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Alginate lyase biosynthesis gene cluster

A technology for alginate lyase and biosynthesis, which is applied in the field of biosynthetic gene clusters of alginate lyase to achieve the effect of enriching material and genetic information

Active Publication Date: 2014-09-10
EAST CHINA UNIV OF SCI & TECH
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

But Kitamikado et al found that V.alginolyticus ATCC17749 strain can produce extracellular alginate lyase (Tseng C-H., Yamaguchi K., Nishimura M., Kitamikado M. Alginate Lyase from Vibrioalginolyticus ATCC17749[J]. Nippon Suisan Gakkaishi, 1992, 58(11):2063-2067), and isolated and purified an alginate lyase with a molecular weight of 47kDa that specifically degrades polymannuronic acid fragments, and found that there were more than one alginate lyase in this strain ( Kitamikado M, TsengCH, Yamaguchi K, Nakamura T.Two types of alginate lyases[J].Appl EnvironMicrobiol, 1992,58(8):2474-2478), but there has been no research on V.alginolyticus ATCC17749 alginate lyase since then There are reports

Method used

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  • Alginate lyase biosynthesis gene cluster

Examples

Experimental program
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Embodiment 1

[0031] Embodiment 1, the cloning of alginate lyase gene cluster

[0032] 1.1 Cloning of alginate lyase gene

[0033] A series of primers were designed and synthesized for amplifying alginate lyase genes algV1, algV2, algV3, algV4 and algV5 from the genome of V. alginolyticus ATCC17749. The sequences and characteristics of the primers are shown in Table 1.

[0034] Table 1. Primer characteristics for PCR amplification of algV1-algV5 genes

[0035]

[0036]

[0037] Note: ggatcc is the BamHI restriction site; catatg is the NdeI restriction site; ccatgg NcoI restriction site

[0038] Then, use the bacterial genome extraction kit to extract the genomic DNA of V. alginolyticus ATCC17749 as a template for PCR amplification.

[0039] The total volume of the PCR reaction is 50 μL, and the reaction components are: 10×Taq DNA polymerase buffer 5 μL, 25 mmol / L dNTP 4 μL, 5 pmol / μL forward and reverse primers 10 μL each, DNA template 2.5 μL, Taq DNA polymerase 1.0 μL, Make up...

Embodiment 2

[0052] Example 2. Activity identification of alginate lyase

[0053] 2.1 Expression of alginate lyase in Escherichia coli

[0054] The pET28a-algV1, pET28a-algV2, pET28a-algV3, pET28a-algV4 and pET28a-algV5 expression plasmids obtained in Example 1.1 were respectively transformed into Escherichia coli BL21(DE3) competent cells for gene expression. The recombinant Escherichia coli containing the expression plasmid was inoculated in LB liquid medium containing kanamycin (final concentration 50 μg / mL) and cultured for 12-16 hours, and the above-mentioned primary culture was inoculated in 30 mL of In LB liquid medium containing kanamycin (final concentration 50 μg / mL), culture at 37°C for about 2.5h to OD 600 When =0.6~0.8, add IPTG (final concentration: 1mmol / L) to induce expression at 16°C for 8.5h. The bacteria were collected, and the expression of each gene in E. coli was detected by SDS-PAGE electrophoresis.

[0055] Taking the thalli before induction as a negative contr...

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Abstract

The invention discloses an alginate lyase biosynthesis gene cluster which comprises five alginate lyase encoding genes: algV1, algV2, algV3, algV4 and algV5.The gene cluster is derived from Vibrio alginolyticus ATCC17749. The contained all gene and protein information related to the alginate lyase biosynthesis, provided by the invention, can help people to understand the biosynthesis mechanism of the alginate lyase so as to provide a great deal of substance and genetic information for the large-scale application of the alginate lyase.

Description

technical field [0001] The invention belongs to the field of microbial genetic engineering, in particular to the biosynthetic gene cluster of alginate lyase. Background technique [0002] Alginate, also known as alginate, is one of the main components of the cell wall of brown algae. Certain bacterial families such as Azotobacter and Pseudomonas also produce alginic acid as capsular polysaccharides of bacteria (Gregory L, Larry HK.Characterization of the exocellular polysaccharides from Azotobacter chroococcum[J]. Carbohydrate Research, 1988, 181 (1): 143-152; Alferd L, Russell SJ. A polysaccharide resembling alginic acid from a Pseudomonas microorganism [J]. Nature, 1964, 204 (10): 187-188). The alginic acid molecule is composed of two residues of α-L-guluronic acid (G) and β-D-mannuronic acid (M) connected by 1,4-bonds. It is found that the alginic acid molecule is composed of 3 A modular structure, namely: poly-gulucuronic acid segment (Poly-gulutonate, PG), homo-poly-m...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/60C12R1/63
Inventor 叶江胥晴晴张惠展
Owner EAST CHINA UNIV OF SCI & TECH
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