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Coding genes and applications of alginate lyases

A technology of alginate lyase and coding gene, which is applied in the direction of lyase, application, genetic engineering, etc., can solve the problems that are not suitable for large-scale industrial application and poor thermal stability, and achieve the purpose of improving industrial application value and thermal stability Good performance and high catalytic efficiency

Active Publication Date: 2018-12-04
QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, most of the currently known alginases are medium and low temperature enzymes, and their thermal stability is not good, so they are not suitable for large-scale industrial applications. Major Factors in Drug Transformation

Method used

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  • Coding genes and applications of alginate lyases
  • Coding genes and applications of alginate lyases
  • Coding genes and applications of alginate lyases

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1, the extraction of the genomic DNA of the marine thermophile Defluviitalea phaphyphila sp.Alg1 strain:

[0048] Defluviitalea phaphyphila sp.Alg1[Ji S Q,Wang B,Lu M,etal. Defluviitalea phaphyphila sp.nov.,a novel thermophilic bacterium that degrades brown algae[J].Applied and environmental microbiology,2016,82(3 ):868-877.] inoculated into liquid medium BMS, cultured in an anaerobic test tube at 60°C for 2 days; took 10 mL of the cultured bacteria solution, centrifuged at 12,000×g for 5 min, and collected the bacterial precipitate; buffered with TE Wash twice with liquid to remove the medium residue, collect the bacteria by centrifugation, and extract the genome of the bacteria with the bacterial genome DNA extraction kit according to the method of the TIANGEN Bacterial Genome Extraction Kit. Finally, the DNA sample was dissolved in sterile deionized water at 60°C to obtain genomic DNA.

[0049] The components of the above-mentioned liquid medium BMS are as f...

Embodiment 2

[0055] Cloning of alginate lyase gene and its expression and purification in Escherichia coli strain BL21(DE3):

[0056] 1) According to the genes of three alginate lyases AlgAT0, AlgAT1, AlgAT5, design the following primers:

[0057] AlgAT0:

[0058] Forward primer F: 5'-ATGAATGTTTACGCTACTTCTACTGAAAC-3';

[0059] Reverse primer R: 5'-ATTATCTATACCTACATCTGACGAAGTTAAAGG-3';

[0060] AlgAT1:

[0061] Forward primer F: 5'-GCAAACTATGAAACTTATGATGGTTTTAAAGTT-3';

[0062] Reverse primer R: 5'-TTGTATTGGAAGTAATACTGGTCCTGCTGGATT-3';

[0063] AlgAT5:

[0064] Forward primer F: 5'-CGGAATTCATGAAGGGAAGATTAAAAAAAATGGT-3' (EcoR I);

[0065] Reverse primer R: 5'-CCGCTCGAGACTATGGGTTACTACTAGATTATAAATTTC-3' (Xho I);

[0066] What is underlined in the above-mentioned forward primer is the restriction endonuclease EcoR I site, and what the reverse primer is underlined is the restriction endonuclease Xho I site. The bases shown in bold are protected bases;

[0067] Using the genomic DNA obta...

Embodiment 3

[0167] Embodiment 3, analysis of enzymatic characteristics of alginate lyase:

[0168] a. Substrate specificity assay of alginate lyase

[0169] Purify the above-mentioned examples to obtain alginate lyase AlgAT0, AlgAT1, and AlgAT5 proteins, respectively take 50 μl of enzymes with a concentration of 2 μg / ml, and add them to acetic acid-sodium acetate containing 2 g / L of different substrates (PolyM, PolyG, PolyMG) In the buffer solution (200mM acetic acid-acetic acid sodium salt buffer solution, pH5.8), react at 70°C for 3 minutes, and measure the change value of its OD235nm under the ultraviolet spectrophotometer with water bath circulating heating (see image 3 ). One enzyme activity unit is defined as the value change of 0.1 value per minute of OD235nm. The specific enzyme activity is defined as the ratio of the enzyme activity to the corresponding protein amount.

[0170] Depend on image 3 The results show that AlgAT0 has a specific enzyme activity of 3592U / mg to sodi...

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Abstract

The invention relates to alginate lyases, and in particular, relates to coding genes and applications of three algin-degrading alginate lyases. The alginate lyases comprise alginate lyases AlgAT0, AlgAT1 and AlgAT5 respectively. The coding genes of the alginate lyases have the base sequences of SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 respectively. The alginate lyase genes are cloned into escherichia coli and pichia pastoris by a genetic engineering method. The AlgAT0, AlgAT1 and AlgAT5 are cloned into an expression vector of pichia pastoris; after fermentation condition optimization, fermentation is performed for 120 h, the concentrations of extracellular proteins are 0.312 g / L, 1 g / L and 9.39 g / L respectively, and the enzyme activities are 64666.67 U / mL, 126666.67 U / mL and 136025.6 U / mLrespectively. An escherichia coli recombinant strain and a pichia pastoris strain capable of producing alginase are obtained. The recombinant enzymes have stable properties and can be used for algin conversion with high added value, the enzyme activities of the three enzymes are much higher than values those reported so far, and the three enzymes have quite good potential for industrial application.

Description

technical field [0001] The present invention relates to alginate lyase, specifically the coding gene of three kinds of alginate degrade alginate lyase and application thereof. Background technique [0002] As the representative of the third generation of sustainable bio-energy, algae does not contain lignin, so it is conducive to the release of monosaccharides, fast reproduction, low energy consumption, no occupation of cultivated land and fresh water, no food conflicts and many other advantages. The first-generation bio-energy raw materials (represented by grains) and the second-generation bio-energy raw materials (represented by straw, etc.) have broad application prospects. At present, the total production of macroalgae in the world is 25 million tons per year, and China accounts for 53.97% of it. It is the largest producer in the world, followed by Indonesia, South Korea, Japan, and Malaysia. The total production of these Asian countries accounts for 96.27% of the total....

Claims

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Application Information

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IPC IPC(8): C12N15/60C12N9/88C12N15/70C12P19/12C12P19/02C12P19/00
CPCC12N9/88C12N15/70C12P19/00C12P19/02C12P19/12
Inventor 李福利苏航冀世奇吕明
Owner QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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