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Alkaline beta-mannase, encoding genes thereof and application of encoding genes

A mannanase, alkaline technology, applied in the field of genetic engineering, can solve the problems of low enzyme activity, low expression, poor stability, etc., and achieve the effect of high activity, high activity and strong stability

Active Publication Date: 2016-07-13
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, a number of alkaline β-mannanase genes from bacteria have been cloned and expressed, but they still face the problems of low enzyme activity, poor stability, and low expression in alkaline or high temperature conditions for industrial applications. Can not meet the requirements of practical industrial applications

Method used

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  • Alkaline beta-mannase, encoding genes thereof and application of encoding genes
  • Alkaline beta-mannase, encoding genes thereof and application of encoding genes
  • Alkaline beta-mannase, encoding genes thereof and application of encoding genes

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preparation example Construction

[0048] The method for preparing alkaline β-mannanase provided by the invention comprises: cultivating the recombinant strain provided by the invention, inducing the expression of the gene encoding alkaline β-mannanase; separating and purifying the expressed alkaline β-mannanase Glycanase. The culture conditions are conventional culture conditions, such as using LB medium (the solvent is water, the solute and its final concentration are respectively: Tryptone10g / L, yeast extract 5g / L, NaCl10g / L), at 35-37°C Grow to OD 600 is 0.6. Since the recombinant strain provided by the invention contains the gene encoding the alkaline β-mannanase, it can efficiently express the alkaline β-mannanase. After culturing, the high-purity alkaline β-mannanase can be obtained through separation and purification. Methods known to those skilled in the art can be used for separation and purification (for example, adding isopropyl-β-d-thiogalactopyranoside (IPTG) to the culture medium to a final co...

Embodiment 1

[0055] Acquisition of alkaline β-mannanase and its coding gene

[0056] (1) Cloning of the gene encoding alkaline β-mannanase (ManA)

[0057] Take the Bacillus clausii S10 isolated from the alkaline hot spring sample in Inner Mongolia, use the genome extraction kit to extract the total DNA of the Bacillus clausii S10, and measure the purity of the DNA with a UV spectrophotometer. The result is: A260 / A280 = 1.88, A260 / A230 = 2.13. 10 μl of the total DNA solution (about 50 μg DNA) was taken, partially digested with restriction endonuclease Sau3AI, and a 2-8 kb DNA fragment was recovered by agarose gel electrophoresis. Then carry out ligation reaction, 4°C ligation reaction for 16 hours, the ligation system is as follows (20 μl):

[0058]

[0059]

[0060] Transform competent Escherichia coli DH5α with the product of the ligation reaction, and then apply it to a solid at pH 8.0 containing 60 μg / ml ampicillin (Amp), 20 μg / ml IPTG, 40 μg / ml galactoside (X-gal) and 0.5% konj...

Embodiment 2

[0076] Detection of Enzymatic Properties of Alkaline β-Mannanase (ManA)

[0077] (1) Standard enzyme activity assay method

[0078] Take 10 μl of the ManA enzyme solution obtained in Example 1 (diluted to 1.5 μg / ml) and 190 μL of glycine-NaOH buffer solution with a pH value of 9.5 containing 0.4% locust bean gum, mix well, react at 75°C for 10 minutes, and add 200 μL Dinitrosalicylic acid solution (DNS) terminated the reaction (edited by Zhang Longxiang et al., "Biochemical Experimental Methods and Techniques", Higher Education Press, 1996), and then measured the absorbance at 540nm after reacting in a boiling water bath for 5 minutes.

[0079] (2) Determination of the optimum pH value and pH stability of ManA

[0080] At 75°C, the ManA enzyme solution was subjected to enzymatic reactions in buffer solutions with different pH values ​​(pH5.5-11.5) to determine its optimum pH value. The remaining conditions were the same as (1), and the buffer solution used was pH5.5 -7.5Na ...

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Abstract

The invention discloses alkaline beta-mannase as shown in (a) or (b).The alkaline beta-mannase as shown in (a) is formed by the amino acid sequence as shown in SEQ ID No.1 or SEQ ID No.3. (b) is protein derived from (a) with unchanged enzyme activity, and is obtained by carrying out substituting, deleting or adding one or more amino acids on the amino sequence shown in SEQ ID No.1 or SEQ ID No.3 , or b is protein with amino sequence of SEQ ID No.1 or SEQ ID No.3 with tags on the amino terminal and / or carboxyl terminal. The invention further discloses genes capable of encoding the alkaline beta-mannase, a recombinant vector containing the genes, a recombinant strain containing the recombination vector and application of the genes, the recombinant vector and the recombinant strain.The invention also discloses a method for preparing the alkaline beta-mannase and a composition for degrading the mannase.The alkaline beta-mannase is high in heat resistance, activity and stability.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a heat-resistant alkaline β-mannanase, its encoding gene and its application. Background technique [0002] Plant hemicellulose is the most abundant polysaccharide compound in nature after cellulose, and mannan is one of the main components of hemicellulose, widely present in plant cell walls, seed endosperms, plant gums (carob gum, locust bean gum, etc.) In bean gum, guar gum), it is the main component of the cell wall of all leguminous plants, mainly in the form of glucomannan, galactomannan and galactoglucomannan. β-mannanase is a hemicellulolytic enzyme capable of degrading the main chain of mannan. It degrades β-1,4-D-mannosidic bonds in an endo-cutting manner to generate mannan oligosaccharides or mannan polysaccharides. β-mannanase has been widely used in many fields such as medicine, food, feed, papermaking, textile, printing and dyeing, washing, petroleum exploration,...

Claims

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Application Information

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IPC IPC(8): C12N9/42C12N15/56C12N15/70C12N15/75C12N15/81C12N1/19C12N1/21C12P19/14C12R1/19C12R1/125C12R1/865
Inventor 马延和周成薛燕芬
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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