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Modification of xylanase to improve thermophilicity, alkophilicity and thermostability

A technology of xylanase and polycanase, applied in the field of xylanase, can solve the problem of not developing thermophilic and basophilic family 11 xylanase

Inactive Publication Date: 2004-12-01
NAT RES COUNCIL OF CANADA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0062] Therefore, despite extensive efforts on Family 11 xylanases, no modified Family 11 xylanases with significantly improved thermophilicity and basophilicity have been developed.

Method used

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  • Modification of xylanase to improve thermophilicity, alkophilicity and thermostability
  • Modification of xylanase to improve thermophilicity, alkophilicity and thermostability
  • Modification of xylanase to improve thermophilicity, alkophilicity and thermostability

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0138] Construction of Trichoderma Reesei modified xylanase NI-TX

[0139] Basic recombinant DNA methods such as plasmid preparation, restriction enzyme digestion, polymerase chain reaction, oligonucleotide phosphorylation, ligation, transformation, and DNA hybridization are based on well-established methods well known to those skilled in the art (Sung et al. ., 1986) or according to the method recommended by the manufacturer of the enzyme or kit. Buffers for many enzymes are supplied as part of a kit or reconstituted according to the manufacturer's recommendations. Restriction enzymes, T4 polynucleotide kinase and T4 DNA ligase were purchased from New England BioLabs LTD, Mississauga, Ont. GeneAmp PCR Kit was purchased from Perkin-Elmer. The starting plasmid pXYbc, a pUC-type plasmid inserted with the Bacillus circulans xylanase gene, had been prepared and published before (Sung et al, 1993; Campbell et al, US Patent 5,405,769, April 11, 1995 date authorized). A common E....

Embodiment 2

[0305] Construction of mutant xylanase NI-BX from Bacillus circulans

[0306]Modifications NI-TX2, NI-TX3, NI-TX7, NI-TX8 and NI-TX9 of the fungal Trichoderma xylanase are also reproduced in the bacterial Bacillus circulans xylanase (BcX). The starting plasmid pXYbc has been prepared and published before (Sung et al, 1993; Campbell et al, US Patent 5,405,769, issued April 11, 1995), and is only briefly described here. Following the same procedure described above for pXyTv(3-190), assemble the coding circular by enzymatic phosphorylation with T4 DNA kinase and ligation of overlapping synthetic oligonucleotides into a linearized plasmid by T4 DNA ligase. Synthetic gene for Bacillus xylanase (BcX) (Figure 4) (Sung et al, 1993).

[0307] A. Construction of plasmid pNI-BX1

[0308] The modification method of NI-BX1 has been used in the preparation of NI-TX2. Mutant NI-BX1 is a modified version of BcX, which replaces the (1-22) region of BcX with the Tfx (1-31) sequence. The con...

Embodiment 3

[0370] Generation and analysis of modified xylanases

[0371] (A) Production of xylanase

[0372] The culture conditions were the same as well-established methods for the production of other E. coli expressed xylanases. 5 ml of an overnight culture in 2YT medium (16 g yeast extract, 10 g tryptone, 5 g NaCl, 1 L water) containing ampicillin (100 mg / L) was added to 2YT medium containing ampicillin (1 L). Shake culture at 37° C. (200 rpm), and harvest the cells after 16 hours.

[0373] (B) Purification of modified xylanase

[0374] To prepare protein samples from cells, 10 g of cell samples were first ground with 25 g of aluminum powder to prepare cell extracts. After grinding into a homogeneous mixture, add a small amount (5 mL) of ice-cold buffer A (10 mM sodium acetate, pH 5.5, for the BcX mutant) or buffer B (10 mM sodium acetate, pH 4.6, for the TX mutant) , grinding the mixture vigorously between each addition. The mixture was centrifuged at 8000xg for 30 minutes to re...

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Abstract

Producing a xylanase enzyme of superior performance in the bleaching of pulp. More specifically, a modified xylanase of Family 11 that shows improved thermophilicity, alkalophilicity, and thermostability as compared to the natural xylanase. The modified xylanases contain any of three types of modifications: (1) changing amino acids 10, 27, and 29 of Trichoderma reesei xylanase II or the corresponding amino acids of another Family 11 xylanase, where these amino acids are changed to histidine, methionine, and leucine, respectively; (2) substitution of amino acids in the N-terminal region with amino acids from another xylanase enzyme. In a preferred embodiment, substitution of the natural Bacillus circulans or Trichoderma reesei xylanase with a short sequence of amino acids from Thermomonospora fusca xylanase yielded chimeric xylanases with higher thermophilicity and alkalophilicity; (3) an extension upstream of the N-terminus of up to 10 amino acids. In a preferred embodiment, extension of the N-terminus of the xylanase with the tripeptide glycine-arginine-arginine improved its performance.

Description

[0001] This application is a divisional application of a Chinese patent application with an application date of September 9, 1997, an application number of 97116504.1, and an invention title of "Modification for Improving Thermophilicity, Basophilicity and Thermostability of Xylanase". technical field [0002] The field of the invention is the modification of proteins by protein engineering. In particular, the present invention relates to modified xylanases resistant to high temperature and high pH. Xylanases are used to enhance bleaching of pulp to produce white paper. The present invention enables the production of xylanases with the enhanced bleaching ability associated with family 11 xylanases, but which remain active at high temperature and pH, and are more suitable for the operational needs of pulp mills than currently used xylanases . Background technique [0003] Since 1991, xylanases have been used to enhance bleaching of pulp to produce bright white paper. These...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09C12N9/24C12N15/00C12N15/56C12R1/09C12R1/885D21C5/00D21C9/10
CPCC12Y302/01032C07K2319/00C12N9/24C12N9/248C12Y302/01008D21C5/005
Inventor 宋·L·荣矢口诚石川和彦
Owner NAT RES COUNCIL OF CANADA
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