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Optimized high-temperature resistant mannanase MAN5gy, and preparation method and application thereof

A technology of mannanase and high temperature resistance, applied in the field of genetic engineering and enzyme engineering, can solve problems such as unsatisfactory effect and loss of mannanase enzyme activity, achieve wide pH stability, reduce anti-nutritional effect, and high The effect of enzyme activity

Active Publication Date: 2014-01-22
QINGDAO GENYUAN BIOLOGICAL TECH GRP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

A key problem in the application of mannanase in the feed industry is that it needs to have better high temperature resistance. Feed granulation needs to be carried out in a high temperature environment, and the activity of mannanase that is not resistant to high temperature will be greatly lost
At present, many researchers have studied the heat resistance of mannanase from different aspects, such as screening heat-resistant phytase-producing strains and using modern molecular biology methods such as protein engineering to obtain high-temperature-resistant mannanase. Sugar enzyme, the effect is not very ideal

Method used

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  • Optimized high-temperature resistant mannanase MAN5gy, and preparation method and application thereof
  • Optimized high-temperature resistant mannanase MAN5gy, and preparation method and application thereof
  • Optimized high-temperature resistant mannanase MAN5gy, and preparation method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Embodiment 1, the acquisition of Aspergillus niger mannanase protein mutant gene

[0026] 1. Select Aspergillus niger Aspergillus niger The amino acid sequence of the mannanase in is shown in SEQ ID NO.1.

[0027] SEQ ID NO. 1:

[0028] mklssslltlaslalanvstalpkaspapstsssaastsfastsglqftidgetgyfagtnsywigfltdnadvdlvmghlkssglkilrvwgfndvtsqpssgtvwyqlhqdgkstintgadglqrldyvvssaeqhdikliinfvnywtdyggmsayvsayggsgetdfytsdtmqsayqtyiktvverysnssavfawelaneprcpscdtsvlynwiektskfikgldadrmvcigdegfglnidsdgsypyqfseglnftmnlgidtidfgtlhlypdswgtsddwgngwitahgaackaagkpclleeygvtsnhcsvegswqktalsttgvgadlfwqygddlstgkspddgntiyygtsdyqclvtdhvaaidsa

[0029] Among them, the enzyme gene encodes 383 amino acids, and the N-terminal 21 amino acids are its predicted signal peptide sequence "mklssslltlaslalanvsta".

[0030] The genome sequence of the gene encoding the above-mentioned mannanase derived from Aspergillus niger is shown in SEQ ID NO. 2.

[0031]Atgaagctctccagctccctcctcaccctggctagcctggcgctggcca...

Embodiment 2

[0043] Example 2 Construction of Aspergillus niger mannanase engineering bacteria

[0044] The yeast expression vector used is pPICZαA, and the host cell for constructing the vector is Escherichia coli DH5α. Design expression primers with restriction sites based on the synthetic mannanase gene sequence:

[0045] Upstream Expression Primer 5'-GGG GAATTC ctgccgaaagcctcccctgc-3' (SEQ ID No: 6)

[0046] Downstream expression primer 5'-GGG GCGGCCGC TTAGGCGCTATCAATAGCAGC-3' (SEQ ID No: 7)

[0047] Carry out PCR amplification to the cloning plasmid, and when the full-length mannanase gene is obtained, it is subjected to double enzyme digestion ( Eco R I+ not 1), simultaneously the expression vector pPICZαA is carried out double enzyme cutting ( Eco R I+ not I), after purifying and recovering the digested product, the two were ligated overnight at 16°C with T4 DNA ligase, and the ligated product was transformed into DH5α competent cells to obtain the recombinant express...

Embodiment 3

[0049] Example 3 Induced Expression of Recombinant Mannanase

[0050] Inoculate the monoclonal strains with high viability screened in 30 mL of BMGY culture medium, shake at 30 °C and 200 rpm until the OD value is 2-4, about 1 day, inoculate in a 1 L shake flask containing 400 mL of BMGY for shaking After culturing for 48 hours, the cells were collected by centrifugation. Then resuspend in 200mL BMMY medium, shake culture at 200 rpm at 30°C. After 72 hours of methanol induction, the supernatant enzyme solution was collected by centrifugation. Carry out SDS-PAGE protein electrophoresis detection, the target protein band is as follows figure 2 shown. Take part of the supernatant enzyme solution and dilute to an appropriate multiple to measure the activity of mannanase.

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Abstract

The invention provides an optimized high-temperature resistant mannanase MAN5gy, and a preparation method and application thereof. According to the invention, mutation of mannanase MAN5gy from Aspergillus niger is achieved to obtain an amino acid sequence shown as in a SEQ ID NO.3, and the invention provides a coding gene man5gy for encoding the above ala mannanase. The mutated mannanase provided by the invention has the following properties: optimal pH value of 4.0 and optimum temperature of 65 DEG C. The mannanase is suitable for feed industry and has synergetic effect with xylanase, so as to effectively eliminate or reduce the anti-nutrition function caused by the increase of viscosity. In the oil and natural gas exploitation industry, mannanase can well degrade galactomannan to enhance flow velocity of oil or gas. Due to the high temperature (>80 DEG C) in the oil exploitation process, the high-temperature resistant mannanase can play a good role.

Description

technical field [0001] The invention belongs to the field of genetic engineering and enzyme engineering, and in particular relates to an optimized high-temperature-resistant mannanase MAN5gy and its preparation method and application. Background technique [0002] Mannan and xylan are the two most important polysaccharides in hemicellulose, and mannan dominates in cork and some special tissues such as fruits and seeds. Mannan is a linear polysaccharide polymerized by β-1,4-mannose sugar bonds. According to the composition of the main chain, it can be divided into two categories: linear mannan and glucomannan. Mannanase (EC3.2.1.78) is a kind of endohydrolase that catalyzes the hydrolysis of β-1,4-mannan glycosidic bonds. It has a wide range of substrates and belongs to hemicellulase. [0003] Mannanase has a wide range of sources. Mannanase in plants exists in storage organs such as seeds and fruits; it is also found in some lower animals such as marine molluscs: snails, b...

Claims

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Application Information

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IPC IPC(8): C12N9/42C12N15/56C12N15/63A23K1/165
CPCA23K20/189C12N9/2494C12Y302/01078
Inventor 张大伟杜彦龙
Owner QINGDAO GENYUAN BIOLOGICAL TECH GRP
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