential scanning calorimetry (DSC) profiles for variant subtilisin according to the invention (7150) and subtilisin wild-type at a protein concentration of 3.0 mg/ml (scan rate 60°C/hr.). Samples were scanned in 50 mM Tris-HCl, pH 8.0, 50 mM KCl with either 10mM EDTA or 10mM CaCl 2 .
  • DSC differential scanning calorimetry
  • FIG. 8 is a graphic illustration showing thermal stability of variant subtilisins according to the invention and subtilisin wild-type at 70°C in 10 mM CaCl 2 , 50 mM Trie- HCl, 50 mM NaCl at pH 8.0.
  • the gene Prior to mutation of a gene coding for an enzyme of interest, the gene generally is first isolated from its natural source and cloned in a cloning vector.
  • mRNA which is transcribed from the gene of interest can be isolated from the source cell and converted into cDNA by reverse transcription for insertion into a cloning vector.
  • a cloning vector can be a phage or plasmid, and generally includes a replicon for autonomous replication of the vector in a microorganism independent of the genome of the microorganism.
  • a cloning vector advantageously includes one or more phenotypic markers, such as DNA coding for antibiotic resistance, to aid in selection of microorganisms transformed by the vector.
  • Procedures for insertion of DNA or cDNA into a vector for cloning purposes are well known in the art. These procedures generally include insertion of the gene of interest into an opened restriction endonuclease site in the vector, and may involve addition of homopolymeric tails of deoxynucleotides to the ends of the gene and linking the gene to opened ends of a cloning vector having complementary homopolymeric tails.
  • a gene of interest present in a cloning vector can be mutated in accordance with the method of this invention.
  • a gene of interest to be treated according to the invention is present in an expression vector.
  • An expression vector generally falls under the definition of a cloning vector since an expression vector usually includes the components of a typical cloning vector, namely, one or more replicons as defined above, and one or more phenotypic markers for selection purposes. Additionally, an expression vector includes control sequences encoding a promoter, operator, ribosome binding site and translation initiation signal. For expression under the direction of the control sequences, a target gene to be treated according to the invention is operably linked with the control sequences in the proper reading frame.
  • An expression vector containing the DNA sequence to be targeted can be a phage or a plasmid, with plasmids being preferred.
  • a method for mutating a cloned DNA segment and thereafter identifying mutants includes the step of providing a single-stranded target region in the cloned DNA segment.
  • the target region can include the entire DNA segment of interest, or a portion thereof.
  • the targeted portion can be randomly or specifically selected.
  • the target region of the DNA segment of interest is rendered single-stranded by any suitable method known in the art, such as the method disclosed in shortle et al., Proc. Natl. Acad. Sci. USA, 75:2170-2174 (1978).
  • Another method involves providing a single-stranded copy of a plasmid containing the DNA segment of interest, and annealing to the single-stranded copy a DNA fragment containing plasmid sequences but not including a segment complementary to the target region. This procedure creates a gapped duplex molecule with a single-stranded target region.
  • One or more mutations are introduced into the single-stranded target region by treating the target region with a chemical or biological mutagenizing agent (mutagen) capable of introducing mutations into single-stranded DNA.
  • a chemical or biological mutagenizing agent capable of introducing mutations into single-stranded DNA.
  • One such chemical mutagen is sodium bisulfite which causes G-C to A-T transitions only (wherein G, C, A or T refer respectively to guanine, cytosine, adenine and thymine).
  • Other chemical or biological mutagens, such as hydroxylamine, nitrous acid. formic acid and hydrazine are suitable and can be used according to this invention.
  • Mutations are introduced at a controllable level, advantageously from one to about five changes per molecule, and a library of mutated plasmid-borne DNA is produced (e.g., by incubating DNA in 4M Na-bisulfite, pH 6.0 for 5 to 30 minutes).
  • the library is large enough to generate up to 10 5 -10 6 different variants upon transformation into a microorganism, since a desired mutation may occur infrequently.
  • mutations are introduced into the cloned DNA by replication of the cloned DNA in a mutator strain of E. coli.
  • a mutator strain of E. coli such as a Mut D strain of E. coli which produces a range of mutations due to its error prone DNA polymerase.
  • Mut D E. coli containing plasmid are grown at 37°C in 250 ml culture in rich media containing 50 mg/ml thymine to an optical density at 650 nm of 0.6.
  • the plasmid is amplified by the addition of 175 ug/ml chloramphenicol and continued incubation for 16 hours at 37°C.
  • the target region is rendered double-stranded by filling in the single-stranded region using DNA polymerase I (Klenow fragment) or reverse transcriptase, see, e.g., Shortle et al., supra.
  • DNA polymerase I Klenow fragment
  • reverse transcriptase see, e.g., Shortle et al., supra.
  • a microorganism is transformed with the mutated double-stranded DNA present in an expression vector, the mutated DNA being operably linked to control sequences capable of directing expression of the mutated DNA in the transformed microorganism.
  • the transformed microorganism then is cultivated under protein-producing conditions including necessary nutrients and physiologically acceptable pH and temperature, such that the mutated DNA is expressed to form an expression product.
  • the method according to this invention can be used to mutate serine proteases to enhance certain characteristics, particularly thermal stability.
  • a protease is a catalyst for the cleavage of peptide bonds.
  • a serine protease is an enzyme which catalyzes the hydrolysis of peptide bonds in which there is an essential serine residue at the active site. Serine proteases can be inhibited by phenylmethanesulfonylfluoride and by diisopropylfluorophosphate.
  • a subtilisin is a serine protease produced by Gram positive bacteria or fungi. The amino acid sequences of seven subtilisins are known. These include five subtilisins from Bacillus strains (subtilisin BPN', subtilisin Carlsberg, subtilisin DY, subtilisin amylosacchariticus, and mesenticopeptidase).
  • subtilisin thermitase from Thermoactinomyces vulgaris is also known. (Meloun et al., "Complete primary structure of thermitase from thermoactinomyces vulgaris and its structural features related to the subtilisin-type proteanases, " FEBS Lett. 183:195-200 (1985) )
  • the amino acid sequences from two fungal proteases are known: proteinase K from Tritirachium album (Jany et al., "Proteinase K from Tritirachium albam Limber,” Biol. Chem. Hoppe-Seyler 366:485-492 (1985)) and thermomycolase from the thermophilic fungus, Malbranchea pulchella (Gaucher et al., "Endopeptidases: Thermomycolin,” Methods Enzvmol. 45:415-433 (1976)).
  • subtilisin material is a proteinaceous material which contains a subtilisin as its active ingredient.
  • any serine protease is a subtilisin which has at least 30%, preferaby 50%, and more preferably 80% amino acid sequence homology with the sequences referenced above for subtilisin BPN', subtilisin Carlsberg, subtilisin DY, subtilisin amylosacchariticus, mesenticopeptidase, thermitase, proteinase K, and thermomycolase.
  • subtilisin material is a proteinaceous material which contains a subtilisin as its active ingredient.
  • any serine protease is a subtilisin which has at least 30%, preferaby 50%, and more preferably 80% amino acid sequence homology with the sequences referenced above for subtilisin BPN', subtilisin Carlsberg, subtilisin DY, subtilisin amylosacchariticus, mesenticopeptidase, thermitase, proteinase K, and thermomycolas
  • B. subtilis is transformed by an expression vector carrying the mutated DNA. If expression is to take place in a secreting microorganism such as B. subtilis, a single sequence may follow the translation initiation signal and precede the DNA sequence of interest. The signal sequence acts to transport the expression product to the cell wall where it is cleaved from the product upon secretion.
  • control sequences as defined above is intended to include a signal sequence, when it is present.
  • transformed B. subtilis is cultivated in the presence of a filter material (such as nitrocellulose) to which the secreted expression product (e.g., enzyme) binds.
  • a filter material such as nitrocellulose
  • the secreted expression product e.g., enzyme
  • filter bound expression product is subjected to conditions which distinguish expression product of interest from wild-type expression product.
  • the filter-bound expression product can be subjected to conditions which would inactivate a wild-type expression product.
  • the treated expression product can thereafter be contacted with a substrate of the enzyme, and enzyme activity with the substrate identifies an expression product with enhanced stability and thereby a desired mutation.
  • homologous serine proteases from other microorganisms may be mutated and screened according to this invention.
  • These homologous serine proteases may include, but are not limited to, those from other Bacillus strains such as subtilisin Carlsberg from Bacillus licheniformis. subtilisin DY, subtilisin amylosachariticus, and mesentericopeptidase.
  • Fungal proteases, such as protease K and thermomycolase may also be used, as well as mammalian proteases produced in a bacterial host.
  • subtilisin gene from a Bacillus species including the natural promoter and other control sequences is cloned into a plasmid vector containing replicons for both E. coli and B. subtilis. a selectable phenotypic marker, and the M13 origin of replication for production of single-stranded plasmid DNA upon superinfe ⁇ tion with helper phage IR1.
  • Single-stranded plasmid DNA containing the cloned subtilisin gene is isolated and annealed with a DNA fragment containing vector sequences but not the coding region of subtilisin, to create, a gapped duplex molecule.
  • the mutagens, nitrous acid and formic acid may also be used to produce mutant libraries. Because these chemicals are not as specific for single-stranded DNA as sodium bisulfite, the mutagenesis reactions are performed according to the following procedure. The coding portion of the subtilisin gene is cloned into M13 phage by standard methods and single-stranded phage DNA prepared. The single-stranded DNA is then reacted with 1M nitrous acid pH 4.3 for 15-60 minutes at 23°C or 2.4M formic acid for 1-5 minutes at 23°C. These ranges of reaction times produce a mutation frequency of from 1 in 1000 to 5 in 1000.
  • a universal primer is annealed to the M13 DNA and duplex DNA is synthesized using the mutagenized single-stranded DNA as a template so that the coding portion of the subtilisin gene becomes fully double-stranded.
  • the coding region can be cut out of the M13 vector with restriction enzymes and ligated into an unmutagenized expression vector so that mutations occur only in the restriction fragment.
  • the variant library is used to transform B. subtilis which will both express and secrete subtilisin.
  • the subtilisin-bearing plasmid advantageously contains a high copy B. subtilis replicon and is capable of producing subtilisin at a high level in a Bacillus host.
  • a protease deficient B. subtilis strain is transformed with the variant plasmid library and plated out as follows: A nitrocellulose filter is placed on a nutrient base in a petri dish, and a cellulose acetate filter is placed on top of the nitrocellulose.
  • Colonies are grown on the cellulose acetate, and subtilisin from individual colonies is secreted through the cellulose acetate onto the nitrocellulose filter where it is stably bound. Subtilisin from hundreds of colonies is bound to a single filter allowing subsequent screening of thousands of different variants by processing multiple filters.
  • the. filters are incubated at 70°C for 30 minutes in buffer solution to inactivate substantially all wild-type subtilisin activity.
  • Variant subtilisins of enhanced stability retain activity after this heating step.
  • higher temperatures of longer incubation times must be used to inactivate the background activity.
  • the heat-treated filter then is soaked in a solution containing Tosyl-L-Arg methyl ester (TAME) (Sigma) and the pH indicator phenol red (Kodak). Because TAME is a substrate for subtilisin it is cleaved in zones on the filter containing variant subtilisins which remain active after thermal treatment. As cleavage occurs, protons are released in the reaction and cause phenol red to change in color from red to yellow in areas retaining protease activity.
  • the filters could be treated at high pH, with denaturants, oxidizing agents, or under other conditions which normally inactivate an enzyme such as subtilisin, to find resistant variants.
  • Variants with altered substrate specificity could be screened by replacing TAME with other substrates which are normally not cleaved by wild-type subtilisin.
  • the gene coding for a subtilisin material contains serine or aspartic acid at amino acid position 218 and the subtilisin gene may also contain one or more additional amino acid substitions.
  • additional amino acid svibstitutions in the cloned mutant 218-substituted subtilisin gene are the following random mutated subtilisin variants produced by the indicated strains:
  • genes for other serine proteases or other types of enzymes may be mutagenized to produce products with enhanced characteristics.
  • subtilisin BPN' for example, to improve other proteases which are closely related, subtilisin Carlsberg for example. Closeness of relation is measured by comparison of amino-acid sequences. There are many methods of aligning protein sequences, but the differences are only manifest when the degree of relatedness is quite small. The methods described in Atlas of Protein Sequence and Structure. Margaret 0. Dayhoff editor, Vol. 5 Supplement 2, 1976, National Biomedical Research Foundation, Georgetown University Medical Center, Washington, DC, p. 3 ff., entitled SEARCH and ALIGN, define relatedness.
  • subtilisin Carlsberg has only 274 amino acids, while subtilisin BPN' has 275 amino acids. Aligning the two sequences shows that Carlsberg has no residue corresponding to ASN56 of subtilisin BPN'. Thus the amino acid sequence of Carlsberg would appear very different from BPN' unless a gap is recorded at location 56. Therefore, one can predict with high degree of confidence that substituting SER for ASN at location 218 of subtilisin Carlsberg will increase thermal stability, provided that the residues in Carlsberg are numbered by homology to BPN'.
  • subtilisins When one of the two homologous subtilisins has a gap, one must infer that the structures are different at that locality. Examples of such differences are well known in the art. Because of these local differences, one should not transfer stabilizing mutations if either subtilisin has a gap at, or immediately adjacent, to the site of the mutation. Therefore, after aligning the amino acid sequences, those mutations at or next to gaps are deleted from the list of desirable mutations and the mutation is not made.
  • the stabilizing mutations found by the random mutagenesis method reveal that the structure of the enzyme being studied is not optimal at that location.
  • the random method usually changes very few bases within the gene. Usually only one base is changed within a given codon; thus, one cannot go from any one amino acid to all other amino acids.
  • oligonucleotide-directed mutagenesis can be used to introduce each of the remaining 18 amino acids at that site. These mutants can then be tested for improved properties.
  • the mutant subtilisin material of this invention can be used as an additive to washing preparations, such as detergents, which are used for cleaning, in particular for cleaning clothes.
  • the mutant subtilisin material of this invention is more thermally stable than wild-type subtilisin material and thus does not lose activity as rapidly as wild-type when stored in solution with detergents or when subjected to high heat during use in cleaning.
  • By use of the mutant subtilisin material of this invention as an additive in washing preparations the removal of proteinaceous stains on fabric is improved.
  • the amount of mutant subtilisin material that may be used as an additive to washing preparations are well known in the art, or may readily be ascertained by routine experimentation. The optimal range of enzyme concentration will, of course, be related to the cost of the enzyme and the amount of cleaning needed.
  • the invention is illu ⁇ trated by the following examples which are not intended to be limiting.
  • subtilisin gene from Bacillus amyloliquefaciens including the natural promoter sequences was isolated (Vasantha et al. (1984) J. Bacteriology 159:811-819) and cloned into a vector (pGX4330, Figure 1) containing the beta-lactamase gene and replicon from pBR322 for growth in E. coli; the kanamycin nucleotidyl transferase gene and replicon from pUBHO for growth in B. subtilis; and the M13 origin of replication for production of single-stranded plasmid DNA upon superinfection with helper phage IR1.
  • One ug of single-stranded pGX4330 was mixed with 1 ug of double-stranded pGX4330 cut with BamHI and Sall in 50 mM NaCl, 50 mM Tris-HCl pH 8.0, 10 mM MgCl 2 in a volume of 15 ul.
  • the DNA was heated to 90°C in a boiling water bath for 5 minutes and allowed to anneal at 60°C for 10 minutes.
  • the gapped duplex was reacted with sodium bisulfite pH 6.0 in a volume of 400 ul at 37°C. Reaction time was varied from four to twenty minutes to produce an average of one to five mutations per gene.
  • subtilisin In the presence of calcium, which greatly stabilizes subtilisin, samples were incubated at 65°C and at time intervals up to 90 minutes, aliquots were removed and activity was measured at 37°C using Azocoll as a substrate. In the presence of EDTA timepoints were taken after incubation at 45°C. Variants whose half-time of thermal inactivations was greater than 150% of wild-type subtilisin were of interest and further characterized. To confirm that stable subtilisin phenotypes resulted from plasmid-borne mutations, plasmid from positive colonies was purified and used to retransform B. subtilis. In almost all cases, the retransformed B. subtilis behave as the original isolate.
  • Subtilisin activity was assayed by monitoring the hydrolysis of 1.0 mM solutions of the substrate, succinyl(L)-Ala-(L)-Ala-(L)-Pro-(L)-Phe-p-nitroanilide (SAAPF-pNA (Calbiochem)), in 50 mM Tris-HCl (pH 8.0), 50 mM KCl at 25.0°C.
  • SAAPF-pNA succinyl(L)-Ala-(L)-Ala-(L)-Pro-(L)-Phe-p-nitroanilide
  • Differential scanning calorimetry data was obtained with a Hart Scientific instrument interfaced with an IBM personal computer (model XT) and controlled with DSC Software (Hart Scientific) and the Xenix operating system (Microsoft-Santa Cruz Operations). The temperature was increased from the starting point of 20°C to 90°C at a rate of 60°C/hr. The protein concentration was 3.0 mg/ml.
  • subtilisin in thin layers of polyacrylamide was performed using known techniques, see, e.g. Winter et al. (1977) LKB Application Note 250. Ready-made acrylamide gels, Ampholine PAG plates (pH 3.0 - 9.5) were purchased from LKB, and the calibration standard ⁇ (pH 5.0 - 10.5) were purchased from Pharmacia. The subtilisin samples were inactivated with phenylmethylsulfonylfluoride (PMSF) before. loading onto the gels in order to prevent autolysis during electrofocusing.
  • PMSF phenylmethylsulfonylfluoride
  • subtilisin 7150 was found to have an isoelectric point of 8.8, essentially indistinguishable from that of the wild-type enzyme. Only a minor contaminant with an isoelectric point of 8.3 was detected. This level of homogeneity is comparable to that of the wild type.
  • the specific activity of subtilisin 7150 towards a peptide substrate was observed to be 120 ⁇ 5 Units/mg. This is 50% higher than that observed for the wild type: 80 ⁇ 5 Units/mg, an average for more than two dozen separate isolates. This difference is due to small changes in the kinetic parameters for hydrolysis of the substrate rather than a more highly purified preparation.
  • subtilisin 7150 was further demonstrated for a variety of other conditions.
  • thermodynamic parameters for the unfolding reaction of subtilisin were obtained through the u ⁇ e of differential scanning calorimetry.
  • the results obtained for subtilisin 7150 and the wild-type enzyme are shown in Figure 7.
  • the unfolding transition for subtilisin 7150 was found to occur at 80.7 ⁇ 0.1°C in 50 mM Tris-HCl pH 8.0, 50 mM KCl, and 10 mM CaCl 2 some 2.4° higher than that observed for wild-type under identical conditions.
  • the denaturation temperature of subtilisin 7150 was 62.8°C, about 4°C higher than wild-type subtilisin.
  • the enzymes were incubated at a concentration of 4,000 ADU/gm in the non-phosphate based, U.S. heavy duty liquid detergent Wisk ® (a registered trademark of Lever Bros. Co., Inc., N.Y., N.Y.) adjusted to pH 10.0.
  • the solutions were incubated at 25°C for 56 days under these conditions.
  • a mutant designated GX8315 was constructed using three single-point mutations. The three point mutations were all found by screening randomly mutagenized subtilisin as described above in Example II. The three mutations were Asn218---
  • Mutant GX8315 (Asn218--->Ser, Gly131--->Asp, and Thr254 --->Ala) loses half its activity every
  • a mutant designated GX7164 was made by oligonucleotide-directed mutagenesis with aspartic acid at location 218. This was done because substitution of serine for asparagine at location 218 had been shown to stabilize subtilisin BPN'.
  • Subtilisin 7164 has a half-life for thermal inactivation which is 1.9 times as large as wild-type subtilisin.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Immunology (AREA)
  • Medicinal Chemistry (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Plant Pathology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

On provoque la mutation d'un ADN cloné en créant une région-cible monobrin dans un segment d'ADN cloné, et en introduisant une mutation dans la région-cible monobrin en traitant cette région-cible avec un agent mutagène chimique ou biologique pouvant introduire des mutations dans un ADN monobrin. La région cible mutée est ensuite transformée en double brin et un micro-organisme est transformé avec l'ADN muté à double brin présent dans un vecteur d'expression. Le micro-organisme transformé est cultivé dans des conditions où l'ADN muté est exprimé pour former un produit d'expression, et le produit d'expression est criblé pour identifier la mutation désirée dans le segment d'ADN. Sont décrites des subtilisines mutantes présentant une stabilité thermique améliorée.
EP19870901901 1986-02-12 1987-02-12 Procede de mutagenese et de criblage et produit obtenu. Ceased EP0260299A4 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US82854586A 1986-02-12 1986-02-12
US828545 1986-02-12

Publications (2)

Publication Number Publication Date
EP0260299A1 true EP0260299A1 (fr) 1988-03-23
EP0260299A4 EP0260299A4 (fr) 1988-11-24

Family

ID=25252124

Family Applications (1)

Application Number Title Priority Date Filing Date
EP19870901901 Ceased EP0260299A4 (fr) 1986-02-12 1987-02-12 Procede de mutagenese et de criblage et produit obtenu.

Country Status (4)

Country Link
EP (1) EP0260299A4 (fr)
JP (1) JPS63502959A (fr)
DK (1) DK531887D0 (fr)
WO (1) WO1987005050A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6403692B1 (en) 2001-04-19 2002-06-11 Dow Global Technologies Inc. Filled thermoplastic composition

Families Citing this family (61)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4990452A (en) * 1986-02-12 1991-02-05 Genex Corporation Combining mutations for stabilization of subtilisin
DE3750916T2 (de) * 1986-04-30 1995-06-01 Genencor Int Mutante einer nicht menschlichen Carbonyl-Hydrolase, für diese kodierende DNS-Sequenzen und Vektoren und durch diese Vektoren transformierte Wirte.
NZ221627A (en) * 1986-09-09 1993-04-28 Genencor Inc Preparation of enzymes, modifications, catalytic triads to alter ratios or transesterification/hydrolysis ratios
ATE371029T1 (de) * 1987-04-06 2007-09-15 Novozymes As Regelung von elektrostatischen interaktionen an metallionen-bindungsstellen zur stabilisierung von proteinen
US4914031A (en) * 1987-04-10 1990-04-03 Amgen, Inc. Subtilisin analogs
DK6488D0 (da) * 1988-01-07 1988-01-07 Novo Industri As Enzymer
US5324653A (en) * 1988-02-11 1994-06-28 Gist-Brocades N.V. Recombinant genetic means for the production of serine protease muteins
US6287841B1 (en) 1988-02-11 2001-09-11 Genencor International, Inc. High alkaline serine protease
CN1056187C (zh) 1988-02-11 2000-09-06 金克克国际有限公司 新的蛋白水解酶及其在洗涤剂中的应用
US5116741A (en) * 1988-04-12 1992-05-26 Genex Corporation Biosynthetic uses of thermostable proteases
DK316989D0 (da) * 1989-06-26 1989-06-26 Novo Nordisk As Enzymer
US5665587A (en) * 1989-06-26 1997-09-09 Novo Nordisk A/S Modified subtilisins and detergent compositions containing same
US5658871A (en) * 1989-07-07 1997-08-19 Lever Brothers Company, Division Of Conopco, Inc. Microbial lipase muteins and detergent compositions comprising same
US6271012B1 (en) * 1989-10-11 2001-08-07 Genencor International, Inc. Protease muteins and their use in detergents
EP0471410A3 (en) * 1990-08-15 1992-07-01 Unilever Nv Structured liquid detergent compositions containing subtilisin mutants
US5482849A (en) * 1990-12-21 1996-01-09 Novo Nordisk A/S Subtilisin mutants
ES2090481T3 (es) * 1991-05-01 1996-10-16 Unilever Nv Composiciones detergentes que contienen enzimas estabilizadas.
US5858757A (en) * 1991-05-01 1999-01-12 Novo Nordisk A/S Stabilized enzymes and detergent compositions
US5340735A (en) * 1991-05-29 1994-08-23 Cognis, Inc. Bacillus lentus alkaline protease variants with increased stability
GB9114734D0 (en) * 1991-07-09 1991-08-28 Univ London Process for modifying proteins
US6440717B1 (en) 1993-09-15 2002-08-27 The Procter & Gamble Company BPN′ variants having decreased adsorption and increased hydrolysis
US6436690B1 (en) * 1993-09-15 2002-08-20 The Procter & Gamble Company BPN′ variants having decreased adsorption and increased hydrolysis wherein one or more loop regions are substituted
US6599730B1 (en) 1994-05-02 2003-07-29 Procter & Gamble Company Subtilisin 309 variants having decreased adsorption and increased hydrolysis
US6455295B1 (en) 1995-03-08 2002-09-24 The Procter & Gamble Company Subtilisin Carlsberg variants having decreased adsorption and increased hydrolysis
IL117350A0 (en) 1995-03-09 1996-07-23 Procter & Gamble Proteinase k variants having decreased adsorption and increased hydrolysis
US6475765B1 (en) 1995-03-09 2002-11-05 Procter & Gamble Company Subtilisin DY variants having decreased adsorption and increased hydrolysis
US7018793B1 (en) * 1995-12-07 2006-03-28 Diversa Corporation Combinatorial screening of mixed populations of organisms
WO1998020116A1 (fr) * 1996-11-04 1998-05-14 Novo Nordisk A/S Variants de subtilase et compositions
AU4772697A (en) 1996-11-04 1998-05-29 Novo Nordisk A/S Subtilase variants and compositions
CN100593036C (zh) 1997-08-29 2010-03-03 诺沃奇梅兹有限公司 蛋白酶变体及组合物
WO1999011769A1 (fr) 1997-08-29 1999-03-11 Novo Nordisk A/S Variants de la protease et compositions
DE19743683A1 (de) * 1997-10-02 1999-04-08 Basf Ag Verfahren zur Änderung der Substratspezifität von Enzymen
US6773907B2 (en) 1997-11-21 2004-08-10 Peter Kamp Hansen Subtilase enzymes
DE69830743T2 (de) 1997-11-21 2006-04-27 Novozymes A/S Protease-varianten und zusammensetzungen
US6780629B2 (en) 1997-11-21 2004-08-24 Novozymes A/S Subtilase enzymes
US6908757B1 (en) 1998-03-26 2005-06-21 The Procter & Gamble Company Serine protease variants having amino acid deletions and substitutions
EP1082442A1 (fr) 1998-03-26 2001-03-14 The Procter & Gamble Company Variants de serine-proteases ayant des substitutions d'acides amines
EP1803817B1 (fr) 1998-12-18 2011-04-06 Novozymes A/S Enzymes substitulases des sous-groupes I-S1 et I-S2 ayant un résidu d'acide amine additionnel dans une région boucle de site actif
EP1183340B1 (fr) 1999-05-20 2008-09-03 Novozymes A/S Enzymes subtilases des sous-groupes i-s1 et i-s2 comprenant au moins un reste d'acide amine additionnel entre les positions 126 et 127
ATE408679T1 (de) 1999-05-20 2008-10-15 Novozymes As Subtilase enzyme der i-s1 und i-s2 untergruppen mit mindestens einer zusätzlichen aminosäure zwischen position 127 und 128
EP1183343B2 (fr) 1999-05-20 2013-11-27 Novozymes A/S Enzymes de subtilase des sous-groupes i-s1 et i-s2 possedant au moins un residu supplementaire d'acide amine entre les positions 125 et 126
AU4393000A (en) 1999-05-20 2000-12-12 Novozymes A/S Subtilase enzymes of the i-s1 and i-s2 sub-groups having at least one additionalamino acid residue between positions 128 and 129
AU4392700A (en) 1999-05-20 2000-12-12 Novozymes A/S Subtilase enzymes of the i-s1 and i-s2 sub-groups having at least one additionalamino acid residue between positions 129 and 130
WO2000071685A1 (fr) 1999-05-20 2000-11-30 Novozymes A/S Enzymes subtilases des sous-groupes i-s1 et i-s2 renfermant au moins un residu d'acide amine supplementaire entre les positions 132 et 133
US6946128B1 (en) 1999-07-22 2005-09-20 The Procter & Gamble Company Protease conjugates having sterically protected epitope regions
CA2379712A1 (fr) 1999-07-22 2001-02-01 The Procter & Gamble Company Variants de protease subtilisine possedant des suppressions et des substitutions d'acides amines dans certaines regions de sites antigeniques
CN1377406A (zh) 1999-07-22 2002-10-30 宝洁公司 具有空间保护的切割位点的蛋白酶缀合物
MXPA02000840A (es) 1999-07-22 2002-07-30 Procter & Gamble Variantes de proteasa de subtilisina que tienen substituciones de aminoacidos en regiones de epitopes definidas.
US6727085B2 (en) 1999-12-15 2004-04-27 Fanoe Tina Sejersgaard Subtilase variants having an improved wash performance on egg stains
US6777218B1 (en) 2000-03-14 2004-08-17 Novozymes A/S Subtilase enzymes having an improved wash performance on egg stains
JP2004305175A (ja) 2003-04-10 2004-11-04 Kao Corp アルカリプロテアーゼ
EP1623029B1 (fr) 2003-05-07 2008-09-03 Novozymes A/S Enzymes de subtilisine variant (subtilases)
EP1678296B1 (fr) 2003-10-23 2011-07-13 Novozymes A/S Protease a stabilite amelioree dans les detergents
JP4787571B2 (ja) * 2004-10-08 2011-10-05 花王株式会社 アルカリプロテアーゼ
PT2007942E (pt) 2006-04-14 2014-10-07 Danisco Us Inc Tratamento de têxteis num passo
JP2009534022A (ja) 2006-04-20 2009-09-24 ノボザイムス アクティーゼルスカブ 卵のシミに対する洗浄能力が向上したサビナーゼ変異体
WO2008134343A1 (fr) 2007-04-30 2008-11-06 Danisco Us Inc., Genencor Division Utilisation d'hydrolysats de protéine pour stabiliser des formulations détersives de métalloprotéase
SG148934A1 (en) 2007-06-11 2009-01-29 Novozymes As A process for combined biopolishing and bleach clean-up
WO2012022777A1 (fr) 2010-08-19 2012-02-23 Novozymes A/S Procédé de criblage d'une sporulation induite
WO2020099491A1 (fr) 2018-11-14 2020-05-22 Novozymes A/S Composition de soin buccal comprenant un polypeptide ayant une activité dnase
EP4199897A2 (fr) 2020-08-24 2023-06-28 Novozymes A/S Composition de soin bucco-dentaire comprenant une fructanase

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2423541A1 (fr) * 1978-04-18 1979-11-16 Pasteur Institut Procede de tri parmi une population de micro-organismes, notamment de bacteries, de ceux qui sont porteurs ou producteurs d'un constituant intracellulaire particulier, notamment d'un fragment d'acide nucleique determine
EP0108303A1 (fr) * 1982-11-01 1984-05-16 Miles Laboratories, Inc. Procédé pour la détection et l'isolation d'un micro-organisme
EP0138075A1 (fr) * 1983-09-16 1985-04-24 Synergen Associates, Inc. Méthode pour la génération et détection de variantes d'enzymes à thermostabilité et activité élevée
WO1987004461A1 (fr) * 1986-01-15 1987-07-30 Amgen ANALOGUES DE SUBTILISINE THERMIQUEMENT STABLE ET STABLE AU pH, ET LEUR PROCEDE DE PRODUCTION

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2423541A1 (fr) * 1978-04-18 1979-11-16 Pasteur Institut Procede de tri parmi une population de micro-organismes, notamment de bacteries, de ceux qui sont porteurs ou producteurs d'un constituant intracellulaire particulier, notamment d'un fragment d'acide nucleique determine
EP0108303A1 (fr) * 1982-11-01 1984-05-16 Miles Laboratories, Inc. Procédé pour la détection et l'isolation d'un micro-organisme
EP0138075A1 (fr) * 1983-09-16 1985-04-24 Synergen Associates, Inc. Méthode pour la génération et détection de variantes d'enzymes à thermostabilité et activité élevée
WO1987004461A1 (fr) * 1986-01-15 1987-07-30 Amgen ANALOGUES DE SUBTILISINE THERMIQUEMENT STABLE ET STABLE AU pH, ET LEUR PROCEDE DE PRODUCTION

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
GENETIC TECHNOLOGY NEWS, vol. 6, no. 5, May 1986, pages 3-4; "Genex engineers detergent enzymes" *
J. CELL. BIOCHEM., SUPPL., no. 10, part A, 1986, page 271, no. E101; P. BRYAN et al.: "Protein engineering of subtilisin: proteases of enhanced stability", & SYMPOSIUM ON PROTEASES IN BIOLOGICAL CONTROL AND BIOTECHNOLOGY, 15TH ANNUAL UCLA, Los Angeles, 9th-15th February 1986 *
PROC. NATL. ACAD. SCI. USA, vol. 82, February 1985, pages 747-750; T. ALBER et al.: "A genetic screen for mutations that increase the thermal stability of phage T4 lysozyme" *
PROTEINS: STRUCTURE, FUNCTION AND GENETICS, vol. 1, 10th November 1986, pages 326-334; P.N. BRYAN et al.: "Proteases of enhanced stability: characterization of a thermostable variant of subtilisin" *
SCIENCE, vol. 229, no. 4719, 20th September 1985, pages 1193-1201; D. BOTSTEIN et al.: "Strategies and applications of in vitro mutagenesis" *
See also references of WO8705050A1 *
THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 260, 5th December 1985, pages 15298-15303, The American Society of Biological Chemists, Inc., US; M. MATSUMURA et al.: "Screening for thermostable mutant of kanamycin nucleotidyltransferase by the use of a transformation system for a thermophile, Bacillus stearothermophilus" *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6403692B1 (en) 2001-04-19 2002-06-11 Dow Global Technologies Inc. Filled thermoplastic composition

Also Published As

Publication number Publication date
JPS63502959A (ja) 1988-11-02
DK531887A (da) 1987-10-12
WO1987005050A1 (fr) 1987-08-27
EP0260299A4 (fr) 1988-11-24
DK531887D0 (da) 1987-10-12

Similar Documents

Publication Publication Date Title
US4980288A (en) Subtilisin with increased thermal stability
EP0260299A1 (fr) Procede de mutagenese et de criblage et produit obtenu
US5013657A (en) Subtilisin mutations
JP2594533B2 (ja) 変異ザブチリシンを製造する方法
JP2726799B2 (ja) 変異ズブチリシン
US5543302A (en) Proteases of altered stability to autolytic degradation
US4990452A (en) Combining mutations for stabilization of subtilisin
US5118623A (en) Bleach stable enzymes
US5246849A (en) Thermally stable serine proteases
EP0870833B1 (fr) Genes de proteases ultrathermostables
NZ228424A (en) Protease-deficient gram-positive bacteria and their use as host organisms for the production of recombinant products
Rollence et al. Engineering thermostability in subtilisin BPN'by in vitro mutagenesis
EP0956344A1 (fr) MUTANTS DE LACCASE $i( MYCELIOPHTHORA) ET $i(SCYTALIDIUM) A STABILITE AMELIOREE
Remacle et al. Suppression of a+ 1 T mutation by a nearby substitution in the mitochondrial cox1 gene of Chlamydomonas reinhardtii: a new type of frameshift suppression in an organelle genome
JPH0994100A (ja) 細菌同定法及びキット

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE CH DE FR GB IT LI LU NL SE

17P Request for examination filed

Effective date: 19880223

A4 Supplementary search report drawn up and despatched

Effective date: 19881124

17Q First examination report despatched

Effective date: 19910802

RAP1 Party data changed (applicant data changed or rights of an application transferred)

Owner name: ENZON LABS INC.

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION HAS BEEN REFUSED

18R Application refused

Effective date: 19931209

RIN1 Information on inventor provided before grant (corrected)

Inventor name: PANTOLIANO, MICHAEL, W.

Inventor name: BRYAN, PHILIP, N.

Inventor name: ROLLENCE, MICHELE, L.