A gene encoding endo-β-1,4-mannanase and its preparation and application
A technology of mannanase and gene, which is applied in the field of endo-β-1,4-mannanase coding gene and its preparation and application, and can solve the problems of weak degradation activity and unsuitability for large-scale application
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0055] Example 1 Endo-β-1,4-mannanase full-length gene cloning
[0056] Genomic DNA of Paenibacillus polymyxa was extracted according to the operation steps of Genomic DNA Purification Kit (Thermo, LOT 00105781). After performing multiple sequence alignment analysis on the endo-β-1,4-mannanase gene sequence in The National Center for Biotechnology Information (NCBI) database, a degenerate primer ppman-F: 5'-CGGACGCATATGGAATCARCTMMCTTGGTCAC-3' was designed; ppman-R: 5'-GACGAGCTCGAGCTCCGCTTYATTYTTGGABRAAGTA-3', using the extracted Paenibacillus polymyxa genomic DNA as a template to amplify the gene sequence encoding the mature protein of endo-β-1,4-mannanase (excluding the signal peptide gene). The PCR reaction conditions were: 94°C for 2 min, 1 cycle; 94°C for 30 s, 55°C for 30 s, 72°C for 2 min, 30 cycles; 72°C for 5 min, 1 cycle. PCR products were analyzed by agarose gel electrophoresis (see figure 1 ), the target gene was recovered by cutting the gel, connected to the pro...
Embodiment 2
[0057] Example 2 Endo-β-1,4-mannanase gene sequence analysis
[0058]The sequencing results were analyzed using Basic Local Alignment Search Tool (BLAST) in the GenBank database, DNAMAN software was used for multiple sequence alignment, and Vector NTI was used to analyze sequence information.
[0059] The coding region of the obtained endo-β-1,4-mannanase gene (named ppman) is 1896 bp long, and its nucleotide sequence is shown in SEQ ID NO 1. ppman encodes 631 amino acids and a stop codon, its amino acid sequence is shown in SEQ ID NO 2, the theoretical molecular weight of the protein is 70.11kDa, and the predicted isoelectric point is 4.97. The amino acid encoded by ppman contains two carbohydrate binding modules CBM (Carbohydrate Binding Module) 35 and a GH26 family domain, and its domain characteristics are more similar to those of GH26 family members, thus indicating that ppman is a member of the GH26 family.
Embodiment 3
[0060] Recombinant expression and purification of embodiment 3ppman gene in escherichia coli
[0061] In order to facilitate the recombinant expression of the gene, NdeI and XhoI restriction sites were respectively introduced into the designed upstream and downstream primers. The PCR cleaning product ppman and the expression vector pET21a were double-digested with NdeI and XhoI respectively. 4 DNA ligase connection (ligation system: (5μLT 4 DNA Ligase 0.5μL, 10×T 4 DNA LigaseBuffer 0.5 μL, pET21a 2 μL, PCR product 2 μL), ligation conditions: overnight at room temperature. ). Take 5 μL of the ligation product to transform E.coli TOP10 competent cells, spread on solid Luria-Bertani medium containing 100 μg / mL ampicillin, and culture at 37°C for 12-16h. Pick a single clone, use degenerate primers for colony PCR verification, insert the correctly amplified single clone into liquid Luria-Bertani medium containing 100 μg / mL ampicillin, and extract the plasmid; use endonucleases ...
PUM
Property | Measurement | Unit |
---|---|---|
molecular weight | aaaaa | aaaaa |
Abstract
Description
Claims
Application Information
- R&D Engineer
- R&D Manager
- IP Professional
- Industry Leading Data Capabilities
- Powerful AI technology
- Patent DNA Extraction
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