Xylosidase Xyl43B with high xylose tolerance, and gene and application thereof

A xylosidase and tolerance technology, applied in the field of genetic engineering, can solve the problem that β-xylosidase has not been paid enough attention, and achieve the effect of good xylose tolerance and high activity

Active Publication Date: 2013-09-04
SHANDONG LONGKETE ENZYME PREPARATION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In view of the wide and important uses of xylanase, people have systemati

Method used

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  • Xylosidase Xyl43B with high xylose tolerance, and gene and application thereof
  • Xylosidase Xyl43B with high xylose tolerance, and gene and application thereof
  • Xylosidase Xyl43B with high xylose tolerance, and gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 Cloning of Humicola sp.L8 Xylosidase Encoding Gene xyl43B

[0045] Extraction of Humicola sp.L8 genomic DNA:

[0046] Centrifuge the bacteria cultured in the liquid for 3 days, put them into a mortar, add 2mL extract, grind for 5min, then put the grinding solution in a 50mL centrifuge tube, lyse in a water bath at 65°C for 20min, mix well every 10min, and Centrifuge at 10,000 rpm for 5 min at 4°C. The supernatant was extracted in phenol / chloroform to remove impurities, and then an equal volume of isopropanol was added to the supernatant. After standing at room temperature for 5 minutes, centrifuge at 10,000 rpm for 10 minutes at 4°C. The supernatant was discarded, the precipitate was washed twice with 70% ethanol, dried in vacuo, dissolved by adding an appropriate amount of TE, and stored at -20°C for later use.

[0047] Design of expression primers P1, P2 for 43 family xylosidase genes

[0048] P1:5'-GGG CATATG CCCCAAGTCCGTAACCCCTATCC-3';

[0049] P2:5'...

Embodiment 2

[0052] The preparation of embodiment 2 recombinant xylosidase

[0053] The expression vector pPET-28a was subjected to double enzyme digestion (Ecoli I+Not I), and at the same time, the gene xyl43B encoding xylosidase was double enzyme digested (NdeI+HindIII) to cut out the gene fragment encoding mature xylosidase and the expression vector pPET -28a connection to obtain the recombinant plasmid pPET-xyl43B containing the xylosidase gene xyl43B and transform Escherichia coli BL21 (DE3) to obtain the recombinant Escherichia coli strain BL21 / xyl43B.

[0054] Take the BL21 strain containing the recombinant plasmid, inoculate it in 300mL LB (50μg / mL kanamycin) culture medium, shake and culture at 220rpm at 37°C for 2-3h (OD 600 After reaching 0.6), add a final concentration of 0.6mM IPTG and induce at 30°C 180rpm for 5-8h. The expression level of recombinant xylosidase was 4.7U / mL. SDS-PAGE results ( figure 1 ) showed that the recombinant xylosidase was expressed in Escherichia c...

Embodiment 3

[0055] The activity analysis of embodiment 3 recombinant xylosidases

[0056] Determination of xylosidase activity: measure the amount of p-nitrophenol produced by enzymatic hydrolysis of substrate pNPX at 405 nm. Reaction steps: Mix 250 μL of 2mM pNPX substrate with 150 μL of buffer, add 100 μL of appropriately diluted enzyme solution, react at 50°C for 10 min, add 1.5 mL of 1M Na2CO3 to terminate the reaction, and measure the OD value at 405 nm.

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Abstract

The invention relates to the field of the genetic engineering, and in particular relates to xylosidase Xyl43B with high xylose tolerance, and a gene and an application thereof. The invention provides xylosidase Xyl43B which is from humicola sp.L8, and an amino acid sequence of xylosidase Xyl43B is shown as SEQ ID No.1. The invention provides a coding gene xyl43B for coding xylosidase. The xylosidase has the following properties that the xylose tolerance is high (ki value is 292mM), the xylosidase is stable at the preferable condition that pH is 7.0 and between 5.5 and pH 10.0, and the preferable temperature is 50DEG C and xylosidase has good thermal stability at the temperature of 50DEG C. As a novel zymin, the xylosidase can be widely applied to the food, feed and energy industries.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular, the invention relates to a high xylose tolerance xylosidase Xyl43B and its gene and application. Background technique [0002] As the main component of plant cell walls, hemicellulose is the most abundant carbohydrate after cellulose in terrestrial plants. Improving its biodegradability has far-reaching biological significance. Therefore, hemicellulose is currently used as a sustainable The potential of energy has been paid more and more attention. Among them, xylan is the representative main component of hemicellulose, the second most abundant polysaccharide after cellulose in nature, and the easiest type of hemicellulose to extract, degrade and utilize. Since the main chain of xylan is formed by connecting xylose with β-1,4 glycosidic bonds, and the side chains are modified by various substituents, its degradation requires the synergistic action of main chain hydrolase and bra...

Claims

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Application Information

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IPC IPC(8): C12N9/42C12N15/56C12N15/63C12N1/21C12N1/19C12R1/19C12R1/645
Inventor 王兴吉郭庆文刘文龙张杰
Owner SHANDONG LONGKETE ENZYME PREPARATION
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