Novel beta-mannase and gene from 113th family of alicyclobacillus acidoterrestris and application thereof

A technology of mannanase and acidophilic thermostable bacteria, applied in the field of genetic engineering, can solve the problems of poor thermal stability, low expression level, inappropriate pH range, etc., and achieve good thermal stability

Active Publication Date: 2014-06-11
WUHAN SUNHY BIOLOGICAL +1
View PDF4 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The properties of β-mannanase that have been reported so far all have some defects, such as inappropriate pH range, poor thermal stability, low expression level, etc., which cannot meet the needs of practical applications.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Novel beta-mannase and gene from 113th family of alicyclobacillus acidoterrestris and application thereof
  • Novel beta-mannase and gene from 113th family of alicyclobacillus acidoterrestris and application thereof
  • Novel beta-mannase and gene from 113th family of alicyclobacillus acidoterrestris and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0077] The screening and separation of embodiment 1 Alicyclobacillus

[0078] Fill a 100mL Erlenmeyer flask with 7# liquid medium, adjust the pH to 2.0, and sterilize it for use. Take about 0.5 g of the soil sample of the high hot spring flow out of the refrigerator at 4°C in a sterile operating table, put it in the above-mentioned Erlenmeyer flask, and culture it statically at 60°C for 24 hours to carry out enrichment culture.

[0079] Separation by gradient dilution method: Take 1 mL of enriched and cultured bacterial solution, add it into a test tube filled with 9 mL of sterile saline, mix well, and then make 10 -1 bacterial suspension. Then gradually dilute to 10 according to the multiple relationship of 10 -7 . take 10 -5 、10 -6 Each 100 μL of the gradient soil suspension was applied to a 7# solid medium plate, and cultured in a 60°C incubator.

[0080] After 2-3 days, colonies were grown, and 26 bacterial strains were randomly selected according to the differences ...

Embodiment 2

[0081] Example 2 Extraction of Alicyclobacillus Genome and Identification of 16s DNA

[0082] Genomic DNA extraction: take the bacterial solution cultured at 60°C for 2 days and centrifuge at 10,000rpm for 2min. Take 100 mg of bacterial cells and add 500 μL of sterile water to wash, and centrifuge to get the precipitate. The precipitate was resuspended in 500 μL extract mixture, incubated at 37°C for 60 min, and centrifuged at 10,000 rpm for 10 min to remove the precipitate. The supernatant was extracted sequentially with equal volumes of phenol, phenol:chloroform, and chloroform. Take the upper layer solution and add 0.6-1 times the volume of isopropanol to precipitate at room temperature for 10 minutes. Centrifuge at 12000rpm for 15min. The precipitate was washed with 70% ethanol, centrifuged slightly, dried and dissolved in 30 μL sterile water for later use.

[0083] Identification of 16s DNA: 16S rDNA universal primers 27F, (5-AGAGTTTGATCCTGGCTCAG-3) and 1492R, (5-GGTT...

Embodiment 3

[0084] Example 3 Cloning of Alicyclobacillus β-mannanase Encoding Gene man113A

[0085] The degenerate primers were designed and synthesized according to the conserved sequence of the β-mannanase gene of the 113th family, and the degenerate PCR amplification was carried out using the extracted Alicyclobacillus genome as a template. The PCR reaction parameters are: 95°C for 5min; 94°C for 30sec, 50~45°C for 30sec, 72°C for 30sec, 12 cycles (the annealing temperature drops by 1°C after each cycle); 94°C for 30min, 45°C for 30sec, 72°C 30sec, 30 cycles; 10min at 72°C. The amplified products were detected and analyzed by electrophoresis, and fragments of appropriate size were recovered for sequencing. Analysis of sequencing results showed that the obtained gene fragment of about 200bp was a β-mannanase gene fragment.

[0086] According to the known sequence, the upstream and downstream specific primers were respectively designed by TAIL-PCR method, and the entire sequence of the...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

PropertyMeasurementUnit
molecular weightaaaaaaaaaa
molecular weightaaaaaaaaaa
molecular weightaaaaaaaaaa
Login to view more

Abstract

The invention relates to the field of gene engineering, and in particular relates to a novel beta-mannase and gene from 113th family of alicyclobacillus acidoterrestris and application thereof. The beta-mannase MAN113A amino with an acid sequence shown as a SEQ ID NO.1 provided by the invention has the following advantages: wide pH (5-8) scope, good thermal stability and strong protease resistance. All these advantages mean that the novel beta-mannase has better application value in fields such as feed, food and pharmacy than the previously reported mannase.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a new β-mannanase derived from the 113th family of acidophilus thermostable bacteria, a gene and an application thereof. 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) is a class of endohydrolase that can hydrolyze mannan (including isomannan) containing mannosidic ...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/42C12N15/56C12N15/63C12N1/19C12N1/21C12N1/15C12R1/84C12R1/865C12R1/19C12R1/66C12R1/885C12R1/01
Inventor 詹志春陶纯长方萍
Owner WUHAN SUNHY BIOLOGICAL
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap