A urease structural gene and its expression and application

Abstract

The invention discloses a high-activity urease structural gene and its expression and application, belonging to the technical field of genetic engineering and biomineralization. The invention connects the high-activity urease structural gene UreABCEFGD-mut obtained by site-directed mutagenesis to the expression vector pRS426, and then introduces the recombinant plasmid pRS426-UreABCEFGD-mut into E.coli BL21 (DE3), and heterologously expresses the high-activity urease structural gene. The recombinant urease bacteria can produce urease protein with high-efficiency urea hydrolysis activity, and is applied to biomineralization. The urease produced by the expression of the recombinant urease bacteria is about 1.2 times more active than the urease produced by the original bacteria, which can effectively improve the ability of Escherichia coli to hydrolyze urea, and can effectively solve the problem of low urease activity in the application of microbial induced calcium carbonate precipitation.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering and biomineralization, and specifically relates to a urease structural gene and its expression and application. Background Art

[0002] Concrete is currently the most widely used building material, but it will inevitably produce cracks due to internal and external factors during its use, such as early temperature cracking, shrinkage cracking, and later load cracking. The presence of cracks will lead to instability and reduced durability of building components or the whole. Biomineralized materials are a new type of adhesive that is environmentally friendly, highly compatible with materials, and highly durable. They can be used in environments such as construction projects and cultural relics restoration. Among them, microbial restoration materials are relatively efficient and low-toxic, and have good application prospects.

[0003] Microbial mineralization technology is a method of using microorganisms to convert natural chemicals into mineral precipitation. Most of its applications use urease-type bacteria, such as Bacillus pasteurii (Bacillus pasteurii or Sporosarcina pasteurii). This microorganism is the mineralizing strain with the strongest known biomineralization ability and the highest efficiency in decomposing urea. Its urease is the most active among the known microbial ureases. The urease gene can encode apoproteins ureA, ureB, ureC and auxiliary proteins ureD, ureE, ureF, ureG. Various auxiliary proteins can promote the assembly of nickel active centers in urease, and active holo-ure (ABC) 3 can hydrolyze urea to generate bicarbonate ions or carbonate ions, which further combine with free metal cations such as calcium ions to generate biomineralized precipitates with cementitious properties.

[0004] The calcium carbonate precipitate produced by microorganisms is often not a single calcium carbonate powder, but actually a biological adhesive material containing calcium carbonate, which is more suitable for use in environments where enhanced bonding effects are required, such as soil solidification and cultural relic restoration. In existing technologies, Bacillus pasteurianus is used directly as a raw material for biomineralization. Its urease and the mechanism of forming bacterial spores can be used as a biocement material or additive under extreme conditions such as high pH and high calcium. However, the growth mechanism of Bacillus pasteurianus is complex and lengthy, and its amplification culture also has certain problems. In addition, the urease activity in the application of microbial induced calcium carbonate precipitation is low and it is difficult to meet the requirements of industrial use. Summary of the invention

[0005] In view of the problem of low urease activity in the application of microbial induced calcium carbonate precipitation in the prior art, the purpose of the present invention is to propose a high-activity urease structural gene and its expression and application, and to enhance its urea hydrolysis activity by modifying the urease gene.

[0006] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] The first aspect of the present invention is to provide a high-activity urease structural gene, the nucleotide sequence of which is shown in SEQ ID NO.1;

[0008] It should be noted that the expression vector or cloning vector containing the urease structural gene provided by the present invention all fall within the scope of protection claimed by the present invention.

[0009] The second aspect of the present invention is to provide a recombinant vector comprising a high-activity urease structural gene.

[0010] In some embodiments, the vector is a pRS426 plasmid.

[0011] The third aspect of the present invention is to provide a transformant comprising a highly active urease structural gene.

[0012] In some embodiments, the host cell is E. coli BL21(DE3).

[0013] The fourth aspect of the present invention is to provide a recombinant urease bacteria comprising a highly active urease structural gene.

[0014] The fifth aspect of the present invention is to provide a method for constructing a recombinant urease bacteria, comprising the following steps:

[0015] The high-activity urease structural gene was cloned into the pRS426 plasmid to construct the recombinant plasmid pRS426-UreABCEFGD-mut;

[0016] The recombinant plasmid pRS426-UreABCEFGD-mut was transformed into E. coli BL21 (DE3) to obtain recombinant urease bacteria.

[0017] The sixth aspect of the present invention is to provide a method for preparing highly active urease, wherein the recombinant urease bacteria are inoculated into LB liquid culture medium and cultured to an OD of 600 =0.6-0.8, add IPTG with a final concentration of 1mM for induction culture. 600 =2.0-2.2 when the bacteria are harvested.

[0018] In some embodiments, the LB liquid medium contains 50 μM NiCl2 and 100 μg / mL Amp.

[0019] The seventh aspect of the present invention is to provide the use of recombinant urease bacteria in biomineralization.

[0020] The specific steps of the present invention are:

[0021] (1) The urease gene GenBank: X78411.1 from Bacillus pasteurianus, whose urease sequence number is EC3.5.1.5, was selected, and the target gene fragment was subcloned into the pRS426 plasmid to construct the vector pRS426-UreABCEFGD, and the ureD gene in pRS426-UreABCEFGD was subjected to site-directed mutagenesis. The mutant gene is shown in SEQ ID NO.1, and pRS426-UreABCEFGD-mut was constructed;

[0022] (2) Take the lyophilized plasmid pRS426-UreABCEFGD-mut and dissolve it in double distilled water to obtain a plasmid solution. Take the plasmid solution and introduce it into competent cells E. coli BL21 (DE3). After ice bath, heat shock reaction is completed in a water bath. Then ice bath, add antibiotic-free LB medium, shake culture in a metal bath, centrifuge, retain the supernatant, resuspend, and apply it to LB solid medium. Invert and culture overnight to obtain a single colony. Take a single colony and activate culture in the corresponding resistance LB liquid medium to obtain recombinant urease bacteria E. coli BL21 (DE3) / pRS426-UreABCEFGD-mut;

[0023] (3) Transfer the recombinant urease bacteria to LB liquid medium and culture on a shaking platform until OD 600 =0.6-0.8, add IPTG with a final concentration of 1mM for induction culture, and culture on a shaking platform until OD 600 =2.0-2.2, and the bacteria were harvested to obtain the mut bacterial solution overexpressing urease.

[0024] The present invention has the following beneficial effects:

[0025] The invention performs site-directed mutagenesis on the ureD gene in the urease gene (GenBank: X78411.1, the urease sequence number is EC 3.5.1.5) derived from Bacillus pasteurianus to obtain a high-activity urease structural gene with a nucleotide sequence as shown in SEQ ID NO.1, and then uses Escherichia coli as a chassis cell to heterologously express the high-activity urease structural gene to obtain a recombinant urease bacterium capable of producing urease protein with high-efficiency urea hydrolysis activity, and the recombinant urease bacterium is applied to biomineralization.

[0026] The urease activity of the recombinant urease bacteria provided in the present application is enhanced by about 1.2 times compared with the original strain, which can effectively improve the ability of Escherichia coli to hydrolyze urea and effectively solve the problem of low urease activity in the application of microbial induced calcium carbonate precipitation. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a scanning electron microscope image of biological calcium carbonate particles (50 μm);

[0028] Figure 2 Energy level diagram scanned for the surface of biological calcium carbonate;

[0029] Figure 3 This is a scanning electron microscope image of bacterial pores in biological calcium carbonate particles (10 μm);

[0030] Figure 4 This is a scanning electron microscope image of larger particles of calcium carbonate solid. DETAILED DESCRIPTION

[0031] The present invention will be further described in detail below in conjunction with the accompanying drawings. The embodiments described with reference to the accompanying drawings are exemplary and intended to explain the present invention, but should not be construed as limiting the present invention.

[0032] The first aspect of the present invention is to provide a high-activity urease structural gene, the nucleotide sequence of which is shown in SEQ ID NO.1;

[0033] It should be noted that the expression vector or cloning vector containing the urease structural gene provided by the present invention all fall within the scope of protection claimed by the present invention.

[0034] The second aspect of the present invention is to provide a recombinant vector containing a high-activity urease structural gene, wherein the vector is a pRS426 plasmid.

[0035] The third aspect of the present invention is to provide a transformant comprising a high-activity urease structural gene, wherein the host cell is E. coli BL21 (DE3).

[0036] The fourth aspect of the present invention is to provide a recombinant urease bacteria comprising a highly active urease structural gene.

[0037] The fifth aspect of the present invention is to provide a method for constructing a recombinant urease bacteria, comprising the following steps:

[0038] The high-activity urease structural gene was cloned into the pRS426 plasmid to construct the recombinant plasmid pRS426-UreABCEFGD-mut;

[0039] The recombinant plasmid pRS426-UreABCEFGD-mut was transformed into E. coli BL21 (DE3) to obtain recombinant urease bacteria.

[0040] The sixth aspect of the present invention is to provide a method for preparing highly active urease, wherein the recombinant urease bacteria are inoculated into LB liquid culture medium and cultured to an OD of 600 =0.6-0.8, add IPTG with a final concentration of 1mM for induction culture. 600 =2.0-2.2, and the bacteria were harvested in LB liquid medium containing 50 μM NiCl2 and 100 μg / mL Amp.

[0041] The seventh aspect of the present invention is to provide the use of recombinant urease bacteria in biomineralization.

[0042] Example 1

[0043] 1. Constructing a recombinant urease bacteria containing a highly active urease structural gene, comprising the following steps:

[0044] The urease gene from Bacillus pasteurianus was selected by searching GenBank, and the urease gene GenBank: X78411.1 with the urease sequence number EC3.5.1.5 and the total gene length of 5300 bp was cloned into the pRS426 plasmid to construct the vector pRS426-UreABCEFGD;

[0045] The ureD gene in the vector pRS426-UreABCEFGD was subjected to site-directed mutagenesis to construct a plasmid pRS426-UreABCEFGD-mut. The nucleotide sequence of the highly active urease structural gene obtained after site-directed mutagenesis is shown in SEQ ID NO.1;

[0046] The plasmid pRS426-UreABCEFGD-mut was dissolved in 30 μL double distilled water, and 2 μL of the plasmid solution was introduced into competent cells E. coli BL21 (DE3) (manufacturer: Beijing Qingke Biotechnology Co., Ltd.), and the heat shock reaction was completed by ice bathing for 20 minutes and then bathing at 42°C for 90 seconds, followed by ice bathing for 3 minutes, and then adding 600 μL of antibiotic-free LB medium and shaking cultured in a metal bath at 37°C for 42 minutes, and then centrifuged at 8000 rpm / 5 min, leaving 100 μL of the supernatant for resuspending, and smearing on LB solid medium containing 50 μM NiCl2 and 100 μg / mL Amp, and inverted culture overnight to obtain a single colony;

[0047] A single colony was taken and activated and cultured in 20 mL of LB liquid culture medium containing the corresponding resistance to obtain the recombinant urease bacteria E. coli BL21 (DE3) / pRS426-UreABCEFGD-mut, which was numbered as mut and stored at -80°C in a 20% glycerol system.

[0048] 2. Expression of highly active urease, comprising the following steps:

[0049] The recombinant urease bacteria E. coli BL21 (DE3) / pRS426-UreABCEFGD-mut was transferred to LB liquid medium containing 50 μM NiCl2 and 100 μg / mL Amp, and cultured in a shaking incubator at 200 rpm / 37 ° C for 2.5 h until OD 600=0.6-0.8, add 1 mM IPTG for induction culture, and culture at 220 rpm / 16 ° C shaking incubator for 20 h. 600 =2.0-2.2, and the bacteria were harvested to obtain a bacterial solution containing highly active urease, which was named mut bacterial solution.

[0050] Comparative Example 1

[0051] The vector pRS426-UreABCEFGD was transformed into E. coli BL21 (DE3) to prepare the strain E. coli BL21 (DE3) / pRS426-UreABCEFGD as a control group. The preparation method was similar to that in Example 1. The bacterial solution containing urease that had not been subjected to site-directed mutagenesis obtained by fermentation culture of the strain E. coli BL21 (DE3) / pRS426-UreABCEFGD was named ori bacterial solution.

[0052] Test Example 1: Identification of urease activity

[0053] The mut bacterial solution provided in Example 1 was qualitatively analyzed by phenol red indicator, and the bacterial solution changed from yellow to purple-red, indicating obvious urease activity.

[0054] Take 5 mL of the mut bacterial solution of Example 1 in a 50 mL centrifuge tube, add a stationary solution containing 330 mM calcium chloride and urea, and continue to add 50 μM NiCl2 and 100 μg / mL Amp to obtain a mixed solution, place the mixed solution in a 200 rpm / 37°C shaker for 20 hours, centrifuge and filter after the reaction is completed, and dry at 60°C until there is no obvious weight change to obtain a composite precipitate. The recombinant urease bacteria can hydrolyze urea and further react with calcium ions to form calcium carbonate precipitate. Figure 1 It can be seen that the average size of the biogenic calcium carbonate particles obtained is 100 μm. Figure 2 It can be seen that the energy level diagram of its surface scan shows that its surface is covered with some organic matter metabolized by bacteria; Figure 3 It can be seen that at a magnification of 10 μm, the presence of bacterial pores can be observed; Figure 4 It can be seen that biological calcium carbonate can form larger solid agglomerates when combined with bacteria.

[0055] Test Example 2: Urease activity test

[0056] Take 5mL of mut bacterial solution and 5mL of ori bacterial solution and centrifuge at 8000rpm / 15min to obtain bacterial cells. The bacterial cells are resuspended in an equal volume of Tris-Cl buffer and crushed by high pressure to obtain crude enzyme solutions, thereby obtaining mut crude enzyme solutions and ori crude enzyme solutions, which are tested by urease activity test kit (Cat. No.: ADS-F-N015). Urease activity is shown in Tables 1 and 2, where a urease activity unit is defined as 1μg NH3-N produced per milliliter of liquid per minute (1μg NH3·min -1 mL -1 ), it can be seen from the table that the urease activity of the mutant strain mut is about 1.2 times higher than that of the original strain ori, which indicates that the recombinant urease bacteria E. coli BL21 (DE3) / pRS426-UreABCEFGD-mut provided by the present invention has improved the ability of Escherichia coli to hydrolyze urea.

[0057] Table 1

[0058]

[0059] Note: UE = (ΔA578nm + 0.0015) × 4

[0060] Table 2

[0061]

[0062] Note: UE = (ΔA578nm + 0.0015) × 4

[0063] The above are only some embodiments of the present invention. For those skilled in the art, several modifications and improvements can be made without departing from the creative concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A urease structural gene, characterized in that: Its nucleotide sequence is shown in SEQ ID NO.

1.

2. A recombinant vector comprising the urease structural gene according to claim 1.

3. The recombinant vector according to claim 2, characterized in that The vector is the pRS426 plasmid.

4. A transformant comprising the urease structural gene according to claim 1, characterized in that: The host cell is E. coli BL21 (DE3).

5. A recombinant urease bacteria comprising the urease structural gene according to claim 1.

6. A method for constructing the recombinant urease bacteria according to claim 5, characterized in that: The following steps are involved: The urease structural gene of claim 1 is cloned into the pRS426 plasmid to construct the recombinant plasmid pRS426-UreABCEFGD-mut; The recombinant plasmid pRS426-UreABCEFGD-mut was transformed into E. coli BL21 (DE3) to obtain recombinant urease bacteria.

7. A method for preparing urease, characterized in that: The steps include: The recombinant urease bacteria according to claim 5 were inoculated into LB liquid culture medium and cultured until OD 600 =0.6-0.8, add IPTG with a final concentration of 1mM for induction culture. 600 =2.0-2.2 when the bacteria are harvested.

8. The method according to claim 7, characterized in that The LB liquid medium contained 50 μM NiCl 2 and 100 μg / mL Amp.

9. Use of the recombinant urease bacteria according to claim 5 in biomineralization.

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