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Alginate lyase AlyPL17, truncation and application of alginate lyase AlyPL17 and truncation

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

Active Publication Date: 2021-08-27
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This scenario limits the bioenergy conversion of alginate

Method used

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  • Alginate lyase AlyPL17, truncation and application of alginate lyase AlyPL17 and truncation
  • Alginate lyase AlyPL17, truncation and application of alginate lyase AlyPL17 and truncation
  • Alginate lyase AlyPL17, truncation and application of alginate lyase AlyPL17 and truncation

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Experimental program
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Effect test

Embodiment 1

[0027] Example 1: Culture and Genome Extraction of Pedobacter hainanensis NJ-02.

[0028] The alginate lyase AlyPL17 involved in the present invention is derived from bacterial strain Pedobacter hainanensis NJ-02, and the preservation number of the adopted bacterial strain Pedobacter hainanensis NJ-02 is CCTCCM2016579 (the strain information is disclosed in B.Zhu, et al., Int.J. Biol. Macromol. (2017)). 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 alginate lyase, for example, the sequence identity of AlyPL17 and Alg17c from Saccharophagus degradans 2-40 is 44%, The sequence identity with AlgL derived from Sphingomonas sp.MJ-3 reached 43%. It indicated that AlyPL17 is a novel PL17 family alginate lyase.

[0029] The formulation of the culture medium used is:

[0030] LB medium: tryptone 10g, yeast extract 5g, NaCl 10g, pH7.0. Solid medium supplemented with ...

Embodiment 2

[0032] Spread the preserved strains on the solid selection medium, and cultivate the activated colonies in the liquid selection medium. Take 4mL of the cultured bacterial solution, put it in a 2mL tube, centrifuge at 5000g for 10min. Discard the supernatant, resuspend the bacteria with 180 μL of digestion solution (Digestion Solution), then add 20 μL of proteinase K, mix well, and incubate at 56°C for 30 min. Add 20 μL of RNaseA, mix thoroughly, and place at room temperature for 10 minutes. Add 200 μL of Lysis Solution and mix quickly, the process should not exceed 15s. Add 400 μL of 50% (v / v) ethanol solution and mix well. Transfer the above solution to an adsorption column, let it stand for 1min, centrifuge at 6000g for 1min, discard the waste liquid, and transfer the adsorption column to a new 2mL tube. Add 500μL wash buffer I, centrifuge at 8000g for 1min, and discard the waste liquid. Add 500μL wash buffer II, centrifuge at 8000g for 1min, and discard the waste liquid...

Embodiment 3

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

[0045] Design primers 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] Perform PCR amplification to obtain the full-length sequences of AlyPL17, AlyPL17-N and AlyPL17-C genes. The PCR conditions were: pre-denaturation at 94°C for 3 minutes, followed by 30 cycles of 94°C for 30s, 55°C for 30s, and 72°C for ...

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Abstract

The invention discloses alginate lyase AlyPL17 and a truncation thereof. The invention further clones and transforms genes of the AlyPL17 and the truncation thereof onto an escherichia coli expression vector to obtain an escherichia coli recombinant strain capable of heterologous expression, and the strain is used for heterologous expression of corresponding alginate lyase. Results of enzymatic property characterization show that the optimum temperature of the AlyPL17 and the truncation thereof is 45 DEG C, the optimum pH is 9.0, and the AlyPL17 has high specific activity. From the aspect of action mode, the whole enzyme AlyPL17 of the enzyme adopts a mixed action mode, and truncation bodies at two ends adopt an incision action mode, which indicates that the unique action mode of the enzyme is caused by the synergistic effect of the truncation bodies at the two ends. The invention has great reference value for analyzing the alginate lyase in a mixed action mode, and the obtained alginate lyase AlyPL17 can be used as a potential tool for converting a brown algae resource into a biological energy source.

Description

technical field [0001] The invention belongs to the field of genetic engineering technology and protein expression, in particular to the cloning, expression and immobilization of alginate lyase gene. Background technique [0002] Global energy is increasingly tense, and biomass energy has become a research hotspot. The large-scale application of corn and other grains as the first generation of biomass resources will bring 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 the cell wall of brown algae, alginate is an acidic a...

Claims

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Application Information

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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 UNIV OF TECH
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