CN111172068B - Construction method and application of whole-cell hybrid system with cell periplasm photosensitization - Google Patents
Construction method and application of whole-cell hybrid system with cell periplasm photosensitization Download PDFInfo
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Abstract
The invention belongs to the technical field of biological-inorganic composite materials, and relates to a construction method of a periplasmic photosensitized whole-cell heterozygous system, which comprises the following steps: selecting bacterial cells capable of expressing biological enzymes in periplasm of cells, inoculating the bacterial cells into a corresponding culture medium, activating under an aerobic condition, and then mixing a bacterial solution and a culture solution according to the ratio of 1: transferring the bacteria to a new culture bottle in a ratio of 100, and carrying out anaerobic treatment to enable the bacteria to express biological enzyme in the periplasm of the cells after the concentration of the bacteria reaches 0.4-0.6; then, adding a semiconductor nano photosensitizer under an anaerobic condition, and placing the semiconductor nano photosensitizer in a constant-temperature shaking table at 30 ℃ for cell hybridization; and finally, centrifuging and washing the solution, and then re-suspending the whole cell hybrid system with the photosensitization of the periplasm of the cell into a new reaction buffer solution. The invention is constructed by semiconductor nano particles with broad spectrum absorption range and cells expressing biological enzyme in the periplasm of cells, is used for realizing the high-efficiency conversion of solar energy-chemical energy, and has good application prospect in the fields of environment, energy and the like.
Description
Technical Field
The invention belongs to the technical field of biological-inorganic composite materials, relates to cell hybridization, and particularly relates to a construction method and application of a periplasmic photosensitized whole cell hybridization system.
Background
Semiconductor photocatalytic nano materials are increasingly paid attention to by people due to the potential application value of the semiconductor photocatalytic nano materials in the aspects of solving energy crisis, environmental problems and the like due to good light capture capacity, stability and catalytic activity. However, the semiconductor photocatalytic nano material has few catalytic active sites and slow catalytic reaction, and further application of the semiconductor photocatalytic nano material is restricted. To compensate for this deficiency, in recent years, biological enzymes have been increasingly used for solar-chemical energy conversion due to their specificity and high efficiency. The semiconductor catalytic material is combined with the biological enzyme, the biological enzyme is used as a high-efficiency catalytic active site, and the semiconductor material is used as a photosensitizer, so that the high-efficiency conversion of solar energy-chemical energy is realized. Although the inorganic-biological enzyme hybrid has high catalytic performance, the biological enzyme itself has complex purification steps, low yield and sensitivity to oxygen, so that the application is limited.
In recent years, a new idea is provided for constructing a novel inorganic-biological hybrid system by using whole cells capable of expressing biological enzymes to replace the biological enzymes. Compared with pure biological enzymes, cells have the advantages of easy availability, stability and self-repair. Based on this, honda et al utilized TiO 2 Photocatalyst andE.coliinorganic-biological whole cell hybrid systems (Honda, Y.; hagiwara, H.; ida, S.; ishihara, T.) were constructed for the first time.Angew. Chem. Int. Edit.2016, 55, 8045-8048), realizing high-efficiency hydrogen production performance by water decomposition. TiO 2 2 As a photosensitizer, electrons excited by light are transmitted to biological enzyme of cells through a transmembrane process of an electron mediator methyl viologen, and solar energy conversion is realized on the biological enzyme; the disadvantages are that the transmembrane transmission process of the electrons is slow, and additional energy is consumed, so that the transfer efficiency of the electrons is not high. To overcome this problem, poppeyer et al proposed an intracellular photosensitizing whole cell system (Zhang, h.; liu h.; tian, z.; lu, d.; yu, y.; cestellos-Blanco, s.; sakimoto, k.; yang, P).Nat. Nanotechnol. 2018, 13, 900-905) which grow the gold nanoclusters into cells, so that the light excitation of the photosensitizer (Au nanocluster) and the transfer process of electrons are carried out in cytoplasm, and the electron transfer efficiency is effectively improved. It should be noted that in this system, due to the complexity of the components in the cytoplasm, the transmission of light is disturbed and the absorption of light by the photosensitizing agent is affected.
In bacterial cells, a periplasm exists between the outer and inner cell membranes. It is closer to the outer membrane than to the cytoplasm, which can reduce interference in light transmission and facilitate light absorption by the photosensitizing agent. In addition, the small space can ensure that the biological enzyme has higher local concentration, increase the contact of the photosensitizer and the biological enzyme and accelerate catalysisAnd (4) carrying out a reaction. The inventors have constructed a whole cell hybrid system with photosensitization of the periplasm of the cell. Through regulation and control, the photocatalysis nano material (such as TiO) with good biocompatibility is obtained 2 , NaTaO 3 , KTaO 3 , Bi 3 TaO 7 , WO 3 , Bi 2 O 3 , BiVO 4 , CdS, CuIn 2 S 4 /ZnS QDs, g-C 3 N 4 Etc.) to cells that express the biological enzyme in the periplasm of the cell (e.g.Shewanella oneidensis，Desulfovibrio gigas, Desulfovibrio vulgaris, Desulfovibrio desulfuricans, Desulfovibrio baculatusEtc.); meanwhile, effective light absorption and electron transfer are realized, and efficient solar energy-chemical energy conversion is realized.
To date, no report has been found on the construction of a whole-cell hybrid system with photosensitization of the periplasm of the cell.
Disclosure of Invention
The invention aims to provide a universal construction method of a whole-cell heterozygous system for the photosensitization of periplasm of cells, which is used for realizing the high-efficiency conversion of solar energy and chemical energy.
Technical scheme
A construction method of a whole-cell hybrid system with photosensitization of periplasm of cells comprises the following steps:
A. selecting bacterial cells capable of expressing biological enzymes in a periplasm of cells, inoculating the bacterial cells into a corresponding culture medium, activating under an aerobic condition, and then mixing a bacterial liquid and a culture solution (LB/MOPS) according to the ratio of 1:100 (v/v) ratio into a fresh flask, to be measured for 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 cells;
B. after the expression of the biological enzyme in the anaerobic process is finished, adding a semiconductor nano photosensitizer under the anaerobic condition, and placing the semiconductor nano photosensitizer in a constant-temperature shaking table at 30 ℃ for cell hybridization;
C. after completion of the hybridization, the solution was centrifuged, washed, and the whole cell hybridization system with photosensitization of the periplasm of the cells was resuspended in a new reaction buffer solution.
In the preferred embodiment of the inventionIn step A, the bacterial cell isShewanella oneidensis, Desulfovibrio gigas, Desulfovibrio vulgaris, Desulfovibrio desulfuricans, Desulfovibrio baculatusEtc. of。
In the preferred embodiment of the present invention, the anaerobic treatment in 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. In this process, the bacteria express biological enzymes (hydrogenases) under anaerobic conditions, and the activity of the enzymes is verified by enzyme activity assays. Of course, different anaerobic treatments exist for different strains, and the anaerobic treatment process in the prior art needs to be determined according to the strains.
In a preferred embodiment of the present invention, the semiconductor nano-photosensitizer in step B comprises 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 g-C 3 N 4 Quantum dots, and the like. The semiconductor photosensitizers can be characterized by X-ray diffraction. Nanoparticles with too large a particle size can affect the rate of endocytosis or diffusion into the cell, and therefore it is important to control the size of the nanoparticles. The particle size of the semiconductor nano photosensitizer is preferably not more than 20nm.
In the preferred embodiment of the invention, 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.
The theoretical content of the added conductor nano photosensitizer is 1 mg/mL, and when the retention time is 1 h, the content of the photosensitizer carried by cells in bacteria is the highest and reaches 28%, and the photocatalytic hydrogen production efficiency is the best.
In the step A, the hydrogenase expression is detected by a method of adding methyl viologen and electron donor to detect enzyme activity.
In step B of the invention, the nanoparticles or quantum dots with smaller size can be gradually entered into the cells by the bacterial cells through endocytosis or diffusion. After being specifically absorbed by living cells, the nano-particles or quantum dots containing the surface ligands are chemically connected with molecules with biological recognition, such as peptides, antibodies, nucleic acids or small molecule ligands. The concentration and time of the semiconductor nano photosensitizer can be controlled, and the content of the semiconductor nano photosensitizer entering cells can be adjusted. When the concentration is too low, the content of the nano particles or quantum dots which can be diffused or endocytosed into cells is too low, so that the carrying rate of the nano particles or quantum dots in the cells is too low; when the concentration of the nanoparticles or the quantum dots is too high, the nanoparticles or the quantum dots are agglomerated together, and the content of the nanoparticles or the quantum dots entering the cells is also influenced. Therefore, the concentration and time of the nanoparticles or the quantum dots are reasonably regulated and controlled, so that the carrying rate of the nanoparticles or the quantum dots in cells can be optimal, and the photocatalytic efficiency of a hybrid system can be optimal. The intracellular content of the photosensitizing agent was measured by a plasma inductively coupled spectrometer (ICP).
Another object of the present invention is to provide a whole-cell hybrid system with photosensitization of periplasm of cells prepared by the method of the present invention, which can be applied to visible light catalysis hydrogen production.
Photocatalytic hydrogen production performance of CIZS QDs/SW periplasm photosensitization whole-cell hybrid system
And (3) testing the photocatalytic hydrogen production performance of the CIZS QDs/SW periplasm photosensitization whole-cell hybrid system by using a closed gas circulation system. In a glove box, ascorbic acid (100 mM) was added as a cavitating sacrificial agent to the prepared hybrid system, followed by filling into a reaction flask. A300W xenon lamp is used as a light source, and ultraviolet light and infrared light are filtered by a cut-off filter (lambda is more than 420 and less than 780 nm). The quartz reactor was then connected to a gas circulation system and evacuated to ensure that the reaction system was anaerobic. The reaction temperature was controlled at 37 ℃ using a circulating water bath. Dark reaction was performed for 1 hour before light irradiation to check if the device was air-leaking. Carrying out photocatalytic reaction at room temperature for 9 hours, setting gasAutomatic sampling procedure for phase chromatography, detecting H produced every hour 2 Peak area of (a), and generation of H by gas chromatography analysis 2 The content of (a).
In order to compare with the yield of the photosensitizer alone, the concentration of the photosensitizer in the whole cell hybrid system of the cell periplasmic photosensitizer was measured by ICP and the content thereof was calculated. The photo-catalytic hydrogen production performance of the photo-catalytic hydrogen production system is measured by adopting the same mass of the photo-sensitizer for experiment.
Advantageous effects
The invention is constructed by semiconductor nano particles with broad spectrum absorption range and cells expressing biological enzyme in periplasm of cells, and is a method which has simple preparation process and low cost and can realize high-efficiency solar-chemical energy conversion. The invention provides a universal construction method of a whole-cell heterozygous system for the photosensitization of periplasm of cells, which is used for realizing the high-efficiency conversion of solar energy and chemical energy and has good application prospect in the fields of environment, energy sources and the like.
Drawings
FIG. 1 is an X-ray diffraction pattern of CIZS QDs prepared.
FIG. 2 is a transmission electron microscope (a) and a high resolution transmission image (b) of CIZS QDs prepared.
FIG. 3 is a scanning electron micrograph (a)/transmission electron micrograph (b) and partial transmission electron micrograph (c) of the prepared SW.
FIG. 4 is a comparison chart of hydrogen production performance of the constructed CIZS QDs/SW periplasm photosensitization whole cell heterozygous system.
Detailed Description
The present invention will be described in detail below with reference to examples to enable those skilled in the art to better understand the present invention, but the present invention is not limited to the following examples.
Unless otherwise defined, terms (including technical and scientific terms) used herein should be construed to have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It will be further understood that terms used herein should be interpreted as having a meaning that is consistent with their meaning in the context of this specification and the relevant art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.
Example 1
CIZS QDs/Shewanella oneidensisConstruction of periplasmic photosensitization whole-cell hybrid system
Taking a small amountShewanella oneidensisThe MR-1 (SW) strain was inoculated into Luria Bertani (LB) medium containing yeast (5 g/L), tryptone (10 g/L) and sodium chloride (10 g/L), and cultured overnight under aerobic conditions in a 30 ℃ shaker (180 rpm); then inoculating 100 mul of culture solution into 200 mL of improved LB culture medium (LB +100 mM MOPS, pH 7.2), and continuously carrying out aerobic culture on a shaking table at 30 ℃ for 3-4 h; by measuring OD 600 Monitoring cell growth as cell concentration (OD) 600 ) When the concentration reaches 0.4-0.6, adding 20mM sodium lactate, 10mM cysteine and 25mM sodium fumarate, and transferring to an anaerobic serum bottle; the serum bottle was purged with nitrogen to remove oxygen from the broth, capped with a butyl rubber stopper, sealed, placed in a 30 ℃ shaker, and anaerobically incubated for 20h.
After the above-described completion of anaerobic culture, 100 mg of CIZS QDs were added (anaerobic conditions) to the SW cell broth medium and cultured anaerobically for an additional 1 hour to ensure that the CIZS QDs were carried into the interior of the cells by SW. And finally, centrifuging the cell bacteria liquid culture medium for 5 minutes at the rotating speed of 5000 rpm, washing, collecting cell precipitates and suspending the cell precipitates in a Tris buffer solution to obtain the CIZS QDs/SW periplasmic photosensitization whole-cell hybrid system.
The content of the photosensitizer carried by the cells in the prepared CIZS QDs/SW periplasm photosensitization whole-cell hybrid system is 28%, and the hydrogen production efficiency is 490 mu mol after 9 h of reaction under visible light.
Example 2
CIZS QDs/Desulfovibrio vulgarisConstruction of periplasmic photosensitization whole cell hybrid system
Taking small amountDesulfovibrio vulgarisInoculating the strain into bicarbonate buffer culture medium containing yeast powder, peptone, trace minerals, vitamins and NaH 2 CO 3 、 NH 4 Cl、NaH 2 PO 4 KCl, 60% lactic acid syrup and MgSO 4 、Na 2 SO 4 Adding lactic acid and ferric citrate (III) as electron donor and electron acceptor, respectively, and culturing overnight in 35 deg.C shaking table (180 rpm) under aerobic condition; 100 mg of CIZS QDs were then added to the mixtureDesulfovibrio vulgarisAdditional anaerobic incubation for 1 hour to ensure that the CIZS QDs are coveredDesulfovibrio vulgarisCarry into the interior of the cell; finally, the cell culture solution is centrifuged for 5 minutes at 5000 rpm, washed, the cell pellet is collected and resuspended in bicarbonate buffer to obtain CIZS QDsDesulfovibrio vulgarisA periplasmic photosensitized whole cell hybrid system.
The obtained CIZS QDsDesulfovibrio vulgarisThe content of the photosensitizer carried by the cells in the cell periplasm photosensitization whole cell heterozygous system is 23 percent, and the hydrogen production efficiency is 426 mu mol after 9 hours of reaction under visible light.
Example 3
CdS/Desulfovibrio gigasConstruction of periplasmic photosensitization whole-cell hybrid system
Taking small amountDesulfovibrio gigasInoculating the strain into a culture medium containing lactic acid and sulfate as carbon and energy, and culturing overnight in a shaker (180 rpm) at 35 deg.C under aerobic condition; measuring OD 600 Cell growth was monitored and when the cells reached late exponential growth 100 mg CdS was added (anaerobic conditions) toDesulfovibrio gigasPerforming anaerobic culture for 1 hr in cell culture solution to ensure CdS to be absorbedDesulfovibrio gigasCarry into the interior of the cell; finally, centrifuging the cell culture solution culture medium for 5 minutes at the rotating speed of 5000 rpm, washing, collecting cell precipitates and suspending the cell precipitates in MOPS buffer solution to obtain CdS-Desulfovibrio gigasA periplasmic photosensitized whole cell hybrid system.
Prepared CdS/inDesulfovibrio gigasThe content of a photosensitizer carried by cells in the cell periplasm photosensitization whole cell heterozygous system is 18 percent, and the hydrogen production efficiency is 386 mu mol after 9 hours of reaction under visible light.
The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications of equivalent structures and equivalent processes performed by the present invention or directly or indirectly applied to other related technical fields are included in the 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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