Recombinant engineering bacterium for displaying carbonic anhydrase on surface as well as construction method and application thereof

文档序号：1932585 发布日期：2021-12-07 浏览：15次中文

阅读说明：本技术 表面展示碳酸酐酶的重组工程菌及其构建方法与应用 (Recombinant engineering bacterium for displaying carbonic anhydrase on surface as well as construction method and application thereof ) 是由贾晓强朱银壮刘亚茹于 2021-08-04 设计创作，主要内容包括：本发明涉及表面展示碳酸酐酶的重组工程菌及其构建方法与应用。包括构建基于冰核蛋白的表面展示碳酸酐酶的重组工程菌,将碳酸酐酶重组工程菌接种到培养基中振荡培养,加入IPTG和ZnSO-(4)诱导碳酸酐酶的表达,收集的表面展示碳酸酐酶重组菌制备全细胞催化剂,并用于CO-(2)矿化沉积CaCO-(3)。避免了常规表达胞内酶存在外膜屏障导致催化效率低等缺陷,将酶展示到细胞表面,既保留了细胞内酶的代谢潜能,又让酶底物和酶促反应产物不需要穿过膜屏障可以与酶直接接触,降低底物和产物传质阻力,提高全细胞催化剂酶活力。本发明制备酶活力更高、更加稳定新型全细胞生物催化剂有望实现CO-(2)矿化沉积CaCO-(3)以捕集CO-(2),为工业应用奠定基础。(The invention relates to a recombinant engineering bacterium for displaying carbonic anhydrase on the surface, a construction method and application thereof. Comprises constructing recombinant engineering bacteria of surface display carbonic anhydrase based on ice nucleoprotein, inoculating the recombinant engineering bacteria of carbonic anhydrase into culture medium, shake culturing, adding IPTG and ZnSO 4 Inducing expression of carbonic anhydrase, collecting recombinant bacteria with surface display carbonic anhydrase to prepare whole-cell catalyst, and using the whole-cell catalyst in CO 2 Mineralizing deposited CaCO 3 . Avoids the defects of low catalytic efficiency and the like caused by the outer membrane barrier existing in the conventional expression intracellular enzyme, displays the enzyme on the cell surface, and not only ensures the stabilityThe metabolic potential of enzyme in the cell is reserved, and the enzyme substrate and the enzymatic reaction product can directly contact with the enzyme without penetrating through a membrane barrier, so that the mass transfer resistance of the substrate and the product is reduced, and the enzyme activity of the whole-cell catalyst is improved. The novel whole-cell biocatalyst prepared by the invention has higher enzyme activity and is more stable, and CO is expected to be realized 2 Mineralizing deposited CaCO 3 To capture CO 2 And lays a foundation for industrial application.)

1. A construction method of recombinant engineering bacteria with carbonic anhydrase displayed on the surface; the method is characterized by comprising the following steps:

(1) amplifying nucleotide sequences encoding an N-terminal domain of Iciclenin (INPN) and Carbonic Anhydrase (CA) by Polymerase Chain Reaction (PCR), respectively;

(2) fusing INPN and CA in an enzyme digestion connection mode, cloning a fused CA surface display module to an expression vector in an enzyme digestion connection mode, and constructing a recombinant expression vector;

(3) and (3) transforming the recombinant expression vector into a host cell escherichia coli BL21(DE3), and screening positive recombinant bacteria by colony PCR or enzyme digestion after plasmid extraction to obtain the recombinant engineering bacteria with the carbonic anhydrase displayed on the surface.

2. The construction method of the recombinant engineering bacteria with carbonic anhydrase displayed on the surface according to claim 1; wherein the carbonic anhydrase in step (1) is selected from the group consisting of alpha-type carbonic anhydrase (HPCA) of Helicobacter pylori 26695 and alpha-type carbonic anhydrase (SazCA) of sulfurhydronium azoense.

3. The construction method of the recombinant engineering bacteria with carbonic anhydrase displayed on the surface according to claim 1; wherein the nucleotide sequence of the coded ice nucleoprotein N-terminal structural domain (INPN) in the step (1) is shown as SEQ ID No. 1; the nucleotide sequence of carbonic anhydrase (HPCA) is shown in SEQ ID No. 2; the nucleotide sequence of carbonic anhydrase (SazCA) is shown in SEQ ID No. 3.

4. The construction method of the recombinant engineering bacteria with carbonic anhydrase displayed on the surface according to claim 1; the method is characterized in that the fusion mode in the step (2) comprises the following steps: the INPN and the CA are connected by two front-end sub-repetitive sequences (RE) in the middle repetitive domain of the ice nucleoprotein, and the nucleotide sequence of the INPN and the CA is shown as SEQ ID No. 4; the INPN is connected with the CA through a Linker (GGGGS); the INPN and the CA are fused and connected by a front two sub-repetitive sequences and a linker in the middle repetitive domain of the ice nucleoprotein.

5. The construction method of the recombinant engineering bacteria with carbonic anhydrase displayed on the surface according to claim 1; characterized in that, the expression vector in the step (2) comprises: plasmid pET-28a, pET-32a fused with the solubility-aiding protein TrxA and pET-22b fused with the PelB signal peptide.

6. The recombinant engineered bacteria for surface display of carbonic anhydrase constructed by the construction method of any of claims 1 to 5, comprising HPCA surface display engineered bacteria: e-28a-I_LH, recombinant plasmid p28a-I contained_LThe nucleotide sequence of H is SEQ ID No. 5; e-28a-I_RH, recombinant plasmid p28a-I contained_RThe nucleotide sequence of H is SEQ ID No. 6; e-28a-I_RLH, recombinant plasmid p28a-I contained_RLThe nucleotide sequence of H is SEQ ID No.7 and the SazCA surface display engineering bacterium: e-28a-I_RLS, recombinant plasmid p28a-I contained_RLThe nucleotide sequence of S is SEQ ID No. 8; e-22b-I_RLS, contained recombinant plasmid p22b-I_RLThe nucleotide sequence of S is SEQ ID No. 9; e-32a-I_RLS, contained recombinant plasmid p32a-I_RLThe nucleotide sequence of S is SEQ ID No. 10.

7. The preparation method of the whole-cell catalyst by using the recombinant engineering bacteria for surface display of carbonic anhydrase of claim 5, which comprises the following steps:

(1) inoculating the obtained recombinant engineering bacteria with the carbonic anhydrase displayed on the surface into a culture medium, and culturing overnight to activate bacteria;

(2) transferring the seed liquid into a new culture medium, and performing shake culture until the seed liquid is OD₆₀₀Adding inducer IPTG with final concentration of 0.2-1.0mM and inducer ZnSO with final concentration of 0.0-2.0mM when the concentration is 0.6-0.8%₄And continuously culturing at 15-37 ℃ for 6-60h to induce the carbonic anhydrase expression.

(3) And centrifuging the induced bacterial liquid in a refrigerated centrifuge at 4-8 ℃ and 6000-9000rpm for 7-10min to collect thalli, washing the thalli at least twice by using deionized water, washing the thalli at least twice by using a Tris-HCl buffer solution, and suspending the thalli in a 5-15ml Tris-HCl buffer solution to obtain a whole-cell catalyst sample.

8. The enzymatic properties of the whole-cell catalyst prepared by using the recombinant engineering bacteria for surface display of carbonic anhydrase according to claim 6, wherein: the surface-displayed carbonic anhydrase whole-cell catalyst has improved thermal stability, pH stability, and long-term storage stability compared to free carbonic anhydrase.

9. Use of the recombinant engineered bacteria with carbonic anhydrase surface-displayed in claim 5 for CO, wherein the recombinant engineered bacteria with carbonic anhydrase surface-displayed are used₂Mineralizing catalytic CaCO₃And (4) generation of a precipitate.

Technical Field

The invention relates to a recombinant engineering bacterium for displaying carbonic anhydrase on the surface, a construction method and application thereof, belonging to the field of molecular biology and biotechnology.

Background

Carbon dioxide (CO)₂) Is the main greenhouse gas causing climate change^[1]. Since the industrial revolution, global climate change due to the emission of a large amount of CO2 has attracted much attention due to the use of fossil fuels and large-scale industrial production. CO2₂The increasing concentration has caused global warming and affected the environment and human health. Currently, economic, safe, efficient CO is being researched and developed₂Capture, utilization and storage technology (CCUS) have become a worldwide hotspot^[2]. For capturing CO industrially₂The traditional methods mainly comprise an absorption method, an adsorption method, a low-temperature condensation method and a membrane separation technology. The method has the problems of complex process, high energy consumption, secondary pollution to the environment caused by byproducts and the like. CO Capture Using Carbonic Anhydrase (CA)₂CO being currently the most promising candidate₂One of the technical means of acquisition and utilization^[3]CaCO, end product thereof₃The properties are very stable throughout the geological cycle and are environmentally friendly. However, the pure enzyme has high purification cost, poor stability, high volatility and no recyclability, and the intracellular enzyme system has the defects of low catalytic efficiency of the outer membrane barrier and the like, so that the application of the pure enzyme is limited. Therefore, the CA is displayed on the cell surface by a genetic engineering means, and the construction of a novel whole-cell biocatalyst is expected to solve the existing problems.

Cell surface display (Cell surface display) is a technique in which a protein or a short peptide (target protein) and an outer membrane protein (carrier protein) of a microbial Cell are anchored as a fusion protein to the surface of the microbial Cell by genetic engineering means. Coli has become the most widely used host, mainly due to its complete genetic system and the ability to achieve high density surface display of full-length recombinant proteins. Coli cell surface display is achieved by contacting the target protein (passenger protein) with an anchorAfter fusion of the native surface protein (carrier protein) located in the outer membrane, the carrier protein binds to direct the transport of the target protein through the inner and periplasm of E.coli, stably anchored to the outer membrane of the cell. The nature of the carrier protein determines the orientation of the target protein (i.e., N-or C-terminal fusion) and limits the molecular mass size of the displayed target protein. Ice Nuclear Protein (INP) -based surface display systems capable of achieving higher surface display levels^[4]The INP consists of three functional domains, namely an N-terminal functional domain, a middle repetitive sequence and a C-terminal functional domain, wherein compared with the other two functional domains, the expression level of a target protein displayed by using INP-N as a vector on a single cell is the most, and the enzyme activity of the cell is the highest^[5]. Furthermore, the intermediate repeats of INP are highly degenerate regions that can be used as scalable linkers to achieve display of target proteins at different distances from the cell surface.

Coli is one of the most important and popular choices in the production of recombinant proteins and host cells as expression platforms^[6,7]. Production of carbonic anhydrase by recombinant E.coli is a beneficial and effective option to increase protein production^[8]. At present, the technology for expressing CA in escherichia coli cells is mature, but the technology still has a great defect in practical application due to low catalytic efficiency and instability. Although theoretically CO₂And HCO^3-Belongs to small molecular substances and can freely enter and exit cells, but the contact between intracellular CA and a substrate can still be limited to a great extent due to the resistance action of a cell membrane, and the timely discharge of products can also be limited, so that the catalytic efficiency of CA is reduced. Therefore, if the enzyme is displayed on the cell surface, the metabolic potential of the enzyme in the cell is reserved, and the enzyme substrate and the enzymatic reaction product can be directly contacted with the enzyme without penetrating through a membrane barrier, so that the mass transfer resistance of the substrate and the product is reduced, and the enzyme activity of the whole-cell catalyst is improved. At present, the research on the surface display of carbonic anhydrase is relatively rare, and in the reported research, the whole-cell catalytic activity of the surface display engineering bacteria is low, mainly because the expressed carbonic anhydrase has low autocatalytic activity and unstable enzymological properties. Thus, a variety of tables are utilizedThe carrier shows carbonic anhydrase with high catalytic activity on the cell surface in different fusion modes to construct surface-displayed carbonic anhydrase recombinant engineering bacteria, and the novel whole-cell biocatalyst with higher enzyme activity and more stability can be prepared, so that CO is expected to be realized₂Mineralizing deposited CaCO₃To capture CO₂And lays a foundation for industrial application.

[1]Anderson T R,Hawkins E,Jones P D.CO₂,the greenhouse effect and global warming:from the pioneering work of Arrhenius and Callendar to today's Earth System Models[J].Endeavour,2016,40(3):178-187.

[2]North M,Styring P.Perspectives and visions on CO₂ capture and utilisation[J].Faraday Discussions,2015,183:489-502.

[3]De Simone G,Fiore A D,Capasso C,et al.The zinc coordination pattern in theη-carbonic anhydrase from plasmodium falciparum is different from all other carbonic anhydrase genetic families[J].Bioorganic and Medicinal Chemistry Letters,2015,25(7):1385-1389.

[4]Karami A,Latifi A M,Khodi S.Comparison of the organophosphorus hydrolase surface display using InaVN and Lpp-OmpA Systems in Escherichia coli[J].Journal of Microbiology and Biotechnology,2014,24(3):379-385.

[5]Fan L H,Liu N,Yu M R,et al.Cell Surface display of carbonic anhydrase on Escherichia coli using ice nucleation protein for CO₂sequestration[J].Biotechnology and Bioengineering,2011,108(12):2853-2864.

[6]Kacar B,Ge X L,Sanyal S,et al.Experimental evolution of Escherichia coli harboring an ancient translation protein[J].Journal of Molecular Evolution,2017,84(2-3):69-84.

[7]Schlegel,Susan,Genevaux,et al.Isolating Escherichia coli strains for recombinant protein production[J].Cellular and Molecular Life Sciences,2017,74(1):891-908.

[8]Tan S I,Han Y L,Yu Y J,et al.Efficient carbon dioxide sequestration by using recombinant carbonic anhydrase[J].Process Biochemistry,2018,73(8):38-46.

Disclosure of Invention

In view of the above, the invention provides a recombinant engineering bacterium with carbonic anhydrase displayed on the surface, a construction method and an application thereof, and the feasibility thereof is verified, and the specific technology is as follows:

the invention discloses a construction method of recombinant engineering bacteria with carbonic anhydrase displayed on the surface; the method comprises the following steps:

(1) amplifying nucleotide sequences encoding an N-terminal domain of Iciclenin (INPN) and Carbonic Anhydrase (CA) by Polymerase Chain Reaction (PCR), respectively;

The carbonic anhydrase of step (1) is selected from the group consisting of alpha-type carbonic anhydrase (HPCA) of Helicobacter pylori 26695 and alpha-type carbonic anhydrase (SazCA) of sulfohydronium azorense.

The nucleotide sequence of the coded ice nucleoprotein N-terminal structural domain (INPN) in the step (1) is shown as SEQ ID No. 1; the nucleotide sequence of carbonic anhydrase (HPCA) is shown in SEQ ID No. 2; the nucleotide sequence of carbonic anhydrase (SazCA) is shown in SEQ ID No. 3.

The fusion mode in the step (2) comprises the following steps:

the INPN and the CA are connected by two front-end sub-repetitive sequences (RE) in the middle repetitive domain of the ice nucleoprotein, and the nucleotide sequence of the INPN and the CA is shown as SEQ ID No. 4;

the INPN is connected with the CA through a Linker (GGGGS);

the INPN and the CA are fused and connected by two front-end sub-repetitive sequences and a linker in the middle repetitive domain of the ice nucleoprotein;

preferably, the INPN and CA are fused and connected by the front two subrepeaters and the linker in the middle repetitive domain of the ice nucleoprotein.

The expression vector in the step (2) comprises: plasmid pET-28a, pET-32a fused with the facilitator TrxA and pET-22b fused with the PelB signal peptide, preferably pET-22b fused with the PelB signal peptide.

The recombinant engineering bacteria with the carbonic anhydrase surface display constructed by the construction method comprise HPCA surface display engineering bacteria: e-28a-I_LH, recombinant plasmid p28a-I contained_LThe nucleotide sequence of H is SEQ ID No. 5; e-28a-I_RH, recombinant plasmid p28a-I contained_RThe nucleotide sequence of H is SEQ ID No. 6; e-28a-I_RLH, recombinant plasmid p28a-I contained_RLThe nucleotide sequence of H is SEQ ID No.7 and the SazCA surface display engineering bacterium: e-28a-I_RLS, recombinant plasmid p28a-I contained_RLThe nucleotide sequence of S is SEQ ID No. 8; e-22b-I_RLS, contained recombinant plasmid p22b-I_RLThe nucleotide sequence of S is SEQ ID No. 9; e-32a-I_RLS, contained recombinant plasmid p32a-I_RLThe nucleotide sequence of S is SEQ ID No. 10.

The preparation method of the whole-cell catalyst by displaying carbonic anhydrase recombinant engineering bacteria on the surface comprises the following steps:

(1) inoculating the obtained recombinant engineering bacteria with the carbonic anhydrase displayed on the surface into an LB culture medium, and carrying out overnight culture at 37 ℃ and 220rpm to activate bacteria, thereby obtaining seed liquid;

(2) transferring the seed liquid to a new LB culture medium with an inoculation amount of 1-3%, culturing at 30-37 deg.C and 200-220rpm, and performing shake culture to OD₆₀₀Adding IPTG inducer with final concentration of 0.2-1.0mM and ZnSO with final concentration of 0.0-2.0mM when the concentration is 0.6-0.8%₄After the inducer, the culture is continued for 6 to 60 hours at the temperature of between 15 and 37 ℃ to induce the carbonic anhydrase expression.

(3) And centrifuging the induced bacterial liquid in a refrigerated centrifuge at 4-8 ℃ and 6000-9000rpm for 7-10min to collect thalli, washing the thalli at least twice by using deionized water, washing the thalli at least twice by using a Tris-HCl buffer solution, and suspending the thalli in 5-15mL of Tris-HCl buffer solution to obtain a whole-cell catalyst sample.

Compared with free carbonic anhydrase, the whole-cell catalyst prepared by the recombinant engineering bacteria with the carbonic anhydrase displayed on the surface has improved thermal stability, pH stability and long-term storage stability.

The recombinant engineering bacteria with the surface displaying carbonic anhydrase constructed by the invention is applied to CO₂Mineralizing catalytic CaCO₃A precipitate forms. CaCO generated by recombinant engineering bacteria displaying carbonic anhydrase on surface₃241mg of the precipitate was greater than 173mg produced by intracellular expression of the carbonic anhydrase strain. Surface display of CaCO catalytically produced by the strains at the same time₃The amount of the enzyme is larger than that of the intracellular expression strain E-22b-S, which shows that the sazCA is anchored on the cell outer membrane, the enzyme activity of the whole-cell catalyst is improved, and the catalytic CaCO is improved₃The rate of deposition.

Compared with the prior art, the invention has the beneficial effects that:

(1) the invention utilizes the surface display technology and INPN to display carbonic anhydrase on the surface of escherichia coli cells to obtain the recombinant engineering bacteria with the carbonic anhydrase displayed on the surface, and solves the problems of high purification cost, poor stability, volatility, incapability of recycling and the like of pure carbonic anhydrase.

(2) Compared with recombinant expression intracellular enzyme, the recombinant engineering bacteria with the carbonic anhydrase displayed on the surface constructed by the invention can directly contact with the enzyme without passing through a membrane barrier, thereby reducing the mass transfer resistance of the substrate and the product and improving the activity of the whole-cell catalytic enzyme.

(3) The invention anchors carbonic anhydrase on the cell surface, saves the preparation and purification steps of enzyme, reduces the application cost, and the cell membrane can play the role of immobilized enzyme, so that various performances of the enzyme are more stable. The invention provides a low-cost, simple and convenient method for preparing the carbonic anhydrase whole-cell biocatalyst with high apparent activity.

(4) Application of recombinant engineering bacteria with surface display carbonic anhydrase constructed by the invention to CO₂Mineralizing deposited CaCO₃For actual CO₂Compared with free enzyme, the recombinant engineering bacteria have better stability and can effectively catalyze CO₂Conversion to CaCO by hydration₃And the sediment lays a foundation for the industrial application of the surface display whole-cell biocatalyst.

Drawings

FIG. 1 is a flow chart of construction of HPCA expression vector surface display in different fusion modes

FIG. 2 recombinant plasmid p28a-I_LH、p28a-I_RH and p28a-I_RLH atlas

FIG. 3 is a flow chart of construction of intracellular expression and surface display SazCA expression vector

FIG. 4 recombinant plasmid p28a-S, p22b-S, p32a-S, p28a-I_RLS、p22b-I_RLS and p32a-I_RLS atlas

FIG. 5 immunoblot analysis of cytoplasmic, intracellular membrane and extracellular membrane components of carbonic anhydrase surface display strains

FIG. 6 Effect of different induction temperatures on the Whole cell enzyme Activity of Carbonic anhydrase surface displaying strains and intracellular expressing strains

FIG. 7 different ZnSO₄Effect of concentration on Carbonic anhydrase surface display Strain and intracellular expression Strain Whole cell enzyme Activity

FIG. 8 Effect of different IPTG concentrations on the Whole cell enzyme Activity of Carbonic anhydrase surface displaying strains and intracellular expressing strains

FIG. 9 Effect of different induction times on the Whole cell enzyme Activity of Carbonic anhydrase surface displaying strains and intracellular expressing strains

FIG. 10 is a schematic thermal stability diagram of recombinant carbonic anhydrase whole cell catalyst and free carbonic anhydrase

FIG. 11 schematic pH stability of recombinant carbonic anhydrase whole cell catalyst with free carbonic anhydrase

FIG. 12 schematic long-term stability of recombinant carbonic anhydrase whole-cell catalyst with free carbonic anhydrase

FIG. 13 shows the use of carbonic anhydrase recombinant engineered bacteria for CO₂Mineralizing catalytic CaCO₃Deposition amount

Detailed Description

The invention is described in further detail below with reference to the following figures and specific examples:

the media formulations and solution formulations used in the following examples of the invention are as follows:

LB medium (g/L): 10.0 parts of peptone, 10.0 parts of NaCl, 5.0 parts of yeast extract powder and 7.0-7.2 parts of pH.

100mM IPTG solution: 1.1915g IPTG was dissolved in 48mL of water, the volume was adjusted to 50mL in a volumetric flask, and the solution was sterilized by filtration through a 0.22 μm sterile filter in a clean bench and stored at-20 ℃.

100mM ZnSO₄Solution: 1.4378g of ZnSO4 was dissolved in 48mL of ultrapure water, the volume was adjusted to 50mL in a volumetric flask, and the solution was sterilized by filtration through a sterile filter of 0.22 μm on a clean bench and stored at-20 ℃.

Tris-HCl (pH 8.3) buffer solution: 2.4228g Tris was dissolved in 980ml deionized water, pH adjusted to 8.3 with hydrochloric acid, volume to 1L in volumetric flask and stored at 4 ℃.

PBS (pH 7.4) buffer solution: 0.272g KH₂PO₄，1.136g Na₂HPO₄10.676g NaCl and 0.295g KCl in 980mL deionized water, the volume is constant to 1L in a volumetric flask, and the solution is stored at 4 ℃.

Saturated CO₂Aqueous solution: adding a certain amount of deionized water into a screw bottle, sealing the bottle mouth with a rubber plug with a hole, and mixing with CO₂One end of a rubber tube connected with the gas tank is placed at the bottom of the liquid from a hole of the rubber plug, the screw bottle is always separated from the liquid tank and placed in an ice-water bath, and CO is continuously introduced₂The gas is used for more than 1h, and the pH value of the liquid is measured to reach 3.89 by a pH meter, and the liquid is ready to use after being prepared.

Citric acid-sodium citrate buffer (pH 4-6): firstly, 0.1M of citric acid and sodium citrate mother liquor is prepared respectively, 21.01g of citric acid is accurately weighed and dissolved in 980ml of deionized water, then the volumetric flask is filled with a constant volume to 1L to obtain a mother liquor I, and 29.41g of sodium citrate is accurately weighed and dissolved in 980ml of deionized water, then the volumetric flask is filled with a constant volume to 1L to obtain a mother liquor II. Accurately measuring 13.1mL of mother liquor I and 6.9mL of mother liquor II, and uniformly mixing to obtain a citric acid-sodium citrate buffer solution with the pH value of 4; accurately measuring 8.2mL of citric acid mother liquor and 11.8mL of mother liquor II, and uniformly mixing to obtain a citric acid-sodium citrate buffer solution with the pH value of 5; accurately measuring 3.8mL of citric acid mother liquor and 16.2mL of mother liquor II, and uniformly mixing to obtain the citric acid-sodium citrate buffer solution with the pH value of 6. Stored in a refrigerator at 4 ℃.

Three portions of 2.4228g Tris are accurately weighed, dissolved in 980mL deionized water respectively, the pH is adjusted to 7, 8 and 9 by hydrochloric acid, the volume is fixed to 1L in a volumetric flask, and the solution is stored at 4 ℃.

0.02M glycine-NaOH buffer (10-12): accurately weighing three parts of 1.5015g of glycine and Tris, dissolving in 980mL of deionized water, adjusting the pH to 10, 11 and 12 by using NaOH, fixing the volume to 1L, and storing at 4 ℃.

4wt％CaCl₂Solution: weighing 4g of CaCl₂Then, the mixture was dissolved in 96g of Tris-HCl (pH 8.3) buffer.

The primers and the related sequencing services used in the embodiment of the invention are provided by Beijing optimalaceae Biotechnology Co., Ltd; the operations of plasmid extraction, PCR product purification, agarose gel recovery and the like are carried out according to a corresponding kit provided by Bomaide biotechnology limited; all restriction enzymes and T4 DNA ligase used were purchased from Thermo Fisher.

Example 1 construction of Escherichia coli surface-displaying alpha-type Carboxylic acid anhydrase (HPCA) recombinant engineering bacteria derived from Helicobacter pylori 26695 in different fusion modes

The construction process of the recombinant plasmid of alpha-type carbonic anhydrase (HPCA) from Helicobacter pylori 26695 displayed on the surface of Escherichia coli is shown in FIG. 1, and the recombinant plasmid vector of surface display HPCA is constructed in different fusion modes. The method comprises the following specific steps:

(1) PCR amplification of target gene fragment

E.coli surface display HPCA related gene fragment amplification: plasmid p2-inak is used as a template, an upstream primer is NcoI-INPN-F, a downstream primer is H-lin-INPN-R, an N-terminal sequence INPN of ice crystal nucleoprotein shown in SEQ ID NO.1 introduced with an enzyme cutting site is obtained by high-fidelity polymerase amplification, a termination codon of the INPN is removed, and then flexible connecting peptide FL (GGGGS) is introduced. Plasmid p2-inak is taken as a template, an upstream primer is NcoI-INPN-F, a downstream primer is H-INPN (Re) -R, an N-terminal sequence of ice crystal nucleoprotein introduced into an enzyme cutting site is obtained by high-fidelity polymerase amplification, and simultaneously, the N-terminal sequence has two front-end sub-repetitive sequences (RE) in an INPN middle repetitive domain shown in SEQ ID No.4, a termination codon of the sequence can be selectively removed, and flexible connecting peptide FL (GGGGS) is introduced after the sequence. The plasmid p-HPCA is used as a template, an upstream primer is H-HPCA-F, a downstream primer is XhoI-HPCA-R, a HPCA coding sequence shown in SEQ ID NO.2 introduced with a restriction enzyme cutting site is obtained by high-fidelity polymerase amplification, and a primer sequence used for target fragment amplification is shown as follows:

NcoI-INPN-F：CATGCCATGGATGACCCTGGATAAAGCACTGG, the underlined bases indicate NcoI cleavage sites;

H-lin-INPN-R：CCCAAGCTTCGAACCGCCACCGCCGGTCTGCAAATTCTGCGGCG, bases underlined indicate Hind III sites;

H-INPN(Re)-R：CCCAAGCTTAATTAGATCACTGTGGTTGC, bases underlined indicate Hind III sites;

H-HPCA-F：CCCAAGCTTATGGAGAACACCAAGTGGG, bases underlined indicate Hind III sites;

XhoI-HPCA-R：CCGCTCGAGATTAGTGGTGGTGGTGGTGGTGGCGGGTCTCAGCTGAGC, bases underlined indicate XhoI cleavage sites.

The PCR system for sequence amplification is 50. mu.L, including ddH₂018. mu.L, 2 XPhanta Max Buffer 25. mu.L, dNTP Mix 1. mu.L, Phanta Max Super-Fidelity DNA polymerase 1. mu.L, upstream primer, downstream primer and template 1. mu.L each;

the PCR conditions of sequence amplification are pre-denaturation at 95 ℃ for 30s, denaturation at 95 ℃ for 15s, annealing at 60 ℃ for 15s, and extension at 72 ℃ for 60s, and complete extension at 72 ℃ for 5min after 35 cycles.

(2) Enzyme digestion and purification of target gene fragment

After the PCR amplification product in the step (1) is verified by agarose gel electrophoresis, gel bands corresponding to NcoI-INPN-Lin-Hind III, NcoI-INPN (RE) -FL-Hind III and Hind III-HPCA-XhoI are obtained, and the target band is recovered according to the gel recovery kit instructions. Then, carrying out enzyme digestion on the gene fragment recovered from the gel:

NcoI-INPN-Lin-Hind III, NcoI-INPN (RE) -Hind III and NcoI-INPN (RE) -Lin-Hind III were double digested with NcoI and Hind III, respectively, and Hind III-HPCA-XhoI were double digested with Hind III and XhoI. The digested products were purified according to the PCR product purification kit instructions, the concentration of each DNA solution was determined, and the fragments NcoI-INPN-FL-Hind III, NcoI-INPN (RE) -Hind III and NcoI-INPN (RE) -FL-Hind III recovered after the digestion purification were mixed with the Hind III-HPCA-XhoI fragment in the following ratio of 1: the T4 ligase ligation system was prepared at a ratio of 1. The ligation reaction procedure was: at 22 ℃ for 30 min; 70 ℃ for 5 min. The obtained DNA product was run again and the fusion gene fragments were recovered to obtain HPCA fusion modules INPN-FL-HPCA, INPN (RE) -HPCA and INPN (RE) -FL-HPCA, as shown in FIG. 1, and the HPCA was constructed by connecting the HPCA with INPN in different fusion modes.

(3) Construction of surface display HPCA recombinant expression vector and engineering bacteria

The plasmid pET-28a (+) and the purified and recovered HPCA fusion modules INPN-FL-HPCA, INPN (RE) -HPCA and INPN (RE) -FL-HPCA are subjected to enzyme digestion by restriction enzymes NcoI and XhoI, and the linear plasmid purified and recovered by PCR products and the purified and recovered HPCA fusion modules INPN-FL-HPCA, INPN (RE) -HPCA and INPN (RE) -FL-HPCA are subjected to enzyme digestion according to the following steps of 1: 5, the T4 ligase ligation system was prepared for ligation. The ligation products were transformed into competent cells e.coli BL21(DE3), positive clones were screened by colony PCR, and recombinant plasmids were extracted and sent to beijing jinzhi biotechnology limited for sequencing. The obtained HPCA surface display recombinant plasmid map without any mutation is shown in the attached figure 2, and HPCA surface display expression vectors constructed in different fusion modes are as follows: p28a-I_LH (nucleotide sequence is SEQ ID No.5), p28a-I_RH (nucleotide sequence is SEQ ID No.6) and p28a-I_RLH (the nucleotide sequence is SEQ ID No.7), and the obtained positive clone bacterial strain is the Escherichia coli surface display HPCA recombinant engineering bacterium E-28a-I_LH、E-28a-I_RH and E-28a-I_RLH。

Example 2 construction of recombinant engineering bacteria for intracellular expression and surface display of alpha-carboanhydrase (SazCA) derived from sulfohydrogenium azorense in Escherichia coli

The construction process of the escherichia coli intracellular expression and surface display of the alpha-type carbonic anhydrase (SazCA) recombinant plasmid derived from sulfohydrogenium azorense is shown in fig. 3, and different vectors are used for constructing an intracellular expression and surface display SazCA recombinant plasmid vector. The method comprises the following specific steps:

(1) PCR amplification of target gene fragment

And (3) amplifying the intracellular expression SazCA fragment of the escherichia coli: the method comprises the following steps of taking a plasmid p-SazCA as a template, taking an upstream primer as NcoI-SazCA-F and a downstream primer as XhoI-SazCA-R, amplifying by using high-fidelity polymerase to obtain a SazCA coding sequence shown in SEQ ID NO.3 introduced with an enzyme cutting site, wherein a primer sequence used for amplifying a target fragment is shown as follows:

NcoI-SazCA-F：CATGCCATGGGCGTGGCCGAGGTCCACCACTGGTC, the underlined bases indicate NcoI cleavage sites;

XhoI-SazCA-R：CCGCTCGAGTCAGTGGTGGTGGTGGTGGTGGTTGCTTTCCAGGATGTAGC, bases underlined indicate XhoI cleavage sites.

And (3) displaying amplification of a SazCA related gene fragment on the surface of escherichia coli: using plasmid p2-inak as a template, an upstream primer NcoI-INPN (Re) -F and a downstream primer H-lin-INPN (Re) -R, amplifying by using high-fidelity polymerase to obtain an N-terminal sequence INPN of the ice crystal nucleoprotein introduced with an enzyme cutting site, simultaneously carrying two front-end sub-repetitive sequences (RE) in an INPN middle repetitive domain shown in SEQ ID NO.4, removing a stop codon of the INPN, and introducing a flexible connecting peptide FL (GGGGS) behind the sequence. The method comprises the following steps of taking a plasmid p-SazCA as a template, taking an upstream primer as H-SazCA-F and a downstream primer as XhoI-SazCA-R, amplifying by using high-fidelity polymerase to obtain a SazCA coding sequence shown in SEQ ID NO.3 introduced with an enzyme cutting site, wherein a primer sequence used for target fragment amplification is shown as follows:

NcoI-INPN(Re)-F：CATGCCATGGGCATGACCCTGGATAAAGCACTGG, the underlined bases indicate NcoI cleavage sites;

H-lin-INPN(Re)-R：CCCAAGCTTGCTGCCACCACCACCAATC, bases underlined indicate Hind III sites;

H-SazCA-F：CCCAAGCTTATGGCCGAGGTCCACCACTGGTC, bases underlined indicate Hind III sites;

Xh-SazCA-R：CCGCTCGAGATTAGTGGTGGTGGTGGTGGTGGCGGGTCTCAGCTGAGC bases corresponding to underlining indicate XhoI enzymeA cleavage site.

(2) Enzyme digestion and purification of target gene fragment

And (2) verifying the PCR amplification product in the step (1) by agarose gel electrophoresis to obtain gel bands corresponding to NcoI-SazCA-XhoI, NcoI-INPN-Hind III and Hind III-SazCA-XhoI, and recovering the target band according to the instructions of a gel recovery kit. Then, carrying out enzyme digestion on the gene fragment recovered from the gel: NcoI-SazCA-XhoI was double-digested with NcoI and XhoI, NcoI-INPN-HindIII was double-digested with NcoI and HindIII, and HindIII-SazCA-XhoI was double-digested with HindIII and XhoI. Purifying digestion products according to the instruction of a PCR product purification kit, determining the concentration of each DNA solution, and carrying out enzyme digestion purification on the recovered NcoI-INPN-Hind III fragment and Hind III-SazCA-XhoI fragment according to the following ratio of 1: the T4 ligase ligation system was prepared at a ratio of 1. The ligation reaction procedure was: at 22 ℃ for 30 min; 70 ℃ for 5 min. And (3) running the obtained DNA product again and recovering the fusion gene segment to obtain a SazCA fusion module INPN (RE) -FL-SazCA, wherein the INPN and the SazCA in different intracellular expression and surface display carriers are subjected to fusion connection by two front-end sub-repetitive sequences and a linker in the middle repetitive domain of the ice nucleoprotein.

(3) Recombinant expression vector and construction of engineering bacteria

The plasmids pET-28a (+), pET-22b (+), and pET-32a (+) were digested with restriction enzymes NcoI and XhoI, and the linear plasmids recovered by purification of the PCR products and the gene fragments NcoI-SazCA-XhoI recovered by purification were ligated in the same manner as in 1: 5, the T4 ligase ligation system was prepared for ligation. Coli BL21(DE3), positive clones were screened by colony PCR, and recombinant plasmids were extracted and sent to beijing technologies, inc. The map of the obtained recombinant plasmid for intracellular expression of the SazCA without any mutation is shown in the attached figure 4, and different expression vectors are respectively used for constructing and obtaining the recombinant plasmid for intracellular expression of the SazCA: p28a-S, p22b-S and p32a-S, and the obtained positive clone bacterial strains are the recombinant engineering bacteria E-28a-S, E-22b-S and E-32a-S of the escherichia coli for intracellular expression of the SazCA.

The plasmids pET-28a (+), pET-22b (+), pET-32a (+) and the purified and recovered SazCA fusion module INPN (RE) -FL-SazCA are digested by restriction enzymes NcoI and XhoI, and the linear plasmids purified and recovered by PCR products and the purified and recovered SazCA fusion module INPN (RE) -FL-SazCA are subjected to 1: 5, the T4 ligase ligation system was prepared for ligation. The ligation products were transformed into competent cells e.coli BL21(DE3), positive clones were screened by colony PCR, and recombinant plasmids were extracted and sent to beijing jinzhi biotechnology limited for sequencing. The map of the obtained SazCA surface display recombinant plasmid without any mutation is shown in the attached figure 4, and different expression vectors are respectively used for constructing to obtain the SazCA surface display recombinant plasmid: p28a-I_RLS (nucleotide sequence is SEQ ID No.8), p22b-I_RLS (nucleotide sequence is SEQ ID No.9) and p32a-I_RLS (the nucleotide sequence is SEQ ID No.10), and the obtained positive clone bacterial strain is the recombinant engineering bacterium E-28a-I of the sazCA displayed on the surface of the escherichia coli_RLS、E-22b-I_RLS and E-32a-I_RLS。

Example 3 recombinant surface display of Escherichia coli expression and localization of SazCA

(1) Strain activation: 100 μ L of the engineered Strain E-22b-I was taken from a glycerol tube stored at-20 deg.C_RLS was inoculated into a test tube containing 3mL of LB medium and cultured overnight at 37 ℃ and 220rpm to obtain a seed solution.

(2) Inoculating 2mL of the seed culture solution obtained in the step (1) into 100mL of LB liquid medium, culturing at 37 ℃ and 220rpm to OD₆₀₀When the concentration is 0.6-0.8, adding inducer IPTG with final concentration of 0.4mM and ZnSO with final concentration of 0.5mM₄And the shaker temperature was set at 25 ℃ and after 24h of induction, all cell pellets were collected, washed three times with PBS solution and then resuspended with 10mL PBS.

(3) By means of ultrasoundsThe cells were disrupted by a sonicator for 10 minutes (5 seconds per sonication, 5 seconds pause) and then centrifuged at 8000rpm for 10 minutes to remove incompletely disrupted cell debris. The centrifuged supernatant was collected, transferred to a dedicated centrifuge tube of a Beckmann ultracentrifuge, and centrifuged at 39000rpm for 1h, with cytoplasmic fractions present in the supernatant and membrane fractions in the pellet fraction. With a solution containing 0.01mM magnesium chloride (MgCl)₂) And 2% Triton X-100 PBS buffer to resuspend the pellet and add it to room temperature and incubate for 30min to fully dissolve the membrane components, after which it is centrifuged again at 39000rpm for 1 h. The final precipitate is the outer cell membrane component of the engineering bacteria, and the supernatant is the inner cell membrane component of the engineering bacteria.

(4) The different cell components were analyzed by Western blotting. The expected molecular weight of INPN-SazCA is 52.59 kDa. As shown in FIG. 5, the surface of the sazCA shows the engineered bacterium E-22b-I_RLImmunoblot analysis of the cytoplasm, intracellular membrane and extracellular membrane components of S showed that there was a significant band at greater than 50kDa in the extracellular membrane component, consistent with the size of the INPN-SazCA fusion protein, indicating successful expression of the fusion protein in the carbonic anhydrase surface display strain in the engineered bacteria, and that SazCA and INPN were anchored in the cell' S outer membrane as fusion proteins.

Example 4 preparation of Whole cell catalyst from recombinant engineered bacteria and enzyme Activity analysis thereof

(1) Strain culture and protein induction expression: 100 μ of LSazCA intracellular expression strain E-22b-S and surface display strain E-22b-I were taken from glycerol tubes stored at-20 deg.C_RLInoculating S into a test tube loaded with 3mL of LB culture medium, culturing at 37 ℃ and 220rpm overnight to obtain a seed solution, inoculating 2mL of the seed solution into 100mL of LB liquid culture medium, culturing at 37 ℃ and 220rpm for 110min, and adjusting OD of fermentation liquor in each bottle by using fresh LB culture medium₆₀₀Induction conditions were optimized at 0.6 ± 0.02, and the optimization experiment was as follows: adding 0.2mM inducer IPTG and 0.5mM ZnSO₄Inducing at five temperatures of 15 deg.C, 20 deg.C, 25 deg.C, 30 deg.C and 37 deg.C for 12h, and collecting thallus; ② adding 0.2mM inducer IPTG and adding ZnSO₄The concentration gradient is 0mM, 0.5mM, 1mM, 1.5mM, 2mM, and the mixture is collected after low temperature induction at 25 ℃ for 12hThalli; ③ adding inducer IPTG 0mM, 0.2mM, 0.4mM, 0.6mM, 0.8mM, 1mM and ZnSO₄Inducing at 25 deg.C for 12 hr with 0.5mM, and collecting thallus; fourthly, adding inducer IPTG 0.4mM and ZnSO₄0.5mM, inducing at 25 deg.C for 6h, 12h, 24h, 36h, 48h, 60h, respectively, and collecting thallus.

(2) Preparing a whole-cell catalyst: and centrifuging the induced bacterial liquid in a refrigerated centrifuge at 4 ℃ and 7000rpm for 10min to collect thalli, washing the thalli with deionized water for 2 times, washing the thalli with Tris-HCl buffer solution for 2 times, suspending the thalli in 10mL of Tris-HCl buffer solution to obtain a whole-cell catalyst sample, measuring OD600, and storing the sample in a refrigerator at 4 ℃ for enzyme activity to be measured.

(3) And (3) enzyme activity analysis: and (3) measuring the enzyme activity of the whole-cell catalyst obtained in the step (2) by using an electrode method. Intracellular expression engineering bacteria E-22b-S of SazCA and surface display recombinant engineering bacteria E-22b-I of SazCA_RLThe whole-cell enzyme activities of S under different induction conditions are shown in FIGS. 6-9 (FIG. 6: different induction temperatures; FIG. 7: different ZnSO)₄Concentration; FIG. 8: different IPTG concentrations; FIG. 9: different induction time), the optimal induction temperature of E-22b-S is 25 ℃, the optimal IPTG induction concentration is 0.4mM, and Zn is added to the optimal external source²⁺The concentration is 0.5mM, the optimal induction time is 12h, and the whole-cell enzyme activity is 8.355UmL^-1OD₆₀₀ ^-1. Cell surface display strain E-22b-I_RLThe optimum induction temperature of S is 25 ℃, the optimum IPTG induction concentration is 0.4mM, and Zn is added to the optimum exogenous source²⁺The concentration is selected to be 0.5mM, the optimal induction time is 24h, and the whole-cell enzyme activity is 11.43UmL^-1OD₆₀₀ ^-1. Before the induction guide condition is optimized, the whole-cell enzyme activity of E-22b-S is 6.59UmL^-1OD₆₀₀ ^-1，E-22b-I_RLThe whole-cell enzyme activity of S is 9.475UmL^-1OD₆₀₀ ^-1. Meanwhile, after optimization, the enzyme activity of the surface display strain under the optimal induction condition is still obviously superior to that of the intracellular expression strain. Further proves that carbonic anhydrase is displayed on the cell surface by means of genetic engineering to construct a novel whole-cell catalyst. Not only can retain the metabolic potential of the enzyme in the cell, but also can ensure that the enzyme substrate and the enzymatic reaction product can directly contact with the enzyme without penetrating through a membrane barrierThe contact reduces the mass transfer resistance of the substrate and the product, and improves the enzyme activity of the whole-cell catalyst.

Example 5 surface display of SazCA Whole cell catalyst enzymatic Properties analysis

(1) Thermal stability analysis: placing the whole cell catalyst sample with highest enzyme activity in example 3 in a water bath kettle with the temperature of 25 ℃, 50 ℃ and 70 ℃ for incubation for different time (0h, 1h, 2h, 3h, 4h, 6h and 12h) to take out a proper amount of surface display cells and free enzyme liquid, and adding CO₂For measuring enzyme activity of a substrate, setting the enzyme activity at 0h as 100%, calculating residual enzyme activity of different samples at different temperatures, and obtaining all data through an average value of three independent determinations.

(2) Analysis of pH stability: the whole cell catalyst sample with highest enzyme activity in example 3 is placed in buffer solutions with different pH values (pH value is 4.0-12.0), placed at 4 ℃ for 24 hours, then the enzyme activity is measured in Tris-Hcl with pH value of 8.3, the highest enzyme activity is set as 100%, and E-22b-I is calculated_RLS and free sazCA are relatively enzyme active after treatment at different pH. Wherein the buffer solution is: a citric acid-sodium citrate buffer solution with pH of 4.0-6.0, a Tris-HCl buffer solution with pH of 7.0-9.0, and a glycine-NaOH buffer solution with pH of 10.0-12.0. All data were obtained by averaging three independent determinations.

(3) Analysis of long-term stability: the sample of the whole-cell catalyst having the highest enzymatic activity in example 3 was placed in a thermostatic incubator at 25 ℃ using CO₂Equal amounts of samples were taken as substrates at appropriate times (0d, 2d, 4d, 6d, 10d) for enzyme activity, and all buffers used for long-term stability analysis were sterilized using pre-sterilized 0.22 μm filters to avoid contamination. The enzyme activity measured at 0d was set to 100%, and the residual enzyme activities over time of SazCA and free SazCA displayed on the surface were calculated, and all data were obtained by averaging three independent measurements.

(4) Surface display of enzymatic properties analysis of the SazCA whole-cell catalyst: the thermal stability analysis is shown in FIG. 10, E-22b-I at 25 ℃_RLThe relative residual enzyme activities of S and Free SazCA are almost kept constant, and after heat preservation at 25 ℃ for 12h, E-22b-I_RLThe residual enzyme activity of S is95% of the initial enzyme activity, and the residual enzyme activity of Free-SazCA is 87.2% of the initial enzyme activity; at higher temperature (50 deg.C and 70 deg.C), the enzyme activity of Free-SazCA is decreased at a higher rate along with the extension of water bath time, and after treatment at 50 deg.C for 12h, E-22b-I_RLThe residual relative enzyme activity of S is 72.49 percent, and the relative enzyme activity of Free SazCA is reduced to 43.75 percent; after treatment for 12h at 70 ℃, E-22b-I_RLThe residual enzyme activity of S is above 40%, and the enzyme activity of Free SazCA is reduced to 15.12%. Thus, the whole-cell catalyst E-22b-I containing surface-displayed SazCA_RLS significantly improves its thermal stability at higher temperatures. The pH stability assay is shown in FIG. 11, E-22b-I_RLThe relative enzyme activities of S and Free SazCA show the situation that the relative enzyme activities increase firstly and then decrease with the increase of pH, when the environmental pH is close to 9, the stability of the surface display strain and the Free enzyme is not obviously different, but under the acidic and strong alkaline conditions, the surface display strain E-22b-I_RLS acts as a whole cell catalyst and exhibits higher pH stability than free enzyme. Long term stability analysis is shown in FIG. 12, E-22b-I after 10d_RLThe residual enzyme activity of S is about 75% of the initial enzyme activity, and free SazCA is only 36.3%, so that the long-term storage stability of the enzyme after surface display is also obviously improved.

Example 6 use of Escherichia coli SacCA intracellular expression bacteria and SacCA recombinant surface display bacteria for CO₂Mineralizing deposited CaCO₃

(1) Surface display strain E-22b-I_RLS and intracellular expression strain E-22b-S for CO₂Mineralization, using strain E-22b containing a blank vector as a control: 8mL Tris-HCl was added to a 100mL centrifuge tube, and 1mg carbonic anhydrase or whole cell catalyst was added to the buffer. Adding ice-cold CO prepared in advance into the mixed system₂Saturated solution, after 5min reaction. Cells displaying and expressing sazcA on their surface were removed by centrifugation, and 25mL of CaCl was added to the liquid₂Solution, maintaining the reaction at the desired temperature for 10 min. Placing the water system filter membrane in a vacuum drying oven for 24h in advance, drying and weighing, and recording as W₁Produced CaCO₃Separating the precipitate by vacuum pump filtration, placing in a vacuum drying oven together with the filter membrane for 36h, drying, weighing, and recording as W₂Of substantially mineralized CaCO₃Mass W ═ W₂-W₁。

(2)CaCO₃Deposition amount: CO2₂The mineralization results are shown in FIG. 13, and CaCO is generated by different mineralization systems under the same conditions₃The deposition amount sequence is as follows: e-22b-I_RLS(241mg)>E-22b-S(173mg)>E-22b (88 mg). Surface display of Strain E-22b-I at the same time_RLCaCO produced by S catalysis₃The amount of the enzyme is larger than that of the intracellular expression strain E-22b-S, which indicates that the sazCA is anchored on the outer cell membrane, and the enzyme substrate or the enzymatic reaction product can directly contact with the enzyme without penetrating through the membrane barrier, thereby reducing the mass transfer resistance of the substrate and the product, improving the enzyme activity of the whole-cell catalyst, and further improving the catalysis CaCO₃The rate of deposition.

While the invention has been described in further detail with reference to specific embodiments thereof, it will be apparent to one skilled in the art that various changes and modifications can be made therein without departing from the spirit of the invention, and such changes and modifications are to be considered within the scope of the invention.

Sequence listing

<110> Tianjin university

<120> recombinant engineering bacteria for surface display of carbonic anhydrase, construction method and application thereof

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catgagaatg gtctggtcgg tttactgtgg ggcgctggaa ccagcgcttt tctaagcgtg 180

catgccgatg ctcgatggat tgtctgtgaa gttgccgttg cagacatcat cagtctggaa 240

gagccgggaa tggtcaagtt tccgcgggcc gaggtggttc atgtcggcga caggatcagc 300

gcgtcacact tcatttcggc acgtcaggcc gaccctgcgt caacgtcaac gtcaacgtca 360

acgtcaacgt taacgccaat gcctacggcc atacccacgc ccatgcctgc ggtagcaagt 420

gtcacgttac cggtggccga acaggcccgt catgaagtgt tcgatgtcgc gtcggtcagc 480

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cactactatc acacccagga caaggccgac ctgcagttca agtatgccgc cagcaagccg 180

aaggccgtgt tcttcaccca tcacaccctg aaggccagct tcgagccgac caaccacatc 240

aactaccgtg gccatgacta cgtgctggac aacgtgcact tccatgcgcc aatggagttc 300

ctgatcaaca acaagacccg tccgctgagc gcccacttcg tccacaagga cgccaagggt 360

cgcctgctgg tgctggccat cggcttcgag gaaggcaaag aaaacccgaa cctggacccg 420

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ggcgtggcct ggttcgtgat cgaggaaccg ctggaagtga gcgccaagca gctggccgag 600

atcaagaaac gcatgaagaa cagcccgaac cagcgtccgg tccagccgga ctacaacacc 660

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tacaaggtgc acgccaagct ggaaaagctg cacatcaact acaacaaggc ggtcaacccg 180

gaaatcgtga acaacggcca caccatccag gtgaacgtgc tggaagattt caaactgaac 240

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gtgaacggca agtactaccc gctggaaatg cacctggtgc acaaggacaa ggacggcaac 360

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ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 180

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acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 300

ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 360

ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 420

acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt 480

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tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac tgcaatttat 600

tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa 660

actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc 720

gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga 780

aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc 840

agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac 900

cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac 960

aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat 1020

tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag 1080

tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca 1140

taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac 1200

ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg 1260

tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca 1320

tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcataacac 1380

cccttgtatt actgtttatg taagcagaca gttttattgt tcatgaccaa aatcccttaa 1440

cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga 1500

gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg 1560

gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc 1620

agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag 1680

aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc 1740

agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg 1800

cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac 1860

accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga 1920

aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt 1980

ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag 2040

cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg 2100

gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta 2160

tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc 2220

agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg 2280

tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatatggtgc actctcagta 2340

caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg 2400

ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct 2460

gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag 2520

gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc 2580

gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag 2640

aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt 2700

ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa 2760

acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg 2820

ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg 2880

tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc 2940

tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta 3000

cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca 3060

gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc 3120

ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc 3180

catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa 3240

ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc 3300

gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac 3360

gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca 3420

ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta 3480

atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa 3540

cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat 3600

tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca 3660

ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa 3720

aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt 3780

atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg 3840

cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca 3900

gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta 3960

tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg 4020

agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat 4080

gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct 4140

ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg 4200

catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat 4260

tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc 4320

tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca 4380

gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg 4440

ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt 4500

tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg 4560

catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct 4620

cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga 4680

tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg 4740

ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc 4800

ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg 4860

cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg 4920

gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatctc gatcccgcga 4980

aattaatacg actcactata ggggaattgt gagcggataa caattcccct ctagaaataa 5040

ttttgtttaa ctttaagaag gagatatacc atggatgacc ctggataaag cactggttct 5100

gcgtacctgt gccaataata tggcagatca ttgtggtctg atttggcctg caagcggcac 5160

cgttgaaagc cgttattggc agagcacccg tcgtcatgaa aatggtctgg ttggtctgct 5220

gtggggtgca ggcaccagcg catttctgag cgttcatgca gatgcacgtt ggattgtttg 5280

tgaagttgca gttgcagata ttatcagcct ggaagaacct ggtatggtta aatttccgcg 5340

tgccgaagtt gttcatgttg gtgatcgtat tagcgcaagc cattttatca gcgcacgtca 5400

ggcagatccg gcaagcacca gcacctcaac cagcaccagt acactgaccc cgatgccgac 5460

cgcaattccg acaccgatgc ctgcagttgc cagcgttacc ctgccggttg cagaacaggc 5520

acgtcatgaa gtttttgatg ttgcaagcgt tagcgcagca gcagcaccgg ttaatacact 5580

gccggttacc acaccgcaga atctgcagac cggatccggt ggtggtggca gcatggagaa 5640

caccaagtgg gactacaaga acaaagagaa cggtccgcac cgctgggaca agctgcacaa 5700

ggacttcgag gtgtgcaaga gcggcaagag ccagtcgccg atcaacatcg agcactacta 5760

tcacacccag gacaaggccg acctgcagtt caagtatgcc gccagcaagc cgaaggccgt 5820

gttcttcacc catcacaccc tgaaggccag cttcgagccg accaaccaca tcaactaccg 5880

tggccatgac tacgtgctgg acaacgtgca cttccatgcg ccaatggagt tcctgatcaa 5940

caacaagacc cgtccgctga gcgcccactt cgtccacaag gacgccaagg gtcgcctgct 6000

ggtgctggcc atcggcttcg aggaaggcaa agaaaacccg aacctggacc cgatcctgga 6060

aggcatccag aagaagcaga acttcaaaga ggtggccctg gacgccttcc tgccgaagtc 6120

gatcaactac taccacttca acggcagcct gaccgcgcca ccgtgcaccg aaggcgtggc 6180

ctggttcgtg atcgaggaac cgctggaagt gagcgccaag cagctggccg agatcaagaa 6240

acgcatgaag aacagcccga accagcgtcc ggtccagccg gactacaaca ccgtgatcat 6300

caagagcagc gcggaaaccc gccaccacca ccaccaccac taagaattcg agctccgtcg 6360

acaagcttgc ggccgcactc gagcaccacc accaccacca ctgagatccg gctgctaaca 6420

aagcccgaaa ggaagctgag ttggctgctg ccaccgctga gcaataacta gcataacccc 6480

ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa aggaggaact atatccggat 6540

<210> 10

<211> 6621

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10