Optimized acid-resistant mannase MAN26gy as well as preparation method and application thereof

A technology of mannanase and acid resistance, which is applied in the field of genetic engineering and enzyme engineering, and can solve the problem that the energy utilization rate is only 50-60%.

Active Publication Date: 2014-04-02
QINGDAO GENYUAN BIOLOGICAL TECH GRP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Soybean meal, cotton meal, and rapeseed meal are the most commonly used vegetable protein raw materials, but the energy utilization rate of soybean meal for monogastric poultry (including pigs and chickens) is only 50-60%

Method used

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  • Optimized acid-resistant mannase MAN26gy as well as preparation method and application thereof
  • Optimized acid-resistant mannase MAN26gy as well as preparation method and application thereof
  • Optimized acid-resistant mannase MAN26gy as well as preparation method and application thereof

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Experimental program
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Effect test

Embodiment 1

[0022] 1. Site-directed mutation of GH26β-mannanase gene

[0023] The mannanase MAN26gy of the present invention is derived from Bacillus subtilis (it can be derived from commercially available Bacillus subtilis, such as purchased from the Agricultural Microorganism Culture Collection Center, its number is ACCC01779), the amino acid sequence of mannanase MAN26gy As the following SEQ ID No:1:

[0024] LFKKHTISLLILFLLASAVLAKPIEAHTVSPVNPNAQQTTKAVMNWLAHLPNRTENRVLSGAFGGYSHDTFSMAEADRIRSATG L SPAIYGCDYARGWLETA N IEDSIDVSCNGDLISYWKNGGIPQISLHLANPAFQSGHFKTPITNDQYKKILDSSTAEGKRLNAMLSKIADGLQELENQGVPVLFRPLHEMNGEWFWWGLTSYNQKDNERISLYKQLYKKIYHYMTDTRGLDHLIWVYSPDANRDFKTDFYPGASYVLTALNKPFAFTEVGPQTANGSFDYSLFINAIKQKYPKTIYFLAWNDEWSPAVNKGASALYHDSWTLNKGEIWNGDSLTPIVE。

[0025] Encoding the above-mentioned mannanase gene man26gy derived from Bacillus subtilis, its genome sequence is as follows SEQ ID No: 2:

[0026] Ttgtttaagaaacatacgatctctttgctcattttatttttacttgcgtctgctgttttagcaaaaccaattgaagcgcatactgt...

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Abstract

The invention provides optimized acid-resistant mannase MAN26gy and application thereof. 85-th-site amino acid leucine (L) in an amino acid sequence of mature mannase is modified into aspartic acid (D); 103-th-site amino acid asparaginate (N) is modified into aspartic acid (D). The optimal pH value of the modified mannase is 6.0 which is improved greater in comparison with the original mannase (the optimal pH value being 7.0); the acid resistance of the modified mannase is further improved, so that over 80% of the greatest enzyme activity between the pH values of 4.0 and 7.0 can be kept, and 70% of the greatest enzyme activity also can be kept when the pH value is 3.0. According to the invention, the enzymatic properties of the modified mannase are researched, so that greater experimental data is provided for enzyme application.

Description

technical field [0001] The invention belongs to the field of genetic engineering and enzyme engineering, and specifically relates to an optimized acid-resistant mannanase MAN26gy and its preparation method and application. Background technique [0002] β-mannanase (EC3.2.1.78) is a type of hemicellulase that can hydrolyze mannan. It also exists in some lower animals. The properties of mannanase vary greatly due to different sources. Generally speaking, the molecular weight of β-mannanase in bacteria is mostly between 35kD and 55kD, and the optimum pH value is slightly acidic or neutral, and the optimum pH value of some alkalophilic bacillus can reach 9.0~10.0 . The molecular weight of β-mannanase derived from fungi is about 45kD-55kD, and the optimum pH value is 4.5-5.5. Compared with bacteria, the pH stability and optimum pH value of mannanase derived from fungi are lower, and the heat resistance is also worse than that of bacteria. The molecular weight of mannanase pr...

Claims

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Application Information

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IPC IPC(8): C12N9/42C12N15/56C12N15/63C12N1/21C12N1/19A23K1/165C12R1/19
CPCC12N9/2494C12Y302/01078
Inventor 张大伟杜彦龙张明俊赵志强
Owner QINGDAO GENYUAN BIOLOGICAL TECH GRP
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