Gene pme16A for encoding metalloprotease and application thereof

A technology of metalloprotease and pme16a, which is applied in application, genetic engineering, plant genetic improvement, etc., to achieve good hydrolysis ability and make up for the shortage.

Inactive Publication Date: 2014-01-15
GUANGXI UNIV
View PDF1 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Metalloprotease-producing strains can be screened by pure culture technology, but due to the limitations of various conditions, about 99% of microorganisms in nature cannot be achieved by pu

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
  • Gene pme16A for encoding metalloprotease and application thereof
  • Gene pme16A for encoding metalloprotease and application thereof
  • Gene pme16A for encoding metalloprotease and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0027] Example 1

[0028] A cloning of the gene pme16A encoding a novel metalloprotease, which mainly includes the following steps 1, 2, 3, and 4.

[0029] Step 1: Extract and purify metagenomic DNA from the active sludge of polluted water samples. The method steps are as follows.

[0030] (1) First prepare DNA extraction solution: 100mM sodium phosphate buffer (pH8.0), 1%(w / v) CTAB, 100mM EDTA (pH8.0), 0.3M NaCl, 0.01mM Tris-HCl (pH8.0) );

[0031] (2) PVPP pickling treatment: Weigh 10g PVPP, add 3M HCl, soak for 12h, filter with filter paper, wash and stir PVPP with 20mM potassium phosphate buffer (pH7.4), repeat several times until the suspension reaches neutrality. PVPP filtered and dried at 50℃;

[0032] (3) Weigh 1g of sample into a 15mL centrifuge tube, add 0.25g of acidified PVPP, 4mL of extraction buffer, mix well, and freeze-thaw at 70℃ and liquid nitrogen three times for 30 minutes each time. After the last dissolution, add SDS to a final concentration of 1%, mix upside do...

Example Embodiment

[0051] Example 2

[0052] Construction of pme16A gene expression recombinant plasmid.

[0053] The plasmid pET-32a(+) provided by Novagen in the United States was used as an expression vector and E.coli BL21(DE3)pLysS was used as a host cell for heterologous and efficient expression of target genes.

[0054] According to the pme16A gene sequence, use relevant software to design primers. Forward amplification primer F1: 5’-GC GAATTC ATGCCCCTTTATCAGACCTTC-3’, introducing EcoRI restriction site; reverse amplification primer R1: 5’-AA CTCGAG GGGCGATTCGAGCTC-3', add PstI restriction site. Using pGXM16 as a template, using Pfu DNA polymerase for PCR reaction to amplify the pme16A gene, such as Image 6 Shown. The expression vector pET-32(a) + plasmid was extracted, double-enzyme digested with EcoRI and Pst Ⅰ, and after purification, it was ligated with the pme16A amplified product that was also double-enzyme-cut and purified to construct the expression recombinant plasmid, which was t...

Example Embodiment

[0055] Please refer to the second embodiment Image 6 . Image 6 To express the results of the recombinant plasmid pGXPME16A, 1 is 1kb Ladder Maker, and the fragment sizes from top to bottom are: 10.0kb, 8.0kb, 6.0kb, 5.0kb, 4.0kb, 3.5kb, 3.0kb, 2.5kb, 2.0kb , 1.5kb, 1.0kb, 750bp, 500bp, 250bp; 2 is the result of digesting pGXPME16A with EcoRI and PstI.

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 a gene pme16A for encoding metalloprotease. A nucleotide sequence of the gene pme16A is as shown in a sequence table No.1; a sequence of an amino acid residue of the gene pme16A is as shown in a sequence table No.2. DNA (Deoxyribose Nucleic Acid) in the sequence table 1 is started with an initiation codon ATG and is end with a termination codon TAA from a first nucleotide to a 1461th nucleotide at an end 5'; a complete ORF (nucleotides 1 to 1461) exists; initiation codons ATG of the gene pme16A are from the first to third nucleotides at the end 5'; the termination codons TAA are from the 1459th to 1461th nucleotides at the end 5'. The gene pme16A provides a novel material for industrial production application of the metalloprotease, can effectively make up the current situation of shortage of animal and plant source metalloprotease raw materials and has important research value and application prospect.

Description

technical field [0001] The invention relates to a gene pme16A encoding metalloprotease and its application. Background technique [0002] Metalloprotease (EC3.4.24.-) refers to a class of protease whose active center depends on metal ions. Most metalloproteases mainly rely on divalent cations, such as zinc ions, copper ions, cobalt ions, and manganese ions. where Zn 2+ most common. Metalloproteases are easily and strongly inhibited by metal chelators. Metalloproteases are widely distributed and specific in nature, and have important economic and application values. Due to the different sources of metalloproteases, most of them have their own distinctive characteristics. Generally, it has the characteristics of high temperature resistance or low temperature resistance, resistance to organic solvents, alkali resistance, and heat sensitivity. At present, metalloproteases are mainly used in food, detergent, cosmetics, antineoplastic drugs and disease mechanism research, an...

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): C12N15/57C12N9/50C12N15/10
Inventor 蒋承建黄捷古恒森曾蓉陈高申佩弘武波
Owner GUANGXI UNIV
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