EP0260299A1 - Mutagenesis and screening method and product - Google Patents
Mutagenesis and screening method and productInfo
- 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
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Classifications
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- 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
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- 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
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- 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
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- 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
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- 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.
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.The mutation of a cloned DNA is caused by creating a single-stranded target region in a segment of cloned DNA, and by introducing a mutation in the single-stranded target region by treating this target region with a chemical or biological mutagenic agent which can introduce mutations in single strand DNA. The mutated target region is then transformed into double strand and a microorganism is transformed with the mutated double strand DNA present in an expression vector. The transformed microorganism is cultured under conditions where the mutated DNA is expressed to form an expression product, and the expression product is screened to identify the desired mutation in the DNA segment. Mutant subtilisins are described having improved thermal stability.
Description
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US82854586A | 1986-02-12 | 1986-02-12 | |
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Cited By (1)
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US6403692B1 (en) | 2001-04-19 | 2002-06-11 | Dow Global Technologies Inc. | Filled thermoplastic composition |
Also Published As
Publication number | Publication date |
---|---|
EP0260299A4 (en) | 1988-11-24 |
WO1987005050A1 (en) | 1987-08-27 |
DK531887D0 (en) | 1987-10-12 |
DK531887A (en) | 1987-10-12 |
JPS63502959A (en) | 1988-11-02 |
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