SUBTILISIN NUTATIONS

Field of the Invention

The invention pertains to modified subtilisin enzymes which have enhanced thermal stability and to the genes which encode the subtilisin enzymes.

Background of the Invention:

Proteins are linear polymers of amino acids. Since the polymeri¬ zation reactions which produce proteins result in the loss of one molecule of water from each amino acid, proteins are often said to be composed of amino acid "residues." Natural protein molecules may contain as many as 20 different types of amino acid residues, each of which contains a distinctive side chain. The sequence of amino acids in a protein defines the primary structure of the protein.

Proteins fold into a three-dimensional structure. The folding is determined by the sequence of amino acids and by the protein's en¬ vironment. The remarkable properties of proteins depend directly from the protein's three-dimensional conformation. Thus, this conformation determines the activity or stability of enzymes, the capacity and specificity of binding proteins, and the structural attributes of receptor molecules.

The three-dimensional structure of a protein may be determined in a number of ways. Perhaps the best known way of determining protein structure involves the use of the technique of X-ray crystallography. An excellent general review of this technique can be found in Ph ^y sical Biochemistry. Van Holde, K.E. (Prentice-Hall, NJ (1971) pp221-239) which reference is herein incorporated by reference. Using this technique, it is possible to elucidate three-dimensional structure with remarkable precision. It is also possible to probe the three- dimensional structure of a protein using circular dichroism, light scattering, or by measuring the absorption and emission of radiant energy (Van Holde, Physical Biochemistry. Prentice-Hall, NJ (1971)). Additionally, protein structure may be determined through the use of the techniques of neutron defraction, or by nuclear magnetic resonance (Physical Chemistry. 4th Ed. Moore, W.J., Prentice-Hall, NJ (1972) which reference is hereby incorporated by reference).

The examination of the three-dimensional structure of numerous natural proteins has revealed a number of recurring patterns. Alpha helices, parallel beta sheets, and anti-parallel beta sheets are the most common patterns observed. An excellent description of such protein patterns is provided by Dickerson, R.E., et al . In: The Structure and Action of Proteins. W.A. Benjamin, Inc., CA (1969). The assignment of each amino acid to one of these patterns defines the secondary structure of the protein. The helices, sheets and turns of a protein's secondary structure pack together to produce the three- dimensional structure of the protein. The three-dimensional structure of many proteins may be characterized as having internal surfaces (directed away from the aqueous environment in which the protein is normally found) and external surfaces (which are in close proximity to the aqueous environment). Through the study of many natural proteins, researchers have discovered that hydrophobic residues (such as tryp- tophan, phenylalanine, tyrosine, leucine, isoleucine, valine, or methionine) are most frequently found on the internal surface of protein molecules. In contrast, hydrophilic residues (such as aspar-

tic acid, asparagine, glutamate, glutamine, lysine, arginine, histi- dine, serine, threonine, glycine, and proline) are most frequently found on the external protein surface. The amino acids alanine, glycine, serine and threonine are encountered with equal frequency on both the internal and external protein surfaces.

Proteins exist in a dynamic equilibrium between a folded, ordered state and an unfolded, disordered state. This equilibrium in part reflects the short range interactions between the different segments of the polypeptide chain which tend to stabilize the protein's struc¬ ture, and, on the other hand, those thermodynamic forces which tend to promote the randomization of the molecule.

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. Enzymes have been studied to determine correlations between the three-dimen¬ sional structure of the enzyme and its activity or stability.

The amino acid sequence of an enzyme determines the characteris¬ tics 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 proper¬ ties 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.

Although there may be slight variations in a distinct type of naturally occurring enzyme within a given species of organism, enzymes 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 charac¬ teristics of a naturally occurring or "wild-type" enzyme are not necessarily optimized for utilization outside of the natural environ¬ ment 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.

Summary of the Invention

The invention relates to modified subtilisin enzymes which have increased thermal stability. In addition, the invention pertains to cloned mutant genes coding for a subtilisin material having at least one amino acid substitution which has increased thermal stability over wild-type subtilisin.

Definitions

The following definitions are used in describing the invention.

Protein

A protein is a heteropolymer made by living cells and composed of amino acids. A typical protein comprises 100 to 1000 amino acids. The exact sequence of amino acids determines the structure and func¬ tion of the protein.

Amino acid

Amino acids are naturally occurring compounds that are the build¬ ing blocks of proteins. The natural amino acids are usually ab¬ breviated to either three letters or one letter. The most common amino acids, and their symbols, are given in Table 1. The amino acids are joined head to tail to form a long main chain. Each kind of amino acid has a different side group.

Table 1. Amino acid names and abbreviations.

Amino Acid Three letter code Single letter code

Alanine Ala A

Arginine Arg R

Aspartic acid Asp D

Asparagine Asn N

Cysteine Cys C

Glutamic acid Glu E

Gluta ine Gin Q

Glycine Gly G

Histidine His H

Isoleucine He I

Leucine Leu L

Lysine ys K

Methionine Met M

Phenylalanine Phe F

Proline Pro P

Serine Ser S

Threonine Thr T

Tryptophane Trp W

Tyrosine Tyr Y

Valine Val V

Atom names

All amino acids have the same atoms in the main chain and differ only in the side chains. The main-chain atoms are a nitrogen, two carbons, and one oxygen. The first atom is the nitrogen, called N. The next atom is a carbon and is called the alpha-carbon. Side groups are attached to this alpha-carbon. The alpha-carbon is connected to the carbonyl carbon which is called C. C is connected to the carbonyl

oxygen (called 0) and to the N of the next residue. The side group atoms are given names composed of the symbol for the element (C, 0, N, S), a Greek, letter (alpha, beta, gamma, delta, epsilon, zeta and eta), and perhaps an arabic numeral if the side group is forked.

Detailed Description of the Invention

This invention pertains to subtilisin enzymes that have been modified by mutating the various nucleotide sequences that code for the enzymes. The modified subtilisin enzymes of this invention have enhanced thermal stability.

The subtilisin enzymes of this invention belong to a class of enzymes known as proteases. A protease is a catalyst for the cleavage of peptide bonds. An example of this cleavage is given below.

+H ₂ 0 protease

1

One type of protease is a serine protease. A serine protease will catalyze 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 diisopropylfluoro- phosphate.

A subtilisin is a serine protease produced by Gram positive bacteria or by fungi. The amino acid sequences of seven subtilisins are known. These include five subtilisins from Bacillus strains (sub¬ tilisin BPN', subtilisin Carlsberg, subtilisin DY, subtilisin amylo- sacchariticus, and mesenticopeptidase). (Vasantha et al .. "Gene for alkaline protease and neutral protease from Bacillus amyloliouefaciens contain a large open-reading frame between the regions coding for signal sequence and mature protein," J. Bacteriol. 159:811-819 (1984);

Jacobs et al., "Cloning sequencing and expression of subtilisin

Carlsberg from Bacillus lichenifor is." Nucleic Acids Res. 13:8913- 8926 (1985); Nedkov et al .. "Determination of the complete amino acid sequence of subtilisin DY and its comparison with the primary struc¬ tures of the subtilisin BPN', Carlsberg and a ylosacchariticus," Biol . Chem. Hoppe-Seyler 366:421-430 (1985); Kurihara et al .. "Subtilisin amylosacchariticus," J. Biol. Chem. 247:5619-5631 (1972); and Svendsen et al.. "Complete amino acid sequence of alkaline mesentericopepti- dase," FEBS Lett. 196:228-232 (1986)).

The amino acid sequence of the subtilisin thermitase from Ther¬ moactinomvces vulgaris is also known. (Meloun et al .. "Complete primary structure of thermitase from thermoactinomvces vulgaris and its structural features related to the subtilisin-type proteases," 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)).

These enzymes have been shown to be related to subtilisin BPN', not only through their primary sequences and enzymological properties, but also by comparison of x-ray crystallographic data. (McPhalen et al., "Crystal and molecular structure of the inhibitor eglin from leeches in complex with subtilisin Carlsberg," FEBS Lett. 188:55-58

(1985) and Pahler et al., "Three-dimensional structure of fungal proteinase K reveals similarity to bacterial subtilisin," EMBO J. 2:1311-1314 (1984).)

As used in this invention, the term "mutated or modified sub¬ tilisin enzyme(s)" is meant to include mutated serine proteases that have enhanced thermal stability, and are homologous to the subtilisin enzymes of this invention. The mutated or modified subtilisin enzymes are also described herein as "subtilisin material." As used herein, and under the definition of mutated or modified subtilisin enzyme or subtilisin material, the mutations of this invention may be introduced into any serine protease which has at least 50%, and preferably 80% amino acid sequence homology with the sequences for subtilisin BPN', subtilisin Carlsberg, subtilisin DY, subtilisin amylosacchariticus, mesenticopeptidase, thermitase, proteinase , or ther omycolase and therefore may be considered homologous.

The mutated subtilisin enzymes of this invention have enhanced thermal stability over native or wild-type subtilisin. Thermal stability is a good indicator of the overall robustness of a protein. Proteins of high thermal stability often are stable in the presence of chaotropic agents, detergents, and under other conditions, which tend to inactivate proteins. Thermally stable proteins are therefore expected to be useful for many industrial and therapeutic applica¬ tions, in which resistance to high temperature, harsh solvent condi¬ tions or extended shelf-life is required.

As used herein, resistance to thermal inactivation is measured by resistance to thermal inactivation under two representative sets of conditions. The first is in the presence of 10 mM calcium chloride at 65 ^* C and the second is at 45 ^* C in the presence of 10 mM EDTA, which removes free calcium from solution. Calcium is known to stabilize subtilisin. Measurements of stability under these two extremes of calcium concentration were made because potential commercial uses of stable subtil sins could involve conditions with varying amounts of calcium present. The Tl/2 of wild type BPN' subtilisin is 59 ±3

minutes in 10 mM CaCl at 65 ^* C and 14.4 ±0.05 minutes in 1 mM EDTA at 45*C. The thermal stability of the mutated subtilisin is expressed as a ratio of Tl/2 (mutant) divided by the Tl/2 (wild-type).

The mutated subtilisin enzymes detailed in Table 2 have been found to be thermally stable. The mutated subtilisin enzymes of this invention have at least one specific amino acid substitution that enhances thermal stability. Table 2 shows the strain designation of the host cell secreting the subtilisin. The mutation is the amino acid substitution with the naturally occuring amino acid and position number given first with the arrow to the right indicating the amino acid substitution. The ratio of mutant T to wild-type T^ is given next. Finally, the oligonucleotide is given. The first line in this last column is the number (#) designation which indicates a specific oligonucleotide. The second line, below the oligonucleotide number, is the nucleotide or base pair sequence of the oligonucleotide. The mutations were made using subtilisin BPN'. However, as explained herein these mutations can be introduced at analogous positions in serine proteases using oligonucleotide-directed mutagenesis.

Table 2. Mutated Subtilisin BPN' Enzymes.

Tl/2 compared to wild type enzv e

10 mM 1.0 mM Mutagenic

Strain Mutation CaCl EDTA Oligonucleotide

GX7130 Wild Type 1.0 1.0 —

GX7174 VAL8-ILE 2.0 0.8 #201021-mer

CCT TAC GGC ATC TCA CAA ATT

GX7175 GLY169-ALA 5.9 1.1 #2011 21-mer

GGC TAC CCT GCG AAA TAC CCT

GX7195 TYR217→LYS 3.3 2.7 #1928 19-mer CGG GGC GAA AAA CGG TAC G

GX8303 MET50-PHE 0.76 1.4 #2207 19-mer GAG CCA GCT TCG TTC CTT C

GX8309 SER248-ASP 1=5 0.75 #220824-mer CAA GTC CGC GAC AGA TTA GAA AAC

SER249-ARG #220824-mer

GX8314 GLN206-CYS 2.4 5.1 #2181 19-mer GTA TCT ATC TGT AGC ACG C

GX8330 TYR217→LEU 2.0 1.8 #2331 19-mer

CGG GGC GCT TAA CGG TAC G

GX8336 GLN206-TYR 1.1 1.7 #2422 19-mer GTA TCT ATC TAC AGC ACG C

GX8352 SER63-ASP 6.3 -- #2494 21-mer

GAC AAC AAC GAC CAC GGA ACT

TYR217→LYS #192819-mer

GX8354 GLN271-GLU 1.3 _. #2522 17-mer

CAA CGT AGA AGC GGC AG

GX8363 THR22-LYS 1.3 2.1 #2524 18-mer

AGG CTA CAA AGG ATC AAA

ASN76-ASP #2463 20-mer

CGG CTC TTG ACA ACT CAA TC

GX8376 TYR104-VAL 5.0 1.6 #2332 19-mer

CGG CCA AGT TAG CTG GAT C

GLY128→SER #2338 19-mer GCC TCG GCT CTC CTT CTG G

Using the information of the subtilisn enzyme mutations of Tabl 2, one can improve other proteases which are closely related, sub tilisin Carlsberg for example. Closeness of relation is measured b comparison of amino acid sequences. There are many methods of align¬ ing protein sequences, but the differences are only manifest when th degree of relatedness is quite small. The methods described in Atlas of Protein Sequence and Structure. Margaret 0. Dayhoff editor, Vol. Supplement 2, 1976, National Biomedical Research Foundation, Geor¬ getown University Medical Center, Washington, D.C., p. 3 ff., entitled SEARCH and ALIGN, define relatedness. As is well known in the art, related proteins can differ in number of amino acids as well as iden¬ tity of each amino acid along the chain. That is, there can be dele¬ tions or insertions when two structures are aligned for maximum iden¬ tity. For example, 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 sub¬ tilisin BPN'. Thus the amino acid sequence of Carlsberg would appear, for example, very different from BPN' unless a gap is recorded at location 56. Therefore, one can predict with high degree of confi¬ dence that, substituting TYR for LYS at location 217 of subtilisin Carlsberg will increase thermal stability, provided that the residues in Carlsberg are numbered by homology to BPN'.

When one of the two homologous subtilisins has a gap, one must infer that the structures are different at that position. 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.

One can use this reasoning to transfer all of the thermostable mutations described herein to other homologous serine proteases.

In brief, in order to introduce the enhanced themostable muta¬ tion^) of this invention, the gene coding for the desired subtilisin material generally is first isolated from its natural source and cloned in a cloning vector. Alternat vely, mRNA which is transcribed from the gene of interest can be isolated from the source cell and converted into cD A by reverse transcription for insertion into a cloning vector. A cloning vector can be a phage or pTasmid, 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 in¬ clude insertion of the gene coding for the subtilisin material 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 subtilisin gene can then be mutated by oligonucleotide-directed mutagenesis. Oligonucleotide- directed mutagenesis, also called site-directed mutagenesis, is described in detail in Bryan et al .. Proc. Nat! . Acad. Sci. USA 83:3743-3745 (1986), incorporated herein by reference.

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 im¬ proved. 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. Typically, the amount of mutated subtilisin material added to a washing preparation will be from about 2000 to about 4000 Alkaline Delft Units/gram (ADU/gm) of washing preparation.

The invention is illustrated by the following examples which are not intended to be limiting.

EXAMPLES

Example I Ther ostabilitv Studies

The subtilisin gene from Bacillus amyloliαuefaciens (subtilisin BPN') has been cloned and sequenced previously and expressed at high levels from its natural promoter sequences in Bacillus subtilis (Vasantha et al.. Bacteriol. 159-881 (1984); Wells et al .. Nucleic Acids Res. 11:7911 (1983)). Mutations were introduced in vitro into the plasmid-encoded subtilisin gene and their effect on the thermostability of the altered enzyme was analyzed.

All mutant genes were recloned into a pUBHO based expressed plasmid and used to transform B. subtilis. The B. subtilis strain used as the host contains a chromosomal deletion of its subtilisin gene and therefore produces no background wild type activity. All mutant enzymes were efficiently expressed from this vector and were secreted into the culture medium at a concentration of about 1 g/1. Subtilisin is the major secreted protein in this system and comprises almost 80% of the total extracellular protein.

Purifi ation:

The mutated subtilisin enzymes were purified from cell-free fermentation broths by means of the following three-step purification scheme:

(1) DEAE chromatography of crude fermentation broth. The broth was adjusted to pH 7.0 by addition of solid 2-(N-morpholino)- ethanesulfonic acid (Mes) and loaded onto a bed (13 x 5 cm) of DE-52 cellulose (Whatman) which was previously equilibrated with 20 mM Mes buffer (pH 7.0). Subtilisin washes through unretarded under these conditions.

(2) Acetone fractionation of DEAE eluate. Acetone (-20 ^* C) was stirred with the DEAE eluate at 4 ^* C. Subtilisin precipitates between 50 and 70% acetone. The fraction the precipitates between 0 and 50% acetone was di carded.

(3) SE-53 (Whatman) chromatography of acetone precipitate. The acetone precipitated subtilisin was dissolved in 20 mM Mes buffer (pH 6.0) and loaded onto a column (2.5 x 16 cm) of SE-53 cellulose equilibrated with the same buffer. A linear salt gradient (0 to 0.2 M NaCl) was used to elute the subtilisin.

Fractions containing the highest specific activities were pooled and stored at -20 ^* C either as 70% isopropanol or 50% ammonium sulfate precipitants.

Enzyme Assay.

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 HC1 (pH 8.0), 50 mM KC1 at 25'C. One unit of activity corresponds to the amount of enzyme that hydrolyzes 1 umole of substrate per min. under these conditions. One of the products of hydrolysis, p-nitroanilide, has an extinction coefficient of 8800 M^cπT* at 410 nm, thus allowing easy monitoring of the enzymatic reaction (Delmar et al . , Anal . Bioche . 99:316-320 (1979)). Subtilisin concentrations were estimated

by 280 nm using EQ(.1%) ^B 1.17 (Ottesen & Svendson, Methods in En- zymology (1976), p. 207).

Resistance to Thermal Inactivation:

The mutated subtilisin enzymes were tested for resistance to thermal inactivation in solution. Thermal inactivation studies were performed by incubating a subtilisin solution dissolved in 10 mM CaCl ₂ , 50 πfl Tris-HCl pH 8.0 or 1.0 mM EDTA, 50 mM Tris-HCl, pH 8.0. The presence of CaCl ₂ stabilizes subtilisin. The sample was placed in a glass Durham tube which was immersed in a thermostated circulated water bath equilibrated at 65 ^* C in the presence of 10 mM CaCl2 or 45 ^β C in the presence of 1 mM EDTA. Evaporation from the sample tube was prevented by sealing with Parafilm. Aliquots were removed at various time points and assayed by the assay solution at 25 ^* C. The time zero measurement was the rate of hydrolysis of SAAPF-pNA before the sample is immersed in the temperature bath. All subsequent rates of hydro¬ lysis of substrate were measured after immersion in the bath. Plots of the logarithm of the remaining activity versus time were found, for the most part, to be linear over the course of three half-lives. Thus, a first order rate law is applicable. The rate of loss of activity for subtilisin and the wild-type enzyme from strain 7130 at 65'C in the presence of 10 πfl CaCl ₂ , 50 M C1 , and 50 M Tris-HCl, pH 8.0, was found to have a half-life of 59 + 3 minutes which agrees well with that reported in the literature for similar conditions (Voordouw et al .. Biochemistry 15:3716-3724 (1976)). This rate was assigned the reference point 1.0 for wild-type enzyme. The half-life of thermal inactivation (Tl/2) was measured for the mutated subtilisin enzymes. This information was presented in Table 2, above.

Although the foregoing invention has been described by way of illustration and example for purposes of clarity and understanding, it will be obvious that certain changes and modifications may be prac¬ ticed within the scope of the invention, as limited only the scope of the appended claims.