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

Alginate lyase alypl17, truncated body and application thereof

A technology of algin lyase and alypl17-c, which is applied in the field of genetic engineering technology and protein expression, can solve problems such as limiting the conversion of algin bioenergy, and achieve the effect of high specific activity

Active Publication Date: 2022-06-07
NANJING TECH UNIV
View PDF0 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This scenario limits the bioenergy conversion of alginate

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
  • Alginate lyase alypl17, truncated body and application thereof
  • Alginate lyase alypl17, truncated body and application thereof
  • Alginate lyase alypl17, truncated body and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1: Culture and genome extraction of strain Pedobacter hainanensis NJ-02.

[0028] The alginate lyase AlyPL17 involved in the present invention is derived from the strain Pedobacter hainanensis NJ-02, and the adopted strain Pedobacter hainanensis NJ-02 has a preservation number of CCTCCM2016579 (the strain information is disclosed in B.Zhu, et al., Int.J. Biol. Macromol. (2017) in). After uploading the sequence of AlyPL17 to the NCBI database for sequence comparison, it can be seen that AlyPL17 has a high sequence identity with the PL17 family algin lyase. For example, the sequence identity of AlyPL17 and Alg17c from Saccharophagus degradans 2-40 is 44%, The sequence identity with AlgL derived from Sphingonas sp. MJ-3 reached 43%. It was shown that AlyPL17 is a novel PL17 family alginate lyase.

[0029] The formulation of the medium used was:

[0030] LB medium: tryptone 10 g, yeast extract 5 g, NaCl 10 g, pH 7.0. Solid medium supplemented with 1.5% agar. Au...

Embodiment 2

[0032] Preserved strains are spread on solid selective medium, and activated colonies are cultured in liquid selective medium. Take 4mL of the cultured bacterial solution, put it in a 2mL tube, centrifuge at 5000g for 10min. The supernatant was discarded, the bacteria were resuspended with 180 μL of Digestion Solution, 20 μL of proteinase K was added, and after thorough mixing, the cells were incubated at 56° C. for 30 min. Add 20 μL of RNaseA, mix well, and place at room temperature for 10 min. Add 200 μL of Lysis Solution, mix quickly, and the process should not exceed 15s. Add 400 μL of 50% (v / v) ethanol solution and mix well. Transfer the above solution to the adsorption column, stand for 1 min, centrifuge at 6000g for 1 min, discard the waste liquid, and transfer the adsorption column to a new 2mL tube. Add 500 μL of wash buffer I, 8000 g, centrifuge for 1 min, and discard the waste solution. Add 500 μL of wash buffer II, 8000 g, centrifuge for 1 min, and discard the ...

Embodiment 3

[0044] Example 3: Construction of alginate lyase AlyPL17 and its truncations AlyPL17-N and AlyPL17-C recombinant expression vectors and corresponding expression and purification.

[0045] Primers were designed according to AlyPL17:

[0046] Forward primer: 5'-ATGAGCGCGCAGAGCCAT-3'

[0047] Reverse primer: 5'-TTTGCCGCTTTCCACTTTCA-3'.

[0048] The following amplification primers were designed according to the sequence of AlyPL17-N:

[0049] Forward primer: 5'-ATGAGCGCGCAGAGCCAT-3'

[0050] Reverse primer: 5'-CGCTTTGTTATCCGCCAGAT-3'.

[0051] The following amplification primers were designed according to the sequence of AlyPL17-C:

[0052] Forward primer: 5'-GAACCGTTTAAATATCGCAGCC-3'

[0053] Reverse primer: 5'-TTTGCCGCTTTCCACTTTCA-3'.

[0054] PCR amplification was performed to obtain the full-length sequences of AlyPL17, AlyPL17-N and AlyPL17-C genes. PCR conditions were: pre-denaturation at 94 °C for 3 min, followed by 30 cycles of 94 °C for 30 s, 55 °C for 30 s, 72 °C ...

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
Login to View More

Abstract

The invention discloses an alginate lyase AlyPL17 and its truncated body, and further transforms the gene clone of AlyPL17 and its truncated body into an expression vector of Escherichia coli to obtain a recombinant strain of Escherichia coli capable of heterologous expression, using the strain Heterologous expression of the corresponding alginate lyase. The characterization results of enzymatic properties showed that the optimum temperature of AlyPL17 and its truncation was 45℃, the optimum pH was 9.0, and the enzyme had high specific activity. From the mode of action, the holoenzyme AlyPL17 of this enzyme adopts a mixed mode of action, while the two-terminal truncation adopts an endo-interaction mode, indicating that the unique mode of action of this enzyme is caused by the synergistic action of the two-terminal truncation. The invention has great reference value for analyzing the mixed action mode alginase, and the obtained alginate lyase AlyPL17 can be used as a potential tool for transforming brown algae resources into bioenergy.

Description

technical field [0001] The invention belongs to the field of genetic engineering technology and protein expression, and in particular relates to the cloning and expression of an alginate lyase gene and its immobilization. Background technique [0002] The global energy is increasingly tense, and biomass energy has become a research hotspot. As the first-generation biomass resources, the large-scale application of corn and other grains will bring about problems such as food shortages. However, the second-generation biomass resources such as straw lignin are difficult to utilize due to their complex structure, which limits their industrial application to a certain extent. Marine algae are widely distributed, do not require fresh water resources, do not compete with people for food, and contain fermentable sugars in their components. Therefore, marine algae are gradually becoming the third-generation biomass resources. [0003] As the main component of brown algae cell wall, ...

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): C12N9/88C12N15/60C12N15/70C12N1/21C12P19/02C12P19/12C12P19/00C12R1/19
CPCC12N9/88C12N15/70C12P19/02C12P19/12C12P19/00C12Y402/02Y02E50/10
Inventor 朱本伟李谦姚忠熊强孙芸
Owner NANJING TECH 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