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Recombined alkaline pectinase with high pH stability and specific enzyme activity and construction method thereof

A technology of pectinase and specific enzyme activity, which is applied in the field of biogenetic engineering and can solve the problems of time-consuming, high cost, and large water consumption

Inactive Publication Date: 2014-06-25
HUBEI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In industrial applications, the narrow pH range of enzymes will require more technologies to maintain the stability of the pH in the reaction system, and at the same time, a large amount of acid or alkali will be required for neutralization, resulting in high cost, large water consumption, high energy consumption, It takes a long time and the COD value in the waste liquid is high. At the same time, the research on the high efficiency and versatility of pectinase is also imminent

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  • Recombined alkaline pectinase with high pH stability and specific enzyme activity and construction method thereof
  • Recombined alkaline pectinase with high pH stability and specific enzyme activity and construction method thereof

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Embodiment 1

[0059] A kind of alkaline pectinase with good pH stability and high specific enzyme activity and its encoding gene of the present invention is characterized in that the pectinase gene pel168 contained in GenBank accession number AL009126 corresponds to the amino acid sequence of pectinase PEL168 The accession number is CAB12585.1, design point mutation primers (FP and RP) for site-directed mutagenesis, change the guanine G at position 457 of the published gene into thymine T, and obtain pel168V132F, making it encode the amino acid sequence of the mature peptide Valine V (GTC) at position 132 was mutated into phenylalanine F (TTC) to obtain recombinant alkaline pectinase PEL168V132F.

[0060] The recombinant alkaline pectinase of the present invention and the construction method of its encoding gene,

[0061] Including the following steps:

[0062] 1) PCR method was used to clone the gene pel168 encoding alkaline pectinase;

[0063] 2) Link the alkaline pectinase gene pel168 ...

Embodiment 2

[0076] Medium: LB resistant medium: tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, kanamycin 50 mg / mL;

[0077] Cultivation method: After activating the recombinant strain Rb(DE3)-pET28a-pel168V132F of Example 1 on the LB resistance plate to prepare seeds, transfer it to liquid LB medium at an inoculum size of 1:100, at 37°C, 200 rpm cultured to OD 600 0.6, add the inducer IPTG, and then culture at 18°C, 200rpm for 14-16h.

[0078] The induced bacterial cells were ultrasonically destructed, passed through a HIS column and an ion exchange column for purification and concentration, and a purified pectinase mutant PEL168V132F was obtained.

Embodiment 3

[0080] Alkaline pectinase enzyme activity assay method: take 20 μl of diluted enzyme solution, add 2 ml of 0.2% polygalacturonic acid solution, mix well, and start the enzymatic reaction; The reaction was terminated by mol / L phosphoric acid, and the absorbance value was measured at 235 nm. The blank control is an inactive enzyme solution reaction (ie: first add 3ml of phosphoric acid and mix with the enzyme solution to be tested, then add the substrate to react for 15 minutes). The production of 1 mmol / L reducing sugar per minute is regarded as 1 enzyme activity unit (U).

[0081] Enzyme activity calculation: OD 235×10 6 × enzyme dilution factor × total reaction volume

[0082] ______________________________________

[0083] 10 3 ×t×4600×b×volume of enzyme

[0084] Where: 4600 (L.mol -1 cm -1-- )—Molar absorptivity of unsaturated polygalacturonic acid at 235 nm

[0085] t--(min)—enzymatic reaction time...

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Abstract

The invention discloses recombined alkaline pectinase with good pH stability and high specific enzyme activity and a construction method thereof. A Site-directed mutagenesis technique is adopted for mutagenesis of an alkaline pectinase coding gene pel168 of bacillus subtilis 168 and 457-bit guanine G is transformed into thymine T so that the 132-bit valine V of the mature peptide of the coded pectinase is mutated into phenylalanine F, a mutant gene pel168V132F is expressed in colon bacillus Rosset Blue (DE3) and subjected to inducible expression, bacterium breaking and purification, the purified enzyme is subjected to enzyme activity measurement, and the relative activity of the obtained pectinase mutant PEL168V132F is much higher than that of the original pectinase PEL168 when the pH is 3, 5 and 6; and meanwhile, the specific enzyme activity is 2.25 times that of the original pectinase, thus good foundation is laid for wide application of the alkaline pectinase.

Description

technical field [0001] The invention relates to a recombinant alkaline pectinase with improved pH stability and specific enzyme activity, its coding gene, and its construction method, belonging to the technical field of biological genetic engineering. Background technique [0002] Pectinase is a general term for various enzymes that decompose pectin. Pectinase is divided into acid pectinase and alkaline pectinase according to its optimum pH. The optimum pH of acid pectinase is in the acidic range, and it is widely used in the extraction of fruit juice and wine and feed industry (Wu Minchen, 2006). Alkaline pectinase has an optimum pH of 8.0-10.0, and is currently used in plant drug extraction, tea and coffee fermentation, oil extraction, textile, papermaking, detergent, plant fiber processing, pectin-containing industrial wastewater treatment and biotechnology More and more attention has been paid to the application of such industries, so it has become a research hotspot (...

Claims

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Application Information

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IPC IPC(8): C12N9/88C12R1/19
CPCC12N9/88C12N15/70C12Y402/02002
Inventor 张桂敏徐婷宋江宁马延和
Owner HUBEI UNIV
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