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Exoinulinase Z2-5 with low-temperature activity and gene of exoinulinase Z2-5

A technology for exoinulinase and activity, which is applied in the field of genetic engineering, can solve the problem of less exoinulinase, etc., and achieves the effect of good application potential

Inactive Publication Date: 2012-07-11
YUNNAN NORMAL UNIV
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  • Abstract
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  • Claims
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Problems solved by technology

[0005] Exo-inulinase preparations with low-temperature activity can be applied to low-temperature habitats and low-temperature processing processes, but most of the exo-inulinases reported so far are mesophilic or high-temperature enzymes, and exo-inulinases with low-temperature (0–20°C) activity very little inulinase

Method used

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  • Exoinulinase Z2-5 with low-temperature activity and gene of exoinulinase Z2-5
  • Exoinulinase Z2-5 with low-temperature activity and gene of exoinulinase Z2-5
  • Exoinulinase Z2-5 with low-temperature activity and gene of exoinulinase Z2-5

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Experimental program
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Effect test

Embodiment 1

[0033] Embodiment 1: Sphingobacterium exo-inulinase Z2-5 clone

[0034] Extract the genomic DNA of Sphingomonas: centrifuge the 2-day cultured bacteria liquid to get the bacteria, add 1mL lysozyme, treat at 37°C for 60min, then add the lysate, lyse in a water bath at 70°C for 60min, and mix every 10min. Centrifuge at 10000 rpm for 5 min 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.

[0035] The degenerate primers Inu32F and Inu32R were designed and synthesized according to the conserved amino acid sequence of exo-inulinase (H-N-W-M-N-D-P-N-G and R-D-P-K-V-F-W-H-E-Q-S) (Table 1).

[0036] P...

Embodiment 2

[0040] Embodiment 2: recombinant exo-inulinase Z2-5 preparation of

[0041] by Z2-5InuEF with Z2-5InuER As the primer pair (Table 1), the genomic DNA of Sphingobacterium was used as the template for PCR amplification. The PCR reaction parameters were: denaturation at 94°C for 5 min; then denaturation at 94°C for 30 sec, annealing at 45°C for 30 sec, extension at 72°C for 2 min, and after 40 cycles, incubation at 72°C for 5 min and incubation at 25°C for 3 min. exo-inulinase gene Z2-5 . exo-inulinase gene Z2-5 Connect with the expression vector pEASY-E1 to obtain the exo-inulinase gene Z2-5 The recombinant plasmid pEASY-E1- Z2-5 And transform Escherichia coli BL21(DE3) to obtain recombinant Escherichia coli strain BL21(DE3) / Z2-5 .

[0042] Take the recombinant plasmid pEASY-E1- Z2-5 of E. coli BL21(DE3) strains and pEASY-only

[0043] -E1 empty plasmid E. coli BL21(DE3) strain was inoculated in LB (containing 50 μg / mL Amp) culture medium with 0.1% inocul...

Embodiment 3

[0044] Example 3: Characterization of Purified Recombinant Exo-Inulinase Z2-5

[0045] 1. Activity analysis of recombinant exo-inulinase Z2-5

[0046] The activity determination method of the purified recombinant exo-inulinase Z2-5 in Example 2 adopts the 3,5-dinitrosalicylic acid (DNS) method: the substrate is dissolved in 0.1M buffer solution, and its final concentration is 0.5% (w / v); the reaction system contains 100μL appropriate amount of enzyme solution and 900μL 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, and boil for 5 minutes , after cooling to room temperature, measure the OD value at a wavelength of 540nm. One enzyme activity unit (U) is defined as the amount of enzyme required to decompose the substrate to produce 1 μmol of reducing sugar per minute under the given conditions.

[0047] 2. Determination of optimum pH and pH sta...

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Abstract

The invention relates to exoinulinase Z2-5 with low-temperature activity and a gene of the exoinulinase Z2-5. An amino acid sequence of the exoinulinase Z2-5 from sphingobacterium sp. is shown as SEQIDNO.1. The invention provides the gene Z2-5 encoded with the exoinulinase, and a recombinant vector and a recombinant strain of the exoinulinase gene Z2-5. The exoinulinase has the properties that the optimum pH value is 7; after the exoinulinase is treated by a 0.1 M buffer solution of which the pH value is 9.0 at room temperature for 1 hour, the activity of the exoinulinase also can be kept at over 30 percent; the optimum temperature is 40 DEG C, the enzyme activity of the exoinulinase is about 40 percent at the temperature of 10 DEG C and 50 DEG C, and the enzyme activity of the exoinulinase is about 10 percent at the temperature of below 0 DEG C; after the exoinulinase is treated at the temperature of 50 DEG C for 1 hour, the enzyme activity of exoinulinase also can be kept at over 30percent; and substrates of cane sugar, fructosan and the like can be hydrolyzed, so the exoinulinase belongs to the reaction selectivity of the hydrolysis of a fructose glycosidic bond. By the exoinulinase Z2-5, synanthrin can be hydrolyzed to prepare high fructose corn syrup, so the exoinulinase Z2-5 is used for food industry.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to an exo-inulinase Z2-5 with low-temperature activity and its gene. Background technique [0002] Inulin, also known as inulin, is a mixture of natural fructans, which are connected by fructose molecules through β-2,1 glycosidic bonds, with a degree of polymerization from 2-60, and its terminal is a glucose unit (ETTALI BIM et al. Appl Microbiol Biotechnol, 1987, 26: 13-20.). It is widely found in Jerusalem artichoke ( Helianthus tuber osus ), burdock ( Arctium ) ,endive( Chicory intybus ) and many other undeveloped plants (GoudaM K. Biological Sciences, 2002, 5(5): 589-593.). [0003] Inulinase (Inulinase, EC3.2.1.7), scientific name β-2,1-D-fructanase, also known as β-fructanase, is a kind of enzyme that can degrade inulin fructose and fructooligosaccharides polysaccharide hydrolase. Inulinase has two modes of action, one is the exo-type, which hydrolyzes β-2,...

Claims

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Application Information

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IPC IPC(8): C12N9/24C12N15/56C12N15/63C12N1/21C12N1/19C12R1/01
Inventor 黄遵锡彭沫溱周峻沛唐湘华李俊俊许波
Owner YUNNAN NORMAL UNIV
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