Pullulanase variants and methods for preparing such variants with predetermined properties
First Claim
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1. An isolated pullulanase variant, wherein(a) the variant is at least 50% homologous to SEQ ID NO:
- 2;
(b) the variant comprises an amino acid modification compared to SEQ ID NO;
1 in at least one of the positions corresponding to 95-113, K122P, 130-140, K151P, 157-165, 180, 181, 210, 227, 228, 232-238, 259, 266-272, 286, G293P, 298, 299, 300-314, N315P, 337-339, 353, N374P, 380, 384, 385, 392, 394, 396, 406, 408-429, 442, A446P, 478, 500-507, 515, 526, 534, 543, 544 550, T556P, 557, 563, 568, 573, 576, 583, 627, 659-665, G668P, G672P, 681, 684, 688, 689, 751-755, 732, 736, 740, 760, 767, 770 783, 788, 792, 793, K758C+I914C, T916C+A765C, I897C+S819C, P525C+E499C and H286C+T148C; and
(c) the variant has an improved thermostability as compared to the parent pullulanase.
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Abstract
The inventors have modified the amino acid sequence of a pullulanase to obtain variants with improved properties, based on the three-dimensional structure of the pullulanase Promozyme®. The variants have altered physicochemical properties, e.g. an altered pH optimum, improved thermostability, altered substrate specificity, increased specific activity or an altered cleavage pattern.
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Citations
8 Claims
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1. An isolated pullulanase variant, wherein
(a) the variant is at least 50% homologous to SEQ ID NO: - 2;
(b) the variant comprises an amino acid modification compared to SEQ ID NO;
1 in at least one of the positions corresponding to 95-113, K122P, 130-140, K151P, 157-165, 180, 181, 210, 227, 228, 232-238, 259, 266-272, 286, G293P, 298, 299, 300-314, N315P, 337-339, 353, N374P, 380, 384, 385, 392, 394, 396, 406, 408-429, 442, A446P, 478, 500-507, 515, 526, 534, 543, 544 550, T556P, 557, 563, 568, 573, 576, 583, 627, 659-665, G668P, G672P, 681, 684, 688, 689, 751-755, 732, 736, 740, 760, 767, 770 783, 788, 792, 793, K758C+I914C, T916C+A765C, I897C+S819C, P525C+E499C and H286C+T148C; and
(c) the variant has an improved thermostability as compared to the parent pullulanase. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8)
- 2;
Specification