Construction method of recombinant xylanase XynB with anti-inhibition activity

Abstract

The invention discloses a construction method of a recombinant xylanase XynB with an anti-inhibition activity. The method comprises the steps that whole genome of wheat is extracted, following specific primers are used to amplify active site sequences in third exons of XynB genes of the wheat, gel is excised for DNA recovery by electrophoresis after the DNA fragments are digested with restrictionendonucleases EcoR V and EcoR I and construct recombinant expression vectors with a carrier pET30a+, a recombinant plasmid pET30a-Xyn B is obtained after transformation by transformed bacteria, the recombinant plasmid pET30a-Xyn B is transformed into engineering bacteria, monoclonal colonies are obtained after culturing and transferred to a culture medium for culturing and inducing protein expression to obtain recombinant xylanase XynB; wherein the specific primers are: forward primers: 5'-TC (u) GATATC ( / u) ATGCAGCTGGACAACGCCTT-3', reverse primers: 5'-TC (u) GAATTC ( / u) TGCGTCCGTCTTCCACTCCC-3'. The obtained recombinant xylanase XynB has a strong activity, and unpurified enzyme solution is taken to detect DNS. Birchwood xylan is used as a substrate to calculate, and a specific activity ofthe recombinant xylanase Xyn B can reach to 67.30U / mg.

Description

technical field [0001] The present invention relates to the construction of xylanase XynB, in particular to the construction method of recombinant xylanase XynB with anti-inhibition activity. Background technique [0002] Silage, as a traditional way of preserving overwintering forage crops green and juicy, uses fresh green forage as raw material, utilizes lactic acid bacteria that grow naturally on the surface of the plant, and uses anaerobic fermentation under airtight conditions to remove the soluble carbohydrates on the surface of the plant. It is converted into organic acids such as lactic acid and acetic acid, resulting in a decrease in feed pH, to inhibit the growth and reproduction of spoilage microbial flora, so as to achieve the purpose of maintaining the nutritional characteristics of crops. Xylan, as a poly-five-carbon sugar, is the main structural non-starch polysaccharide in the plant cell wall, and is an important part of plant hemicellulose, accounting for on...

AI Technical Summary

Problems solved by technology

It is gratifying that studies have shown that xylanase inhibitory proteins can inhibit the activity of exogenous xylanases (especially from bacteria and other sources), but they cannot inhibit the activity of plant endogenous xylanases (Dornez et al. a1.,2010)

However, directly extracting and isolating xylanase from plants requires a large number of plants, and the process is complicated, and the obtained xylanase activity is not ideal.

However, at present, there are relatively few studies on the cloning and expression of plant endogenous xylanases.

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