CN111172068B - Construction method and application of periplasmic photosensitized whole-cell hybrid system - Google Patents

Abstract

The invention belongs to the technical field of biological-inorganic composite materials, and relates to a method for constructing a periplasmic photosensitized whole-cell hybrid system, comprising: selecting bacterial cells capable of expressing biological enzymes in the periplasm and inoculating them into corresponding culture medium, After activation under aerobic conditions, transfer the bacteria solution and culture solution to a new culture bottle at a ratio of 1:100. After the bacterial concentration reaches 0.4-0.6, perform anaerobic treatment to express biological enzymes in the periplasm; then , add semiconductor nano-photosensitizer under anaerobic conditions, place in a constant temperature shaker at 30°C for cell hybridization; finally, centrifuge and wash the solution, and then resuspend the periplasmic photosensitized whole-cell hybrid system into a new reaction buffer solution. The invention is constructed by semiconductor nanoparticles with broad-spectrum absorption range and cells expressing biological enzymes in the periplasm to realize efficient conversion of solar energy to chemical energy, and has good application prospects in the fields of environment and energy.

Description

Construction method and application of periplasmic photosensitized whole-cell hybrid system

technical field

The invention belongs to the technical field of biological-inorganic composite materials, and relates to cell hybridization, in particular to a method for constructing a periplasmic photosensitized whole-cell hybrid system and its application.

Background technique

Semiconductor photocatalytic nanomaterials have attracted increasing attention because of their good light-harvesting ability, stability and catalytic activity, and their potential application value in solving energy crisis and environmental problems. However, semiconductor photocatalytic nanomaterials have few catalytic active sites and slow catalytic reactions, which restrict their further applications. In order to make up for this deficiency, in recent years, due to the specificity and high efficiency of biological enzymes, biological enzymes have been gradually applied to the conversion of solar energy to chemical energy. Through the combination of semiconductor catalytic materials and biological enzymes, biological enzymes serve as efficient catalytic active sites, and semiconductor materials serve as photosensitizers, thereby realizing efficient conversion of solar energy to chemical energy. Although this inorganic-biological enzyme hybrid system has high catalytic performance, the purification steps of the biological enzyme itself are complicated, the yield is low, and it is relatively sensitive to oxygen, which limits its application.

In recent years, the use of whole cells that can express biological enzymes to replace biological enzymes has provided a new idea for the construction of new inorganic-biological hybrid systems. Compared with pure biological enzymes, cells have the advantages of easy acquisition, stability and self-healing. Based on this, Honda et al. used TiO ₂ photocatalyst and E.coli to construct the inorganic-biological whole-cell hybrid system for the first time (Honda, Y.; Hagiwara, H.; Ida, S.; Ishihara, T. Angew. Chem. Int. Edit. 2016,55, 8045-8048), to achieve efficient water splitting performance for hydrogen production. TiO ₂ is used as a photosensitizer, and the electrons after photoexcitation are transferred to the biological enzymes of the cells through the transmembrane process of the electron mediator methyl viologen, and the solar energy conversion is realized on the biological enzymes; the disadvantage lies in the transmembrane transfer process of electrons It is slow and consumes extra energy, resulting in inefficient transfer of electrons. To overcome this problem, Yang et al. proposed a whole-cell system for intracellular photosensitization (Zhang, H.; Liu, H.; Tian, Z.; Lu, D.; Yu, Y.; Cestellos-Blanco, S.; Sakimoto, KK; Yang, P. Nat. Nanotechnol. 2018, 13, 900-905), who grew gold nanoclusters into cells so that photoexcitation of photosensitizers (Au nanoclusters) and electronic The transfer process is carried out in the cytoplasm, which effectively improves the electron transfer efficiency. It must be pointed out that due to the complex components in the cytoplasm in this system, the transmission of light will be disturbed, and the light absorption of the photosensitizer will also be affected.

In bacterial cells, the periplasm exists between the outer and inner membranes of the cell. Compared with the cytoplasm, it is closer to the outer membrane, which can reduce interference in light transmission and is more conducive to the light absorption of photosensitizers. In addition, it has a smaller space, which can make the biological enzyme have a higher local concentration, increase the contact between the photosensitizer and the biological enzyme, and accelerate the occurrence of the catalytic reaction. The inventors constructed a whole-cell hybrid system with periplasmic photosensitization. By regulation, biocompatible photocatalytic nanomaterials (such as TiO ₂ , NaTaO ₃ , KTaO ₃ , Bi ₃ TaO ₇ , WO ₃ , Bi ₂ O ₃ , BiVO ₄ , CdS, CuIn ₂ S ₄ /ZnSQDs, gC ₃ N ₄ , etc.) to grow into cells that can express biological enzymes in the periplasm (such as Shewanella oneidensis , Desulfovibrio gigas , Desulfovibrio vulgaris , Desulfovibrio desulfuricans , Desulfovibrio baculatus , etc.); simultaneously achieve effective light absorption and electron transfer, and achieve efficient Solar-to-chemical energy conversion.

So far, there has been no report on the construction of a periplasmic photosensitized whole-cell hybrid system.

Contents of the invention

The purpose of the present invention is to provide a universal method for constructing a periplasmic photosensitized whole-cell hybrid system to realize efficient conversion of solar energy to chemical energy.

Technical solutions

A method for constructing a periplasmic photosensitized whole-cell hybrid system, comprising the steps of:

A. Select bacterial cells capable of expressing biological enzymes in the periplasm and inoculate them into the corresponding medium. After activation under aerobic conditions, mix the bacterial solution and culture solution (LB/MOPS) at a ratio of 1:100 (v/v) The ratio was transferred to a new culture bottle, and after the bacterial concentration (OD ₆₀₀ ) in the medium reached 0.4-0.6, anaerobic treatment was performed to express biological enzymes in the periplasm of the cells;

B. After the expression of the biological enzyme in the anaerobic process is completed, add a semiconductor nano-photosensitizer under anaerobic conditions, and place it in a constant temperature shaker at 30°C for cell hybridization;

C. After the hybridization is completed, the solution is centrifuged and washed, and then the periplasmic photosensitized whole-cell hybrid system is resuspended in a new reaction buffer solution.

In a preferred disclosed example of the present invention, the bacterial cells described in step A are Shewanella oneidensis , Desulfovibrio gigas , Desulfovibrio vulgaris , Desulfovibrio desulfuricans , Desulfovibrio baculatus and the like .

In the preferred disclosed example of the present invention, the anaerobic treatment described in step A, the process is: in the ultra-clean bench, transfer the bacterial solution to a 500 mL anaerobic bottle, and then add lactic acid, fumaric acid and cysteine respectively acid, so that the concentrations in the system were 20 mM, 25 mM and 10 mM respectively; then nitrogen gas was passed into the anaerobic bottle for 30 min, the stopper was closed, and the outermost layer was compacted and sealed with an aluminum cap, and placed at 30 °C shaker, cultivated for 20 h. In this process, bacteria express biological enzymes (hydrogenases) under anaerobic conditions, and the activity of the enzymes is verified by enzyme activity assays. Of course, there are different anaerobic treatments for different bacterial species, and the anaerobic treatment process in the prior art needs to be determined according to the bacterial species.

In the preferred disclosed example of the present invention, the semiconductor nano-photosensitizer described in step B includes TiO ₂ , NaTaO ₃ , KTaO ₃ , Bi ₃ TaO ₇ , WO ₃ , Bi ₂ O ₃ , BiVO ₄ , CdS nanoparticles, CuIn ₂ S ₄ /ZnS and gC ₃ N ₄ quantum dots, etc. The semiconductor nano photosensitizer can be characterized by X-ray diffraction. Nanoparticles that are too large will affect the rate of endocytosis or diffusion into cells, so it is very important to control the size of nanoparticles. The particle size of the semiconductor nano photosensitizer is preferably not greater than 20 nm.

In the preferred disclosed example of the present invention, the content of the semiconductor nano-photosensitizer added in step B in the hybrid system is 0.25-1.25 mg/mL, and the holding time is 0.5-2 h.

The theoretical content of the added conductive nano-photosensitizer was 1 mg/mL, and when the holding time was 1 h, the photosensitizer content carried by the cells inside the bacteria was the highest, reaching 28%, and the photocatalytic hydrogen production efficiency was the best.

In step A of the present invention, hydrogenase expression is detected by adding methyl viologen and an electron donor to detect enzyme activity.

In the step B of the present invention, the smaller-sized nanoparticles or quantum dots will be gradually entered into the cells by the bacterial cells through endocytosis or diffusion. Nanoparticles or quantum dots containing surface ligands are specifically absorbed by living cells, and chemically linked with molecules with biological recognition such as peptides, antibodies, nucleic acids or small molecule ligands. Controlling the concentration and time of the semiconductor nano-photosensitizer can adjust the content of the semiconductor nano-photosensitizer entering the cell. When the concentration is too low, the content of nanoparticles or quantum dots that can diffuse or endocytize into the cell is too low, resulting in a low intracellular carrying rate of nanoparticles or quantum dots; when the concentration of nanoparticles or quantum dots is too high, the nanoparticles or quantum dots Quantum dots will clump together and also affect how much of the nanoparticle or quantum dot gets into the cell. Therefore, rationally adjusting the concentration and time of nanoparticles or quantum dots can optimize the carrying rate of nanoparticles or quantum dots in cells, so that the photocatalytic efficiency of the hybrid system can be optimized. The content of photosensitizer in cells was measured by plasma inductively coupled spectrometer (ICP).

Another object of the present invention is to apply the periplasmic photosensitized whole-cell hybrid system prepared according to the method of the present invention to catalytic hydrogen production with visible light.

Photocatalytic hydrogen production performance of CIZS QDs/SW periplasmic photosensitized whole-cell hybrid system

The photocatalytic hydrogen production performance of CIZS QDs/SW periplasmic photosensitized whole-cell hybrid system was tested using a closed gas circulation system. In the glove box, the prepared hybrid system was added with ascorbic acid (100 mM) as a cavitation sacrificial agent, and then loaded into a reaction vial. A 300W xenon lamp is used as the light source, and ultraviolet light and infrared light are filtered out by cut-off filters (420<λ<780 nm). The quartz reactor was then connected to a gas circulation system and evacuated to ensure that the reaction system was in an anaerobic state. The reaction temperature was controlled at 37°C using a circulating water bath. React in dark for 1 hour before light to check the device for air leaks. The photocatalytic reaction was carried out at room temperature for 9 hours, the automatic sampling program of gas chromatography was set, the peak area of the generated _H2 was detected every hour, and the content of the generated _H2 was analyzed by gas chromatography.

In order to compare with the output of a single photosensitizer, the concentration of the photosensitizer in the periplasmic photosensitizer whole-cell hybrid system was detected by ICP, and its content was calculated. The photosensitizer with the same quality was used to test its photocatalytic hydrogen production performance.

Beneficial effect

The invention is constructed by semiconductor nanoparticles with broad-spectrum absorption range and cells expressing biological enzymes in the periplasm, and is a method with simple preparation process, low cost, and high-efficiency solar-chemical energy conversion. The present invention proposes a method for constructing a universal periplasmic photosensitized whole-cell hybrid system to realize efficient conversion of solar energy to chemical energy, and has good application prospects in the fields of environment and energy.

Description of drawings

Figure 1. X-ray diffraction patterns of the as-prepared CIZS QDs.

Figure 2. Transmission electron microscopy (a) and high-resolution transmission image (b) of the as-prepared CIZS QDs.

Figure 3. SEM (a)/TEM (b) and partial TEM image (c) of the prepared SW.

Figure 4. Comparison of the hydrogen production performance of the constructed CIZS QDs/SW periplasmic photosensitized whole-cell hybrid system.

detailed description

The present invention will be described in detail below in conjunction with the examples, so that those skilled in the art can better understand the present invention, but the present invention is not limited to the following examples.

Unless otherwise defined, terms (including technical and technical terms) used herein should be interpreted as having the same meaning as commonly understood by those skilled in the art to which this invention belongs. It will also be understood that the terms used herein should be interpreted to have a meaning consistent with their meanings in the context of this specification and related art, and should not be interpreted in an idealized or over-the-top form, unless expressly so defined herein .

Example 1

Construction of CIZS QDs/ Shewanella oneidensis periplasmic photosensitized whole-cell hybrid system

Take a small amount of Shewanella oneidensis MR-1 (SW) and insert it into Luria Bertani (LB) containing yeast (5 g/L), tryptone (10 g/L) and sodium chloride (10 g/L) medium, placed in a 30°C shaker (180 rpm) for overnight culture under aerobic conditions; then inoculated 100 μL of culture solution into 200 mL of modified LB medium (LB+100 mMMOPS, pH 7.2), at 30°C Continue aerobic culture on a shaker for 3-4 h; monitor cell growth by measuring OD ₆₀₀ . When the cell concentration (OD ₆₀₀ ) reaches 0.4-0.6, add 20 mM sodium lactate, 10 mM cysteine and 25 mM sodium fumarate, and transferred to an anaerobic serum bottle; the serum bottle was purged with nitrogen to remove the oxygen in the bacterial solution, and then the serum bottle cap was sealed with a butyl rubber stopper, and placed on a shaker at 30°C for anaerobic incubation for 20 h.

After the above-mentioned anaerobic culture was completed, 100 mg CIZS QDs were added (anaerobic conditions) to the SW cell culture medium, and an additional anaerobic culture was performed for 1 hour to ensure that the CIZS QDs were carried by the SW into the cell interior. Finally, the cell culture medium was centrifuged at 5000 rpm for 5 minutes, washed, and the cell pellet was collected and resuspended in Tris buffer to obtain the CIZSQDs/SW periplasmic photosensitized whole-cell hybrid system.

The prepared CIZS QDs/SW periplasmic photosensitized whole-cell hybrid system contained 28% of the photosensitizer carried by the internal cells, and the hydrogen production efficiency was 490 μmol after 9 hours of reaction under visible light.

Example 2

Construction of CIZS QDs/ Desulfovibrio vulgaris periplasmic photosensitized whole-cell hybrid system

Take a small amount of Desulfovibrio vulgaris strain and insert it into bicarbonate buffer medium, which contains yeast powder, peptone, trace minerals, vitamins, NaH ₂ CO ₃ , NH ₄ Cl, NaH ₂ PO ₄ , KCl, 60% Add lactic acid syrup, MgSO ₄ , Na ₂ SO ₄ , add lactic acid and iron (III) citrate as electron donor and electron acceptor respectively, and culture overnight under aerobic conditions at 35°C on a shaker (180 rpm); then Add 100 mg CIZS QDs to Desulfovibrio vulgaris and incubate anaerobically for an additional 1 hour to ensure that CIZS QDs are carried by Desulfovibrio vulgaris into the cell; finally, centrifuge the cell culture medium at 5000 rpm for 5 minutes, wash, collect the cell pellet and re- Suspended in bicarbonate buffer to obtain CIZS QDs/ Desulfovibrio vulgaris periplasmic photosensitized whole-cell hybrid system.

The prepared CIZS QDs/ Desulfovibrio vulgaris periplasmic photosensitized whole-cell hybrid system contained 23% of the photosensitizer carried by the internal cells, and the hydrogen production efficiency was 426 μmol after 9 hours of reaction under visible light.

Example 3

Construction of CdS/ Desulfovibrio gigas periplasmic photosensitized whole-cell hybrid system

Take a small amount of Desulfovibrio gigas strains and insert them into the medium containing lactic acid and sulfate as carbon and energy sources, and place them on a shaker (180 rpm) at 35°C for overnight culture under aerobic conditions; measure OD ₆₀₀ to monitor cell growth, and wait until the cells reach In the late stage of exponential growth, add 100 mg CdS (anaerobic conditions) to the Desulfovibrio gigas cell culture medium and incubate anaerobically for an additional 1 hour to ensure that CdS is carried by Desulfovibrio gigas into the cell interior; finally, the cell culture medium is centrifuged Wash at 5000 rpm for 5 minutes, collect the cell pellet and resuspend in MOPS buffer to obtain a CdS/ Desulfovibrio gigas periplasmic photosensitized whole-cell hybrid system.

The prepared CdS/ Desulfovibrio gigas periplasmic photosensitized whole-cell hybrid system contained 18% of the photosensitizer carried by the internal cells, and the hydrogen production efficiency was 386 μmol after 9 hours of reaction under visible light.

The above is only an embodiment of the present invention, and does not limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made by the description of the present invention, or directly or indirectly used in other related technical fields, shall be the same as The theory is included in the patent protection scope of the present invention.

Claims (5)

1. A method for constructing a periplasmic photosensitized whole-cell hybrid system, comprising the steps of:

A. selecting Shewanella oneidensis MR-1 capable of expressing biological enzyme in periplasm of cells, inoculating into corresponding culture medium, activating under aerobic condition, inoculating the culture solution into improved LB culture medium until the bacterial concentration OD in the culture medium ₆₀₀ After reaching 0.4-0.6, anaerobic treatment is carried out to enable the bacteria to express biological enzyme in the periplasm of the cells;

B. after the biological enzyme expression in the anaerobic process is finished, adding a semiconductor nano photosensitizer CuInS under the anaerobic condition ₂ ZnS, placing in a constant temperature shaking table at 30 ℃ for cell hybridization;

C. after the hybridization is finished, centrifuging and washing the solution, and then re-suspending the whole-cell hybridization system with the photosensitization of the periplasm into a new reaction buffer solution;

the anaerobic treatment of the step A comprises the following steps: transferring the bacterial liquid into a 500mL anaerobic bottle in a super clean bench, and then respectively adding lactic acid, fumaric acid and cysteine to make the concentrations of the lactic acid, the fumaric acid and the cysteine in a system respectively 20mM, 25mM and 10mM; then introducing nitrogen into the anaerobic bottle for 30min, covering a plug, compacting and sealing the outermost layer by using an aluminum cover, placing the anaerobic bottle in a shaking table at 30 ℃, and culturing for 20h.

2. The method of constructing a periplasmic photosensitized whole cell hybrid system according to claim 1, wherein: and B, the particle size of the semiconductor nano photosensitizer is not more than 20nm.

3. The method of constructing a periplasmic photosensitized whole cell hybrid system according to claim 1, wherein: the content of the semiconductor nano photosensitizer added in the step B in the hybrid system is 0.25-1.25 mg/mL, and the retention time is 0.5-2 h.

4. A whole cell hybrid system with photosensitization of periplasm of cells constructed according to the method of any one of claims 1 to 3.

5. Use of a periplasmic photosensitized whole cell hybrid system according to claim 4, wherein: the catalyst is applied to visible light catalysis hydrogen production.

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