Cell-wall degrading enzyme variants

Inactive Publication Date: 2005-11-10
NOVOZYMES AS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0068] The enzyme variant of the invention, especially the pectate lyase variant, is very effective for use in an enzyma

Problems solved by technology

The enzymatic degradation of these rather intensively cros

Method used

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  • Cell-wall degrading enzyme variants

Examples

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example 1

Construction of Pectate Lyase Variant (M1691, F198V)

[0267] The wild-type B. licheniformis pectate lyase encoded by SEQ ID NO: 1 is expressed in B. subtilis from a plasmid denoted pMB541, see Materials and Methods. This plasmid contains a fusion of the signal sequence from B. licheniformis alpha-amylase and the gene encoding the mature protein of B. licheniformis pectate lyase (SEQ ID NO: 2, wild-type pectate lyase), the expression of which is directed by the B. licheniformis alpha-amylase promoter. Further, the plasmid contains the origin of replication, ori, from plasmid pUB110 and the cat gene from plasmid pC194 conferring resistance towards chloramphenicol. A specific mutagenesis vector with a 1.2 kb pUC fragment inserted in the unique PstI restriction site located between the nucleotide sequence coding for the signal sequence and the mature, was prepared. The important features of this vector, denoted pCA134 include an origin of replication derived from the pUC plasmids, the c...

example 2

Fermentation, Purification and Characterization of Bacillus licheniformis Pectate Lyase Variant M169I, F198V

[0272] The clone obtained as described in Example 1 was grown in 25×200 ml BPX media with 10 microliters / ml of kanamycin in 500 ml two baffled shake flasks for 5 days at 37° C. at 300 rpm.

[0273] 140 ml of shake flask culture fluid were diluted to 1000 ml with ion free water and applied to S-Sepharose (50 ml column equilibrated with 25 mM sodium acetate buffer pH 5.5). The pure pectate lyase variant was eluted using a NaCl gradient.

[0274] The pectate lyase variant gave a single band in SDS-PAGE of 35 kDa, exhibited 23 APSU units per mg protein, and a molar extinction coefficient of 57750.

[0275] The buffer of the pure enzyme was changed by size chromatography on a high load Superdex S200 column equilibrated with 0.1M EPPS buffer pH 8.0. DSC (Differential Scanning Calorimetry) was performed using a temperature increase of 1° C. per minute. The pure pectate lyase variant unfo...

example3

Construction, Fermentation, Purification and Characterization of Further Bacillus licheniformis Pectate Lyase Variants

[0276] By using the methods described in Example 1 and 2, the Bacillus licheniformis pectate lyase variants (relative to SEQ ID NO: 2) of Table I below were prepared and subjected to DSC (Differential Scanning Calorimetry) at pH 10 or pH 8 using a temperature increase of 1° C. per minute. The wild-type Bacillus licheniformis pectate lyase (SEQ ID NO: 2) has a DSC unfolding temperature of 60° C. (pH 10) and 70° C. (pH 8).

TABLE IDSC unfoldingtemperature(° C.)Variant no.Substitutions relative to SEQ ID NO: 2pH 10pH 81M169I + F198V + E189H672M169I + F198V + S72I723M169I + F198V + F144V + M167I70.14M169I + F198V + S72I + M265K75.95M169I + F198V + S72I + G203V74.76M169I + F198V + S72I + K83H75.77M169I + F198V + S72T668M169I + F198V + M167I65.69M169I + F198V + S72I + L82I +76.8I102F + L129F + V160F10M169I + F198V + T55P70.811M169I + F198V + S269P68.512D282H + N283P + D2...

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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...

Claims

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Application Information

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IPC IPC(8): C07H21/04C11D3/386C11D7/42C12N1/21C12N9/18C12N9/88C12N15/74
CPCC12N9/88C11D3/38636
Inventor SCHRODER GLAD, SANNEANDERSEN, CARSTENSCHULEIN, MARTINDELA, HANNEFRANDSEN, TORBEN
Owner NOVOZYMES AS
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