High-activity beta-mannanase MAN5A with pH value within range of 2.5-6.5, and gene and application thereof

A technology of mannanase and high activity, which is applied in the field of genetic engineering and can solve problems such as low expression, poor thermal stability, and inappropriate pH range

Active Publication Date: 2013-03-20
黑龙江卫诺恩生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, at home and abroad, although many β-mannanases have been cloned and isolated and their properties are determined, there are some defects in the properties and characteristics of these enzymes, for example, the pH range is not suitable, the thermal stability is poor, and the expression level is low. Meet the needs of practical applications

Method used

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  • High-activity beta-mannanase MAN5A with pH value within range of 2.5-6.5, and gene and application thereof
  • High-activity beta-mannanase MAN5A with pH value within range of 2.5-6.5, and gene and application thereof
  • High-activity beta-mannanase MAN5A with pH value within range of 2.5-6.5, and gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0121] Example 1 Extraction of Penicillium sp WN1 Genomic DNA

[0122] The present invention separates the fungus WN1 from the acid wastewater of the Yunnan tin mine. According to the morphology and ITS sequence, it was identified as Penicillium genus and named WN1 (Penicillium sp.). The strain was preserved on August 11, 2011 in the General Microbiology Center of the China Committee for the Collection of Microbial Cultures (No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101), and its preservation number is: CGMCC No. 5131.

[0123] Penicillium WN1 was cultured in potato juice medium and then inoculated in induction medium ((NH 4 ) 2 SO 4 5g / L, KH 2 PO 4 1g / L, MgSO 4 ·7H 2 O 0.5g / L, FeSO 4 ·7H 2 O 0.01g / L, CaCl 2 0.2g / L, 0.5% konjac flour, 1% soybean meal, 1.5% agarose, pH 5.0) plate, cultivated at 30°C for 5-6 days, and measured the activity of producing β-mannanase. It was determined that...

Embodiment 2

[0125] Example 2 Cloning of the gene man5A encoding the acidophilic fungus Penicillium sp·WN1β-mannanase

[0126] The degenerate primers Man5P1 and Man5P2 were designed and synthesized according to the published conserved sequence of β-mannanase gene (see Table 1). PCR amplification was performed using the total DNA of Penicillium sp.WN1 as a template. The PCR reaction parameters are: 95°C, 5min; 94°C, 30sec, 50-45°C, 30sec, 72°C, 30sec, 12 cycles (the annealing temperature drops by 1°C after each cycle); 94°C, 30min, 45 ℃, 30sec, 72℃, 30sec, 30 cycles; 72℃, 10min. A fragment of about 170bp was obtained, which was recovered and sent to Sanbo Biotechnology Co., Ltd. for sequencing.

[0127] TAIL-PCR primers usp1, usp2, usp3; dsp1, dsp2, dsp3 were designed according to the nucleotide sequence obtained by sequencing (see Table 1). The flanking sequence of the known gene sequence was obtained by TAIL-PCR, and the amplified product was recovered and sent to Sanbo Biotechnology C...

Embodiment 3

[0131] RT-PCR analysis of embodiment 3β-mannanase gene

[0132] Extract Penicillium sp.WN1 total RNA, using Oligo(dT) 20 and reverse transcriptase to obtain a strand of cDNA, then design primers MAN5AF and MAN5AR (see Table 1) for amplifying the open reading frame, amplify the single-stranded cDNA, obtain the cDNA sequence of mannanase, and amplify to obtain the product After recovery, it was sent to Sanbo Biotechnology Co., Ltd. for sequencing, and the sequencing result is shown in SEQ ID NO.4.

[0133] After comparing the genome sequence and cDNA sequence of mannanase, it is found that the gene contains 2 introns, the cDNA is 1308bp long, encodes 435 amino acids and a stop codon, and the N-terminal 20 amino acids are its predicted signals Peptide sequence, the measured amino acid sequence of the mature protein of the gene Man5A has the highest identity of 82% with a predicted endoproteinase-1,4-β-mannosidase derived from Talaromycesstipitatus ATCC 10500 (XP002480621). The ...

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Abstract

The invention relates to the field of genetic engineering, and specifically relates to a high-activity beta-mannanase MAN5A with a pH value within the range of 2.5-6.5, and a gene and an application thereof. The invention provides the novel beta-mannanase MAN5A with an amino acid sequence represented by SEQ ID No.1 or 2. The invention also provides a gene coding the beta-mannanase MAN5A. The nucleotide sequence of the gene is represented by SEQ ID No.3 or 4. The invention also provides a recombinant vector and a recombinant strain containing the gene, and an application thereof. The high-activity beta-mannanase MAN5A with the pH value within the range of 2.5-6 is highly acidophilic, and has a wide action pH range, good heat resistance, and good protease resistance. The beta-mannanase MAN5A can be applied in industries such as feedstuffs, foodstuffs, medicines, and the like. With a technical scheme provided by the invention, beta-mannanase which has excellent property and which is suitable for industrial application can be produced with a genetic engineering method.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular, the invention relates to a beta-mannanase MAN5A with high activity in the range of pH 2.5-6.5, its gene and application. Background technique [0002] Plant cell walls are mainly composed of cellulose, hemicellulose, and lignin. Mannan is an important component of plant hemicellulose. It is a linear polymer connected by β-1,4-D-mannose. The side chains of the polysaccharide mainly contain glucosyl, acetyl and hemicellulose. Lactosyl and other substituent groups. Mannan has high hydrophilicity, absorbs a large amount of water in the digestive tract of monogastric animals, increases the viscosity of the contents of the digestive tract, resists gastrointestinal peristalsis, and directly affects the digestion and absorption of nutrients by animals. [0003] β-mannanase (β-mannanase EC3.2.1.78) is an endohydrolase that hydrolyzes mannan. It degrades the β-1, 4 glycosidic bonds of th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/42C12N15/56C12N15/63C12N1/19C12N1/14C12R1/80C12R1/84
Inventor 张王照邵娜张健康朱世琴董玲丽姜小伟
Owner 黑龙江卫诺恩生物技术有限公司
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