Preparation method of high-temperature xylanase

Abstract

The invention relates to the field of biotechnology. A preparation method of high-temperature xylanase comprises the following steps: (1) preparation of crude enzyme: inoculating Thermobifida halotolerans YIM 90462<T> into liquid seed culture medium with inoculum size by 5 to 10 percent of volume, shaking culturing, transferring the Thermobifida halotolerans YIM 90462<T> into liquid fermentation medium by 5 to 10 percent of volume and obtaining crude enzyme; (2) concentrating the volume of the crude enzyme to 1/6 of original volume thereof by an ultra-filtration membrane package with molecular weight cut-off of 10 KDa; (3) adding ammonium sulfate into the concentrated enzyme to lead the saturation thereof to be 50 percent, obtaining precipitates by centrifugation and dissolving the precipitates in buffer solution; (4) taking supernatant fluid from the obtained liquid by centrifugation, adding the supernatant fluid into a Butyl-Sepharose chromatography column which is balanced by Tris-HCl buffer solution containing 0.3M of ammonium sulfate and having pH of 8.0; (5) taking supernatant fluid from the obtained liquid by centrifugation, adding the supernatant fluid into a Phenyl-Sepharose chromatography column which is balanced by Tris-HCl buffer solution containing 0.3M of ammonium sulfate and having pH of 8.0 and eluting the supernatant fluid with Tris-HCl buffer solution which does not contain ammonium sulfate and has pH of 8.0; (6) adding the supernatant fluid into Q-Sepharose chromatography column which is balanced by Tris-HCl buffer solution having pH of 8.0, collecting eluting peaks successively according to retention time, and remaining active components of the xylanase; and finally obtaining the xylanase with the activation of 573U/ml and purification yield of 9 percent.

Description

technical field [0001] The present invention relates to the field of biotechnology. Background technique [0002] Xylan is a hemicellulose and the second largest natural polysaccharide in terms of content. Xylan-like hemicellulose is a kind of renewable carbohydrates that need to be developed and utilized urgently. The xylose and a small amount of other monosaccharides produced after biodegradation can be used as basic carbon sources to produce various fermented products, including organic Acids, amino acids, single-cell proteins, sugar alcohols, industrial enzymes, solvents or fuel alcohols. The xylanase system mainly includes two enzymes, one is β-1,4-xylanase and the other is β-xylanase, and the two work together to degrade xylan into monosaccharides. Xylanase plays an important role in biotransformation, food, medicine, energy, papermaking, and feed industries. There are many kinds of microorganisms that can produce xylanase: fungi, bacteria, and actinomycetes can all...

AI Technical Summary

Problems solved by technology

Although there are many microorganisms that can produce xylanase, because the xylanase of most microor

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