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Flavobacterium strain and endo-alginate lyase coding gene and its preparation and application

A technology for endocutting alginate and Flavobacterium, applied in lyase, application, food preparation, etc.

Active Publication Date: 2014-10-29
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Alginate lyase gene derived from Flavobacterium has not been reported yet

Method used

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  • Flavobacterium strain and endo-alginate lyase coding gene and its preparation and application
  • Flavobacterium strain and endo-alginate lyase coding gene and its preparation and application
  • Flavobacterium strain and endo-alginate lyase coding gene and its preparation and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Cultivation of a new strain of Flavobacterium sp.S20 and production of alginate lyase

[0034] The strain used is Flavobacterium sp. S20. Pick a single clone of Flavobacterium sp.S20 strain (CGMCC NO.5026) and inoculate it into 50ml of liquid medium, and then culture it on a shaker with a temperature of 30°C and a rotation speed of 220rpmin. After 48 hours, the culture solution was centrifuged to collect Flavobacterium sp.S20 cells and the supernatant culture medium was retained. The bacteria were resuspended with 5 ml of phosphate buffer (20 mM, pH 7.0) and then ultrasonically disrupted.

[0035] The liquid medium formula used is (g / L): beef extract 5g, glucose 15g, yeast extract powder 1.0g, NaCl 5.0g, MgSO 4 ·7H 2 O 0.5g, CaCl 2 0.2g, KH 2 PO 4 1.0g, FeSO 4 ·7H 2 O 0.02g, pH value is 7.0.

[0036] Enzyme activity unit (U) definition: the amount of enzyme required to catalyze sodium alginate to produce 1 μmol reducing sugar per minute. According t...

Embodiment 2

[0037] Example 2 Extraction of Flavobacterium sp.S20 bacterial strain genomic DNA

[0038] Take 2ml of fresh Flavobacterium S20 cultured overnight, and centrifuge (12,000rmp, 3min) to collect the bacteria. Wash the bacteria three times with phosphate buffer (20mM, pH7.0), add 650μl DNA extraction buffer (100mM Tris-HCl; 100mM Na 2 EDTA; 100mM Na 3 PO 4 ; 1.5M NaCl; 1% (w / v) hexadecyltrimethylammonium bromide; pH8.0), after mixing, freeze at -80°C, then place it in a 65°C water bath to thaw, and freeze repeatedly Melt three times. After cooling, add 4 μl lysozyme (100 mg / L) and shake horizontally (37 ° C, 225 rpm) for 30 minutes in a shaker, then add 3 μl proteinase K (20 mg / mL) and continue shaking for 30 minutes, and finally add 50 μl 20% (w / v) Sodium lauryl sulfate, after mixing, keep warm at 65°C for 2h (mix by inverting the centrifuge tube up and down every 20min). Centrifuge at room temperature for 10 min at 12,000 rpm, collect the supernatant, add 500 μl of saturate...

Embodiment 3

[0039] Example 3 Construction of total genomic DNA library of Flavobacterium sp.S20 strain and screening of clones expressing alginate lyase activity from the library

[0040] The genomic DNA of the extracted Flavobacterium S20 strain was randomly digested with Sau3A I enzyme, and the digested product was separated by agarose gel electrophoresis to obtain a 3-10kb DNA fragment. These fragments were ligated with the pGEM11z vector that had been digested with BamHI and the phosphate group at the 5' end was removed with alkaline phosphatase, the ligated product was transformed into E. 3-indole-β-D-galactoside, 40μg / ml), IPTG (isopropyl-β-D-thiogalactopyranoside, 40μg / ml), Amp (ampicillin, 50μg / ml) On a solid plate of Luria-Bertani medium, culture at 37°C for 12-16h. Pick the white Escherichia coli transformants that grew out into the wells of a 96-well culture plate (200 μl LB medium / well), and after culturing at 37° C. for 12 to 16 hours, add 150 μl of 50% glycerol to each cult...

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Abstract

Provided is a gene sequence for endo-alginate lyase Alg2A derived from Flavobacterium sp. strain S20 freshly isolated from soil samples, and a preparation method and application therefor. Also provided is a method of preparing the alginate lyase Alg2A, i.e. a technical method utilizing genetic engineering, in which the Alg2A gene is cloned to an Escherichia coli expression vector to obtain an Escherichia coli recombinant strain that can heterologously express said lyase. Using said strain to heterologously express the prepared alginate lyase Alg2A has the function of decomposing sodium alginate and preparing sodium alginate oligosaccharide. The present invention applies to the chemical, agricultural, and food industries, as well as to livestock feed processing, to the field of medicine, and to the field of genetic engineering of marine algae.

Description

technical field [0001] The invention relates to a gene sequence of an endo-alginate lyase Alg2A, a preparation method and application thereof. The invention also provides the recombinant plasmid and recombinant genetic engineering strain of the endo-alginate lyase. The endo-alginate lyase Alg2A of the present invention can be widely used in the fields of chemical industry, agriculture, food, feed addition, medicine, seaweed genetic engineering and the like. Background technique [0002] The vast ocean is rich in seaweed resources, which are mainly composed of four major categories: cyanobacteria, green algae, red algae and brown algae. Algin is a straight-chain acidic polysaccharide. In the natural state, the main forms of algin in the cell wall of brown algae are water-soluble sodium alginate (sodium alginate), potassium and other alkali metal salts and water-insoluble alginic acid. (alginic acid) and its alginates combined with 2 or more valent metal ions. Sodium algina...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12R1/20A23L17/60
CPCC12N1/20A23L1/337C12R1/20C12N9/88C12P19/00A61K38/00C12N15/63A61P31/04C08B37/0003C08B37/003C08B37/0084C12P19/04C12N1/205C12R2001/20
Inventor 杜昱光黄李淑馨李曙光赵小明曹海龙刘航
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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