CN112725369B

CN112725369B - Gene, recombinant vector, recombinant bacterium of salt-tolerant adenylate cyclase and application of gene

Info

Publication number: CN112725369B
Application number: CN202110163662.0A
Authority: CN
Inventors: 应汉杰; 姜达海; 牛欢青; 陈勇; 柳东; 余斌
Original assignee: Nanjing Tech University
Current assignee: Nanjing Tech University
Priority date: 2021-02-05
Filing date: 2021-02-05
Publication date: 2023-05-05
Anticipated expiration: 2041-02-05
Also published as: CN112725369A

Abstract

The invention discloses a salt-tolerant adenylate cyclase gene, a recombinant vector, recombinant bacteria and application thereof. The nucleotide sequence of the salt-tolerant adenylate cyclase gene is shown as SEQ ID NO.1, the amino acid sequence of the salt-tolerant adenylate cyclase coded by the gene is shown as SEQ ID NO.2, total 334 amino acids are shown, and the theoretical molecular weight is 35.99kDa. The catalytic activity of adenylate cyclase expressed by the recombinant bacterium is enhanced along with the increase of the concentration of sodium chloride, and when the concentration of sodium chloride is 2.2 and M, the catalytic activity of recombinant bacterium cells reaches the maximum, the concentration of an optimal substrate is 30mM, the optimal pH value is 10.0, and the optimal temperature is 40 ℃; the recombinant bacterium is used for synthesizing cAMP through whole cell catalysis, and the yield reaches 96.6%. Compared with the existing reported enzyme catalysis process of adenylate cyclase or whole cell catalysis synthesis of cAMP, the recombinant bacterium has higher stability and catalysis efficiency under the hypertonic condition, and has good industrial application prospect.

Description

Gene, recombinant vector, recombinant bacterium of salt-tolerant adenylate cyclase and application of gene

Technical Field

The invention relates to the technical field of genetic engineering, in particular to recombinant bacteria for expressing salt-tolerant adenylate cyclase and application thereof.

Background

Adenosine cyclophosphate (cyclic adenosine monophosphate, abbreviated as cAMP) is a physiologically active substance widely existing in animals, plants and microorganisms, and is used as a second messenger in organisms, has a certain regulation effect on most enzymatic reactions, and participates in important physiological activities such as carbohydrate metabolism, fat metabolism, nucleic acid synthesis and protein synthesis. The cyclic adenosine monophosphate can promote the survival of cardiac muscle cells in vivo, strengthen the anti-injury, anti-ischemia and anti-hypoxia capabilities of the cardiac muscle cells, is mainly used for treating diseases such as cardiac insufficiency, angina pectoris, myocardial infarction and the like clinically, and has very important application value when being widely used as a feed additive in the production of livestock and poultry products.

At present, the large-scale production of cAMP is mainly performed by chemical synthesis. In addition, the preparation method of cAMP also has the problems of a fermentation method, an enzyme method and the like, wherein the fermentation method has the problems of poor repeatability, unstable yield and the like, and the enzyme catalysis method has the problems of low enzyme content, poor enzyme stability and the like. Whole cell catalysis can avoid operations such as separation and purification of enzyme, is easy for large-scale production, and in natural cell environment, the enzyme can reduce the influence of environmental factors on the enzyme, and is beneficial to maintaining the stability of the enzyme, so that the production of cAMP by using whole cell catalysis is a feasible way and has certain advantages.

Disclosure of Invention

The invention aims to: aiming at the defects of the prior art, the invention provides a salt-tolerant adenylate cyclase gene, a recombinant vector containing the same, recombinant bacteria and application thereof.

To achieve the above object, the present invention provides a gene encoding a salt tolerant adenylate cyclase (Hacya) having a nucleotide sequence as shown in SEQ ID NO.1 and a gene length of 1005bp, which is derived from Haloactinopolyspora alba (obtained from NCBI database).

The invention further provides salt-tolerant adenylate cyclase coded by the gene, the amino acid sequence of which is shown as SEQ ID NO.2, and the total amino acid is 334, and the theoretical molecular weight is 35.99kDa.

The invention also provides a recombinant vector containing the salt-tolerant adenylate cyclase encoding gene. Preferably, the recombinant vector is a fusion expression vector, preferably the starting vector is pET28a, and the salt-tolerant adenylate cyclase gene is inserted between EcoRI and NdeI cleavage sites of pET28 a.

Still further, the present invention provides a recombinant bacterium comprising the recombinant vector, preferably the host bacterium is Escherichia coli BL (DE 3).

The invention also provides application of the recombinant bacterium in preparing cyclic adenosine monophosphate; the method comprises the following steps:

(1) Constructing recombinant bacteria, inoculating the activated recombinant bacteria into LB culture medium, and culturing until OD600 is reached _nm When the bacterial sludge is 0.6-0.8, adding IPTG, inducing expression for 4-15h, and centrifuging to collect bacterial sludge.

(2) Whole cell catalysis: adding substrate ATP, metal ions, sodium chloride, a surfactant and bacterial sludge obtained in the step (1) into Tris-HCl buffer solution, and establishing a whole-cell catalytic reaction system to generate cAMP; or performing ultrasonic crushing and centrifugation on the bacterial sludge obtained in the step (1) to obtain crude enzyme liquid containing salt-tolerant adenylate cyclase, adding ATP, metal ions, sodium chloride and the crude enzyme liquid into Tris-HCl buffer solution, establishing an enzyme catalytic reaction system, and performing enzyme catalytic reaction to generate cAMP.

Wherein, the construction method of the recombinant bacteria is as follows:

1) The salt-tolerant adenylate cyclase encoding gene fragment is inserted between EcoR I and Nde I restriction sites of the hairpin vector pET28a to obtain a recombinant vector pET28a-Hacya;

2) And (3) converting the recombinant vector pET28a-Hacya into E.coli BL21 (DE 3) by a thermal shock method to obtain recombinant bacteria.

In step (1), preferably, the final concentration of IPTG is 0.1-1.2mM, more preferably, the final concentration of IPTG is 0.8mM, and the induction of expression is 8h.

In step (2), the final concentration of the Tris-HCl buffer is 40-120mM, the final concentration of ATP is 10-70mM, and the metal ions are 30-80mM Mg ²⁺ The final concentration of the sodium chloride is 0-5M, the dosage of the surfactant is 0.2-2% v/v (relative to the volume of the catalytic liquid), the dosage of the bacterial sludge is 1-50g/L, and the catalytic condition is pH7.0-11.0 and the reaction is carried out for 4-8h at 25-60 ℃.

Preferably, the surfactant is a nonionic surfactant, a cationic surfactant or an anionic surfactant.

Preferably, the concentration of the Tris-HCl buffer system is 100mM, the concentration of ATP is 30mM, the concentration of metal ions is 50mM, the concentration of sodium chloride is 2.2M, the amount of surfactant is Triton X-100 of 0.5% (v/v), the concentration of bacterial sludge is 5g/L, and the catalytic condition is pH 10.0 and the reaction is carried out at 40 ℃ for 4 hours.

The beneficial effects are that: the invention firstly clones the coding gene of the salt-tolerant adenylate cyclase of H.alba, constructs a recombinant strain for expressing the salt-tolerant adenylate cyclase, the catalytic activity of the adenylate cyclase expressed by the recombinant strain is enhanced along with the improvement of the concentration of sodium chloride, the recombinant strain has the characteristics of salt tolerance and high activity, when the concentration of sodium chloride is 2.2M, the catalytic activity of the recombinant strain cell reaches the maximum (about 7 times), the concentration of an optimal substrate is 30mM, the optimal pH value is 10.0, the optimal temperature is 40 ℃, and the cAMP is synthesized by using the whole cell catalysis of the recombinant strain, so that the yield is 96.6%. Compared with the existing reported enzyme catalysis process of adenylate cyclase or whole cell catalysis synthesis of cAMP, the recombinant bacterium has higher stability and catalysis efficiency under the hypertonic condition, and has good industrial application prospect.

Drawings

FIG. 1 is a pET28a-Hacya plasmid map provided by an embodiment of the present invention;

FIG. 2 is a diagram showing the result of electrophoresis of a recombinant bacterium plasmid extraction provided by the embodiment of the invention, wherein lane M is a 10,000bp Marker, and lane 1 is the result of recombinant bacterium plasmid extraction;

FIG. 3 is a diagram showing the result of protein electrophoresis of a crude enzyme solution of recombinant bacteria according to the embodiment of the present invention, wherein lane M is a standard protein Marker, and lane 1 is a crude enzyme solution;

FIG. 4 is a graph showing the optimal sodium chloride concentration of recombinant bacteria according to the embodiment of the invention;

FIG. 5 is a graph of the optimal temperature results of recombinant bacteria provided by the embodiment of the invention;

FIG. 6 is a graph showing the optimal pH of recombinant bacteria according to an embodiment of the present invention;

FIG. 7 is a graph showing the result of concentration of the optimal substrate of the recombinant bacterium provided by the embodiment of the invention;

FIG. 8 is a diagram showing the process of generating cAMP by using recombinant bacterium catalysis according to the embodiment of the present invention.

Detailed Description

The present invention will be described in further detail with reference to specific examples. Detailed embodiments and specific operations are given, examples will aid in understanding the present invention, but the scope of the present invention is not limited to the following examples.

Example 1 cloning of salt tolerant adenylate cyclase gene and construction of recombinant vector.

PCR was performed using the target gene Hacya (SEQ ID NO. 1) as a template and using the primer 1 (SEQ ID NO. 3) and the primer 2 (SEQ ID NO. 4) in the reaction system of Table 1 below, to amplify the target gene Hacya.

Wherein, primer 1 contains EcoR I cleavage site and primer 2 contains Nde I cleavage site.

The PCR reaction system and the reaction conditions are shown in Table 1.

TABLE 1 PCR reaction System and reaction conditions of Hacya Gene

Vector pET28a was subjected to linear cleavage, and the cleavage system and reaction conditions are shown in table 2.

TABLE 2 cleavage System and reaction conditions for vector pET28a

By ClonExpress ^TM II, carrying out one-step cloning on a recombination reaction system (ClonExpress II One Step Cloning Kit; cat: C112-01/02) to obtain a recombination vector pET28a-Hacya, wherein the reaction system and the reaction conditions are shown in Table 3.

TABLE 3 ClonExpress ^TM II recombination reaction system and reaction conditions

After the completion of the one-step cloning reaction, the reaction tube was immediately cooled in an ice-water bath for 5min, then the ligation product was transformed into E.coli DH5a competent cells (purchased from Biotechnology (Shanghai) Co., ltd.), which were spread on LB agar plates with kanamycin resistance, incubated overnight at 37℃to pick up single colonies, incubated overnight in LB liquid medium, and plasmid DNA miniprep kit (purchased from AxyPrep Co., cat: AP-MN-P-250G) was used to extract plasmids, and positive recombinant plasmids were obtained after the sequencing result was correct. The constructed recombinant plasmid pET28a-Hacya is shown in figure 1.

Wherein, the formula of LB agar plate containing kanamycin resistance is as follows: 10g/L peptone, 5g/L yeast extract, 10g/L sodium chloride, 15-20g/L agar and 50mg/L kanamycin.

The formula of the LB liquid medium is as follows: 10g/L peptone, 5g/L yeast extract and 10g/L sodium chloride.

Example 2 construction of recombinant E.coli BL21 (DE 3) (pET 28 a-Hacya).

The constructed recombinant plasmid pET28a-Hacya was transformed into E.coli BL21 (DE 3) (purchased from Biotechnology (Shanghai) Co., ltd.) by hot-shock method, and the specific procedures were as follows: mu.L of the recombinant plasmid (obtained in example 1) was added to 100. Mu.L of E.coli BL21 (DE 3) competent cell solution, placed on ice for 30min, heat-shocked at 42℃for 90s, and immediately removed from ice for 2min. Adding 900 mu L of LB liquid medium, culturing at 37 ℃ and 200rpm for 40min, and then taking 200 mu L of culture solution to be coated in LB solid medium containing kanamycin, and culturing at 37 ℃ for 12h to obtain single colony which is recombinant bacteria. Single colonies were picked, cultured overnight in LB liquid medium, plasmid DNA miniprep kit (purchased from AxyPrep Co., cat: AP-MN-P-250G) was used to extract transformant plasmids, ecoRI and NdeI were used to double-digest the transformant plasmids, gel electrophoresis was used to verify that positive recombinant bacteria were obtained, and the electrophoresis results are shown in FIG. 2.

Example 3 cultivation of recombinant bacteria containing salt-tolerant adenylate cyclase.

Inoculating the constructed recombinant bacteria toIn LB liquid medium containing 50mg/L kanamycin, culturing overnight at 37℃and 200rpm, then inoculating into 500mL triangular flask containing 100mL LB medium containing 50mg/L kanamycin with 1% (v/v) inoculum size, and standing for OD _600nm When the reaction time reaches 0.6-0.8, IPTG with a final concentration of 0.8mM is added, and the reaction is induced for 8 hours at 30℃and 200 rpm. After the induction is finished, the bacterial sludge is collected by centrifugation at 8000rpm for 10min at 4 ℃.

The bacterial sludge collected above was washed twice with 100mM Tris-HCl (pH 8.0), then resuspended in 20mL 100mM Tris-HCl (pH 8.0), sonicated (200 w, over 3s, stopped for 7s, total 10 min), and centrifuged to obtain a crude enzyme solution containing salt-tolerant adenylate cyclase, and the results of protein electrophoresis are shown in FIG. 3.

Example 4 crude enzyme solution catalyzes the production of cAMP.

The enzymatic reaction system (1 mL) and the reaction conditions were as follows: 100mM Tris-HCl (pH 10), 50mM MgCl ₂ 2.2M sodium chloride, 30mM substrate ATP, at 40℃for 6h, heating at 95℃for 10min for inactivation, centrifuging, and passing the supernatant through a membrane. Detection of product cAMP content by HPLC, in particular

C18 HPLC chromatographic column, 250mm×4.6mm; mobile phase: aqueous triethylamine phosphate (wherein the phosphoric acid content is 0.6% (v/v), pH is adjusted to 6.6 with triethylamine): methanol=75:25; an ultraviolet detector having a wavelength of 254nm; the column temperature is 25 ℃, the flow rate is 0.8mL/min, and the sample injection amount is 20 mu L. The final cAMP production rate was 90.2% by detection.

Example 5 recombinant whole cell catalysis to cAMP.

A 50mL beaker was taken to prepare the following reaction solution with a total volume of 20 mL: the bacterial sludge (5 g/L) obtained in example 3, 100mM Tris-HCl (pH 10), 50mM MgCl ₂ 2.2M sodium chloride, 30mM substrate ATP and 0.5% (v/v) Triton X-100. The reaction was carried out at 40℃and 200rpm for 6 hours, and the pH was controlled to 10.0 by NaOH during the reaction. During and after the reaction, 1mL of the catalyst was taken, heated at 95℃for 10min for inactivation, centrifuged, and the supernatant was subjected to membrane filtration to determine cAMP production by the HPLC detection method in example 4. Upon detection, 28.98mM cAMP was finally produced, with a cAMP production rate of 96.6% (FIG. 8).

Comparative example 1

As a control for the present application, the adenylate cyclase gene Arcea derived from Arthrobacter disclosed in invention patent CN102212538 was cloned into pET28a vector as in example 1, and the recombinant plasmid was introduced into E.coli BL21 (DE 3) competent as in example 2. The bacterial sludge (5 g/L) obtained in the method of example 3 was subjected to catalytic reaction to produce cAMP according to the catalytic reaction system and reaction conditions of example 5, and 9.06mM of cAMP was finally produced by detection, and the cAMP yield was 30.2% (FIG. 8).

EXAMPLE 6 determination of optimum sodium chloride concentration by Whole cell catalysis of recombinant bacteria

The reaction system (1 mL) was measured and the reaction conditions were as follows: the recombinant bacterium obtained in example 3 had a bacterial sludge concentration of 10g/L, tris-HCl (100 mM), ATP (30 mM) and MgCl (50 mM) ₂ Sodium chloride concentrations of 0, 1.0, 1.5, 2.0, 2.2, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0M,0.5% (v/v) Triton X-100, pH 9.0, 35 ℃,30 min of reaction, 10min of heating at 95℃for inactivation, centrifugation, supernatant permeation and cAMP production were measured by HPLC detection method in example 4. As a result, as shown in FIG. 4, the catalytic activity of the recombinant bacterium increased with an increase in sodium chloride concentration, and the activity reached the maximum at a sodium chloride concentration of 2.2M, which was about 7 times the initial activity.

Example 6 determination of optimum temperature for whole cell catalysis by recombinant bacteria.

The reaction system (1 mL) was measured and the reaction conditions were as follows: the recombinant bacterium obtained in example 3 had a bacterial sludge concentration of 10g/L, tris-HCl (100 mM), ATP (30 mM) and MgCl (50 mM) ₂ 2.5M sodium chloride, pH 9.0, temperature of 25, 30, 35, 40, 45, 50, 55, 60 ℃, reaction 30min, heating at 95 ℃ for 10min for inactivation, centrifuging, and passing supernatant through membrane. The results of the detection method in example 4 are shown in FIG. 5, and the optimum temperature for the catalysis of the recombinant bacterium is 40 ℃.

Example 7 determination of optimum pH for whole cell catalysis by recombinant bacteria.

The reaction system (1 mL) was measured and the reaction conditions were as follows: the recombinant bacterium obtained in example 3 had a bacterial sludge concentration of 10g/L, tris-HCl (100 mM), ATP (30 mM) and MgCl (50 mM) ₂ 2.5M sodium chloride, pH7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 35 ℃,30 min of reaction, 10min of heating at 95 ℃ for inactivation, centrifuging, and passing the supernatant through a membrane. As a result of the detection method in example 4, the optimum pH for the catalysis of the recombinant bacterium was 10.0, as shown in FIG. 6.

EXAMPLE 8 determination of Whole-cell catalyzed optimum substrate concentration for recombinant bacteria

The reaction system (1 mL) was measured and the reaction conditions were as follows: the recombinant bacteria obtained in example 3 had a bacterial sludge concentration of 10g/L, tris-HCl final concentration of 100mM, and ATP concentrations of 10, 20, 30, 40, 50, 60, 70mM,50mM MgCl, respectively ₂ 2.5M sodium chloride, pH 9.0, reaction at 35 deg.C for 30min, heating at 95 deg.C for 10min for inactivation, centrifuging, and passing supernatant through membrane. As a result of the detection method in example 4, the concentration of the optimum substrate catalyzed by the recombinant bacterium was 30mM, as shown in FIG. 7.

The method constructs a recombinant escherichia coli for expressing salt-tolerant adenylate cyclase by using a genetic engineering means, and the recombinant escherichia coli still has activity under the condition of high concentration sodium chloride of the adenylate cyclase expressed by the recombinant bacteria. Compared with the existing reported enzyme catalysis process of adenylate cyclase or whole cell catalysis synthesis of cAMP, the recombinant bacterium has higher stability and catalysis efficiency under the hypertonic condition, and has good industrial application prospect.

Sequence listing

<110> university of Nanjing Industrial science

<120> a salt-tolerant adenylate cyclase gene, recombinant vector, recombinant bacterium and use thereof

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ggcaccggta gccggctcac ctccgggtac ctggtggcgc agaagctgct gccggagttc 420

gaacggctgc tgatctatgc ttggcgccga aagctggccg cgtcggtgaa ccggctggtc 480

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gggctcggcg ccgtgtcgac gtacaccgtg agcgcccgcc gctga 1005

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Ala Glu Gly Ala Gly Leu Ser Val Ala Glu Ala Gln Arg Tyr Trp Arg

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Ala Leu Gly Phe Pro Asp Val Gly Asp Glu Gln Ala Phe Thr Val Trp

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Asp Met Glu Ala Leu Arg Ala Val Ala Asp Leu Val Arg Asp Ser Val

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Claims

1. A method for preparing cyclic adenosine monophosphate by utilizing recombinant bacteria under a hypertonic condition is characterized in that the recombinant bacteria comprise a recombinant vector, the recombinant vector comprises a gene with a nucleotide sequence shown as SEQ ID NO.1, the starting vector of the recombinant vector is pET28a, and the host cell of the recombinant bacteria isEscherichia coli BL21 (DE 3); the preparation method of the cyclic adenosine monophosphate comprises the following steps:

(1) Constructing recombinant bacteria containing the gene shown in SEQ ID NO.1, activating, inoculating the activated recombinant bacteria into LB culture medium, and culturing until OD600 is reached _nm When the concentration is 0.6-0.8, adding an inducer IPTG, wherein the final concentration of the IPTG is 0.1-1.2mM, inducing expression is 4-15h, and centrifugally collecting bacterial sludge;

(2) Whole cell catalysis: adding ATP, magnesium chloride, sodium chloride, a surfactant and the bacterial sludge obtained in the step (1) into Tris-HCl buffer solution, establishing a whole cell catalytic reaction system, and carrying out a whole cell catalytic reaction to generate cAMP; the concentration of the Tris-HCl buffer system is 100mM, the concentration of ATP is 30mM, the concentration of magnesium chloride is 50mM, the concentration of sodium chloride is 2.2M, the concentration of the surfactant is 0.5% (v/v) Triton X-100, the dosage of bacterial sludge is 5g/L, the catalysis condition is pH 10.0, and the reaction is 6h at 40 ℃.