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Modification of xylanase to improve thermophilicity, alkophilicity and thermostability

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

Inactive Publication Date: 2010-03-24
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

[0141] Construction of Trichoderma Reesei modified xylanase NI-TX

[0142] 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 provided as part of a kit or reconstituted according to the manufacturer's recommendations. Restriction enzymes, T4 polynucleotide kinase and T4 DNA The ligase was purchased from New England BioLabs LTD, Mississauga, Ont. The GeneAmp PCR kit was purchased from Perkin-Elmer. The starting plasmid pXYbc is a pUC-type plasmid inserted into the Bacillus circulans xylanase gene, here It has been prepared and published before (Sung et al, 1993; Campbell et al, US Patent 5,405,769, issued April 11, 1995). A commo...

Embodiment 2

[0308] Construction of mutant xylanase NI-BX from Bacillus circulans

[0309]Modifications of the fungal Trichoderma xylanase NI-TX2, NI-TX3, NI-TX7, NI-TX8 and NI-TX9 are also reproduced in the bacterial Bacillus circulans xylanase (BcX). The starting plasmid pXYbc is in It has been prepared and published before (Sung et al, 1993; Campbell et al, US patent 5,405,769, authorized on April 11, 1995), and is only briefly described here. It is used for pXyTv (3-190 ) same method, a synthetic gene encoding Bacillus circulans xylanase (BcX) was assembled by enzymatic phosphorylation with T4 DNA kinase and ligation of overlapping synthetic oligonucleotides into a linearized plasmid by T4 DNA ligase ( Figure 4 ) (Sung et al., 1993).

[0310] A. Construction of plasmid pNI-BXI

[0311] The modification method of NI-BX1 has been used in the preparation of NI-TX2. The mutant NI-BX1 is a modified version of BcX, which replaces the (1-22) region of BcX with the Tfx (1-31) sequence. In ...

Embodiment 3

[0373] Generation and analysis of modified xylanases

[0374] (A) Production of xylanase

[0375] The culture conditions are the same as the mature method for the production of xylanase expressed by other Escherichia coli. Dissolve 5 ml in 2YT medium (16 g yeast extract, 10 g tryptone, 5 g NaCl, 1 L) containing ampicillin (100 mg / L) The overnight culture in water) was added to 2YT medium (1L) containing ampicillin. Shaking culture (200rpm) at 37°C, after 16 hours, the cells were harvested.

[0376] (B) Purification of modified xylanase

[0377] To prepare protein samples from cells, first grind 10 g of cell samples 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 BcX mutation body) or buffer B (10 mM sodium acetate, pH 4.6, for the TX mutant), and the mixture was triturated vigorously between each addition. The mixture was centrifuged at 8000 x g...

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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. (3) an extension upstream of the N-terminus of up to 10 amino acids.

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 Patents(China)
IPC IPC(8): C12N9/24C12N9/42C12N15/56C12S3/08D21C9/10C12N15/09C12N15/00C12R1/09C12R1/885D21C5/00
CPCC12Y302/01032C07K2319/00C12N9/24C12N9/248D21C5/005C12Y302/01008
Inventor 宋·L·荣矢口诚石川和彦
Owner NAT RES COUNCIL OF CANADA
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