阅读说明：本技术 Sdr家族氧化还原酶e26作为乳化剂的应用 (Application of SDR family oxidoreductase E26 as emulsifier ) 是由 李霜 陶惟一 黄和 于 2021-01-11 设计创作，主要内容包括：本发明涉及SDR家族氧化还原酶E26作为乳化剂的应用,所述SDR家族氧化还原酶E26的氨基酸序列如SEQ ID NO：1所示。本发明还提供了一种生物乳化剂,将SDR家族氧化还原酶E26与添加剂复配后作为所述生物乳化剂,与添加剂复配后获取的乳化剂乳化性能更稳定。本发明的SDR家族氧化还原酶E26可作为一种高效生物乳化剂使用,可作用于宽泛的pH环境,可耐受高温,可耐受高浓度盐,对烃类和油脂均有显著的乳化效果。(The invention relates to an application of SDR family oxidoreductase E26 as an emulsifier, wherein the amino acid sequence of the SDR family oxidoreductase E26 is shown as SEQ ID NO: 1 is shown. The invention also provides a biological emulsifier, wherein the biological emulsifier is prepared by compounding the SDR family oxidoreductase E26 with an additive, and the emulsifying property of the emulsifier obtained by compounding the biological emulsifier with the additive is more stable. The SDR family oxidoreductase E26 can be used as a high-efficiency biological emulsifier, can act on a wide pH environment, can resist high temperature and high-concentration salt, and has a remarkable emulsifying effect on hydrocarbons and grease.)

Use of an SDR family oxidoreductase E26 as an emulsifier, the amino acid sequence of said SDR family oxidoreductase E26 being as set forth in SEQ ID NO: 1 is shown.

2. Use according to claim 1, wherein the emulsifying substrate is a hydrophobic substance; hydrocarbons and greases are preferred.

3. Use according to claim 1, wherein the temperature in the emulsion reaction system does not exceed 85 ℃.

4. The use according to claim 1, wherein the pH in the emulsion reaction system is in the range of 3 to 11.

5. The use according to claim 1, characterized in that a carrier containing said SDR family oxidoreductase E26 protein is used as an emulsifier to carry out an emulsion reaction with a substrate.

6. The use of claim 5, characterized in that the vector containing the SDR family oxidoreductase E26 protein is a purified protein obtained by isolation and purification of the SDR family oxidoreductase E26 protein after expression in recombinant bacteria; or crushing the recombinant bacterial cell after the protein expression to obtain crude protein, and mixing the crude protein and an emulsification substrate for emulsification reaction.

7. The use according to any one of claims 1 to 6, characterized in that different additives are compounded with SDR family oxidoreductase E26.

8. The use according to claim 7, wherein the compounding additive is selected from one of fructose, sucrose, chitosan, agarose, trehalose, starch, gelatin, xanthan gum and gum arabic.

9. The biological emulsifier is characterized in that different additives are compounded with SDR family oxidoreductase E26 to obtain the biological emulsifier, wherein the amino acid sequence of the SDR family oxidoreductase E26 is shown as SEQ ID NO: 1 is shown.

10. The bio-emulsifier according to claim 9, wherein the additive is selected from one of fructose, sucrose, chitosan, agarose, trehalose, starch, gelatin, xanthan gum, and gum arabic.

Technical Field

The invention belongs to the field of biochemical engineering, and particularly relates to application of SDR family oxidoreductase E26 as a biological emulsifier.

Background

Bioleaching agents are high molecular weight biopolymers produced by a wide variety of microorganisms and are effective in forming and stabilizing oil-in-water emulsions [1 ]. They are amphiphilic complexes composed of polysaccharides, lipopolysaccharides, lipoproteins, proteins, and the like [1-3 ]. Similar to surfactants, biological emulsifiers also increase the bioavailability of poorly soluble substrates, thereby promoting biodegradation of poorly soluble substrates [4 ]. They differ from biosurfactants in that biosurfactants do not significantly reduce surface and interfacial tension [1 ]. Thus, some of the adverse effects of biosurfactants on lowering surface tension, such as cell membrane permeability disruption [5], negative interactions with enzymes or proteins [6] and general cellular toxicity [7], can be avoided by using bioamulsifiers. The biological emulsifier has the advantages of low toxicity, biodegradability, high biocompatibility, renewability and the like, and particularly has good stability under extreme temperature, pH value and salt concentration [3 ]. These properties make them extremely valuable for industrial applications in the fields of petroleum, pharmaceutical, cosmetic and food industries [1 ]. Currently, the potential use of bio-emulsifiers in the petroleum industry has been extensively studied, such as Microbial Enhanced Oil Recovery (MEOR) [8, 9] and bioremediation of contaminated soils or other environmental pollution [10 ]. Despite the potential advantages of biological emulsifiers, practical application of biological emulsifiers is hindered by problems such as low yield and high purification cost. To address these problems, many researchers have struggled to produce and develop more effective bio-emulsifiers.

Although the composition of the biological emulsifier is diverse and complex, studies have shown that the emulsifying activity of the biological emulsifier is highly correlated with its chemical composition [11, 12 ]. One of the most well known and studied biological emulsifiers is the emulsifier Emulsan produced by the Acinetobacter calcoseticus RAG-1 strain, a complex of anionic heteropolysaccharides and proteins [13, 14 ]. The protein in Emulsan was purified and demonstrated to be an esterase which, when mixed with different polysaccharides, increased emulsion stability [15-17 ]. In addition, another biological emulsifier Alasan produced by the strain Acinetobacter radioresistances KA53 is composed of alanine, polysaccharides and proteins. Alasan contains three protein components which are considered as the main emulsifying functional substances, of which the 45-kDa protein has been shown to have the highest emulsifying activity, even 11% higher than the intact Alasan complex [3, 18, 19 ]. Two other classes of proteins have hydrophobic surface activity, namely hydrophobin and Phasin. Hydrophobins are a group of small proteins produced by filamentous fungi that have surface activity. However, the low yields of hydrophobin production, isolation and purification still hinder its use as a bio-emulsifier [20 ]. Phasin protein is located on the surface of Polyhydroxyalkanoate (PHA) particles and is an amphiphilic small protein capable of being combined with a hydrophobic polymer. The Phasin protein PhaR shows high emulsifying capacity, but it is expressed in the form of inclusion bodies, which brings difficulty to downstream separation and extraction [21 ]. In conclusion, proteins play an essential role in the emulsifying activity of the bio-emulsifiers, while polysaccharides play a role as stabilizers in the emulsifying system. Based on this premise, engineered biological emulsifiers can be obtained by appropriate combination of protein and polysaccharide components, potentially providing tailored biological emulsifiers for specific hydrophobic compounds. Furthermore, these combined bio-emulsifiers can be provided at relatively low cost, since the protein and polysaccharide components of the bio-emulsifiers are more easily produced and extracted on a large scale in separate industrial processes. However, few proteins have good emulsifying activity, and research and search for proteins having high-efficiency emulsifying activity are a prerequisite for solving this problem.

1.Uzoigwe C, Burgess JG, Ennis CJ, Rahman PK: Bioemulsifiers are not biosurfactants and require different screening approaches. Front Microbiol. 2015;6:245.

2.Dastgheib SMM, Amoozegar MA, Elahi E, Asad S, Banat IM: Bioemulsifier production by a halothermophilic Bacillus strain with potential applications in microbially enhanced oil recovery. Biotechnol Lett. 2008;30:263-270.

3.Toren A, Navonvenezia S, Ron EZ, Rosenberg E: Emulsifying activities of purified Alasan proteins from Acinetobacter radioresistens KA53. Appl Environ Microb. 2001;67:1102.

4.Wang Y, Wang C, Ren HJ, Jia BL, Zhang LY: Effectiveness of recombinant protein AlnA in enhancing the extractability of polychlorinated biphenyls from contaminated soils. J Hazard Mater. 2014;279:67-74.

5.Deleu M, Lorent J, Lins L, Brasseur R, Braun N, El Kirat K, Nylander T, Dufrene YF, Mingeot-Leclercq MP: Effects of surfactin on membrane models displaying lipid phase separation. BBA-Biomembranes. 2013;1828:801-815.

6.Laha S, Luthy RG: Inhibition of phenanthrene mineralization by nonionic surfactants in soil-water systems. Environ Sci Technol. 1991;25:1920-1930.

7.Lamichhane S, Krishna KCB, Sarukkalige R: Surfactant-enhanced remediation of polycyclic aromatic hydrocarbons: A review. J Environ Manage. 2017;199:46-61.

8.Xia MQ, Fu DF, Chakraborty R, Singh RP, Terry N: Enhanced crude oil depletion by constructed bacterial consortium comprising bioemulsifier producer and petroleum hydrocarbon degraders. Bioresource Technol. 2019;282:456-463.

9.Dong H, Xia WJ, Dong HH, She YH, Zhu PF, Liang K, Zhang ZZ, Liang CF, Song ZZ, Sun SS, Zhang GQ: Rhamnolipids produced by indigenous Acinetobacter junii from petroleum reservoir and its potential in enhanced oil recovery. Front Microbiol. 2016;7:1710.

10.Calvo C, Manzanera M, Silva-Castro GA, Uad I, Gonzalez-Lopez J: Application of bioemulsifiers in soil oil bioremediation processes. Future prospects. Sci Total Environ. 2009;407:3634-3640.

11.Kaplan N, Rosenberg E: Exopolysaccharide Distribution of and Bioemulsifier Production by Acinetobacter calcoaceticus BD4 and BD413. Appl Environ Microb. 1982;44:1335-1341.

12.Kaplan N, Zosim Z, Rosenberg E: Reconstitution of emulsifying activity of Acinetobacter calcoaceticus BD4 emulsan by using pure polysaccharide and protein. Appl Environ Microb. 1987;53:440-446.

13.Rosenberg E, Zuckerberg A, Rubinovitz C, Gutnick DL: Emulsifier of Arthrobacter RAG-1: isolation and emulsifying properties. Appl Environ Microb. 1979;37:402-408.

14.Zosim Z, Fleminger G, Gutnick D, Rosenberg E, Wise G: Effect of protein on the emulsifying activity of emulsan. J Disper Sci Technol. 1989;10:307-317.

15.Shabtai Y, Gutnick DL: Exocellular esterase and emulsan release from the cell surface of Acinetobacter calcoaceticus. J Bacteriol. 1985;161:1176-1181.

16.Bach H, Gutnick DL: A unique polypeptide from the C-terminus of the exocellular esterase of Acinetobacter venetianus RAG-1 modulates the emulsifying activity of the polymeric bioemulsifier apoemulsan. Appl Microbiol Biotechnol. 2006;71:177.

17.Bach H, Gutnick DL: Novel polysaccharide-protein-based amphipathic formulations. Appl Microbiol Biotechnol. 2006;71:34-38.

18.Toren A, Orr E, Paitan Y, Ron EZ, Rosenberg E: The active component of the bioemulsifier alasan from Acinetobacter radioresistens KA53 is an OmpA-like protein. J Bacteriol. 2002;184:165.

19.Toren A, Segal G, Ron EZ, Rosenberg E: Structure-function studies of the recombinant protein bioemulsifier AlnA. Environ Microbiol. 2002;4:257-261.

20.Khalesi M, Gebruers K, Derdelinckx G: Recent Advances in Fungal Hydrophobin Towards Using in Industry. Protein J. 2015;34:243-255.

21.Ma HK, Liu MM, Li SY, Wu Q, Chen JC, Chen GQ: Application of polyhydroxyalkanoate (PHA) synthesis regulatory protein PhaR as a bio-surfactant and bactericidal agent. J Biotechnol. 2013;166:34.。

Disclosure of Invention

The invention aims to provide application of SDR family oxidoreductase E26 as a biological emulsifier.

In order to achieve the technical purpose, the invention adopts the following technical scheme:

use of an SDR family oxidoreductase E26 as an emulsifier, the amino acid sequence of said SDR family oxidoreductase E26 being as set forth in SEQ ID NO: 1 is shown. The nucleotide sequence of the gene for coding the SDR family oxidoreductase E26 is shown as SEQ ID NO: 2, respectively.

The inventor finds that the SDR family oxidoreductase E26 has the function of emulsifying hydrocarbon substances and can form a stable emulsion layer with various hydrocarbons under different conditions.

In the application of the invention, the emulsifying substrate is a hydrophobic substance. As the emulsifier, theoretically, any hydrophobic substance can theoretically serve as an emulsifying substrate. The SDR family oxidoreductase E26 has a good emulsification effect on hydrocarbons and grease in hydrophobic substances, and has excellent properties of wide pH environment tolerance, salt resistance, heat resistance and the like.

In the application of the invention, the emulsifying system is a 4 ml system, and the oil phase is as follows in each ml: the water phase proportion relation is 1: 1.

in the application, a carrier containing SDR family oxidoreductase E26 protein is used as an emulsifier, and is contacted with hydrocarbons for emulsification reaction.

The carrier containing the SDR family oxidoreductase E26 protein is an emulsifier, and can be understood as a purified protein obtained by separating and purifying the protein after the protein is expressed in a recombinant bacterium; alternatively, the recombinant bacterial cells in which the above proteins are expressed may be disrupted to obtain crude proteins (total cellular proteins), and the crude proteins may be mixed with hydrocarbons to carry out an emulsion reaction.

In the application, the SDR family oxidoreductase E26 protein can be compounded with additives, so that the biological emulsifier with lower working concentration and more stable emulsifying property can be obtained.

Wherein, the additive added in a compounding way can be selected from one of fructose, sucrose, chitosan, agarose, trehalose, starch, gelatin, xanthan gum and arabic gum.

The invention also aims to provide a biological emulsifier, which is obtained by compounding different additives with the SDR family oxidoreductase E26; the additive is selected from one of fructose, sucrose, chitosan, agarose, trehalose, starch, gelatin, xanthan gum and arabic gum.

The invention has the beneficial effects of providing a novel high-efficiency biological emulsifier and a compounding mode of the biological emulsifier. The emulsifier of the invention has better emulsification effect on hydrocarbons, and has excellent properties of wide tolerance of pH environment, salt resistance, heat resistance and the like.

Drawings

FIG. 1 is an SDS-PAGE electrophoresis of SDR family oxidoreductase E26 protein purification; m is Marker, Line 1 is E26 crude protein; line 2: E26 pure protein.

FIG. 2 is a schematic representation of the emulsification index of SDR family oxidoreductase E26 to diesel with different surfactants commonly used in the market.

FIG. 3 is a graphical representation of the emulsification index of SDR family oxidoreductase E26 on various hydrophobic substrates.

Fig. 4 is a graphical representation of the emulsification index of different concentrations of SDR family oxidoreductase E26 on diesel.

FIG. 5 is a schematic representation of the emulsification index of SDR family oxidoreductase E26 on diesel under different salinity conditions.

FIG. 6 is a schematic representation of the emulsification index of SDR family oxidoreductase E26 on diesel under different pH conditions.

FIG. 7 is a schematic representation of the emulsification index of SDR family oxidoreductase E26 on diesel under different temperature conditions.

FIG. 8 is a schematic diagram of the emulsification index of SDR family oxidoreductase E26 to diesel after being compounded with different additives.

Detailed Description

The strains and material sources used by the invention are as follows:

wild fungusAeribacillus pallidusSL-1 is owned by the laboratory and has been disclosed in the literature previously published by the team of the inventors of the present application (Weiyi Tao et al.Biodefinition of aliphatic and multicyclic aromatic hydrocarbons by the term of the therapeutic biological therefor-producing Bacillus pallidus strain SL-1. ecological oxidation and Environmental safety 2020; 189). The applicant hereby states that it is guaranteed that the biological material of the present strain is released to the public free of charge within 20 years from the date of filing.

Plasmid pET28 was purchased from Novovovozam Biotech Ltd,E.coliDH5 α was purchased from Baori physicians & Tech Ltd.

The construction methods of the strains in the following examples are all conventional methods, and can be carried out according to a kit and a product instruction; the materials, reagents and the like used in the examples are commercially available unless otherwise specified.

Example 1

This example illustrates recombinant engineered bacteriaEscherichia coliConstruction of BL21-pET-e 26.

From wild species using PCRAeribacillus pallidusAmplifying SDR family oxidoreductase E26 gene E26 from SL-1 genome DNA, connecting E26 gene to expression vector pET28 multiple cloning site, and transforming recombinant plasmid pET28-E26 toE.coli Genetically engineered bacterium obtained from BL21(DE3)Escherichia coli BL21-pET-e26。

The construction method specifically comprises the following steps:

cloning of gene e 26:

according to the wild fungusAeribacillus pallidusE26 gene sequence design primer in SL-1 genome sequencing result:

P1: 5’-CGGGATCCCGATGTTAACAAATCAAGTAGC-3’ (BamH I)

P2: 5’- CCAAGCTTGGTGCAATAGGATATTGTTGAA-3’ (Hind III)

and (3) PCR reaction system: ddH₂O20 muL, 25 muL of high-fidelity enzyme mixture, P12 muL of primer, P22 muL of primer and 2 muL of DNA template. PCR procedure: pre-denaturation at 95 ℃ for 5 min; 30 cycles of reaction were carried out: denaturation at 94 deg.C for 45 s, annealing at 56 deg.C for 30 s, extension at 72 deg.C for 60 s, keeping at 72 deg.C for 10 min, and storing at 4 deg.C.

(2) Recombinant gene engineering bacteriumEscherichia coliBL21-pET-e 26:

the PCR product is purified by a kit, is connected with a pET28a vector by double enzyme cutting of BamH I and Hind III, and is transformed toE.coliBL21(DE3), coating on a kanamycin plate containing 50 mug/mL, selecting positive transformants, performing colony PCR and sequencing identification, and obtaining genetically engineered bacteriaEscherichia coli BL21-pET-e26。

Example 2

This example illustrates the obtention of the SDR family oxidoreductase E26.

(1) Inducible expression of SDR family oxidoreductase E26:

culture medium: LB culture medium: 5 g/L of yeast powder, 10 g/L of peptone and 10 g/L of sodium chloride.

The culture conditions are as follows: the 250 mL conical flask was filled with 50 mL of liquid, cultured at 37 ℃ and 200 rpm, and cultured until OD600 reached 0.6-0.8, and 1 mM IPTG was added to induce expression of SDR family oxidoreductase E26. After culturing at 28 ℃ and 200 rpm for 12 hours, cells were collected by centrifugation at 10000 Xg for 10 min. Resuspending the cells in 50mM Tris-HCl buffer solution, pH 7.4, performing high pressure cell disruption by a high pressure homogenizer, centrifuging at 10000 Xg for 20 min after disruption, and removing the precipitate to obtain supernatant, namely the crude enzyme solution.

(2) Measurement of protein amount: protein amounts were determined using the Brandford method.

Preparing protein standard yeast:

preparing a Coomassie brilliant blue solution: 100 mg of Coomassie Brilliant blue G-250 was dissolved in 50 mL of 95% ethanol, and 100 mL of 85% (v/v) H was added₃PO₄Finally diluting to 1L by using distilled water, and filtering by using filter paper for use; 0.1g/L BSA was prepared: weighing 0.01g BSA, dissolving in 10mL distilled water, and preparing into 0.01, 0.02, 0.03, 0.04, 0.06, 0.08mg/mL with normal saline before use; taking 72 mL centrifuge tubes, numbering (0, 1, 2, 3, 4, 5, 6), adding 0.3 mL of the diluted BSA solution with each concentration into the No. 1-6 centrifuge tubes, and finally adding 1.2 mL of Coomassie brilliant blue solution into each centrifuge tube. 1.2 mL Coomassie Brilliant blue solution and 0.3 mL physiological saline were added to centrifuge tube No. 0. After mixing, the standard curve was determined by measuring A595 using the 0 th tube as a control.

The specific steps of protein detection are as follows:

first, a standard curve equation is made by using the concentration of Bovine Serum Albumin (BSA) standard sample to the OD595 absorbance. Then, 300. mu.L of the enzyme solution diluted properly was added to 1.2 mL of Coomassie Brilliant blue working solution, mixed well, left at room temperature for 15 min, and absorbance was measured at 595 nm. Two replicates of each group were run in distilled water as a blank.

(3) And (3) purifying a crude enzyme solution:

buffer a (binding solution): 100 mM sodium phosphate buffer, 500 mM NaCl, 10 mM imidazole, pH 7.4;

buffer B (eluent): 100 mM sodium phosphate buffer, 500 mM NaCl, 500 mM imidazole, pH 7.4.

Protein purification using Ni-NTA HisTrap FF column: filtering the supernatant of the crude enzyme solution by using a 0.22 mu m microporous membrane for later use; mounting the column on a protein purification instrument, balancing the nickel column by using binding solution with 10 times of volume, and setting the flow rate to be 1.0 mL/min; after balancing, loading a sample and carrying out protein hanging columns; washing the nickel column with 5 times of the volume of the binding solution, and washing away the protein which is not bound with the nickel column; eluting with eluent, and collecting eluent containing target protein; the imidazole was removed by dialysis overnight, and then protein concentration was performed by centrifugation in a 10 kDa ultrafiltration tube and purity was analyzed by SDS-PAGE (FIG. 1).

Example 3

This example illustrates the method of determining the emulsifying activity.

The enzyme solution purified in example 2 was added to a buffer (50 mM Tris-HCl pH 7.8) in a total volume of 2mL in a 5mL glass vial containing different hydrocarbons as the emulsifying agent, mixed with the oil phase at a volume ratio of 1:1, vortexed for 2 minutes, and allowed to stand for 48 hours.

The Emulsification index (EI48) is calculated as the ratio of the height of the emulsifying layer to the total height, multiplied by 100%, to represent the emulsifying activity of the emulsifier.

Emulsion index (EI48) = (height of emulsion layer)/(height of total liquid) × 100%.

Example 4

This example illustrates the comparison of the emulsifying activity of SDR family oxidoreductase E26 of the present invention with the currently used components such as a wide variety of surfactants for diesel fuel.

Selecting SDS, Tween 20, Tween 80, commercial detergent and E26 protein to respectively prepare solutions (50 mM Tris-HCl pH 7.4) with the concentration of 500 mg/L, taking 2mL, adding 2mL diesel oil, carrying out vortex oscillation for 2 minutes, standing for 48h, and observing the emulsification index EI of the solutions₄₈. As shown in FIG. 2, SDS, Tween 20, Tween 80 and E26 protein can well emulsify diesel oil and can maintain the emulsifying activity for a long time at normal temperature. The emulsification index of the E26 protein is even slightly higher than that of SDS with the same concentration, which shows that the E26 protein has better emulsification activity compared with the surfactant widely used in the market.

Example 5

This example illustrates the emulsifying activity of the SDR family oxidoreductase E26 of the present invention on different hydrophobic substrates.

The purified SDR family oxidoreductase E26 was formulated into 200 mg/L solution (50 mM Tris-HCl pH 7.8) and emulsified with equal volume of cyclohexane, n-hexane, n-octane, dodecane, hexadecane, xylene, rosemary oil, soybean oil, kerosene, liquid paraffin, and diesel oil. The results show that the E26 protein has good emulsification effects on the above hydrocarbons except that the emulsifiability of the liquid paraffin is almost zero, and the emulsification indexes are 47.6%, 59%, 41.9%, 52.1%, 57.6%, 61.0%, 14.3%, 40.5%, 61.0%, 3.0% and 54.5% respectively, as shown in FIG. 3.

Example 6

This example illustrates the effect of the concentration of the SDR family oxidoreductase E26 of the present invention on the emulsifying activity of diesel fuel.

The purified SDR family oxidoreductase E26 is respectively prepared into solutions (50 mM Tris-HCl pH 7.4) of 500 mg/L, 300 mg/L, 200 mg/L, 100 mg/L and 50 mg/L, and the solutions are emulsified with diesel oil with the same volume, and the emulsification indexes of the solutions on the diesel oil are compared. As shown in FIG. 4, the results indicate that the emulsifying activity decreases with decreasing concentration of E26 protein. When the concentration of the E26 protein is only 100 mg/L, the emulsification index of the E26 protein to diesel oil is 40.5%, and when the concentration of the E26 protein is higher than 100 mg/L, the emulsification activity is more than 50%.

Example 7

This example illustrates the effect of the SDR family oxidoreductase E26 of the present invention on diesel emulsification activity under different salinity conditions.

The purified SDR family oxidoreductase E26 is respectively dissolved in NaCl and MgSO 30 g/L, 50 g/L, 100 g/L, 150 g/L and 200 g/L₄And CaCl₂A solution in which the SDR family oxidoreductase E26 concentrations were all 200 mg/L (50 mM Tris-HCl pH 7.4) was emulsified with an equal volume of diesel oil, left for 48 hours at room temperature and the emulsification index EI was determined₄₈And judging the salinity resistance of the emulsifier. The results are shown in FIG. 5, with NaCl and MgSO₄Increasing concentrations of E26 proteinThe emulsifying activity is slightly reduced but when MgSO₄The emulsifying activity of the E26 protein was slightly increased at concentrations of 30 g/L and 50 g/L over MgSO₄Further increase in concentration decreases the emulsification index. However, the E26 protein can keep higher emulsifying activity in 3 solutions with different salt concentrations.

Example 8

This example illustrates the effect of the SDR family oxidoreductase E26 of the present invention on diesel fuel emulsification activity at different pH conditions.

Preparing 50 mmoL/L of citrate, Tris-HCl and glycine-NaOH buffer solutions, adjusting the pH values to be pH 3, pH 4, pH 5, pH 6, pH7, pH 8, pH 9, pH 10 and pH 11 respectively, dissolving the purified SDR family oxidoreductase E26 in an upper buffer solution respectively to ensure that the concentration of the SDR family oxidoreductase E26 is 200 mg/L, emulsifying the SDR family oxidoreductase E26 with isovolumetric diesel oil, standing the solution at room temperature for 48 hours, and calculating an emulsification index EI₄₈And judging the pH sensitivity of the SDR family oxidoreductase E26 to the diesel oil emulsification. As shown in FIG. 6, the emulsifying activity of the E26 protein on diesel oil was not affected at pH 3-11.

Example 9

This example illustrates the effect of the SDR family oxidoreductase E26 of the present invention on diesel fuel emulsification activity under different temperature conditions.

Preparing a 200 mg/L solution (50 mM Tris-HCl pH 7.4) of the purified SDR family oxidoreductase E26, carrying out water bath for 1 hour at different temperatures of 45 ℃, 55 ℃, 65 ℃, 75 ℃, 85 ℃ and 95 ℃, cooling to room temperature, mixing and emulsifying with diesel oil with the same volume, standing for 48 hours at room temperature, calculating the emulsification index, and determining the heat resistance of the emulsifier. As shown in the result of FIG. 7, the emulsifying activity of the E26 protein on diesel oil is almost stabilized as the temperature is increased to 85 ℃, but the emulsifying activity of E26 is reduced to 28.6% when the temperature is increased to 95 ℃; the result shows that the E26 protein has better heat resistance as an emulsifier, can endure the high temperature of 85 ℃, and keeps good emulsifying activity and stability.

Example 10

This example illustrates the effect of SDR family oxidoreductase E26 on diesel fuel emulsification activity after it was compounded with various additives.

The purified SDR family oxidoreductase E26 was prepared into a 100 mg/L solution (50 mM Tris-HCl pH 7.4), and various additives (fructose, sucrose, chitosan, agarose, trehalose, starch, gelatin, xanthan gum, gum arabic) were added to the solution to a final concentration of 1g/L, respectively, to perform an emulsification reaction with diesel oil, and the obtained mixture was vortexed for 2 minutes, allowed to stand at room temperature for 48 hours, and then the emulsification activity was calculated. As shown in FIG. 8, all the above additives slightly improved the emulsification activity of E26, with the emulsification stability being most significantly improved by gum arabic, agarose and xanthan gum. Therefore, the invention provides a method for increasing the emulsifying activity of the SDR family oxidoreductase E26 protein, namely, the SDR family oxidoreductase E26 protein is compounded with emulsifying stabilizers such as Arabic gum and xanthan gum.

Sequence listing

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gacgttgcca atgaacagca gatgcaaaag atggtcacag aagtattaga agagtttgga 240

caaatcgata ttctcgtcaa caatgcagga atcggatttt ttaaagaagt ggaagagacg 300

accgtggaag aatgggagcg catctttgcc gtcaatgttc aaggtgtgtt tattggggta 360

aaagccgtgc ttccgcatat gaaagaaaga aaatcgggaa caattattac catttcttcc 420

gatgtcggcc gctacacgat tccgaacggg gcggcataca ccgcgaccaa atacgccgtt 480

caaggatttt ccggttcgct tgcgcaggaa gtaagaaagt acggcattcg cgttggcacg 540

attaacccgg gaatggttga tacgtacttt gctaattcga ttcaaggcgt tccggaaaaa 600

cgcgattggc tgaaagccga agatgtcgca aaagcggtcg tgtatatggc aagcgctcca 660

aagcatatgc ttattgatga gattattctt catccgctca ttcaacaata tcctattgca 720