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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  • 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

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

Example Embodiment

[0047] Example 1. Extraction of genomic DNA from marine thermophilic bacteria Defluviitalea phaphyphila sp. Alg1:

[0048] Defluviitalea phaphyphila sp.Alg1[Ji SQ,Wang B,Lu M,etal. Defluviitalea phaphyphila sp.nov.,a novel thermophilic bacterium thatdegrades brown algae[J].Applied and environmental microbiology,2016,82(3 ):868-877.] Inoculate into liquid medium BMS, culture in anaerobic test tube at 60℃ for 2 days; take 10mL of culture broth, centrifuge at 12,000×g for 5min, collect bacterial pellet; buffer with TE Wash twice to remove the residual medium, and collect the bacteria by centrifugation. Refer to the method of TIANGEN bacterial genome extraction kit, extract the bacterial genome with bacterial genomic DNA extraction kit. Finally, the DNA sample was dissolved in sterile deionized water at 60°C to prepare genomic DNA.

[0049] The components of the above liquid medium BMS are as follows:

[0050] Dipotassium hydrogen phosphate 0.1g / L, potassium dihydrogen phosphate 0.1g / L...

Example Embodiment

[0054] Example 2

[0055] Cloning of algin lyase gene and its expression and purification in E. coli strain BL21(DE3):

[0056] 1) According to the genes of the 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'-CGGAATTCATGAAGGGAAGATTAAAAAAATGGT-3' (EcoRI);

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

[0066] The underlined in the forward primer above is the restriction enzyme EcoR I site, and the underlined in the reverse primer is the restriction enzyme Xho I site. The bold display is the protected base;

[0067] Using the genomic DNA prepared in Example 1 as a template, PCR amplificatio...

Example Embodiment

[0167] Example 3. Enzymatic characteristics analysis of algin lyase:

[0168] a. Determination of substrate specificity of algin lyase

[0169] The alginate lyase AlgAT0, AlgAT1, AlgAT5 proteins were obtained after purification of the above-mentioned examples. 50 μl of the enzyme at a concentration of 2 μg / ml was added to the acetic acid-sodium acetate containing 2 g / L of different substrates (PolyM, PolyG, PolyMG) Buffer (200mM acetic acid-sodium acetate buffer, pH5.8), react at 70°C for 3min, and measure the change in OD235nm under a UV spectrophotometer with circulating heating in a water bath (see image 3 ). An enzyme activity unit is defined as a change of 0.1 value per minute of OD235nm. Specific enzyme activity is defined as the ratio of enzyme activity to the corresponding protein amount.

[0170] by image 3 The results showed that the specific enzyme activity of AlgAT0 to sodium alginate was 3592 U / mg, which was PolyMG specific enzyme activity; the specific enzyme activi...

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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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