High-temperature-resistant polyphosphate kinase mutant and application thereof

Abstract

The invention provides a high-temperature-resistant polyphosphokinase mutant and application thereof, and compared with wild polyphosphokinase from Sulfurovum lithotrophum, the amino acid sequence of the high-temperature-resistant polyphosphokinase mutant is subjected to single-point mutation or combined mutation at the amino acid at the 32nd site, the 35th site, the 61th site, the 66th site, the 130th site, the 133rd site, the 144th site or the 226th site. Compared with a wild enzyme, the high-temperature-resistant polyphosphate kinase mutant provided by the invention has the advantage that the resistance to the high temperature of 60 DEG C is obviously improved. When the high-temperature-resistant polyphosphate kinase mutant constructed by the invention is used for preparing ATP (adenosine triphosphate), the sustainability of ATP synthesis is remarkably improved, the ATP yield can reach 16.1 mM after 5 hours of reaction, and the conversion rate exceeds 80%.

Description

Technical Field

[0001] The invention relates to the technical field of molecular biology and bioengineering, and in particular to a high temperature resistant polyphosphate kinase mutant. Background Art

[0002] Cell-free in vitro synthetic biotechnology has received great attention from researchers in recent years. The core of in vitro synthesis is to establish a multi-enzyme catalytic system, which has many advantages, including being free from the restrictions of host cell life activities; being able to express specific substances that are toxic to cells; having no side effects, and being able to obtain the maximum product yield. Therefore, in vitro synthesis has considerable industrial potential and has been widely used in the production of bulk chemicals, pharmaceutical products, and biofuels.

[0003] ATP-dependent energy-consuming enzyme-catalyzed reactions are widely present in nature. Many enzymatic reactions require a molar ratio of ATP to substrate greater than or equal to 1:1, such as various kinases and ligases (Xu et al. Cell-free synthetic biotechnology—multienzyme catalysis and biosynthesis. Chinese Science: Chemistry, 2015). For industrial production, directly adding a large amount of ATP will greatly increase the cost, and the generated ADP and AMP will bring difficulties to subsequent separation and purification. To solve this problem, ATP regeneration systems are usually used in actual production, among which the energy regeneration system based on polyphosphate kinase-inorganic polyphosphate is the most popular. Inorganic polyphosphate is a very cheap phosphate donor compound that can be prepared by polymerization of phosphoric acid at high temperature. It has the advantages of low price and good stability. Motomura et al. divided polyphosphate kinase into two categories based on phylogenetic analysis and catalytic properties: the first type of polyphosphate kinase is more inclined to consume ATP to prolong the polymerization degree of inorganic polyphosphate rather than regenerate ATP. The second class of polyphosphate kinases includes three subclasses: type 2-I catalyzes ADP and inorganic polyphosphate to produce ATP; type 2-II catalyzes AMP and inorganic polyphosphate to produce ADP; polyphosphate kinases that have both functions are called type 2-III (Motomura et al. A new subfamily of polyphosphate kinase 2 (class IIIPPK2) catalysts both nucleoside monophosphate phosphorylation and nucleoside diphosphate phosphorylation. 2014).

[0004] So far, the research and application of different polyphosphate kinases have achieved certain success, but they still face some common problems, the most prominent of which is that most polyphosphate kinases cannot tolerate high temperatures. Most of the second-class polyphosphate kinases used for ATP regeneration are difficult to remain stable at high temperatures above 50°C, which leads to very limited applications of this energy regeneration system. Therefore, polyphosphate kinases must be engineered to enhance their tolerance to high temperatures. Summary of the invention

[0005] The first object of the present invention is to provide a polyphosphate kinase mutant with enhanced tolerance to high temperature of 60°C, which can significantly improve the production rate and yield of ATP obtained by catalyzing adenosine at a high temperature of 60°C.

[0006] The second object of the present invention is to provide the use of a thermostable polyphosphate kinase mutant in catalyzing the preparation of ATP.

[0007] In order to achieve the above-mentioned object, the present invention provides a thermostable polyphosphate kinase mutant, wherein the amino acid sequence of the mutant is compared with the wild-type polyphosphate kinase derived from Sulfurovum lithotrophicum (NCBI No.: AKF24980.1), and a single point mutation or a combined mutation is performed at the amino acid at position 32, position 35, position 61, position 66, position 130, position 133, position 144 or position 226.

[0008] As a preferred embodiment, the single point mutation is: cysteine ​​(C) at position 32 mutates to leucine (L), and the mutation is conventionally named C32L; leucine (L) at position 35 mutates to proline (F), and the mutation is conventionally named L35F; threonine (T) at position 61 mutates to isoleucine (I), and the mutation is conventionally named T61I; valine (V) at position 66 mutates to lysine (K), and the mutation is conventionally named V66K; threonine (T) at position 130 mutates to isoleucine (I), and the mutation is conventionally named T61I; valine (V) at position 66 mutates to lysine (K), and the mutation is conventionally named V66K; (T) mutates to valine (V), and the mutation is conventionally named T130V; the 133rd asparagine (N) mutates to alanine (A), and the mutation is conventionally named N133A; the 144th threonine (T) mutates to serine (S), and the mutation is conventionally named T144S; the 226th aspartic acid (D) mutates to valine (V), and the mutation is conventionally named D226V; the combined mutation is two or more mutations in the single point mutation.

[0009] As a further preferred embodiment, the combined mutation is: a multi-point combined mutation at position 66 and position 226, V66K and D226V, and the mutant is named M1;

[0010] The multiple combined mutations at positions 32, 35, and 144, C32L, L35F, and T144S, were named M2;

[0011] Multiple combined mutations at positions 61, 130, 133, and 144, T61I, T130V, N133A, and T144S, were named M3;

[0012] The mutant contains multiple combined mutations at positions 32, 35, 66, 130, 144 and 226, namely C32L, L35F, V66K, T130V, T144S and D226V, and is named M4.

[0013] In order to achieve the second purpose of the present invention, the present invention discloses the use of the thermostable polyphosphate kinase mutant in catalytic preparation of ATP, using the thermostable polyphosphate kinase mutant as a catalyst and adenosine as a substrate to prepare ATP.

[0014] As a preferred solution, ADP, MgCl 2 , sodium hexametaphosphate and adenosine are used as substrates, adenosine kinase and AMP phosphotransferase are used as biocatalysts, and the above-mentioned high-temperature-resistant polyphosphate kinase mutant is added to catalyze the preparation of ATP.

[0015] As a further preferred embodiment, the reaction system has a pH of 6 to 8, a temperature of 50 to 60°C, an ADP dosage of 1 to 5 mM, and MgCl 2 The added dosage is 40-60mM, the added dosage of sodium hexametaphosphate is 10-40mM, and the added dosage of adenosine is 10-50mM.

[0016] The present invention also provides a coding gene for a thermostable polyphosphate kinase mutant and a recombinant genetic engineering bacterium containing the coding gene.

[0017] The advantage of the present invention is that the thermostable polyphosphate kinase mutant provided by the present invention has significantly improved resistance to 60°C high temperature compared with the wild-type enzyme. The thermostable polyphosphate kinase mutant constructed by the present invention is used to prepare ATP, which significantly improves the sustainability of ATP synthesis. The mutant obtained by the present invention has an ATP yield of 3.5-16.1mM after 5 hours of reaction, and the highest conversion rate exceeds 80%. The production rate and yield of ATP obtained by catalyzing adenosine at a high temperature of 60°C are greatly improved, and it has good industrial application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 .Expression of polyphosphate kinase, 1: M1 mutant whole cell lysis fluid; 2: M2 mutant whole cell lysis fluid; 3: M3 mutant whole cell lysis fluid; 4: M4 mutant whole cell lysis fluid.

[0019] Figure 2 .Schematic diagram of the preparation of ATP.

[0020] Figure 3 .Diagram of ATP production process monitored by HPLC. DETAILED DESCRIPTION

[0021] The technical scheme in the embodiments of the present invention is clearly and completely described below in conjunction with specific embodiments. The test methods used in the following embodiments are conventional methods unless otherwise specified, and the materials, reagents, etc. used are all commercially available unless otherwise specified. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0022] Example 1. Screening of mutants

[0023] Through ancestral sequence reconstruction, structural analysis and energy calculation, single point mutations that may improve stability are screened. The main judgment criteria are:

[0024] (1) The mutation should eliminate the original force forms that are not conducive to thermal stability, such as electrostatic repulsion, charge accumulation, etc.

[0025] (2) The mutation should not destroy the existing thermally stable force pattern and stable protein structure;

[0026] (3) Mutations should introduce new forms of forces that are beneficial to thermal stability, such as hydrogen bonds, salt bridges, and hydrophobic interactions.

[0027] After the above analysis, the mutants were selected in which cysteine ​​(C) at position 32 was replaced by leucine (L); leucine (L) at position 35 was replaced by proline (F); glutamic acid (E) at position 58 was replaced by serine (S); threonine (T) at position 61 was replaced by isoleucine (I); valine (V) at position 66 was replaced by lysine (K); phenylalanine (F) at position 77 was replaced by isoleucine (I); threonine (T) at position 130 was replaced by valine (V); asparagine (N) at position 133 was replaced by alanine (A); threonine (T) at position 144 was replaced by serine (S); glutamine (Q) at position 173 was replaced by proline (L); and aspartic acid (D) at position 226 was replaced by valine (V).

[0028] Example 2. Construction of mutants

[0029] The pET28a recombinant plasmid containing the wild-type polyphosphate kinase gene (derived from Sulfurovum lithotrophicum, NCBI number: AKF24980.1) was used as a template to perform site-directed mutagenesis on the above sites. The primers were designed using oligo7 software and then PCR site-directed mutagenesis was performed. The primers are shown in Table 1.

[0030] Table 1. Primer sequences (SEQ ID NO.1 to SEQ ID NO.22)

[0031]

[0032]

[0033] Table 2. Reaction system:

[0034] template 5μL DNA polymerase 25μL F Primer 3μL R Primer 3μL <![CDATA[ddH 2 The]]> 14μL Total volume 50μL

[0035] Table 3. PCR cycle process:

[0036] Preheat 98℃5min transsexual 98℃10s annealing 55℃30s extend 72℃1min Final extension 72℃10min

[0037] The denaturation-annealing-extension cycle was repeated 30 times. DNA polymerase was purchased from Nanjing Novozyme Biotechnology Co., Ltd. The PCR product was recovered and detected by agarose (1%) gel electrophoresis, then mixed evenly with the vector, and the two PCR products were connected at 37°C for 15 minutes using homologous recombinase (Shanghai Tolo Biotechnology Co., Ltd.) to obtain a circularized mutant plasmid. The ligation product was transformed into Escherichia coli, and after culture on kanamycin-resistant plates, a single clone was selected for sequencing to verify that the sequence was correct, and a mutant plasmid was obtained. The construction of the combined mutation is based on the pET28a recombinant plasmid containing a single mutation or multiple mutation polyphosphate kinase gene as a template, and is obtained through the same operation as above.

[0038] Example 3. Preparation of pure enzyme

[0039] The plasmid carrying the polyphosphate kinase mutant was transformed into BL21 (DE3) to obtain recombinant bacteria.

[0040] The recombinant bacteria were inoculated into 50 mL of LB containing 50 mg / L kanamycin and cultured at 37°C, 220 rpm for 6-8 h. Subsequently, 2% inoculum was inoculated into 100 mL of LB and cultured at 37°C. 600 When the pH value reaches 0.6-0.8, add IPTG with a final concentration of 0.2 mM, and induce protein expression at 18°C ​​and 220 rpm for 16-18 h. Collect the cells by centrifugation, discard the supernatant, and add an appropriate amount of 50 mM NaH containing 10 mM imidazole and 500 mM NaCl. 2 PO 4The resuspended cells were added to a buffer (pH 8.0). The resuspended cells were added to a high-pressure homogenizer for crushing at a pressure of 700 bar until the bacterial solution became clear. The crushed cells were collected and centrifuged to obtain the supernatant, which was poured into a nickel column and homogenized with NaH containing different concentrations of imidazole. 2 PO 4 Buffer was used for elution, and the eluate at 200 mM imidazole concentration was collected. The eluate was concentrated by ultrafiltration until the residual volume reached about 1 mL, and 50% molecular grade glycerol was added at a volume ratio of 1:1, mixed, and then divided and stored at -80°C for future use. Figure 1 . is the expression of polyphosphate kinase.

[0041] Example 4: Polyphosphate kinase activity and high temperature tolerance test

[0042] The high temperature tolerance was calculated by incubating the enzyme at 60° C. for different time intervals in 100 mM Tris-HCl buffer, pH 7.0, and measuring the residual activity of the enzyme at 60° C. The inactivation rate constant of the enzyme was estimated using a first-order kinetic reaction model.

[0043] lnA=lnA 0 -kt

[0044] A is the relative enzyme activity after heat treatment for t hours; A 0 is the initial relative enzyme activity; k is the inactivation rate constant. The time when the enzyme activity is reduced to half of the initial enzyme activity is recorded as t 1 / 2 .

[0045] The reaction system contained 100 mM Tris-HCl buffer at pH 7.0, 10 mM ADP, 20 mM MgCl 2 , 5mM sodium hexametaphosphate, pure enzyme 0.002g / L, water was used to make up the system to 1mL. The reaction solution was reacted at 60℃ for 2min, sampled, and 1M hydrochloric acid was added at a volume ratio of 1:1 to terminate the reaction. The sample was placed on ice for 10min, then centrifuged at 12000 rpm for 10 minutes, the supernatant was collected, and the ATP content was detected by high performance liquid chromatography. The enzyme activity was calculated based on the ATP concentration obtained by the measurement, and the enzyme activity was defined as the amount of enzyme (g) that produces 1μmol of product per minute.

[0046] Table 4. Enzyme activity results

[0047] Enzymes Mutation site <![CDATA[t 1 / 2 (h)]]> Enzyme activity (U / mg) WT none 2.2 180.5 C32L 32 3.0 166.1 L35F 35 12.5 153.5 E58S 58 1.2 55 T61I 61 7.8 125.2 V66K 66 6.2 123.5 F77I 77 0.6 58 T130V 130 7.9 146.0 N133A 133 4.2 160.3 T144S 144 9.1 110.9 Q173L 173 0.8 156.4 D226V 226 9.8 144.6 M1 V66K / D226V 14.6 100.6 M2 C32L / L35F / T144S 15.5 123.6 M3 T61I / T130V / N133A / T144S 23.5 105.6 M4 C32L / L35F / V66K / T130V / T144S / D226V 28.6 96.7

[0048] Conclusion: The enzyme activities and half-lives of wild-type and mutants were measured and it was found that the thermal stability of C32L, L35F, T61I, V66K, F77I, T130V, N133A, T144S, D226V, M1, M2, M3 and M4 mutants was improved compared with the wild-type. However, the stability of E58S, F77I and Q173L mutants was not improved compared with the wild-type, which ruled out these three single-point mutations.

[0049] Example 5. Preparation of ATP

[0050] The recombinant bacteria expressing adenosine kinase (NCBI No. NP_195950.1) and AMP phosphotransferase (NCBI No. SHF67157.1) preserved in our laboratory were used to prepare pure enzymes according to the method described in Example 3. The reaction diagram is shown in Figure 2 .

[0051] The reaction system contained 100 mM Tris-HCl buffer at pH 7.0, 2 mM ADP, 40 mM MgCl 2 , 20mM sodium hexametaphosphate, 20mM adenosine. Then add 0.1g / L adenosine kinase, 0.1g / L AMP phosphotransferase, and 0.1g / L polyphosphate kinase, with a total volume of 1mL. After reacting at 60℃ for 5 hours, the ATP content was detected by high performance liquid chromatography ( Figure 3 ).

[0052] Table 5. ATP production levels of different polyphosphate kinase mutants

[0053] Enzymes Production rate (mM / h) Yield (mM) Increase the ratio WT 0.70 3.5 100％ C32L 1.52 7.6 217％ L35F 2.48 12.4 354％ E58S 0.8 1.6 45.7％ T61I 2.38 11.9 340％ V66K 2.20 11.0 314％ F77I 0.5 0.9 25.7％ T130V 2.18 10.9 311％ N133A 1.76 8.8 251％ T144S 2.52 12.6 360％ Q173L 0.9 1.7 48.6％ D226V 2.46 12.3 351％ M1 2.82 14.1 403％ M2 3.16 15.8 451％ M3 3.04 15.2 434％ M4 3.22 16.1 460％

[0054] Conclusion: The polyphosphate kinase mutants provided by the present invention include single point mutants and combined mutants. Compared with the wild-type polyphosphate kinase, the three mutants E58S, F77I and Q173L have no improvement in half-life at 60°C compared with the wild-type, so their ATP production level is not improved compared with the wild-type, so they are excluded. The single point mutants and combined mutants of C32L, L35F, T61I, V66K, F77I, T130V, N133A, T144S, and D226V have longer half-lives at 60°C than the wild-type, thereby improving the ATP production level; especially the combined mutants, which show the superposition effect of the thermal stability of the single point mutants. Based on this, the polyphosphate kinase mutants provided by the present invention have better thermal stability and are suitable for catalyzing the production of ATP at higher temperatures.

[0055] The above is only a preferred embodiment of the present invention. It should be pointed out that ordinary technicians in this technical field can make several improvements and modifications without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A thermostable polyphosphate kinase mutant, It is characterized in that Compared with the wild-type polyphosphate kinase from Sulfurovum lithotrophicum, the amino acid sequence of the mutant undergoes single point mutation or combined mutation at the amino acid at position 32, 35, 61, 66, 130, 133, 144 or 226.

2. A thermostable polyphosphate kinase mutant according to claim 1, It is characterized in that The single point mutations are: cysteine ​​at position 32 mutates to leucine; leucine at position 35 mutates to proline; threonine at position 61 mutates to isoleucine; valine at position 66 mutates to lysine; threonine at position 130 mutates to valine; asparagine at position 133 mutates to alanine; threonine at position 144 mutates to serine; aspartic acid at position 226 mutates to valine; the combined mutations are two or more mutations in the single point mutations.

3. A thermostable polyphosphate kinase mutant according to claim 2, It is characterized in that The combined mutations are: a combined mutation at position 66 and position 226; a combined mutation at position 32, position 35 and position 144; a combined mutation at position 61, position 130, position 133 and position 144; a combined mutation at position 32, position 35, position 66, position 130, position 144 and position 226.

4. A gene encoding the thermostable polyphosphate kinase mutant according to any one of claims 1 to 3.

5. A recombinant genetically engineered bacterium comprising the coding gene according to claim 4.

6. Use of the thermostable polyphosphate kinase mutant according to any one of claims 1 to 3 in catalyzing the preparation of ATP.

7. The use according to claim 6, It is characterized in that ADP, MgCl 2 , sodium hexametaphosphate, and adenosine are used as substrates, adenosine kinase and AMP phosphotransferase are used as biocatalysts, and the thermostable polyphosphate kinase mutant described in any one of claims 1 to 3 is added to catalyze the preparation of ATP.

8. The use according to claim 7, It is characterized in that The reaction system is pH 6-8, temperature 50-60°C, ADP addition 1-5 mM, MgCl 2 The added dosage is 40-60mM, the added dosage of sodium hexametaphosphate is 10-40mM, and the added dosage of adenosine is 10-50mM.

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