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Salt-tolerant ethanol-tolerant protease-tolerant surfactant-tolerant exoinulinase, gene thereof, vector and strain

A technology of exo-inulinase and surfactant, which is applied in the field of genetic engineering, can solve the problems of unreported and low enzyme activity, and achieve the effect of good salt resistance, ethanol resistance, protease resistance and surfactant resistance

Inactive Publication Date: 2014-08-13
YUNNAN NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, a salt-tolerant exo-inulinase has been reported (patent application number: 201310275633.9). This enzyme is an alkaline enzyme with very low enzyme activity under acidic conditions; Surfactant exo-inulinase

Method used

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  • Salt-tolerant ethanol-tolerant protease-tolerant surfactant-tolerant exoinulinase, gene thereof, vector and strain
  • Salt-tolerant ethanol-tolerant protease-tolerant surfactant-tolerant exoinulinase, gene thereof, vector and strain
  • Salt-tolerant ethanol-tolerant protease-tolerant surfactant-tolerant exoinulinase, gene thereof, vector and strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1: Cloning of exo-inulinase gene inuAJB13

[0040] Extract the genomic DNA of Sphingomonas: Centrifuge the bacterial liquid cultured for 2 days to get the bacterial cells, add 1mL lysozyme, treat at 37°C for 60min, then add the lysate, the composition of the lyse is: 50mM Tris, 20mM EDTA, Nacl500mM, 2% SDS (w / v), pH 8.0, lysed in a water bath at 70°C for 60 minutes, mixed every 10 minutes, and centrifuged at 10,000 rpm for 5 minutes at 4°C. The supernatant was extracted in phenol / chloroform to remove impurity proteins, and then an equal volume of isopropanol was added to the supernatant. After standing at room temperature for 5 minutes, centrifuge at 10,000 rpm for 10 minutes at 4°C. The supernatant was discarded, the precipitate was washed twice with 70% ethanol, dried in vacuo, dissolved by adding an appropriate amount of TE, and stored at -20°C for later use.

[0041] Table 1. Cloning and expression primers of exo-inulinase gene inuAJB13

[0042]

[0043...

Embodiment 2

[0045] Embodiment 2: Preparation of recombinant exo-inulinase InuAJB13

[0046] Using inuAJB13F and inuAJB13R as a primer pair (Table 1) and Sphingomonas genomic DNA as a template, PCR amplification was performed. The parameters of the PCR reaction were: denaturation at 94°C for 5 min; then denaturation at 94°C for 30 sec, annealing at 61°C for 30 sec, extension at 72°C for 1 min and 30 sec, and after 30 cycles, incubation at 72°C for 10 min. According to the results of PCR, the exo-inulinase gene inuAJB13 was obtained, and a protruding A base was introduced at the 3' end of the gene. The exo-inuAJB13 gene inuAJB13 and the expression vector pEasy-E2 were connected by T-A method to obtain the recombinant expression plasmid pEasy-E2-inuAJB13 containing inuAJB13. Transform pEasy-E2-inuAJB13 into E. coli BL21(DE3) to obtain recombinant E. coli strain BL21(DE3) / inuAJB13.

[0047] Take the recombinant Escherichia coli strain BL21(DE3) / inuAJB13 containing the recombinant plasmid pE...

Embodiment 3

[0048] Example 3: Determination of the properties of the purified recombinant exo-inulinase InuAJB13

[0049] 1. Activity analysis of purified recombinant exo-inulinase InuAJB13:

[0050] Example 2 The activity determination method of the purified recombinant exo-inulinase InuAJB13 adopts the 3,5-dinitrosalicylic acid (DNS) method: the substrate is dissolved in 0.1M buffer to make the final concentration 0.5% (w / v); the reaction system contains 100 μL of appropriately diluted enzyme solution and 900 μL of substrate; after the substrate is preheated at the reaction temperature for 5 minutes, add the enzyme solution and react for 10 minutes, then add 1.5mL DNS to terminate the reaction, boil in water for 5 minutes, and cool After reaching room temperature, measure the OD value at a wavelength of 540nm; 1 enzyme activity unit (U) is defined as the amount of enzyme required to decompose the substrate to produce 1 μmol reducing sugar (calculated as fructose) per minute under given ...

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Abstract

The invention discloses a salt-tolerant ethanol-tolerant protease-tolerant surfactant-tolerant exoinulinase, a gene thereof, a vector and a strain. The exoinulinase InuAJB13 possesses the following properties: the optimum pH is 5.5, 50% or more of enzymatic activity is maintained in the pH scope of 4.0-7.0; the remnant enzyme activity reaches 90% or more after exoinulinase is processed by a buffer with a concentration of 0.1 M and pH of 4.0-7.0 for 1 h; the optimum temperature is 55 DEG C, and exoinulinase has the enzyme activity at 10-70 DEG C; a NaCl solution with a concentration of 0.6-4.5 M is capable of improving the enzyme activity by 0.2-0.6 times; 100% of the enzyme activity can be kept after exoinulinase is processed by a NaCl solution with a concentration of 0.2-4.5 M at 37 DEG C for 60 min; exoinulinase keeps the activity in 10% (V / V) of ethanol; exoinulinase keeps 88% or more of the activity after being processed in 3.0-15.0% (V / V) ethanol at 37 DEG C for 60 min; exoinulinase activity is not influenced or slightly influenced by trypsin, protease K, surfactants, most of metal ions, and commercial liquid laundry detergents; and exoinulinase is capable of hydrolyzing inulin, cane sugar, raffinose, stachyose, beta-2,6-fructan (levan) and soluble starch. The exoinulinase disclosed by the invention is applicable to industries such as feed, foodstuff, washing and biofuels.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a salt-resistant, ethanol-resistant, protease-resistant and surfactant-resistant exo-inulinase and its gene, carrier and bacterial strain. Background technique [0002] Inulin, also known as inulin, is a kind of polyfructose linked by β-2,1 glycosidic bonds. Its reducing end is connected with a glucose group and has a straight chain structure. It mainly exists in Jerusalem artichoke, chicory, Roots or stems of dicotyledonous plants such as dandelion, burdock and artichoke (Roberfroid et al. JNutr, 2007, 137:2493–2502). Jerusalem artichoke is commonly known as Jerusalem artichoke or ghost ginger. It has the advantages of strong disease resistance, high yield, cold resistance, barren resistance, drought resistance, easy planting, and strong adaptability. Its tubers have a large yield and can contain up to 20% fresh weight or 80% dry weight inulin (Kango et al. Food Bio...

Claims

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Application Information

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IPC IPC(8): C12N9/24C12N15/56C12N15/70C12N1/21C12R1/19
CPCC12N9/2402C12Y302/0108
Inventor 黄遵锡周峻沛张蕊唐湘华李俊俊许波丁俊美高雅洁
Owner YUNNAN NORMAL UNIV
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