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Alginate lyase mutant gene, alginate lyase mutant, engineering bacterium containing alginate lyase mutant, construction method and application

A technology of alginate lyase and mutant gene, applied in mutants and its application, in the field of alginate lyase mutant gene, which can solve the problems of inability to carry out recombinant expression and molecular transformation, difficult to find new genes, and change product size, etc. Achieve good industrial application prospects, high activity and stability, and increase the effect of polymerization degree

Active Publication Date: 2021-06-11
WUZHOUFENG AGRI SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Using this strategy, you must have a certain understanding of the relevant gene sequence to design PCR primers, and find new molecules in a certain class of proteins with similar structures or functions, and it is difficult to discover brand new genes
[0004] With the continuous development of high-throughput sequencing technology, more and more microbial genome sequences producing alginate lyase have been determined, which makes it possible to analyze the sequence information through genome mining technology and fish for the gene encoding alginate lyase To become concise and fast, Badur AH et al. sequenced the genome of a strain of bacteria Vibrio splendidus from the ocean. Through analysis, it was found that the strain contained 4 genes encoding alginate lyase, and the gene was cloned and tested in the large intestine. Recombinant expression in Bacillus Escherichia coli BL21 (DE3) (Badur et alAppl.Environ.Microbiol.2015.81:1865-1873); most of the alginate lyases obtained so far are alginate cleavages that specifically degrade homopolymannuronic acid Among the enzymes, a small number of alginate lyases have the activity of degrading guluronic acid, and the alginate lyases with broad substrate specificity are very rare, only the alginate lyase Aly-SJ02 from Pseudoalteromonassp. (Li Jianwei , Marine Drugs, 2011, 21:1374-80), alginate lyase AlyIH from Isoptericola halotolerans in termite gut (Dou Wenfang et al., Carbohydrate Polymers, 2013, 98: 1476-82) and other enzymes with broad substrate specificity The activity is low, and the stability is poor, wherein AlyIH has only obtained the purified enzyme, has not obtained its coding gene, and cannot carry out recombinant expression and molecular transformation; and most alginate lyases (including CN 107099545 ​​A) activity is low, the Oligosaccharides are mostly 2 to 4 sugars
According to previous literature reports, many enzymes have two loops, which are easy to form a cleft that binds to the substrate. Changing the amino acid residues near the loop is a better strategy to explore the properties of the enzyme (R.He, A.C. Reyes, T.L. Amyes, J.P. Richard, Enzyme Architecture: The Role of a Flexible Loop in Activation of Glycerol-3-phosphate Dehydrogenase for Catalysis of Hydride Transfer, Biochemistry-Us 57(23)(2018)3227-3236), it is possible to change the size of the product

Method used

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  • Alginate lyase mutant gene, alginate lyase mutant, engineering bacterium containing alginate lyase mutant, construction method and application
  • Alginate lyase mutant gene, alginate lyase mutant, engineering bacterium containing alginate lyase mutant, construction method and application
  • Alginate lyase mutant gene, alginate lyase mutant, engineering bacterium containing alginate lyase mutant, construction method and application

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Experimental program
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Effect test

Embodiment 1

[0026] Embodiment 1: the cultivation and identification of bacterial strain Vibrio sp.NJU-03

[0027] The strains used are derived from seaweed samples collected from the Yellow Sea. After isolation, a strain NJ-04 producing alginate lyase is obtained. The formula of the selective medium used is:

[0028] Beef extract 5g, glucose 15g, yeast extract powder 1.0g, NaCl 5.0g, MgSO 4 ·7H 2O 0.5g, CaCl 2 0.2g, KH 2 PO 4 1.0g, FeSO 4 ·7H 2 O 0.02g, pH value is 7.0. For solid medium add 1.5% agar.

[0029] Introduce the colony into 100mL liquid medium for enrichment culture, and culture at 30°C and 150rpm for 36h. 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, then add 20 μL of proteinase K, mix thoroughly, 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...

Embodiment 2

[0030] Example 2: Cloning and Identification of Alginate Lyase Mutant mAlgNJU-03 Encoding Gene

[0031] According to the deduced alginate lyase mutant gene sequence in the Vibrio sp.JCM 19052 gene, the following amplification primers were designed: the upstream primer forward primer (5'-ATGAAAAGCAAGTTAGTCAA-3'), and the downstream primer reviseprimer (5'-CGGGAATTCATACCCGTCG-3' ). Mutation primer: upstream primer forward primer (5'-GCGCATGAACCTAACGAAGACGCGTCATCAGATCCAGAG-3'), and downstream primer revise primer (5'-CTGGATCTGATGACGCGTCTTCGTTAGGTTCATGCGCAAAG-3'). Using the extracted strain genome as a template, the full-length sequence of the alginate lyase mutant mAlgNJU-03 gene was obtained by overlapping PCR amplification.

[0032] 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 2 minutes, and finally extension at 72°C for 10 minutes. Agarose gel electrophoresis showed a specific band at about...

Embodiment 3

[0036] Example 3: Construction of alginate lyase mAlgNJU-03 recombinant expression vector.

[0037] According to the complete sequence of alginate lyase mutant gene design upstream primer (5'-CGC GGATCC ATGAAAAGCAAGTTAGTCAA-3') and downstream primer (5'-CCG CTCGAG CGGGAATTCATACCCGTCG-3'), mutation primer: upstream primer forward primer (5'-GCGCATGAACCTAACGAAGACGCGTCATCAGATCCAGAG-3'), and downstream primer revise primer (5'-CTGGATCTGATGACGCGTCTTCGTTAGGTTCATGCGCAAAG-3'). The full-length sequence of the alginate lyase mutant mAlg NJU-03 gene was obtained by overlapping PCR amplification. 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 2 minutes, and finally extension at 72°C for 10 minutes. The PCR product was digested with NdeI and XhoI and recovered; the Escherichia coli expression vector pET21a(+) was also digested with NdeI and XhoI and recovered, connected with the above-mentioned target g...

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Abstract

The invention discloses an alginate lyase mutant gene, an alginate lyase mutant, an engineering bacterium containing the alginate lyase mutant as well as a construction method and application. The invention further discloses a technical method of genetic engineering, the mutant gene of the novel alginate lyase is cloned to an escherichia coli expression vector to obtain an escherichia coli recombinant strain capable of heterologously expressing the alginate lyase, and the alginate lyase mutant mAlgNJU-03 prepared by heterologously expressing the strain has the functions of degrading sodium alginate to prepare sodium alginate oligosaccharide and changing the polymerization degree of the oligosaccharide. The alginate lyase mutant mAlgNJU-03 provided by the invention can be widely applied to the fields of chemical engineering, agriculture, food, feed additives, medicines, seaweed genetic engineering and the like.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a mutant gene of alginate lyase, mutants and applications thereof. Background technique [0002] my country has a vast sea area, which contains rich marine biological resources, especially seaweed resources. Brown algae mainly include kelp, wakame, etc. The alginate rich in the cell wall is a linear acidic polysaccharide. In the natural state, the main existence state of alginate is water-soluble sodium alginate (sodium alginate), potassium alginate Other alginates and water-insoluble alginic acid (alginic acid). Sodium alginate (trade name: sodium alginate) or other alginates currently on the market are mainly obtained from brown algae. Studies have found that the oligosaccharides obtained by degrading sodium alginate have various biological activities, such as immune regulation, growth promotion, induction of plant resistance and improvement of protein...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/60C12N9/88C12N15/70C12N1/21C12P19/00C12R1/19
CPCC12N9/88C12N15/70C12P19/00C12Y402/02003C12Y402/02011
Inventor 李峰张志凯迟艳王学江于蒙爱
Owner WUZHOUFENG AGRI SCI & TECH
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