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Method for increasing phytoene dehydrogenase in vitro reaction rate

A technology of phytoene and lycopene, which is applied in the field of bioengineering, can solve problems such as slow reaction rate, and achieve the effects of reducing oxidative loss, improving accuracy, and improving enzymatic reaction rate.

Inactive Publication Date: 2013-03-13
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The present invention aims at the defect of slow reaction rate of the existing enzyme in vitro reaction system, and provides a method for effectively increasing the in vitro reaction rate of phytoene dehydrogenase

Method used

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  • Method for increasing phytoene dehydrogenase in vitro reaction rate
  • Method for increasing phytoene dehydrogenase in vitro reaction rate
  • Method for increasing phytoene dehydrogenase in vitro reaction rate

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1: Ultrasonic mixing method to establish phytoene dehydrogenase in vitro reaction

[0027] (1) Construction of recombinant Escherichia coli expressing phytoene dehydrogenase

[0028] The phytoene dehydrogenase gene (GenBank No. CP000661) of Rhodobacter sphearoides strain preservation number ATCC No. 17025 was amplified by PCR, and connected to Escherichia coli through NdeI and SalI restriction sites A recombinant plasmid was constructed on the expression vector pET-22b (Novagen, Germany), and the recombinant plasmid was transformed into Escherichia coli BL21 (DE3) competent cells to obtain recombinant Escherichia coli expressing phytoene dehydrogenase;

[0029] (2) Preparation of cell disruption solution

[0030] Centrifuge the 200mL recombinant Escherichia coli culture fluid expressing phytoene dehydrogenase prepared in step (1) at 12000rpm for 5 minutes to collect the bacterial cells; resuspend the collected Escherichia coli cells in 10mL, pH7.9 In 100mmol / L...

experiment example 1

[0034] Experimental example 1: The reaction system is not mixed with ultrasonic waves.

[0035] Take 400 μL of the recombinant Escherichia coli disrupted liquid prepared in the example, add the same amount of phytoene acetone solution, glucose, glucose oxidase and catalase as in the example, and make the volume to 500 μL with buffer; mix well Afterwards, 30° C. was protected from light, and the closed shaking reaction was carried out. The reaction conditions, reaction time, reaction termination, and product extraction and detection were the same as in Example 1.

[0036] The above-mentioned reaction system does not need ultrasonic mixing, and the main product in the in vitro reaction system is streptosporine, and a trace amount of lycopene is generated (attached figure 2), the total yield of carotenoid products was 0.557±0.143μg·mL -1 h -1 .

experiment example 2

[0037] Experimental Example 2: Add soybean lecithin emulsion to the control reaction system

[0038] Get 400 μL of recombinant Escherichia coli disrupted solution prepared in the examples, add the same amount of phytoene acetone solution, glucose, glucose oxidase and catalase as in the examples, add 10 μL of 40 mg / mL emulsified soybean lecithin, The volume was adjusted to 500 μL with buffer solution; 30° C. was protected from light and the reaction was shaken in an airtight manner. The reaction conditions, reaction time, reaction termination, and product extraction and detection were the same as in Example 1.

[0039] The above control reaction system was added to the external reaction of soybean lecithin emulsion, and the total yield of the product was 0.540±0.225μg·mL -1 h -1 , similar to Experimental Example 1, soybean lecithin in the crude enzyme solution has no obvious effect on improving the reaction rate.

[0040] The preparation method of the above-mentioned emulsifi...

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Abstract

The invention relates to a method for increasing water-organic double-phase enzymatic reaction rate, in particular relates to a method for increasing phytoene dehydrogenase in vitro reaction rate and belongs to the technical field of biological engineering. The method comprises the following steps of: adding phytoene acetone solution, glucose, glucose oxidase and hydrogenperoxidase into crushed coarse enzyme solution to ultrasonically mix uniformly; performing dark airtight oscillating reaction at the temperature of between 28 and 30 DEG C; adding methanol; performing warm bath at the temperature of between 55 and 60 DEG C for 15 to 20 minutes; adding petroleum ether with the boiling range of 30 to 60 DEG C to oscillate to extract reaction products; collecting the upper layer of a petroleum ether layer; and performing vacuum rotary evaporation to obtain the product. A phytoene dehydrogenase in vitro reaction system is optimized by utilizing the mechanical effects such as homogeneity, dispersion, emulsification and the like of ultrasonic wave, so probability of a substrate contacting the enzyme is improved, the enzymatic reaction rate is obviously increased, reaction time is shortened, oxidation loss of the product is reduced, and accuracy of data is improved.

Description

technical field [0001] The invention relates to a method for increasing the rate of water-organic two-phase enzymatic reaction, in particular to a method for increasing the in vitro reaction rate of phytoene dehydrogenase, belonging to the technical field of bioengineering. Background of the invention [0002] Phytoene desaturase (phytoene desaturase 1.14.99.-) is a key enzyme in the synthesis and metabolism of carotenoids, which can catalyze colorless phytoene to generate colored carotenoids. Enzymes are widely found in plants and microorganisms. According to the number of dehydrogenation, there are at most 6 kinds of products of phytoene dehydrogenase, which are hexahydrophytoene, ζ-carotene, streptoerythrin, lycopene, 3,4-dide Phytoene, 3,4,3',4'-tetradehydrolycopene, among which phytoene dehydrogenase with streptosporine and lycopene as products is the most common. Almost all carotenoids naturally occurring in nature are produced with streptosporin or lycopene as inter...

Claims

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Application Information

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IPC IPC(8): C12P23/00C12N15/53C12N15/70C12N1/21C12R1/19
Inventor 肖敏张金华卢丽丽
Owner SHANDONG UNIV
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