Pullulanase variants and polynucleotides encoding same
First Claim
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1. A pullulanase variant comprising a substitution at one or more positions corresponding to positions 368, 393, 492, 610, 631, and 632, of the polypeptide of SEQ ID NO:
- 3, wherein the pullulanase variant has;
(a) pullulanase activity,(b) at least 85%, but less than 100% sequence identity to the polypeptide of SEQ ID NO;
3,(c) has increased thermoactivity compared to the pullulanase of SEQ ID NO;
3; and
(d) the substitution is selected from the group consisting of;
(i) a substitution at a position corresponding to position 368 with Ala, Arg, Asp, Cys, Glu, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val,(ii) a substitution at a position corresponding to position 393 with Ala, Arg, Asp, Cyt, Glu, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val,(iii) a substitution at a position corresponding to position 492 with Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Trp, Tyr or Val,(iv) a substitution at a position corresponding to position 610 with Ala, Arg, Asp, Cys, Glu, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val,(v) a substitution at a position corresponding to position 631 with Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Trp, Tyr or Val,(vi) a substitution at a position corresponding to position 632 with Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Thr, Trp, Tyr or Val.
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Abstract
The present invention relates to pullulanase variants comprising substitutions of the parent pullulanase at one or more positions corresponding to positions 393, 143, 150, 243, 244, 345, 346, 368, 370, 373, 381, 382, 385, 387, 402, 429, 430, 431, 432, 456, 486, 492, 610, 624, 631, 632, 665 and 699 of the polypeptide of SEQ ID NO: 3. The present invention also relates to polynucleotides encoding the variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of using the variants.
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Citations
28 Claims
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1. A pullulanase variant comprising a substitution at one or more positions corresponding to positions 368, 393, 492, 610, 631, and 632, of the polypeptide of SEQ ID NO:
- 3, wherein the pullulanase variant has;
(a) pullulanase activity, (b) at least 85%, but less than 100% sequence identity to the polypeptide of SEQ ID NO;
3,(c) has increased thermoactivity compared to the pullulanase of SEQ ID NO;
3; and(d) the substitution is selected from the group consisting of; (i) a substitution at a position corresponding to position 368 with Ala, Arg, Asp, Cys, Glu, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val, (ii) a substitution at a position corresponding to position 393 with Ala, Arg, Asp, Cyt, Glu, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val, (iii) a substitution at a position corresponding to position 492 with Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Trp, Tyr or Val, (iv) a substitution at a position corresponding to position 610 with Ala, Arg, Asp, Cys, Glu, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val, (v) a substitution at a position corresponding to position 631 with Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Trp, Tyr or Val, (vi) a substitution at a position corresponding to position 632 with Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Thr, Trp, Tyr or Val. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11)
- 3, wherein the pullulanase variant has;
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12. A pullulanase variant, which has:
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(a) at least 90% sequence identity to the polypeptide of SEQ ID NO;
28, wherein the pullulanase variant has(b) pullulanase activity, (c) increased thermoactivity compared to the pullulanase of SEQ ID NO;
3, and(d) the following amino acid residues at the following positions corresponding to the positions of SEQ ID NO;
3(i) a glycine residue at position 368, (ii) an alanine residue at position 393, (iii) a glutamic acid residue at position 431, (iv) a phenylalanine residue at position 432, (v) an alanine or serine residue at position 492, (vi) an arginine residue at position 610, (vii) a serine residue at position 624, (viii) a serine residue at position 631, and (ix) a cysteine residue at position 632. - View Dependent Claims (13, 14, 15, 16, 17)
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18. A pullulanase variant comprising a variant catalytic domain, wherein the variant catalytic domain comprises a substitution at one or more positions corresponding to positions 368, 393, 492, 610, 631, and 632 of the polypeptide of SEQ ID NO:
- 3, wherein;
(a) the pullulanase variant has pullulanase activity, (b) the variant catalytic domain at least 85%, but less than 100% sequence identity to amino acids 330 to 828 of SEQ ID NO;
3,(c) the pullulanase variant has increased thermoactivity compared to the pullulanase of SEQ ID NO;
3; and(d) the substitution is selected from the group consisting of; (i) a substitution at a position corresponding to position 368 with Ala, Arg, Asp, Cys, Glu, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val, (ii) a substitution at a position corresponding to position 393 with Ala, Arg, Asp, Cyt, Glu, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val, (iii) a substitution at a position corresponding to position 492 with Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Trp, Tyr or Val, (iv) a substitution at a position corresponding to position 610 with Ala, Arg, Asp, Cys, Glu, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val, (v) a substitution at a position corresponding to position 631 with Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Trp, Tyr or Val, (vi) a substitution at a position corresponding to position 632 with Ala, Arg, Asn, Asp, Cys, Glu, Gln Gly, His, Ile Leu, Lys, Met, Phe, Pro, Thr, Trp, Tyr or Val. - View Dependent Claims (19, 20, 21, 22, 23, 24, 25, 26, 27, 28)
- 3, wherein;
Specification