Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

High-temperature resistant acidic beta-mannase Man-L30 and gene and application thereof

A mannanase and high temperature-resistant technology, applied in the field of genetic engineering, can solve the problems of unstable composition of enzymatic hydrolysis products, high production cost, low fermentation enzyme activity and the like

Active Publication Date: 2014-04-23
北京科为博生物科技有限公司
View PDF4 Cites 3 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, most of the researches of relevant domestic scientific research institutes are limited to the laboratory stage. The reported fermentation enzyme activity is low, the composition of enzymatic hydrolyzate is unstable, the production cost is high, and it is difficult to obtain a competitive advantage.

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
  • High-temperature resistant acidic beta-mannase Man-L30 and gene and application thereof
  • High-temperature resistant acidic beta-mannase Man-L30 and gene and application thereof
  • High-temperature resistant acidic beta-mannase Man-L30 and gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0101] Example 1 Cloning of Bacillus licheniformis CRVAB001β-mannanase encoding gene man-L30

[0102] Extraction of Bacillus licheniformis CRVAB001 genomic DNA:

[0103] (1) Take 0.5-2mL culture liquid, centrifuge at 10000rpm for 30s, absorb the supernatant as much as possible, and collect the bacteria;

[0104] (2) Add 200 μL buffer RB to the EP tube to resuspend, centrifuge at 10,000 rpm for 30 s, and discard the supernatant;

[0105] (3) For Gram-positive bacteria: add 120 μL lysozyme, mix by inverting, and bathe in 37℃ water for 30-60min;

[0106] (4) Centrifuge at 12000rpm for 2min, discard the supernatant and resuspend the cells in 180μL buffer RB by shaking or pipetting;

[0107] (5) Add 20 μL of RNase A (25 mg / mL) solution, vortex and mix, and place at room temperature for 5-10 minutes;

[0108] (6) Add 800 μL of binding solution CB, then add 100 μL of isopropanol, and vortex immediately to mix thoroughly, at this time flocculent precipitation may appear;

[0109] ...

Embodiment 2

[0120] Example 2 Preparation of recombinant β-mannanase Man-L30

[0121] Design synthetic expression primers according to the obtained gene sequence of β-mannanase Man-L30:

[0122] P3: 5'-CGGAATTCCATACAGTATCTCCAGTAAAT-3';

[0123] P4: 5'-CTGCGGCCGCTTCTGCAATTGGTGTTAAA-3'.

[0124] The total DNA of Bacillus licheniformis CRVAB001 was re-amplified by PCR to obtain the β-mannanase Man-L30 gene with a recombination restriction site. The expression vector pPIC9 was subjected to double enzyme digestion (EcoR I+Not I), and at the same time, the gene man-L30 encoding β-mannanase was subjected to double enzyme digestion (EcoR I+Not I) to obtain the mature β-mannanase The gene fragment of the carbohydrase was connected with the expression vector pPIC9 to obtain the recombinant plasmid pPIC-man-L30 containing the Bacillus licheniformis CRVAB001 gene man-L30 and transform Pichia pastoris GS115 by electroporation to obtain the recombinant Pichia pastoris strain GS115 / man-L30 .

[0125]...

Embodiment 3

[0126] Example 3 Analysis of Enzymatic Properties of Recombinant β-Mannanase

[0127] The DNS method was used to analyze the activity of the β-mannanase of the present invention. The specific method is as follows: at pH 4.5, 55°C, 1 mL of reaction system includes 100 μL of appropriate diluted enzyme solution, 900 μL of substrate, reacted for 10 minutes, added 1.5 mL of DNS to terminate the reaction, and boiled for 5 minutes. After cooling, the OD value was measured at 540 nm. Definition of β-mannanase activity unit: Under certain conditions, the amount of enzyme required to decompose β-mannan to generate 1 μmol reducing sugar per minute is 1 activity unit (U).

[0128] (1) Optimum pH and pH stability of β-mannanase Man-L30

[0129] The recombinant expressed β-mannanase Man-L30 was subjected to enzymatic reaction at different pH to determine its optimum pH. The buffer used is pH0.5-2.2 KCI-HCl buffer, pH2.2-8.0 disodium citric acid monohydrogen phosphate series buffer and pH...

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
Login to View More

Abstract

The invention relates to the field of genetic engineering, and in particular relates to high-temperature resistant acidic beta-mannase Man-L30 and a gene and application thereof. The high-temperature resistant acidic beta-mannase Man-L30 is characterized in that the amino acid sequence is as shown in SEQ ID No.1 or 2. The high-temperature resistant acidic beta-mannase gene man-L30 encodes the high-temperature resistant acidic beta-mannase Man-L30 as disclosed in claim 1 and has the nucleotide sequence as shown in SEQ ID No.4 or 5. The beta-mannase Man-L30 also has the characteristics of being resistant to high temperature, high in pH (Power of Hydrogen) stability, high in protease resistance, etc.; the pH is preferably 5.5; the enzyme can remain more than 65% of activity under the pH of 2.5 to 6.5; the temperature is preferably 50 DEG C, and the antipepsin and trypsin processing capacity is relatively high under such temperature. The high-temperature resistant acidic beta-mannase Man-L30 is applicable to the feeds, foods, medicines and other industrial fields.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular, the invention relates to a high-temperature-resistant acidic β-mannanase Man-L30 and its gene and application. Background technique [0002] β-mannanase (β-mannanase EC3.2.1.78) is a hemicellulolytic enzyme that degrades β-1,4 glycosidic bonds in an endo-cutting manner, and the non-reducing end of the degradation product is mannose. Products include glucomannan, galactomannan, and β-mannan. The source of β-mannanase is very wide, including plants, bacteria, fungi, actinomycetes and even molluscs. Mannan is the hemicellulose with the most widespread distribution and the highest content in plant feed materials except cellulose and xylan, and there is a large amount of amyloid accumulation in many plants. The digestive enzymes of livestock, poultry and fish do not contain mannanase, and the conversion of such substances requires the addition of exogenous enzymes. Adding mannanase...

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 Applications(China)
IPC IPC(8): C12N9/42C12N15/56C12N15/81C12N1/19C12R1/10C12R1/84C12R1/865C12R1/78
CPCC12N9/2494C12Y302/01078
Inventor 吴培均李富伟罗建杰李兆勇
Owner 北京科为博生物科技有限公司
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

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

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com