Cell-wall degrading enzyme variants

Abstract

The present invention relates to variants of a cell-wall degrading enzyme having a beta-helix structure, which variant has at least one substituent in a position determined by identifying all residues potentially belonging to a stack; characterizing the stack as interior or exterior; characterizing the stack as polar, hydrophobic or aromatic / heteroaromatic based on the dominating characteristics of the parent or wild-type enzyme stack residues and / or its orientation relative to the beta-helix (interior or exterior); optimizing all stack positions of a stack either to hydrophobic aliphatic amino acids, hydrophobic aromatic or polar amino acids by allowing mutations within one or all positions to amino acids belonging to one of these groups; measuring thermostability of the variants by DSC or an application-related assay such as a Pad-Steam application test; and selecting the stabilized variants. Variants of a wild-type parent pectate lyase (EC 4.2.2.2) having the conserved amino acid residues D111, D141 or E141, D145, K165, R194 and R199 when aligned with the pectate lyase comprising the amino acid sequence of SEQ ID NO: 2 are preferred.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application is a divisional of U.S. application Ser. No. 10 / 403,192 filed Mar. 31, 2003, which is a divisional of application Ser. No. 09 / 910,505 filed Jul. 19, 2001, now U.S. Pat. No. 6,607,902, which claims priority or the benefit under 35 U.S.C. 119 of Danish application nos. PA 2000 01117, PA 2001 00705 and PA 2001 00734 filed Jul. 19, 2000, May 4, 2001 and May 10, 2001, respectively, and U.S. provisional application No. 60 / 290,724 filed May 14, 2001, respectively, the contents of which are fully incorporated herein by reference.BACKGROUND OF THE INVENTION [0002] 1. Field of the Invention [0003] The present invention relates to variants of microbial cell-wall degrading enzymes, more specifically to variants of enzymes having a pectinase structure similar to that of Bacillus licheniformis enzymes exhibiting pectate lyase activity as their major enzymatic activity in the neutral and alkaline pH ranges; to a method of producing su...

AI Technical Summary

Benefits of technology

[0056] The inventors have now found that certain amino acid substitutions in cell-wall degrading enzymes having a structure including a beta-helix result in enzyme variants having improved performance in the neutral or alkaline pH range, especially improved thermostability when determined by DSC (Disc Scanning Calorimetry) or by a Pad-Steam application test.

[0057] In a preferred embodiment of the invention, variants of the Bacillus licheniformis pectate lyase (EC 4.2.2.2) encoded by SEQ ID NO: 1 exhibit improved properties over the parent pectate lyase, the improved properties being advantageous when the enzyme is applied industrially. The inventors have provided such variants by having succeeded in identifying certain positions in the protein sequence in which positions the naturally occurring amino acid residue may be substituted or deleted or in which positions one or more amino acid residues may be inserted with the purpose of providing an improved pectate lyase variant, and have further provided a method of constructing cell-wall degrading enzyme variants with improved performance in industrial applications.

[0065] In comparison with the wild-type cell-wall degrading enzyme, especially a wild-type pectate lyase, it is contemplated that the variant of the invention exhibits increased thermal stability, either due to further stabilization of the beta-helix structure of the protein by amino acid substitution in positions within the aliphatic and aromatic stacks of amino acid side chains, or to further stabilization of the binding cleft or the C-terminal turn. Increased thermostability of an enzyme is indeed very useful in many industrial applications which advantageously can be carried out at a temperature above the temperature optimum for the enzymatic activity of the wild-type enzyme.

[0068] The enzyme variant of the invention, especially the pectate lyase variant, is very effective for use in an enzymatic scouring process in the preparation of cellulosic material e.g. for proper response in subsequent dyeing operations.

Problems solved by technology

The enzymatic degradation of these rather intensively cross-linked polysaccharide structures is not a simple process.

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