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Application of sdr family oxidoreductase e26 as emulsifier

A reductase, E26 technology, applied in the directions of oxidoreductase, resistance to vector-borne diseases, liquid carbon-containing fuel, etc., to achieve the effect of tolerance to pH environment, good emulsification effect, and wide pH environment

Active Publication Date: 2022-05-17
NANJING TECH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, few proteins have good emulsifying activity, research and exploration of proteins with high-efficiency emulsifying activity is the premise to solve this problem

Method used

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  • Application of sdr family oxidoreductase e26 as emulsifier
  • Application of sdr family oxidoreductase e26 as emulsifier
  • Application of sdr family oxidoreductase e26 as emulsifier

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] This example illustrates the recombinant engineered bacteria Escherichia coli Construction of BL21-pET-e26.

[0051] Using PCR technology, from wild bacteria Aeribacillus pallidus The SDR family oxidoreductase E26 gene e26 was amplified from the SL-1 genomic DNA, and the e26 gene was connected to the multiple cloning site of the expression vector pET28, and the recombinant plasmid pET28-e26 was transformed into E. coli Genetically engineered bacteria obtained from BL21(DE3) Escherichia coli BL21-pET-e26.

[0052] The above construction methods specifically include:

[0053] Cloning of gene e26:

[0054] According to wild mushroom Aeribacillus pallidus Design primers for the e26 gene sequence in the SL-1 genome sequencing results:

[0055] P1: 5'-CGGGATCCCGATGTTAACAAATCAAGTAGC-3' (BamH I)

[0056] P2: 5’- CCAAGCTTGGTGCAATAGGATATTGTTGAA-3’ (Hind III)

[0057] PCR reaction system: ddH 2 O 20 µL, High Fidelity Enzyme Mix 25 µL, Primer P1 2 µL, Primer P2 2 ...

Embodiment 2

[0061] This example illustrates the acquisition of the SDR family oxidoreductase E26.

[0062] (1) Induced expression of SDR family oxidoreductase E26:

[0063] Medium: LB medium: yeast powder 5 g / L, peptone 10 g / L, sodium chloride 10 g / L.

[0064] Culture conditions: 50 mL of liquid in a 250 mL Erlenmeyer flask, culture temperature 37°C, 200 rpm, culture until OD600 reaches 0.6-0.8, add 1 mM IPTG to induce the expression of SDR family oxidoreductase E26. After culturing for 12 hours at 28°C and 200 rpm, the cells were collected by centrifugation at 10,000×g for 10 min. The cells were resuspended in 50 mM Tris-HCl buffer, pH 7.4, and the cells were crushed by a high-pressure homogenizer, centrifuged at 10,000×g for 20 min, and the supernatant was discarded, which was the crude enzyme solution.

[0065] (2) Determination of protein amount: the protein amount was measured by the Brandford method.

[0066] Production of protein standard song:

[0067] Prepare Coomassie Brilli...

Embodiment 3

[0075] This example illustrates the emulsifying activity assay method.

[0076] Using different hydrocarbons as emulsification objects, in a 5mL volumetric glass bottle, add the purified enzyme solution in Example 2 to the buffer solution (50 mM Tris-HCl pH7.8), the total volume is 2 mL, and mix with the oil phase Mix at a volume ratio of 1:1, vortex for 2 minutes, and let stand for 48 hours.

[0077] Emulsification index (EI48) is calculated by multiplying the ratio of the height of the emulsified layer to the total height by 100% to represent the emulsifying activity of the emulsifier.

[0078] Emulsification index (EI48) = (emulsion layer height) / (total liquid height) × 100%.

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Abstract

The present invention relates to the application of SDR family oxidoreductase E26 as an emulsifier. The amino acid sequence of the SDR family oxidoreductase E26 is shown in SEQ ID NO:1. The present invention also provides a biological emulsifier, which is made by compounding SDR family oxidoreductase E26 with additives, and the emulsifier obtained after compounding with additives has more stable emulsifying performance. The SDR family oxidoreductase E26 of the present invention can be used as a high-efficiency biological emulsifier, can act in a wide pH environment, can withstand high temperature, can tolerate high-concentration salt, and has a significant emulsifying effect on hydrocarbons and oils .

Description

technical field [0001] The invention belongs to the field of biochemical industry, and in particular relates to the application of SDR family oxidoreductase E26 as a biological emulsifier. Background technique [0002] Bioemulsifiers are high molecular weight biopolymers produced by a wide variety of microorganisms that are effective in forming and stabilizing oil-in-water emulsions [1]. They are amphiphilic complexes composed of polysaccharides, lipopolysaccharides, lipoproteins and proteins [1-3]. Similar to surfactants, bioemulsifiers can also increase the bioavailability of insoluble substrates, thereby promoting the biodegradation of insoluble substrates [4]. They differ from biosurfactants in that bioemulsifiers do not significantly reduce surface and interfacial tension [1]. Therefore, for some adverse effects of biosurfactants to reduce surface tension, such as disrupting the permeability of cell membranes [5], negative interactions with enzymes or proteins [6] and...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/02C10L1/32
CPCC10L1/32Y02A50/30
Inventor 李霜陶惟一黄和
Owner NANJING TECH UNIV
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