Recombinant vector containing Jerusalem artichoke fructan 1-exohydrolase gene and application thereof in producing alcohol by fermentation

A kind of technology of hydrolase and fructan, applied in the field of genetic engineering

Inactive Publication Date: 2015-11-11
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are few reports on the use of hydrolytic enzymes from higher plants to transfer alcohol to yeast

Method used

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  • Recombinant vector containing Jerusalem artichoke fructan 1-exohydrolase gene and application thereof in producing alcohol by fermentation
  • Recombinant vector containing Jerusalem artichoke fructan 1-exohydrolase gene and application thereof in producing alcohol by fermentation
  • Recombinant vector containing Jerusalem artichoke fructan 1-exohydrolase gene and application thereof in producing alcohol by fermentation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] The thermal stability of the Jerusalem artichoke Ht1-FEHs of embodiment 1 recombination

[0036] The temperature stability of the purified recombinant Ht1-FEHs enzyme was determined by putting the recombinant enzyme in a water bath at different temperatures (4, 30, 35, 40, 50, 60, 70°C) for 2 hours, then placing the recombinant enzyme on ice to restore the low temperature, and then Mix with the most suitable substrate 1-kestose, pH 6, react overnight at 35°C, and determine the remaining enzyme activity. The temperature stability of the enzyme was determined by taking the enzyme activity of the recombinant enzyme at 4°C as a reference.

[0037] The result is as figure 1 As shown, the purified recombinant enzyme Ht1-FEHII still maintains more than 80% of its activity after being incubated at a temperature below 40°C for 2 hours, indicating that the enzyme is very stable at room temperature, while the enzyme activity of Ht1-FEHI at a temperature above 35°C will decrease. ...

Embodiment 2

[0038] The pH stability of the recombinant Jerusalem artichoke Ht1-FEHs of embodiment 2

[0039] Mix the purified recombinant proteins Ht1-FEHI and II with buffers of different pH (3, 4, 5, 5.5, 6, 6.5, 7, 8), and measure the remaining enzyme activity after standing at 0°C for 24 hours to evaluate the recombinant Enzyme stability to pH. The pH stability of inulinase was determined with reference to the enzyme activity placed at the optimum pH6. Method for measuring remaining enzyme activity: 1-kestose was used as substrate, pH 6, reacted overnight at 35°C, and the change of enzyme activity under the influence of different pH was measured. The results show (such as figure 2 ), the enzyme activities of the recombinant proteins Ht1-FEHI and II were relatively stable between pH 4 and 8.

Embodiment 3

[0040] The effect of different metal compounds of embodiment 3 on the activity of recombinant Jerusalem artichoke Ht1-FEHs

[0041] Mix the purified recombinant proteins Ht1-FEHI and II with different metal compounds, the final ion concentration is 1.0mM, and place it at 0°C for 60min to measure the inulinase activity. The metal ions include ZnSO 4 , CuSO 4 .5H 2 O, MgSO 4 .5H 2 O, FeCl 3 , CaCl 2 , KCl, MnCl 2 , LiCl, FeCl 2 , NaCl and CoCl 2 etc., the control is the enzyme activity of the recombinant enzyme added with the same volume of distilled water, and the influence of metal ions on the enzyme activity is judged according to the remaining enzyme activity.

[0042] The result is as image 3 As shown, 1.0mM Li + , Ca 2+ 、K + 、Na + , Mg 2+ and Co 2+ There are different degrees of activation of recombinase Ht1-FEHI and II, while Fe 2+ and Cu 2+ It has a certain inhibitory effect on the enzyme activity of recombinase 1-FEHs, indicating that they can change t...

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Abstract

The invention discloses a recombinant vector containing Jerusalem artichoke fructan 1-exohydrolase gene and application thereof in producing alcohol by fermentation. The recombinant plasmid is prepared by the following steps: inserting a Saccharomyces cerevisiae 3-phosphoglyceric kinase gene PGK1 promoter between EcoRV and SpeI enzyme digestion sites of an expression vector pUG6, inserting a CYC1-terminator-fused Jerusalem artichoke fructan 1-exohydrolase gene Ht 1-FEHs between SpeI and HpaI enzyme digestion sites of the expression vector pUG6, and inserting an 18SrDNA sequence segment of Saccharomyces cerevisiae genome between XbaI and BstEII enzyme digestion sites of the expression vector pUG6. The invention also discloses application of the recombinant plasmid in producing alcohol by fermentation. The Jerusalem artichoke inulase genes Ht-FEHI and Ht-FEHII can promote fermentation of the Saccharomyces cerevisiae to produce alcohol, thereby providing a new method for enhancing the alcohol yield of Saccharomyces cerevisiae fermentation.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and relates to a recombinant carrier containing Jerusalem artichoke fructan 1-exohydrolase gene and its application in alcohol production by fermentation. Background technique [0002] Plant fructan is a kind of carbohydrate with low content in general plants. It is formed by connecting multiple fructosyl groups with glycosidic bonds. It is only found in the tubers of Jerusalem artichoke (Helianthus tuberosus); chicory (Cichoriumintybus), burdock (Arctium lappal) and chicken feet Roots of grass (Dactylis glomeratus); corms of lilies (Lilium brownii) and onions (Alliumcepa); and tissues of a few plants such as ryegrass (Loliumperenne), barley (Hordeumvulgare) and wheat (Triticumaestivum) stems and leaves. The inulin (Inulin) extracted from Jerusalem artichoke is a mixture of fructans, the number of its monosaccharide group (polymerization degree) is between 3-50, and inulin accounts for 20% of t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/81C12N1/19C12P7/06C12R1/865
CPCY02E50/10
Inventor 梁明祥张茜许欢欢
Owner NANJING AGRICULTURAL UNIVERSITY
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