Mutant proteolytic enzymes and method of production

Details Mutant proteolytic enzymes and method of production Mutant proteolytic enzymes and method of production

2000-05-31

2002-06-11

Nashed, Nashaat T. (Department: 1652)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving hydrolase

C435S219000, C435S220000, C435S221000, C435S024000, C702S019000

Reexamination Certificate

active

06403331

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to mutant proteolytic enzymes having improved properties relative to the wild-type enzyme, to genetic constructs which code for the mutant proteolytic enzymes, to methods of predicting mutations which enhance the stability of the enzyme, and to methods of producing the mutant proteolytic enzymes.
2. Description of the Related Art
Subtilisins
are a family of extracellular proteins having molecular weights in the range of 25,000-35,000 daltons and are produced by various Bacillus species. These proteins function as peptide hydrolases in that they catalyze the hydrolysis of peptide linkages in protein substrates at neutral and alkaline pH values.
Subtilisins
are termed serine proteases because they contain a specific serine residue which participates in the catalytic hydrolysis of peptide substrates. A
subtilisin
enzyme isolated from soil samples and produced by
Bacillus lentus
for use in detergent formulations having increased protease and oxidative stability over commercially available enzymes under conditions of pH 7 to 10 and at temperature of 10 to 60° C. in aqueous solutions has been disclosed in copending patent application Ser. No. 07/398,854, filed on Aug. 25, 1989. This
B. lentus
alkaline protease enzyme (BLAP, vide infra) is obtained in commercial quantities by cultivating a
Bacillus licheniformis
ATCC 53926 strain which had been transformed by an expression plasmid which contained the wild type BLAP gene and the
B. licheniformis
ATCC 53926 alkaline protease gene promoter.
Industrial processes generally are performed under physical conditions which require highly stable enzymes. Enzymes may be inactivated by high temperatures, pH extremes, oxidation, and surfactants. Even though
Bacillus subtilisin
proteases are currently used in many industrial applications, including detergent formulations, stability improvements are still needed. Market trends are toward more concentrated detergent powders, and an increase in liquid formulations. Increased shelf stability and oxidative stability, with retention of catalytic efficiency are needed. It is therefore desirable to isolate novel enzymes with increased stability, or to improve the stability of existing enzymes, including
subtilisin
proteases such as BLAP.
The stability of a protein is a function of its three dimensional structure. A protein folds into a three dimensional conformation based upon the primary amino acid sequence, and upon its surrounding environment. The function and stability of a protein are a direct result of its three dimensional structure.
A large body of information has been published which describes changes in enzyme properties as a result of alterations in the primary amino acid sequence of the enzyme. These alterations can result from random or site specific alterations of the gene which expresses the enzyme using genetic engineering techniques. Random approaches mutagenize total cellular DNA, followed by selection for the synthesis of an enzyme with improved properties. This approach requires neither knowledge of the three dimensional structure of the enzyme, nor any predictive capability on the part of the researcher. Site directed mutagenesis, on the other hand, requires a rational approach for the introduction of amino acid changes. In this approach one or more amino acids may be replaced by other residues by altering the DNA sequence which encodes the protein. This can be accomplished using oligonucleotide directed in vitro mutagenesis. The following references teach site-directed mutagenesis procedures used to generate specific amino acid substitution(s): Hines, J. C., and Ray, D. S. (1980) Gene 11:207-218; Zoller, M. J., and Smith, M. (1982) Nucleic Acids Res. 10:6487-6500; Norrander, J., et al. (1983) Gene 26:101-106; Morinaga, Y., et al. (1984) Bio/Technology 2:636-639; Kramer, W., et al. (1984) Nucleic Acids Res. 12:9441-9456; Carter, P., et al. (1985) Nucleic Acids Res. 13:4431-4443; Kunkel, T. A. (1985) Proc. Natl. Acad. Sci.
USA 82:488-492; Bryan, P., et al. (1986) Proc. Natl. Acad. Sci. USA 83:3743-3745.
A rational approach may or may not require knowledge of a protein's structure. For example, patent application WO 89/06279 describes the comparison of the primary amino acid sequence of different
subtilisins
while contrasting differences in physical and chemical properties. The primary amino acid sequences of the different
subtilisins
are aligned for the greatest homology, while taking into account amino acid insertions, deletions, and total number of amino acids.
Currently, the amino acid sequences of at least 10
subtilisin
proteases have been published. Eight of these
subtilisins
were isolated from species of Bacilli, and include
subtilisin
168 (Stahl, M. L., and Ferrari, E. (1984) J. Bacteriol. 158:411-418),
subtilisin
BPN′ (Vasantha, N., et al., (1984) J. Bacteriol. 159:811-819),
subtilisin
Carlsberg (Jacobs, M., et al. (1985) Nucleic Acids Res. 13:8913-8926),
subtilisin
DY (Nedkov, P., et al. (1985) Biol. Chem. Hoppe-Seyler 366:421-430),
subtilisin
amylosacchariticus (Kurihara, M., et al. (1972) J.Biol. Chem. 247:5619-5631),
subtilisin
mesenticopeptidase (Svendsen, I., et al. (1986) FEBS Lett. 196:228-232),
subtilisin
147 and
subtilisin
309 (Hastrup et al. (1989) WO 89/06279),
subtilisin
PB92 (Van Eekelen et al. (1989) EP 0328229), and
subtilisin
BLAP (Ladin, B., et al. (1990) Society for Industrial Microbiology Annual Meeting, Abstract P60). The remaining two
subtilisin
sequences are thermitase from the fungus
Thermoactinomyces vulgaris
(Meloun, B., et al. (1985) FEBS Lett. 183:195-200), and proteinase K from the fungus
Tritirachium album limber
(Jany, K. -D., and Mayer, B. (1985) Biol. Chem. Hoppe-Seyler 366:485-492).
Methods for obtaining optimum alignment of homologous proteins are described in Atlas of Protein Sequence and Structure, Vol. 5, Supplement 2 (1976) (Dayhoff, M. O., ed., Natl. Biomed. Res. Found., Silver Springs, Md.). This comparison is then used to identify specific amino acid alterations which might produce desirable improvements in the target enzyme. Wells, J. A., et al. (1987) Proc. Natl. Acad. Sci. USA 84:1219-1223, used primary sequence alignment to predict site directed mutations which affect the substrate specificity of a
subtilisin
. Using the alignment approach WO 89/06279 teaches the construction of mutant
subtilisins
having improved properties including an increased resistance to oxidation, increased proteolytic activity, and improved washing performance for laundry detergent applications. Patent applications WO 89/09819, and WO 89/09830 teach improvement in the thermal stability of
subtilisin
BPN′ by the introduction of one or more amino acid changes based on the alignment of the primary amino acid sequences of
subtilisin
BPN′ with the more thermal stable
subtilisin
Carlsberg. From hereon, amino acids will be referred to by the one or three letter code as defined in Table 1.
TABLE 1
One and Three Letter Code for Amino Acids
A = Ala = Alanine
C = Cys = Cysteine
D = Asp = Aspartic acid or aspartate
E = Glu = Glutamic acid or glutamate
F = Phe = Phenylalanine
G = Gly = Glycine
H = His = Histidine
I = Ile = Isoleucine
K = Lys = Lysine
L = Leu = Leucine
M = Met = Methionine
N = Asn = Asparagine
P = Pro = Proline
Q = Gln = Glutamine
R = Arg = Arginine
S = Ser = Serine
T = Thr = Threonine
V = Val = Valine
W = Trp = Tryptophan
Y = Tyr = Tyrosine
Rational mutational approaches may also predict mutations which improve an enzyme property based upon the three dimensional structure of an enzyme, in addition to the alignment of primary amino acid sequences described above. One method for determining the three dimensional structure of

Affiliated with

Also associated with

Rating

Mutant proteolytic enzymes and method of production does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Mutant proteolytic enzymes and method of production, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Mutant proteolytic enzymes and method of production will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-2957613