Acidic beta-glucanase P-Bglu16A, and gene and application thereof

A technology of p-bglu16a and ppic9-p-bglu16a, which is applied to acid beta-glucanase P-Bglu16A and its gene and application fields, and can solve problems such as limited application effect and the like

Active Publication Date: 2014-04-09
北京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

[0005] β-glucanase is the earliest enzyme used in the feed industry. It mainly comes from the 16 family β-glucanase of Bacillus. The pH of its action is neutral, which limits its application effect in feed

Method used

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  • Acidic beta-glucanase P-Bglu16A, and gene and application thereof
  • Acidic beta-glucanase P-Bglu16A, and gene and application thereof
  • Acidic beta-glucanase P-Bglu16A, and gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0115] The isolation of the genomic DNA of embodiment 1 Penicillium sp.WN1 bacterial strain

[0116] 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.

[0117] 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, 1% corn cob powder, 1% bran, 1.5% agarose, pH5.0) plate, cultivated at 30°C for 5-6d, and measured its activity of producing β-glucanase. It w...

Embodiment 2

[0119] Example 2 Cloning of the gene encoding Penicillium sp.WN1β-glucanase

[0120] The degenerate primers GH16F: TGCGGTAYNTGGCCNGC and GH16R: CCGGCCCANTBNCCRCARAA were designed and synthesized according to the conserved sequence (CGTWPA and FCGDWAG) of the 16th family of glycoside hydrolases β-glucanase, where: Y=C / T, R=A / G, B =G / C / T, N=A / T / G / C

[0121] Genomic DNA of Penicillium sp.WN1 was used as template, and GH16F and GH16R were used as primers for PCR amplification. The PCR reaction parameters are: 95°C, 5min; 94°C, 30sec, 49°C, 30sec, 72°C, 1min, after 30 cycles; 72°C, 10min, 4°C heat preservation. Agarose electrophoresis detection. The obtained fragment with a length of about 580bp was recovered and ligated with pEASY-T3 vector and sequenced. The 588bp nucleotide sequence of the conserved region was obtained.

[0122] In order to obtain the full-length sequence of the coding gene, according to the nucleotide sequence of the conserved region obtained by sequencing,...

Embodiment 3

[0126] Example 3 Isolate total RNA from Penicillium Peniciilium sp.WN1 and synthesize cDNA

[0127]Penicillium sp.WN1 was grown under conditions inducing β-glucanase, and based on the determination of β-glucanase activity in the culture supernatant, mycelia were harvested by filtration at several time points and immediately lysed. Freeze in nitrogen and grind in a mortar. And the total RNA of Penicillium sp.WN1 was extracted according to the instructions purchased from Promega RNA extraction kit.

[0128] The first-strand cDNA was synthesized using the reverse transcription kit purchased from Toyobo Co., Ltd. and following the operating instructions. Then designed primers Pbglu F: 5′-ATGCGTCCTTCGACTCTCCTTCAACTGGC-3′, and PbgluR: 5′-CTAGCTTGAGTAGACCTTGAGAGAGTTGATGAG-3′ to obtain the cDNA sequence encoding β-glucanase P-Bglu16A. After the amplified product was recovered, it was sent to Sanbo Biotechnology Co., Ltd. for sequencing. The sequencing result is shown in SEQ ID NO.4....

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Abstract

The invention relates to the field of genetic engineering, and specifically relates to an acidic beta-glucanase P-Bglu16A, and a gene and an application thereof. The invention provides the novel acidic beta-glucanase P-Bglu16A with an amino acid sequence represented by SEQ ID No.1 or 2. The invention also provides a gene coding the acidic beta-glucanase P-Bglu16A. The nucleotide sequence of the gene is represented by SEQ ID No.3, 4, or 5. The invention also provides a recombinant vector and a recombinant strain containing the gene, and an application thereof. The acidic beta-glucanase P-Bglu16A has a wide action pH range (pH 3.5-6.0), an optimal pH of 5.0, an optimal temperature of 50-55 DEG C, and good stability and heat resistance under acidic condition. The acidic beta-glucanase P-Bglu16A can be applied in industries such as feedstuffs, brewing, winemaking, baking, and the like. With a technical scheme provided by the invention, beta-glucanase 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 an acidic β-glucanase P-Bglu16A and its gene and application. Background technique [0002] β-glucan is the main component of the cell wall of cereal plants, especially abundant in the endosperm cell walls of barley, oats, wheat, and rice (Buliga et al. Carbohydr Res 1986, 157: 139-156). β-glucan is a linear molecule formed by connecting more than 1200 pyranoid D-glucose residues with β-1, 4 and 1, 3-glycosidic bonds. The unique molecular structure of β-glucan makes it soluble in water in the form of high molecular weight, and the solution viscosity is very high, which brings a lot of inconvenience to industrial production. For example: β-glucan absorbs water and swells and sticks in the digestive tract, making it the main anti-nutritional factor that limits the effective utilization of nutrients in wheat feed. High molecular weight barley β-glucan can ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/24C12N15/56C12N15/63C12N1/19C12N1/15C12N15/81C12N15/80C12R1/84C12R1/865C12R1/80
Inventor 张健康张王照朱世琴邵娜姜小伟董玲丽
Owner 北京卫诺恩生物技术有限公司
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