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Xylanases with enhanced thermophilicity and alkalophilicity

一种木聚糖酶、聚糖酶的技术，应用在木聚糖酶领域，能够解决嗜热性和嗜碱性不利、酶活性降低等问题

Inactive Publication Date: 2005-11-16
NAT RES COUNCIL OF CANADA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these mutations, including N11D, also have adverse effects on the thermophilic and basophilic xylanases, resulting in reduced activity of the enzyme at higher temperatures and neutral-alkaline pH compared to native TrX II

Method used

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  • Xylanases with enhanced thermophilicity and alkalophilicity
  • Xylanases with enhanced thermophilicity and alkalophilicity
  • Xylanases with enhanced thermophilicity and alkalophilicity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0133] Embodiment 1: Construction Trichoderma reesei mutant xylanase

[0134] Basic recombinant DNA methods, such as plasmid preparation, restriction enzyme digestion, polymerase chain reaction, oligonucleotide phosphorylation, ligation, transformation, and DNA hybridization, were performed according to well-established protocols well known to those skilled in the art (e.g., Sung et al., 1986 ), or as recommended by the manufacturer of the enzyme or kit. Buffers for many enzymes are already provided as part of the kit or produced according to the manufacturer's directions. Restriction enzymes, T4 polynucleotide kinase and T4 DNA ligase were purchased from New England BioLabs Ltd, Mississauga, Ont. GeneAmp PCR reagent kit was purchased from Perkin-Elmer. The precursor plasmid pXYbc, a pUC-type plasmid with an inserted Bacillus circulans xylanase gene, has been previously prepared and published (Sung et al., 1993; Campbell et al., U.S. Patent No. 5,405,769). A commonly used E...

Embodiment 2

[0323] Example 2: Qualitative mutant xylanase

[0324] 2-1: Production of xylanase

[0325] Culture conditions consisted of adding 2YT medium with ampicillin to 2YT medium with ampicillin (100 mg / L) from a 5 ml culture inoculated overnight in 2YT medium (16 g bacto-tryptone, 10 g yeast extract, 5 g NaCl, 1 L water) (1L). Cultures were grown at 37°C with shaking (200 rpm). After 16 hours, cells were harvested.

[0326] 2-2: Purification of mutant xylanase

[0327] Protein samples were prepared from cells by first producing a cell extract prepared by grinding 10 g of cell paste with 25 g of alumina powder. After grinding to a smooth mixture, 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) was added , and the mixture was triturated vigorously during the addition of buffer. The mixture was centrifuged at 8000 xg for 30 minutes to remove alumina and cell debris.

[032...

Embodiment 3

[0341] Example 3: Thermophilicity of mutant xylanases

[0342] Thermophilicity was tested by testing the effect of different temperatures on the enzymatic hydrolysis of soluble xylan by different mutant xylanases.

[0343] The analysis procedure is similar to the standard analysis, the difference is the incubation temperature and time. Xylanase (15 μg / mL) in 50 mM sodium citrate buffer, pH 5.5, and soluble birch wood xylan substrate were mixed and incubated at different temperatures in a circulating water bath. After 30 minutes of incubation, the amount of reducing sugars released from xylan was determined by HBAH analysis and calculated as relative activity, with the value at 40°C representing 100%.

[0344] Figure 3 shows the effect of temperature on the hydrolysis of xylan by TrX-HML-75A105H-125A129E-144R (TrX-HML-AHAE-R). This mutant xylanase showed moderately improved enzymatic activity at higher temperatures compared to the precursor without the H144R mutation (TrX-HML...

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Abstract

The present invention provides a xylanase, or a modified xylanase enzyme comprising at least one substituted amino acid residue at a position selected from the group consisting of amino acid 11, 116, 118, 144 and 161, the position determined from sequence alignment of the modified xylanase with Trichoderma reesei xylanase II amino acid sequence. The xylanases described herein exhibit improved thermophilicity, alkalophilicity, expression efficiency, or a combination thereof, in comparison to a corresponding native xylanase.

Description

[0001] The present invention relates to xylanases. More particularly, the present invention relates to xylanases and modified xylanases with improved performance under high temperature and high pH conditions. Background of the invention [0002] Xylanases are a group of enzymes with wide commercial use. Xylanase is mainly used in the biological bleaching of pulp in the paper industry. In addition, xylanases are used as clarifiers in fruit juices and wines, and as enzyme preparations in the washing of precision instruments and semiconductors (eg, U.S. Patent No. 5,078,802), and they are also used to improve the digestion of poultry and pig feed sex. [0003] In pulp production in the paper industry, fibrous raw materials are subjected to high temperature and pressure treatment in the presence of chemicals. This process converts the fibers into pulp, known as pulping. After pulping, the pulp is bleached. Xylanases are used to enhance bleaching of pulp. The xylanase treatme...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/24D21C5/00D21C9/10
CPCC12Y302/01008D21C5/005C12N9/24D21C9/1036C12N9/2482C12N9/248
Inventor 宋永霖
Owner NAT RES COUNCIL OF CANADA
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