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

Beta-1, 4-endo-chitosanase (Aus CsnA) gene cloning and preparation of recombinase

A chitosanase and gene technology, applied in the field of bioengineering, can solve problems such as no reports, and achieve the effects of large-scale industrial production, high catalytic activity and thermal stability

Inactive Publication Date: 2012-03-28
JIANGNAN UNIV
View PDF0 Cites 2 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the research on chitosanase mainly focuses on bacteria and actinomycetes, and there is no report on the cloning and expression of the chitosanase gene (Aus csnA) derived from the fungus Aspergillus usamii.

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
  • Beta-1, 4-endo-chitosanase (Aus CsnA) gene cloning and preparation of recombinase
  • Beta-1, 4-endo-chitosanase (Aus CsnA) gene cloning and preparation of recombinase
  • Beta-1, 4-endo-chitosanase (Aus CsnA) gene cloning and preparation of recombinase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Cloning of Example 1 Aus csnA 3' end mRNA sequence

[0036] The first strand of cDNA was synthesized by reverse transcription with Oligo dT-Adaptor Primer as primers; the first round of PCR was performed with M13 Primer M4 and Csn-F1 as primers, and the reaction conditions were: 94°C for 2min, 30 cycles (94°C 30s, 52°C for 30s, 72°C for 90s), 72°C for 10min; use M13 Primer M4 and Csn-F2 as primers for the second round of PCR, and the reaction conditions are: 94°C for 2min, 30 cycles (94°C for 30s, 52°C 30s, 72°C 90s), 72°C 10min. The two rounds of PCR products were analyzed by 1% agarose gel electrophoresis, and the target band was recovered and ligated with pUCm-T (pUCm-T-csnA3′), transformed into JM109, and then sent to Shanghai Sangon for sequencing.

Embodiment 2A

[0037] Cloning of embodiment 2 Aus csnA 5' end mRNA sequence

[0038] The first round of PCR was performed using the Outer Primer and Csn-RE1 of the 5′-Full RACE Kit as primers. The reaction conditions were: 94°C for 3min, 30 cycles (94°C for 30s, 55°C for 30s, 72°C for 60s), 72°C 10 min; the second round of PCR was performed using Inner Primer and Csn-RE2 as primers, and the reaction conditions were: 94°C for 3 min, 30 cycles (94°C for 30s, 55°C for 30s, 72°C for 60s), and 72°C for 10 min. The two rounds of PCR products were analyzed by 1% agarose gel electrophoresis, and the target band was recovered and ligated with pUCm-T (pUCm-T-csnA5′), transformed into JM109, and then sent to Shanghai Sangon for sequencing.

Embodiment 3

[0039] Example 3 Cloning of Aus csnA 5' end promoter sequence

[0040] Using the processed A.usamii E001 genomic DNA as a template, the first round of PCR used T-PrimerF and Csn-RE1 as primers, and the reaction conditions were: 94°C for 4min, 30 cycles (94°C for 30s, 55°C for 30s, 72°C 60s), 72°C for 10min; the second round of PCR uses T-PrimerF and Csn-RE2 as primers, and the reaction conditions are: 94°C, 4min, 30 cycles (94°C for 30s, 55°C for 30s, 72°C for 60s), 72°C 10min. The two rounds of PCR products were analyzed by 1% agarose gel electrophoresis, and the target band was recovered by tapping the gel and ligated with pUCm-T (pUCm-T-csnAP), transformed into JM109, and then sent to Shanghai Sangon for sequencing.

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

No PUM Login to View More

Abstract

The invention provides a cloning method of complete mRNA and DNA sequences of a novel endo-chitosanase gene originated from Aspergillus usamii E001 strain. The nucleotide sequences are respectively SEQ ID NO:1 and SEQ ID NO:2. It shows through bioinformatics analysis that the endo-chitosanase belongs to glucoside hydrolase family 75 and is named as Aus CsnA. The amino acid sequence is SEQ ID NO:3 and the corresponding gene is named as Aus csnA. The invention also discloses an Aus CsnA engineering bacteria construction method and a recombinant Aus CsnA highly expression and purification method. The most appropriate operation temperature and pH of the prepared recombinant Aus CsnA are respectively 50 DEG C and 5.0, and the recombinant Aus CsnA is stable below 50 DEG C when pH is 4.0-7.0. The invention has great industrial production potential and economical values.

Description

technical field [0001] The present invention relates to the cloning of the complete mRNA and DNA sequence of a chitosanase gene derived from Aspergillus usamii (Aspergillus usamii) E001 strain, the construction of chitosanase engineering bacteria and the preparation of recombinant chitosanase The method for high-efficiency expression and purification belongs to the technical field of bioengineering. Background technique [0002] Chitosan (chitosan), also known as deacetylated chitin, soluble chitin, polychitosan, chitosan, polyglucosamine, etc., is a product obtained by deacetylating chitin, usually chitin When the degree of deacetylation exceeds 70%, it is called chitosan. Chitosan widely exists in shells of shrimps, crabs, insects, and cell walls of fungi and algae. The obtained chitosan oligosaccharide not only has the advantages of good water solubility and easy absorption, but also has many unique physiological activities and functional properties in recent years. Ch...

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
IPC IPC(8): C12N15/56C12N9/42C12N15/10C12N1/19C12N15/81C12R1/66C12R1/84
Inventor 邬敏辰胡蝶史红玲陈忠法李剑芳
Owner JIANGNAN UNIV
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