Use of xoxf-type mdh of methylotrophs for adsorption and / or separation of lanthanides

Abstract

The present application relates to the field of biological environmental protection, and more particularly to the application of XoxF type MDH of methylotrophic bacteria in adsorption and / or separation of lanthanide series elements. The present application provides the application of XoxF type MDH of methylotrophic bacteria in adsorption and / or separation of lanthanide series elements. The present application provides a method for inducing the activity of XoxF type MDH in methylotrophic bacteria by using lanthanide series elements in rare earth tailings as a cofactor, and the method can be used as a selective adsorbent for La, Ce, Pr and Nd, which can provide a new source for the application of high-value REE products.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological environmental protection, and in particular to the application of XoxF-type MDH of methylotrophic bacteria in adsorption and / or separation of lanthanide elements. BACKGROUND

[0002] The unique physicochemical properties of rare earth elements (REEs) give them a revolutionary transformation in optics, electronics, magnetism and other high-tech fields. However, traditional chemical methods are technically complex, designed with dangerous chemicals, and have limited separation efficiency of rare earths, leading to irreversible environmental pollution. The use of microorganisms and biomolecules and other environmentally friendly and widely applicable biological material tools has high selectivity and high fidelity, and is more promising for the separation of REEs. However, due to the low efficiency of biological materials in extracting high-performance REE materials from ores, there are still major challenges in industrial applications. Therefore, it is necessary to develop a new generation of biological synthetic materials for biological casting to obtain high-value REEs.

[0003] Methanol dehydrogenase (MDH) is considered to be a key and essential enzyme in the methanol metabolism of Methylorubrum extorquens strains. Recent studies have shown that there is XoxF-type MDH in M. extorquens AM1, which is assisted by lanthanide elements. And it was found that XoxF-type MDH is widely distributed in the genomes of many bacteria. It can be actively used and designed to rely on lanthanide-dependent MDH for methanol biocatalysis. SUMMARY

[0004] Therefore, the present application provides the application of XoxF-type MDH of methylotrophic bacteria in adsorption and / or separation of lanthanide elements. The present application provides a method for inducing XoxF-type MDH activity in methylotrophic strains using lanthanide elements in rare earth tailings as cofactors, and can be used as a selective adsorbent for La, Ce, Pr and Nd, which can provide a new source for high-value REE product applications.

[0005] In order to achieve the above-mentioned application purposes, the present application provides the following technical solutions:

[0006] The present application provides the application of XoxF-type MDH of methylotrophic bacteria in adsorption and / or separation of lanthanide elements.

[0007] In some embodiments of the present application, in the above-mentioned application, the methylotrophic bacteria include M. extorquens AM1.

[0008] In some embodiments of the present application, in the above-mentioned application, the lanthanide elements include one or more of La, Ce, Pr or Nd.

[0009] The present application also provides a recombinant strain of methylotrophic bacteria, which overexpresses XoxF-type MDH.

[0010] In some embodiments of the present application, in the above-mentioned recombinant strain, the XoxF-type MDH is overexpressed by expressing a lanthanide transport cluster.

[0011] In some embodiments of the present application, in the above-mentioned recombinant strain, the expression module comprises a promoter and a lanthanide transport cluster.

[0012] In some embodiments of the present application, in the above-mentioned recombinant strain, the promoter comprises P_mxaF / P_LA.

[0013] In some embodiments of the present application, in the above-mentioned recombinant strain, the lanthanide transport cluster has:

[0014] (1) the nucleotide sequence shown in SEQ ID NO: 1; or

[0015] (2) a nucleotide sequence encoding the same protein as the nucleotide sequence shown in (1), but different from the nucleotide sequence shown in (1) due to the degeneracy of the genetic code; or

[0016] (3) a nucleotide sequence obtained by substitution, deletion or addition of one or more nucleotides to the nucleotide sequence shown in (1) or (2), and a nucleotide sequence functionally identical or similar to the nucleotide sequence shown in (1) or (2); or

[0017] (4) a nucleotide sequence having at least 90% sequence homology with the nucleotide sequence shown in (1), (2) or (3).

[0018] The present application also provides a XoxF-type MDH mutant, which has:

[0019] (5) the amino acid sequence shown in SEQ ID NO: 2; or

[0020] (6) a sequence obtained by substitution, deletion, addition and / or replacement of one or more amino acids in the amino acid sequence shown in (5); or

[0021] (7) a sequence having more than 90% homology with the amino acid sequence shown in (5) or (6).

[0022] The present application also provides a preparation comprising the above-mentioned recombinant strain and / or the above-mentioned XoxF-type MDH mutant and an acceptable adjuvant.

[0023] In some embodiments of the present application, the sequence of the above-mentioned pET-25b plasmid is shown in SEQ ID NO: 7:

[0024]

[0025]

[0026]

[0027] The application also provides the use of the above-mentioned recombinant strain and / or the above-mentioned XoxF-type MDH mutant in adsorption and / or separation of lanthanide elements.

[0028] The application also provides the use of the above-mentioned recombinant strain and / or the above-mentioned XoxF-type MDH mutant in preparation of a lanthanide ligand.

[0029] The application provides the use of XoxF-type MDH of methylotrophic bacteria in adsorption and / or separation of lanthanide elements. The application provides that the lanthanide elements in rare earth tailings are used as an auxiliary factor to induce the activity of XoxF-type MDH in a methylotrophic bacterial strain, and can be used as a selective adsorbent of La, Ce, Pr and Nd, which can provide a new source for high-value-added REE product applications. BRIEF DESCRIPTION OF DRAWINGS

[0030] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or prior art description.

[0031] Figure 1 SDS-PAGE analysis of purified MDH of strain AM1 grown on methanol / tailings powder; wherein: 1 shows a protein Marker; 2 shows the purified MDH obtained. DETAILED DESCRIPTION

[0032] The application discloses the use of XoxF-type MDH of methylotrophic bacteria in adsorption and / or separation of lanthanide elements.

[0033] It should be understood that the expression "one or more of" individually includes each object recited after the expression and various different combinations of two or more of the recited objects, unless otherwise understood from the context and usage. The expression "and / or" in combination with three or more recited objects should be understood to have the same meaning, unless otherwise understood from the context.

[0034] The terms "comprising", "having" or "including", including the use of their grammatical synonyms, should generally be understood to be open-ended and non-limiting, for example, not excluding other unrecited elements or steps, unless otherwise specifically stated or understood from the context.

[0035] It should be understood that the order of steps or order for performing certain actions is immaterial so long as the application remains operable. Moreover, two or more steps or actions can be conducted simultaneously.

[0036] The use of any and all examples, or exemplary language herein, for example, only the intention to better describe the application, and unless otherwise claimed, does not limit the scope of the application. Any language in the specification should not be interpreted as indicating any non-claimed element essential to the practice of the application.

[0037] In addition, the numerical ranges and parameters regarding the present application are approximate, and the numerical values in specific examples have been presented herein as precisely as possible. However, any numerical value inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing procedures. Therefore, unless otherwise indicated, it is intended that all ranges, amounts, values and percentages used herein are "about" modified. Herein, "about" generally means within ±10%, ±5%, ±1%, or ±0.5% of a particular value or range.

[0038] The present application provides the following technical solutions:

[0039] (1) Cultivation of microorganisms: M. extorquens AM1 strain (ATCC 14718 / DSM1338 / JCM2805 / NCIMB9133; commercial strain, item number TS179122) was cultured in MP medium at 30°C, 220 rpm for 2 days.

[0040] (2) Treatment of rare earth tailings: The rare earth tailings (Baotou rare earth tailings) were washed several times with ultrapure water, and the collected rare earth tailings were dried, weighed and sterilized.

[0041] (3) Obtain methylotrophic strains activated XoxF type MDH: M. extorquens AM1 strain was cultured in MP liquid medium, and rare earth tailings (see Table 1 for specific components of tailings) and methanol (as carbon source) were added. And obtain the bacterial precipitate by filtration and centrifugation.

[0042] Table 1 Element composition and proportion of rare earth tailings

[0043]

[0044]

[0045] Table 2 Rare earth element composition and proportion of rare earth tailings

[0046]

[0047] (4) Washing of the bacterial pellet: Washing with acetate buffer and resuspending with the same.

[0048] (5) Preparation of bacterial lysate: Lysing the bacterial cells with ultrasonic cell disruptor, removing the cell debris by centrifugation, precipitating the impurities by adding solid ammonium sulfate, discarding the precipitate by centrifugation and collecting the supernatant.

[0049] (6) Purification of XoxF-type MDH: The collected supernatant is subjected to primary purification by hydrophobic chromatography column, and the collected flow sample is subjected to secondary purification by cation exchange column (SP column). The obtained active substance presents a single band by SDS-PAGE.

[0050] (7) Determination of MDH activity: The enzyme activity of the purified MDH is determined by PES and DCPIP. PES is used to replace physiological EA cytochrome. The detection mixture (DCPIP and PES) is subjected to short-term heating to reduce the automatic bleaching of free radicals. In the preparation process, the light should be reduced to the minimum. Finally, the enzyme activity is measured by spectrophotometer. And the enzyme activity is calculated by formula, and the results are shown in Table 3. The purified XoxF-type MDH still has high methanol catalysis (enzyme activity: 1.205 U / mg).

[0051] (8) Detection of rare earth content in MDH: The content and types of rare earth elements in MDH are detected by ICP-MS. The purified XoxF-type MDH only contains La, Ce, Pr and Nd, etc. This shows that the XoxF-type MDH in M. extorquens AM1 has the ability of selective adsorbent and direct utilization of lanthanide elements in tailings.

[0052] (9) Modification of the chassis of methylotrophic microorganisms: By editing the lanthanide element utilization and transport channel of the cell of the methylotrophic microorganism, the modified microorganism has the ability of selective adsorption and specific utilization of lanthanide elements. By changing the amino acid of XoxF-type MDH combined with lanthanide elements, the specific combination of XoxF-type MDH with a kind of lanthanide element is realized. This cell factory not only can realize the selective adsorption and separation of lanthanide elements, but also can be used as a functional lanthanide ligand for advanced biosynthesis.

[0053] In the embodiments 1-9 of the present application, the raw materials and reagents used can be purchased from the market.

[0054] The present application is further described below in combination with examples:

[0055] Example 1: Cultivation of methylotrophic strain

[0056] A single colony of M. extorquens AM1 was picked and inoculated in 20 mL of MP liquid medium with 0.5% (m / v) of succinate as carbon source. After 2 days of incubation at 30°C, 300 rpm, the bacterial concentration reached 1 x 10 8 CFU / mL, 10 mL of this bacterial solution was added to 100 mL of MP liquid medium with 0.5% (m / v) of succinate. After incubation at 30°C, 300 rpm until the logarithmic phase, this bacterial solution was used as seed solution.

[0057] Example 2 Treatment of rare earth tailings

[0058] The rare earth tailings were washed several times with ultrapure water until the rare earth tailings were washed to be sandy (quickly settled in water), the collected rare earth tailings were dried and weighed (1.0 g / portion), and sterilized.

[0059] Example 3 Methanotrophic strain co-cultivation with tailings

[0060] 10 mL of seed solution with a concentration of 1 x 10 8 CFU / mL obtained in Example 1 was added to 1000 mL of MP liquid medium, and 1.0 g of rare earth tailings obtained in Example 2 and 0.5% (v / v) of methanol as carbon source were added, and then the mixture was incubated until the OD 600 was 2.0. The removal of the tailings was achieved by decanting and filtering. The bacterial cells were obtained by centrifugation at 6000 rpm / min for 20 min.

[0061] Example 4 Washing of bacterial cell precipitate

[0062] The bacterial precipitate obtained in Example 3 was washed three times with 25 mM acetic acid buffer at pH 5.0, and the precipitate was resuspended with the buffer.

[0063] Example 5 Preparation of bacterial lysate

[0064] After the bacterial cells obtained in Example 4 were lysed by cell sonicator under the condition of ice-water mixed bath, the cell debris was removed by centrifugation at 4°C, 12000 rpm for 10 min.

[0065] The conditions of cell sonication were as follows: power was 300 W, working for 5 s, stopping for 5 s, and the total time of sonication was 30 min.

[0066] Solid ammonium sulfate was added for the precipitation of impurities, and the precipitate was removed by centrifugation at 4°C, 12000 rpm for 10 min.

[0067] Example 6 Purification of XoxF-type MDH

[0068] The hydrophobic chromatography column was pre-equilibrated with 25 mM pH 5.0 acetate buffer containing 1.2 M ammonium sulfate, and the supernatant collected in Example 5 was loaded into the hydrophobic chromatography column. Gradient elution was performed using 25 mM pH 5.0 acetate buffer containing 1.2-0.7 M ammonium sulfate, and the flow-through sample was collected for primary purification.

[0069] The flow-through sample collected was further passed through a cation exchange column (SP column). The cation exchange column was pre-equilibrated with 25 mM pH 5.0 acetate buffer. The flow-through sample collected was loaded into the equilibrated SP column, and gradient elution was performed using NaCl at a concentration gradient of 0-400 mM to obtain purified MDH.

[0070] The purified MDH was detected by SDS-PAGE, and only a single band was shown in the result, indicating that the purified MDH was relatively pure.

[0071] Example 7: Determination of MDH activity

[0072] The enzyme activity of the purified MDH was determined by PES and DCPIP. PES was used to replace physiological EA cytochrome. 200 μL of 100 mM PES solution, 1 mL of 2 mM DCPIP solution, and 600 μL of 500 mM NH4Cl solution were taken, and the volume was supplemented to 10 mL with pH 9.0 Tris-HCl buffer to serve as the determination solution.

[0073] 500 μL of the determination solution was mixed with 400 μL of pH 9.0 Tris-HCl buffer in a centrifuge tube, and allowed to equilibrate in the dark at 30°C for at least 2 min. 200 μL of the mixture was taken into an enzyme plate, and 4 μL of enzyme solution at a concentration of 200 μM was added. The OD 600 nm was determined for 2 min. 50 μL of 1 M MeOH was added, and the OD 600 nm was determined, and the enzyme activity was calculated using the formula.

[0074] Enzyme activity calculation formula:

[0075] Specific enzyme activity (μmol / min·mg) = enzyme unit U (μmol / mol) / enzyme amount (mg)

[0076] Enzyme unit U (μmol / min) = -1 x [measured initial rate of slope] / [epsilon (cm·M) x path length of cell (cm)] 10^6

[0077] The results show that the purified MDH still has a high enzyme activity, and the enzyme activity is 1.21 U / mg (as shown in Table 3).

[0078] Table 3 Activity of XoxF type MDH

[0079]

[0080] Example 8 Determination of rare earth species and content in MDH

[0081] The content and species of rare earth elements in MDH were detected by ICP-MS. The results are shown in Table 4. The XoxF type MDH contains only La, Ce, Pr and Nd, and the contents are 265.19 μg / g, 574.01 μg / g, 54.37 μg / g and 146.87 μg / g, respectively.

[0082] Table 4 Content of rare earth elements in MDH

[0083]

[0084] Example 9 Modification of methanotrophic microbial chassis

[0085] (1) Increasing expression of existing genes

[0086] The plasmid pCM110 (Gene Bank: AF327718.1) was used as a vector for modification. First, the high-efficiency promoter P_mxaF / P_LA was introduced into the plasmid.

[0087] The specific method is to cut the vector plasmid pCM110 with a restriction enzyme, and at the same time cut both ends of the high-efficiency promoter P_mxaF / P_LA with the same restriction enzyme to form the same sticky ends, and use DNA ligase to insert the target gene fragment into the plasmid cut, to form the plasmid pCM110 with the introduced high-efficiency promoter P_mxaF / P_LA. Subsequently, the lanthanide transport cluster (lut, Ln utilization and transport, MexAM1_META1p1778 to MexAM1_META1p1787) was introduced into the plasmid pCM110 with the high-efficiency promoter P_mxaF / P_LA, to obtain a lanthanide transport cluster expression vector recombinant pCM110 with the high-efficiency promoter P_mxaF / P_LA control.

[0088] Lanthanide transport cluster sequence controlled by high-efficiency promoter P_mxaF / P_LA:

[0089]

[0090]

[0091]

[0092]

[0093]

[0094]

[0095] (as shown in SEQ ID NO: 1)

[0096] The above-mentioned recombinant pCM110 is introduced into M. extorquens AM1 competent cells by using the electric shock transformation method, and the lanthanide series element transport system is efficiently expressed in situ, so that the modified microorganism has high selective adsorption and specific utilization ability for lanthanide series elements. The results are shown in Table 5, and the modified microorganism has selective utilization for lanthanide series elements. The XoxF type MDH contains only La, Ce and Nd and other three rare earth elements, and their contents are 297.21 μg / g, 602.34 μg / g and 106.16 μg / g, respectively. Compared with the XoxF type MDH in the M. extorquens AM1 strain before modification, the adsorption of Pr element is reduced, which shows that this method can improve the specific adsorption effect of M. extorquens AM1 strain on lanthanide series elements.

[0097] Table 5 Content of rare earth elements in engineered MDH

[0098]

[0099] (2) Modification based on existing proteins

[0100] By directed evolution of enzymes, their performance can be improved. Methanol dehydrogenase XoxF (NC_012988.1 (2405310..2407115)) is a type of methanol dehydrogenase with lanthanum and other light rare earth elements as metal core, which can catalyze the dehydrogenation process of methanol under mild conditions. Based on XoxF (NC_012988.1 (2405310..2407115)), directed evolution can improve its catalytic activity or change the binding specificity of its binding domain.

[0101] The genome of M. extorquens AM1 was subjected to PCR amplification using primers (primer sequence: upper primer-TAYGCCGAYGGCAAGSTGST; (as shown in SEQ ID NO: 3) lower primer-CCGTCRTARTCCCAYTGRTCGAA (as shown in SEQ ID NO: 4)) to obtain the gene sequence of XoxF. The XoxF sequence obtained by amplification was used as a template, and by using a low-fidelity DNA polymerase, Mg 2+ , Mn 2+ Error-prone PCR was performed, and a mutagenesis library of gene XoxF was obtained by gel recovery.

[0102] The primer sequence used in error-prone PCR was: upper primer-CATATGTAYGCCGAYGGCAAGST GST; (as shown in SEQ ID NO: 5) lower primer-CCGTCRTARTCCCAYTGRTCGAAGAGCTC (as shown in SEQ ID NO: 6).

[0103] The error-prone PCR product and the pET-25b plasmid were double-digested by Nde I and Xho I restriction endonucleases, and were ligated by T4 ligase to obtain the recombinant plasmid pET-25b-XoxF. The recombinant plasmid was transformed into plasmid into Escherichia coli BL21 (DE3) competent cells (commercialized competent cells, product number EC0114) for preservation and library construction.

[0104] Based on the PES-DCPIP enzyme activity test method, a high-throughput screening was performed on the constructed Escherichia coli BL21 XoxF gene mutation library, and a mutant strain with strong enzyme activity was isolated. LC-MS / MS was used to sequence the XoxF mutant protein therein, and the sequence of the high-activity methanol dehydrogenase was obtained, and the screening process was completed. The results are shown in Table 6, and the enzyme activity of the mutant XoxF type MDH screened is increased by 30%, and the enzyme activity is 1.573 U / mg (the original enzyme activity is 1.21 U / mg).

[0105] Table 6 Activity of engineered XoxF type MDH

[0106]

[0107] Amino acid sequence of XoxF type mutant MDH:

[0108] (as shown in SEQ ID NO: 2)

[0109] By introducing high-activity methanol dehydrogenase into the existing rare earth biological system, a new cell factory is constructed to realize efficient expression of high-activity protein. This cell factory can not only realize selective adsorption and separation of lanthanide elements, but also can be used as a functional lanthanide ligand for advanced biosynthesis.

[0110] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. An XoxF-type methanol dehydrogenase mutant, characterized in that, Its amino acid sequence is shown in SEQ ID NO:

2.

2. A formulation, characterized in that, Includes the XoxF type methanol dehydrogenase mutant as described in claim 1 and acceptable adjuvants.

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