US5324653A — Recombinant genetic means for the production of s…

Claim 1

Independent Claim 158 words

A method for obtaining a serine protease mutein having at least one improved property relative to a corresponding wild-type protease for use in detergents which comprises the steps of: mutagenizing a cloned gene encoding a mature PB92 protease obtained from Bacillus novo species PB92, where said mature PB92 protease has an amino acid sequence as shown in FIG. 4; introducing said gene into a host strain whereby a transformed host strain is obtained; growing said transformed host whereby said mutant gene is expressed and a serine protease mutein differing from said wild-type protease by no more than three amino acid substitutions corresponding to a residue position selected from the group consisting of 116, 126, 127, 128, 160, 166, 169, and 216, is identified by recovering said mutein and screening it for at least one improved property selected from the group consisting of: (1) wash performance, and (2) retained wash performance and improved stability against oxidizing agents in detergents.

Claim 2

Independent Claim 158 words

A method for obtaining a serine protease mutein having at least one improved property relative to a corresponding wild-type protease for use in detergents which comprises the steps of: mutagenizing a cloned gene encoding a mature PB92 protease obtained from Bacillus novo species PB92, where said mature PB92 protease has an amino acid sequence as shown in FIG. 4; introducing said gene into a host strain whereby a transformed host strain is obtained; growing said transformed host whereby said mutant gene is expressed and a serine protease mutein differing from said wild-type protease by no more than three amino acid substitutions corresponding to a residue position selected from the group consisting of 116, 126, 127, 128, 160, 166, 169, and 216, is identified by recovering said mutein and screening it for at least one improved property selected from the group consisting of: (1) wash performance, and (2) retained wash performance and improved stability against oxidizing agents in detergents.

Claim 3

Dependent Claim 12 words

The method according to claim 1, wherein said residue position is 216.

Claim 4

Dependent Claim 15 words

The method according to claim 2, wherein said amino acid substitution is to a serine.

Claim 5

Dependent Claim 91 words

An isolated DNA segment consisting essentially of a region encoding a serine protease mutein characterized as differing by no more than three amino acid substitutions corresponding to a residue position selected from the group consisting of 116, 126, 127, 128, 160, 166, 169, and 216 from a wild-type PB92 protease obtained from Bacillus novo species PB92, and having, relative to said wild-type PB92 protease, at least one improved property selected from the group consisting of: (1) wash performance, and (2) retained wash performance and improved stability against oxidizing agents in detergents.

Claim 6

Dependent Claim 91 words

An isolated DNA segment consisting essentially of a region encoding a serine protease mutein characterized as differing by no more than three amino acid substitutions corresponding to a residue position selected from the group consisting of 116, 126, 127, 128, 160, 166, 169, and 216 from a wild-type PB92 protease obtained from Bacillus novo species PB92, and having, relative to said wild-type PB92 protease, at least one improved property selected from the group consisting of: (1) wash performance, and (2) retained wash performance and improved stability against oxidizing agents in detergents.

Claim 7

Dependent Claim 116 words

An isolated DNA segment consisting essentially of a region encoding a serine protease mutein characterized as differing by no more than three amino acid substitutions corresponding to a residue position selected from the group consisting of 87, 116, 126, 127, 128, 160, 166, 169, 212 and 216, wherein said mutein has a substitution at amino acid 116 from glycine to a higher molecular weight non-polar aliphatic amino acid as compared to a wild-type PB92 protease obtained from Bacillus novo species PB92, and having, relative to said wild-type PB92 protease, at least one improved property selected from the group consisting of: (1) wash performance, and (2) retained wash performance and improved stability against oxidizing agents in detergents.

Claim 8

Dependent Claim 116 words

An isolated DNA segment consisting essentially of a region encoding a serine protease mutein characterized as differing by no more than three amino acid substitutions corresponding to a residue position selected from the group consisting of 87, 116, 126, 127, 128, 160, 166, 169, 212 and 216, wherein said mutein has a substitution at amino acid 116 from glycine to a higher molecular weight non-polar aliphatic amino acid as compared to a wild-type PB92 protease obtained from Bacillus novo species PB92, and having, relative to said wild-type PB92 protease, at least one improved property selected from the group consisting of: (1) wash performance, and (2) retained wash performance and improved stability against oxidizing agents in detergents.

Claim 9

Dependent Claim 112 words

An isolated DNA segment consisting essentially of a region encoding a serine protease mutein characterized as differing by no more than three amino acid substitutions corresponding to a residue position selected from the group consisting of 87, 116, 126, 127, 128, 160, 166, 169, 212, and 216, wherein said mutein has a substitution at amino acid 116 from glycine to valine, isoleucine or leucine as compared to a wild-type PB92 protease obtained from Bacillus novo species PB92, and having, relative to said wild-type PB92 protease, at least one improved property selected from the group consisting of: (1) wash performance, and (2) retained wash performance and improved stability against oxidizing agents in detergents.

Claim 10

Dependent Claim 112 words

An isolated DNA segment consisting essentially of a region encoding a serine protease mutein characterized as differing by no more than three amino acid substitutions corresponding to a residue position selected from the group consisting of 87, 116, 126, 127, 128, 160, 166, 169, 212, and 216, wherein said mutein has a substitution at amino acid 116 from glycine to valine, isoleucine or leucine as compared to a wild-type PB92 protease obtained from Bacillus novo species PB92, and having, relative to said wild-type PB92 protease, at least one improved property selected from the group consisting of: (1) wash performance, and (2) retained wash performance and improved stability against oxidizing agents in detergents.

Claim 11

Dependent Claim 112 words

An isolated DNA segment consisting essentially of a region encoding a serine protease mutein characterized as differing by no more than three amino acid substitutions corresponding to a residue position selected from the group consisting of 87, 116, 126, 127, 128, 160, 166, 169, 212, and 216, wherein said mutein has a substitution at amino acid 160 to glycine or an anionic amino acid as compared to a wild-type PB92 protease obtained from Bacillus novo species PB92, and having, relative to said wild-type PB92 protease, at least one improved property selected from the group consisting of: (1) wash performance, and (2) retained wash performance and improved stability against oxidizing agents in detergents.

Claim 12

Dependent Claim 112 words

An isolated DNA segment consisting essentially of a region encoding a serine protease mutein characterized as differing by no more than three amino acid substitutions corresponding to a residue position selected from the group consisting of 87, 116, 126, 127, 128, 160, 166, 169, 212, and 216, wherein said mutein has a substitution at amino acid 160 to glycine or an anionic amino acid as compared to a wild-type PB92 protease obtained from Bacillus novo species PB92, and having, relative to said wild-type PB92 protease, at least one improved property selected from the group consisting of: (1) wash performance, and (2) retained wash performance and improved stability against oxidizing agents in detergents.

Claim 13

Dependent Claim 110 words

An isolated DNA segment consisting essentially of a region encoding a serine protease mutein characterized as differing by no more than three amino acid substitutions corresponding to a residue position selected from the group consisting of 87, 116, 126, 127, 128, 160, 166, 169, 212, and 216 wherein said mutein has a substitution at amino acid 166 to an anionic amino acid as compared to a wild-type PB92 protease obtained from Bacillus novo species PB92, and having, relative to said wild-type PB92 protease, at least one improved property selected from the group consisting of: (1) wash performance, and (2) retained wash performance and improved stability against oxidizing agents in detergents.

Claim 14

Dependent Claim 110 words

An isolated DNA segment consisting essentially of a region encoding a serine protease mutein characterized as differing by no more than three amino acid substitutions corresponding to a residue position selected from the group consisting of 87, 116, 126, 127, 128, 160, 166, 169, 212, and 216 wherein said mutein has a substitution at amino acid 166 to an anionic amino acid as compared to a wild-type PB92 protease obtained from Bacillus novo species PB92, and having, relative to said wild-type PB92 protease, at least one improved property selected from the group consisting of: (1) wash performance, and (2) retained wash performance and improved stability against oxidizing agents in detergents.

Claim 15

Dependent Claim 111 words

An isolated DNA segment consisting essentially of a region encoding a serine protease mutein characterized as differing by no more than three amino acid substitutions corresponding to a residue position selected from the group consisting of 87, 116, 126, 127, 128, 160, 166, 169, 212, and 216, wherein said mutein has a substitution at amino acid 169 to a non-polar aliphatic amino acid as compared to a wild-type PB92 protease obtained from Bacillus novo species PB92, and having, relative to said wild-type PB92 protease, at least one improved property selected from the group consisting of: (1) wash performance, and (2) retained wash performance and improved stability against oxidizing agents in detergents.

Claim 16

Dependent Claim 111 words

An isolated DNA segment consisting essentially of a region encoding a serine protease mutein characterized as differing by no more than three amino acid substitutions corresponding to a residue position selected from the group consisting of 87, 116, 126, 127, 128, 160, 166, 169, 212, and 216, wherein said mutein has a substitution at amino acid 169 to a non-polar aliphatic amino acid as compared to a wild-type PB92 protease obtained from Bacillus novo species PB92, and having, relative to said wild-type PB92 protease, at least one improved property selected from the group consisting of: (1) wash performance, and (2) retained wash performance and improved stability against oxidizing agents in detergents.

Claim 17

Dependent Claim 109 words

An isolated DNA segment consisting essentially of a region encoding a serine protease mutein characterized as differing by no more than three amino acid substitutions corresponding to a residue position selected from the group consisting of 116, 126, 127, 128, 160, 166, 169, and 216 wherein said mutein has a substitution from a neutral amino acid to an anionic amino acid as compared to a wild-type PB92 protease obtained from Bacillus novo species PB92, and having, relative to said wild-type PB92 protease, at least one improved property selected from the group consisting of: (1) wash performance, and (2) retained wash performance and improved stability against oxidizing agents in detergents.

Claim 18

Dependent Claim 109 words

An isolated DNA segment consisting essentially of a region encoding a serine protease mutein characterized as differing by no more than three amino acid substitutions corresponding to a residue position selected from the group consisting of 116, 126, 127, 128, 160, 166, 169, and 216 wherein said mutein has a substitution from a neutral amino acid to an anionic amino acid as compared to a wild-type PB92 protease obtained from Bacillus novo species PB92, and having, relative to said wild-type PB92 protease, at least one improved property selected from the group consisting of: (1) wash performance, and (2) retained wash performance and improved stability against oxidizing agents in detergents.

Claim 19

Dependent Claim 88 words

An isolated DNA segment consisting essentially of a region encoding a serine protease mutein characterized as differing by no more than three amino acid substitutions corresponding to a residue position selected from the group consisting of 126, 127, and 128 as compared to a wild-type PB92 protease obtained from Bacillus novo species PB92, and having, relative to said wild-type PB92 protease, at least one improved property selected from the group consisting of: (1) wash performance, and (2) retained wash performance and improved stability against oxidizing agents in detergents

Claim 20

Dependent Claim 88 words

An isolated DNA segment consisting essentially of a region encoding a serine protease mutein characterized as differing by no more than three amino acid substitutions corresponding to a residue position selected from the group consisting of 126, 127, and 128 as compared to a wild-type PB92 protease obtained from Bacillus novo species PB92, and having, relative to said wild-type PB92 protease, at least one improved property selected from the group consisting of: (1) wash performance, and (2) retained wash performance and improved stability against oxidizing agents in detergents

Claim 21

Dependent Claim 42 words

The isolated DNA segment according to claim 11, wherein said mutations are at amino acid 126, from serine to any other non-hydroxylated amino acid; at amino acid 127 to any other acid; and at amino acid 128 to any other amino acid.

Claim 22

Dependent Claim 70 words

An isolated DNA segment consisting essentially of a region encoding a serine protease mutein characterized as differing by no more than three amino acid substitutions corresponding to a residue position selected from the group consisting of 87, 116, 126, 127, 128, 160, 166, 169, 212, and 216, wherein said mutein has a substitution at amino acid 216 from methionine to an amino acid other than a sulfur containing amino acid.

Claim 23

Dependent Claim 70 words

An isolated DNA segment consisting essentially of a region encoding a serine protease mutein characterized as differing by no more than three amino acid substitutions corresponding to a residue position selected from the group consisting of 87, 116, 126, 127, 128, 160, 166, 169, 212, and 216, wherein said mutein has a substitution at amino acid 216 from methionine to an amino acid other than a sulfur containing amino acid.

Claim 24

Dependent Claim 22 words

The isolated DNA segment according to claim 13, wherein said amino acid other than a sulfur containing amino acid is a serine.

Claim 25

Dependent Claim 125 words

An isolated DNA segment consisting essentially of a region encoding a serine protease mutein characterized as differing by no more than three amino acid substitutions corresponding to a residue position selected from the group consisting of 87, 116, 126, 127, 128, 160, 166, 169, 212 and 216 wherein said serine protease mutein has an amino acid sequence as shown in FIG. 4 except that said amino acid sequence comprises a mutation at least of amino acid 128 as compared to a wild-type PB92 protease obtained from Bacillus novo species PB92, and having, relative to said wild-type PB92 protease, at least one improved property selected from the group consisting of: (1) wash performance, and (2) retained wash performance and improve stability against oxidizing agents in detergents.

Claim 26

Dependent Claim 125 words

An isolated DNA segment consisting essentially of a region encoding a serine protease mutein characterized as differing by no more than three amino acid substitutions corresponding to a residue position selected from the group consisting of 87, 116, 126, 127, 128, 160, 166, 169, 212 and 216 wherein said serine protease mutein has an amino acid sequence as shown in FIG. 4 except that said amino acid sequence comprises a mutation at least of amino acid 128 as compared to a wild-type PB92 protease obtained from Bacillus novo species PB92, and having, relative to said wild-type PB92 protease, at least one improved property selected from the group consisting of: (1) wash performance, and (2) retained wash performance and improve stability against oxidizing agents in detergents.

Claim 27

Dependent Claim 46 words

An isolated gene encoding a serine protease mutein which as an amino acid sequence as shown in FIG. 4 except that said amino acid sequence has a mutation selected from the group consisting of (M216S); (M216Q); (S160D); (S160G, N212D); (S160D, M216Q); (S160D, M216S); and (A166D, M169I).

Claim 28

Dependent Claim 46 words

An isolated gene encoding a serine protease mutein which as an amino acid sequence as shown in FIG. 4 except that said amino acid sequence has a mutation selected from the group consisting of (M216S); (M216Q); (S160D); (S160G, N212D); (S160D, M216Q); (S160D, M216S); and (A166D, M169I).

Claim 29

Dependent Claim 72 words

An isolated DNA segment encoding a serine protease mutein, wherein said mutein has an amino acid sequence as shown in FIG. 4 except that said amino acid sequence has a mutation selected from the group consisting of: (a) S at position 160 to C, D, G, or N; (b) A at position 166 to D; (c) M at position 169 to I; (d) M at position 216 to Q or S; and.

Claim 30

Dependent Claim 72 words

An isolated DNA segment encoding a serine protease mutein, wherein said mutein has an amino acid sequence as shown in FIG. 4 except that said amino acid sequence has a mutation selected from the group consisting of: (a) S at position 160 to C, D, G, or N; (b) A at position 166 to D; (c) M at position 169 to I; (d) M at position 216 to Q or S; and.

Claim 31

Dependent Claim 74 words

An isolated DNA segment encoding a mutant proteolytic enzyme wherein said enzyme has an amino acid sequence as shown in FIG. 4 except that said amino acid sequence has a mutation selected from the group consisting of: (G116V, S126V, P127E, S128K); (G116V, S126G, P127Q, S128I); (G116V, S126L, P127N, S128V); (G116V, S126L, P127Q, S128A); (G116V, S126R, P127S, S128P); (G116V, S126F, P127L, S128T); (S126M, P127A, S128G); (S126M, P127A, S128G, S160D); and (G116V, S126N, P127S, S128A, S160D).

Claim 32

Dependent Claim 74 words

An isolated DNA segment encoding a mutant proteolytic enzyme wherein said enzyme has an amino acid sequence as shown in FIG. 4 except that said amino acid sequence has a mutation selected from the group consisting of: (G116V, S126V, P127E, S128K); (G116V, S126G, P127Q, S128I); (G116V, S126L, P127N, S128V); (G116V, S126L, P127Q, S128A); (G116V, S126R, P127S, S128P); (G116V, S126F, P127L, S128T); (S126M, P127A, S128G); (S126M, P127A, S128G, S160D); and (G116V, S126N, P127S, S128A, S160D).

Claim 33

Dependent Claim 86 words

An isolated DNA segment encoding a serine protease mutein, wherein said mutein has an amino acid sequence as shown in FIG. 4 except that said amino acid sequence has a mutation selected from the group consisting of: (a) G at position 116 to V; (b) S at position 126 to F, H, L, M, Q, R, or V; (c) P at position 127 to A, E, L, M, N, Q, R, or Y; (d) S at position 128 to A, G, K, P, T, or V.

Claim 34

Dependent Claim 86 words

An isolated DNA segment encoding a serine protease mutein, wherein said mutein has an amino acid sequence as shown in FIG. 4 except that said amino acid sequence has a mutation selected from the group consisting of: (a) G at position 116 to V; (b) S at position 126 to F, H, L, M, Q, R, or V; (c) P at position 127 to A, E, L, M, N, Q, R, or Y; (d) S at position 128 to A, G, K, P, T, or V.

Claim 35

Dependent Claim 75 words

An isolated gene encoding a serine protease mutein which has an amino acid sequence as shown in FIG. 4 except that said amino acid sequence has a mutation selected from the group consisting of (G116V, S126V, P127E, S128K); (G116V, S126G, P127Q, S128I); (G116V, S126L, P127N, S128V); (G116V, S126L, P127Q, S128A); (G116V, S126V, P127M); (G116V, S126H, P127Y); (G116V, S126R, P127S, S128P); (G116V, S126F, P127Q); (G116V, S126F, P127L, S128T); (S126M, P127A, S128G): and (S126M, P127A, S128G, S160D).

Claim 36

Dependent Claim 75 words

An isolated gene encoding a serine protease mutein which has an amino acid sequence as shown in FIG. 4 except that said amino acid sequence has a mutation selected from the group consisting of (G116V, S126V, P127E, S128K); (G116V, S126G, P127Q, S128I); (G116V, S126L, P127N, S128V); (G116V, S126L, P127Q, S128A); (G116V, S126V, P127M); (G116V, S126H, P127Y); (G116V, S126R, P127S, S128P); (G116V, S126F, P127Q); (G116V, S126F, P127L, S128T); (S126M, P127A, S128G): and (S126M, P127A, S128G, S160D).

Claim 37

Dependent Claim 33 words

An isolated gene encoding a serine protease mutein which has an amino acid sequence as shown in FIG. 4 except that said amino acid sequence has a mutation (G116V, S126N, P127S, S128A, S160D).

Claim 38

Dependent Claim 33 words

An isolated gene encoding a serine protease mutein which has an amino acid sequence as shown in FIG. 4 except that said amino acid sequence has a mutation (G116V, S126N, P127S, S128A, S160D).

Claim 39

Dependent Claim 29 words

The isolated DNA segment according to claim 4, wherein said DNA segment is further included on a vector, said vector comprising sequences for the expression of said DNA segment.

Annuity Stage	Window Opens	Payment Deadline	Surcharge Period Until	Estimated Fee	Status
3.5 Years (Year 4)	1997-06-27	1997-12-27	1998-06-28	$2000.00	Window Closed / Lapsed
7.5 Years (Year 8)	2001-06-27	2001-12-27	2002-06-28	$3760.00	Window Closed / Lapsed
11.5 Years (Year 12)	2005-06-27	2005-12-27	2006-06-28	$7700.00	Window Closed / Lapsed

Recombinant genetic means for the production of serine protease muteins

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