A kind of Bacillus subtilis polyphosphatase Bs-PPK and its application

Abstract

The present invention discloses a Bacillus subtilis polyphosphatase Bs-PPK and its application. The polyphosphatase derived from Bacillus subtilis in the present invention Bs The gene sequence of ‑PPK is shown in SEQ ID NO: 1. The amino acid sequence of the polyphosphatase is shown in SEQ ID NO: 2. The polyphosphatase derived from Bacillus subtilis in the present invention Bs ‑PPK has the ability to efficiently synthesize ATP: ADP 6 mM, phosphate donor sodium hexaphosphate 9 mM, Mg 2+ The substance was 5 mM and 92.34% ATP was generated after 10 min of reaction at pH 7.0 and 30°C. The enzyme could be reused and still retained 50% enzyme activity after 5 times of reuse.

Description

Technical Field

[0001] The invention relates to the field of polyphosphatase, and in particular to a Bacillus subtilis polyphosphatase Bs-PPK and an application thereof. Background Art

[0002] Adenosine triphosphate (ATP) is a nucleotide composed of three main components: adenine, D-ribose, and a chain of three phosphate groups bound to D-ribose. The chemical energy of ATP is stored in the bonds between the phosphate groups. In most cases, ATP is converted to adenosine diphosphate (ADP), releasing energy and an inorganic phosphate ion. ADP can be further converted to AMP, releasing energy and PRPP. In biocatalysis, ATP is the most commonly used energy donor and is used in a variety of cascade reactions. However, ATP is expensive, and it is not economically feasible to directly add it to catalyze the synthesis of biologically active substances on a large scale. In addition, a large amount of byproduct ADP is produced in the reaction system after ATP conversion and energy supply. The accumulation of ADP will inhibit the key enzymes in the biocatalytic reaction, reduce the conversion efficiency or cause the reaction to proceed in the reverse direction. Therefore, the use of an ATP regeneration system is crucial to reduce the cost of biocatalytic reactions, increase substrate conversion rates, and inhibit reverse reactions.

[0003] Poly phosphate kinase (PPK) can use polyphosphate (Poly P) as a phosphate donor to carry out ATP synthesis and regeneration reactions. Compared with other ATP regeneration systems, such as acetyl kinase-acetyl phosphate (the phosphate donor is acetyl phosphate, which needs to be prepared and is unstable) and pyruvate kinase-phosphoenolpyruvate system (phosphoenolpyruvate is expensive), the required substrate - polyphosphate - is cheap and stable, and is more suitable for industrial scale-up.

[0004] PPK (EC2.7.4.1) is mainly divided into two families, PPK1 and PPK2, which catalyze the reversible transfer of the γ-phosphoryl group of ATP or GTP to Poly P. Compared with PPK1, PPK2 can use Poly P as a donor to achieve the phosphorylation of ADP / GDP to ATP / GTP. At the same time, the rate of PPK2 generating ATP / GTP is 75 times that of its reverse reaction, which is much higher than PPK1. PPK2 uses short-chain Poly P as a substrate and is mainly used for the catabolism of Poly P; when the content of Poly P is sufficient, a polymerization reaction can occur between Poly P to increase the apparent activity of PPK2 in the body by 10 times, so PPK2 kinase can be used to develop a Poly P-dependent ATP regeneration system for nucleotides and is used in various catalytic reactions that require the participation of ATP.

[0005] PPK2 and PPK1 have low sequence similarity and are highly conserved. The PPK2 family can be divided into three subtypes: I, II, and III according to the sequence functional characteristics and developmental analysis of PPK2. PPK2-I type mainly catalyzes the phosphorylation of ADP to ATP, PPK2-II type prefers to catalyze the phosphorylation of AMP to ADP, and PPK2-III type is active on both substrates, ADP and AMP, and can achieve phosphorylation from AMP to ADP and ADP to ATP at the same time, making it a bifunctional biocatalyst. Summary of the invention

[0006] In order to solve the problems of unstable production enzyme properties and high synthesis cost in the existing ATP synthesis process, the main purpose of the present invention is to provide a Bacillus subtilis polyphosphatase Bs -PPK.

[0007] Another object of the present invention is to provide the use of the above-mentioned polyphosphatase in ATP regeneration.

[0008] To achieve the above objectives, the present invention is implemented through the following technical solutions.

[0009] A Bacillus subtilis polyphosphatase Bs-PPK, which is a protein of (a) or (b):

[0010] (a) a gene fragment consisting of the base sequence shown in SEQ ID NO: 1;

[0011] (b) The base sequence in (a) is codon optimized and adapted to obtain a gene fragment derived from (a) suitable for a Saccharomyces cerevisiae vector.

[0012] Furthermore, the amino acid sequence of the gene fragment in (b) is shown in SEQ ID NO: 2.

[0013] Furthermore, the polyphosphatase Bs - Application of PPK in ATP regeneration, the Bacillus subtilis polyphosphatase Bs -PPK catalyzes ADP and sodium hexaphosphate to produce ATP within 10 min.

[0014] Furthermore, polyphosphatase Bs -PPK is prepared by using Bacillus subtilis with a deposit number of GDMCC No: 1.3763, which was deposited in Guangdong Provincial Microbiological Culture Collection Center on December 7, 2022; the taxonomic name of the Bacillus subtilis is: Bacillus subtilis, The deposit address is Building 59, No. 100, Xianlie Middle Road, Guangzhou; after culturing in Sc liquid medium containing glucose, it is transferred to Sc liquid medium containing galactose for induction culture, and the bacterial cells collected by centrifugation are polyphosphatase Bs -PPK whole cell enzyme preparation.

[0015] Furthermore, the culture medium is a Sc liquid culture medium; the glucose-containing Sc liquid culture medium comprises 1.5 wt %-2.5 wt % glucose, 0.67 wt %-1.34 wt % yeast-amino-free nitrogen source and 2 wt %-2.5 wt % acid-hydrolyzed casein; the galactose-containing Sc liquid culture medium comprises 2 wt %-2.5 wt % galactose, 0.67 wt %-1.34 wt % yeast-amino-free nitrogen source and 2 wt %-2.5 wt % acid-hydrolyzed casein.

[0016] Furthermore, the Bacillus subtilis is cultured in the Sc liquid medium containing glucose by: culturing in the Sc liquid medium containing glucose at 28-30° C. and 180-200 rpm for 18-24 h; and the induction culture is specifically: induction culture at 20-25° C. for 60-72 h.

[0017] Furthermore, the centrifugation conditions are 5000-90000 rpm, preferably 9000 rpm, at 0-8°C for 8-10 min, preferably at 4°C for 10 min.

[0018] Polyphosphatase Bs -PPK expression method, comprising the following steps:

[0019] Based on the conservation of the PPK enzyme sequence of Bacillus, primers were designed and cloned from Bacillus subtilis GDMCC1.3763. Bs-PPK enzyme gene sequence.

[0020] Construction of recombinant plasmid pYD1- Bs -PPK, recombinant polyphosphatase via the surface display system of Saccharomyces cerevisiae EBY100 Bs -PPK was displayed on the cell surface of Saccharomyces cerevisiae to obtain a whole-cell enzyme catalyst.

[0021] The present invention obtains a new polyphosphatase from the deposited strain Bacillus subtilis GDMCC1.3763 Bs -PPK can convert 6 mM ADP into 5.54 mM ATP within 10 min, with an ATP molar yield of 92.34%. It can efficiently catalyze ADP and sodium hexaphosphate to generate ATP, building an efficient and stable ATP regeneration system.

[0022] The present invention designs primers based on the conservative sequence of the Bacillus PPK enzyme, uses the Bacillus subtilis genome as a template, and clones a polyphosphatase gene Bs- PPK (SEQ ID NO: 1), and translated according to the codon table of Bacillus subtilis to obtain the amino acid sequence (SEQ ID NO: 2), and the gene was optimized for codon preference to obtain the sequence SEQ ID NO: 3.

[0023] right Bs- The gene sequence SEQ ID NO: 1 and the amino acid sequence SEQ ID NO: 2 of PPK were analyzed and compared, and the results showed that: Bs -PPK is a completely new enzyme.

[0024] Bacillus subtilis polyphosphatase of the present invention Bs -PPK amino acid sequence SEQ ID NO: 2 and Bacillus Bacillus sp . The phosphate-nucleotide phosphotransferase of SJS has 97.55% similarity; Metabacillus sp . FJAT-52054 has 95.10% similarity to the PPK2 family polyphosphate kinase; Metabacillus flavus The PPK2 family polyphosphate kinase has 94.76% similarity with Bacillus Bacillus sp .FJAT-42376 has 93.71% similarity to the PPK2 family polyphosphate kinase.

[0025] Among them, the one with the highest similarity Bacillus spCompared with the phosphate-nucleotide phosphotransferase of SJS, the differences are mainly reflected in: Q5K, K11N, D18N, A154K, L167M, K252E and N277D. These amino acid differences are distributed on both sides of the sequence.

[0026] It can be seen from this that the Bacillus subtilis polyphosphatase disclosed in the present invention Bs -PPK is a novel polyphosphatase that is highly conserved in Bacillus.

[0027] Compared with the prior art, the present invention has the following advantages and technical effects:

[0028] 1. The present invention determines that Bacillus subtilis polyphosphatase Bs -PPK gene sequence and amino acid sequence, through sequence comparison and analysis, it can be seen that the Bacillus subtilis disclosed in the present invention Bs- PPK enzyme is a new type of polyphosphatase.

[0029] 2. Bacillus subtilis polyphosphatase disclosed in the present invention Bs -PPK can be effectively displayed on the surface of Saccharomyces cerevisiae cells, effectively improving the application and recovery efficiency of the enzyme.

[0030] 3. Bacillus subtilis polyphosphatase Bs -PPK has the ability to efficiently catalyze the conversion of ADP into ATP. 3 mg / mL whole-cell enzyme preparation can convert 6 mM ADP into 5.54 mM ATP within 10 min, and the ATP molar generation rate is 92.34%.

[0031] 4. Bacillus subtilis polyphosphatase Bs -PPK has a molar ATP production rate of more than 90% at 30-45°C and pH 5.5-8.5, and 5 mM Mg is added 2+ After five times of repeated recycling, the whole cell enzyme preparation still has 50% enzyme activity, which provides a basis for the subsequent industrial and efficient production of ATP regeneration system.

[0032] In summary, the present invention discloses Bs -PPK enzyme can efficiently and stably convert ADP to ATP and has important application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Example 2 Bacillus subtilis polyphosphatase Bs -Conservation analysis of the amino acid sequences of PPK and PPK enzymes from other Bacillus species.

[0034] Figure 2is the Bacillus subtilis polyphosphatase in Example 2 Bs -PPK and Bacillus sp . Comparison analysis of the secondary structures of PPK enzymes from SJS.

[0035] Figure 3 is the Bacillus subtilis polyphosphatase in Example 2 Bs -PPK and Bacillus sp . Comparison analysis of the tertiary structure of PPK enzymes from SJS.

[0036] Figure 4 It is a schematic diagram of the surface display and catalytic reaction of Bacillus subtilis polyphosphatase Bs-PPK in Example 3.

[0037] Figure 5 is the Bacillus subtilis polyphosphatase in Example 3 Bs - Laser confocal microscopy results showing the successful display of PPK on the cell surface of Saccharomyces cerevisiae EBY100.

[0038] Figure 6 is the Bacillus subtilis polyphosphatase in Example 3 Bs -Flow cytometric analysis results showing the successful display of PPK on the cell surface of Saccharomyces cerevisiae EBY100 cells.

[0039] Figure 7 The standard curves of ATP and ADP measured at a wavelength of 260 nm in Example 4 are shown in Figure 4, wherein a) is the ATP standard curve and b) is the ADP standard curve.

[0040] Figure 8 In Example 4 Bs -Results of liquid phase detection of PPK whole-cell enzyme catalytic activity.

[0041] Figure 9a~Figure 9c In Example 4, respectively Bs -Comparison of the effects of temperature, pH, and metal ions on enzyme activity in the PPK whole-cell enzymatic property assay.

[0042] Fig.10 In Example 5 Bs -Verification of the reproducible recovery times of PPK whole cell enzyme preparation. DETAILED DESCRIPTION

[0043] The present invention is further described in detail below in conjunction with embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0044] If no specific experimental conditions are specified in the following embodiments, conventional experimental conditions or experimental conditions recommended by the reagent company are generally followed. The materials and reagents used, unless otherwise specified, are all reagents and materials obtained from commercial sources.

[0045] Example 1 Amplification of polyphosphatase gene in Bacillus subtilis GDMCC1.3763

[0046] The following steps are involved:

[0047] 1. Cultivation: Cultivate Bacillus subtilis in 20 mL LB liquid medium at 37°C and 180 rpm until the mid-logarithmic growth period. Bacillus subtilis The GDMCC1.376 suspension was used as the seed solution and inoculated into 100 mL LB liquid culture medium at a ratio of 5%. After culturing at 37°C and 180 rpm for 16 h, the culture solution was centrifuged at 8000 rpm and 4°C for 10 min. The bacterial cells were washed twice with pH 7.0 buffer and resuspended with an appropriate amount of sterile water to obtain a bacterial solution.

[0048] LB medium (g / L): yeast extract 5, tryptone 10, NaCl 10, pH 7.0.

[0049] 2. The bacterial solution obtained in step 1 was used to extract total DNA using the Ezup column-based bacterial genomic DNA extraction kit (Sangon Biotechnology, Shanghai).

[0050] 3. The sequences of polyphosphate kinase (PPK2) of Bacillus were collected in the NCBI database (https: / / www.ncbi.nlm.nih.gov). After conservative sequence analysis, the sequences of polyphosphate kinase (PPK2) of Bacillus were analyzed. Bacillus sp. HMSC76G11(Accession Number:OHR67094)and Bacillus subtilis The primers were designed using the PPK2 gene sequence of JRS10 (Accession Number: CUB59476) as a template, as shown in the following table. The total DNA of Bacillus subtilis GDMCC1.376 was used as a template, and PCR amplification was performed using primers 1-4 (Table 1) in sequence. The PCR amplification program was as follows: PCR program: 95 ℃ pre-denaturation for 1 min → [95 ℃ denaturation for 30 s → 50 ℃ annealing for 30 s → 72 ℃ extension for 1 min] for 35 cycles → 72 ℃ extension for 10 min. The amplified sequences were spliced, and the flanking sequences at both ends of the above target nucleic acid sequence were amplified using the High-efficiency Tail-PCR technology. The obtained flanking sequences were sequenced and spliced ​​to obtain the nucleotide sequence of the enzyme (SEQ ID NO: 1). According to the Bacillus subtilis B. subtilis subsp. The 168 codon table was translated to give the amino acid sequence (SEQ ID NO: 2).

[0051] Table 1 For cloning Bs -Primer sequences for the PPK gene

[0052]

[0053] SEQ ID NO: 1 is:

[0054] ATGAACGTGAACAAATATAAAATTGAAGGAAACAAAGAAATTAAACTGAAAAACTATCCGACATCAGAAGACCACGGATTTTCAGAAGAAGAACTGCTGGACCACCACATTCCGGAATCAATTTCAAAACTGAAAGACCTGCACNNNAAACTGCACGCGGACGAAAAAAAAGGAATTGTGGTGGTGCTGCAAGCGATGGACGCGGCGGGAAAAGACGAAGCGATTTCATATATTTTTTCAAACCTGAACGCGCAAGGACTGAAAACAACATCATTTCAAAAACCGTCAGACACAGAAAGAAACCACGACTATCTGNNNAGAATTAGAGAAGGACTGCCGGAAAGAGGACAAGTGGGAATTCTGAACAGATCACACTATGAAGACGTGATTGCGCCGAGAGTGCACGACCTGCTGGAAGAAGGAACAATTCCGGACGACAAAGACGAACACGAAGTGNNNAAAATGAGATATAGACAAATTAAAGACTTTGAAAGATATATGGTGGAAAACGGATTTGAAGTGGTGAAATTTTTTTTTAACATGTCAAAAGAAGAACAAAAAAACAGACTGCTGGAAAGAATGAAAAACCCGAAAAAAAACNNNGAATTTTCATTTTCAGACGTGGAAGAAAGAGAACACNNNGACTCATATCAACACATTTTTGAAGACATGCTGAACAACACATCAACAGAATATGCGCCGNNNCACATTCTGCCGGCGGACGACGAANNNTATTCAAGATATATTATTACAGAAACAATGATTGAATGTCTGGAAAAAATTGACCCGCAATTTCCGGAAATTTCAGAAGAAAACAGAAAAAAACTGGACGAATATATTGAAAAACTGGAAAAAGAA。

[0055] Example 2 Bacillus subtilis polyphosphatase Bs- PPK amino acid sequence comparison and analysis

[0056] Bacillus subtilis polyphosphatase Bs-The PPK amino acid sequence SEQ ID NO: 2 was compared by BLAST sequence comparison. Bs- PPK and Bacillus Bacillus sp .SJS has 97.55% similarity to the phosphate-nucleotide phosphotransferase of Parabacillus sp. Metabacillus sp . FJAT-52054 has 95.10% similarity to the PPK2 family polyphosphate kinase; Metabacillus flavus The PPK2 family polyphosphate kinase has 94.76% similarity with Bacillus Bacillus sp . FJAT-42376 has 93.71% similarity to the PPK2 family polyphosphate kinase.

[0057] DNAman software was used to identify polyphosphatase from Bacillus subtilis Bs -PPK amino acid sequence was subjected to sequence conservation analysis, and the results were as follows Figure 1 As shown, Figure 1 Analysis software DNAman, sequence source: Bacillus sp .HMSC76G11 (accession number: OHR67094), Bacillus sp . SJS (accession number: WP_035407176), Bacillus sp . FJAT-42376 (accession number: WP_123913790), Bacillus sp .FJAT-27264 (accession number: WP_066367508.1). Figure 1 It can be seen that polyphosphatases from different sources have a high degree of sequence conservation, and the black shaded parts in the figure are all highly conserved sequence fragments.

[0058] in, Bs- Compared with the phosphate-nucleotide phosphotransferase of Bacillus, which has the highest similarity, the amino acid differences of PPK are shown in Table 2:

[0059] Table 2 Bs Amino acid differences between -PPK and phosphate-nucleotide phosphotransferases from Bacillus

[0060]

[0061] Its secondary structure is compared with Figure 2 As shown, the black part is the Ω ring, the red part is the α helix, and the blue part is the β fold. Figure 2 It can be seen that the amino acid differences between the two are mainly concentrated in the random coil, which does not affect the main secondary structure (α helix and β fold). From the 3D structure simulation ( Figure 3 ) The results show that the spatial structures of the two are highly similar, which indicates that Bs -PPK also has PPK2 enzyme activity. Figure 3 Middle, left Bacillus sp . PPK enzyme from SJS, on the right Bs -PPK.

[0062] It can be seen from this that the Bacillus subtilis polyphosphate kinase disclosed in the present invention Bs- The PPK enzyme is highly conserved among Bacillus species and is a completely new PPK2 enzyme.

[0063] SEQ ID NO: 2 is:

[0064] MNVNKYKIEGNKEIKLKNYPTSEDHGFSEEELLDHHIPESISKLKDLHWK

[0065] LHADEKKGIVVVLQAMDAAGKDEAISYIFSNLNAQGLKTTSFQKPSDTERNHDYLWRIREGLPERGQVGILNRSHYEDVIAPRVHDLLEEGTIPDDKDEHEVWKMRYRQIKDFERYMV ENGFEVVKFFFNMSKEEQKNRLLERMKNPKKNWEFSFSDVEEREHWDSYQHIFEDMLNNTSTEYAPWHILPADDEWYSRYIITETMIECLEKIDPQFPEISEENRKKLDEYIEKLEKE.

[0066] Example 3 Bacillus subtilis polyphosphatase Bs- Construction of PPK surface carrier on yeast

[0067] Saccharomyces cerevisiae Saccharomyces cerevisiae EBY100 and its supporting surface display system as Bs- The expression vector of PPK belongs to the α-lectin system, which is composed of the core subunit agglutinin α (Agα) 1 and the binding subunit Agα2. The C-terminus of the core subunit Agα1 is covalently bound to the cell wall glucan, while the binding subunit Agα2 is connected to the core subunit Agα1 through a disulfide bond. Bs PPK enzyme was fused to Agα2 at its N-terminus to achieve surface display in yeast cells.

[0068] like Figure 4The polyphosphatase gene Bs-PPK was cloned from Bacillus subtilis GDMCC1.3763 ​​and transferred into plasmid pYD1 to construct surface display recombinant plasmid pYD1-Bs-PPK. Among them, PGAL1 is a galactose promoter, AGA2 is a lectin α binding subunit, one end of which is connected to the core subunit AGA1 in the EBY100 gene of Saccharomyces cerevisiae by a disulfide bond, and the other end is connected to the displayed target protein, Bs-PPK gene is Bacillus subtilis polyphosphatase gene, TRP1 is a tryptophan screening tag used to screen successfully transformed transformants, AmpR is an ampicillin resistance gene, indicating that the transformant has ampicillin resistance, and ori indicates the replication origin on the plasmid. The Bs-PPK enzyme displayed on the surface of the Saccharomyces cerevisiae cell wall has the ability to synthesize ATP and catalyzes the substrate ADP and sodium hexaphosphate poly6 to synthesize ATP.

[0069] In order to improve the expression level of the target protein in EBY100, based on the codon preference of Saccharomyces cerevisiae, the amino acid sequence was not changed. Bs- The PPK gene was codon optimized. The optimization was mainly focused on the 5' region to improve the translation initiation efficiency; the high GC content sequence was optimized; the repetitive sequence region was optimized; and the specified restriction endonuclease sites were excluded. Finally, the optimized Bs -PPK gene sequence (SED ID NO.3).

[0070] After optimization Bs -PPK gene with EcoR I and Xol Ⅰ restriction site, a Flag tag was added to the 5' end, and inserted into the plasmid vector pYD1. The surface display vector pYD1- Bs -PPK schematic diagram Figure 4 shown.

[0071] The recombinant plasmid was transferred into Escherichia coli DH5α by electroporation and amplified, and the recombinant plasmid was extracted by alkaline lysis and verified by double enzyme digestion. The competent cells of Saccharomyces cerevisiae EBY100 were prepared by LiAc method and transferred into the constructed recombinant plasmid pYD1- Bs -PPK, after transformation, samples were spread on MDL screening plates and cultured inverted at 30°C for 2-4 days until single colonies appeared.

[0072] Positive transformants on the MDL plate were picked up, cultured in Sc liquid medium (containing 0.67wt% YNB, 2wt% glucose, 2wt% casamino acids) at 30°C, 180rpm for 24h, collected by centrifugation, resuspended in Sc liquid medium (containing 0.67wt% YNB, 2wt% galactose, 2wt% casamino acids), induced at 20°C, 180rpm for 60-72h, collected by centrifugation for further identification and screening.

[0073] The EBY- Bs -PPK yeast was collected by centrifugation at 8000 r / min for 5 min, washed three times with PBS buffer, resuspended in 1 mg / mL BSA solution, blocked overnight, and stored at 4°C for later use; Bs -PPK bacterial solution 2OD 600 , centrifuged at 4°C and 8000 r / min for 5 min, and then washed once with PBS; resuspended the cells in 1 mL of PBS containing 1 mg / mL BSA, added primary antibody (mouse anti-Flag antibody) according to the dilution ratio, incubated at 37°C for 2 h, and mixed once every 30 min; centrifuged and washed the cells once with PBS, resuspended the cells in PBS containing 1 mg / mL BSA, added fluorescent secondary antibody (rabbit anti-mouse antibody conjugated to fluorescein isothiocyanate (FITC)) according to the dilution ratio, incubated at 37°C for 1 h in the dark, and mixed once in the middle; washed the cells three times with 1 mL PBS (without BSA), and resuspended the cells with 1 mL PBS; took the same amount of empty bacterial cells EBY100-pYD1 as a control, and performed immunofluorescence staining according to the same steps. After immunofluorescence staining, add 10 µL of the stained bacterial solution on the slide and cover the slide. Excite the fluorescent dye with 495 nm wavelength of excitation light under a fluorescence microscope and observe and take pictures with an oil immersion lens. Use the stained empty bacteria as a negative control. Figure 5 As shown, Figure 5 The fluorescent dye was excited by 495 nm wavelength excitation light under a fluorescence microscope. The positive sample EBY- Bs -PPK showed green fluorescence, while the negative control EBY-pYD1 did not show green fluorescence. Bs -PPK cells showed bright green fluorescence, while the negative control EBY100-pYD1 showed no green fluorescence, indicating that EBY- Bs -PPK expresses the PPK enzyme localized on the surface of Saccharomyces cerevisiae EBY100.

[0074] Take 500 μL of the stained bacterial solution, fully disperse the bacterial cells and use them for flow cytometry analysis. Use the empty bacterial cells EBY100-pYD1 stained under the same conditions as the negative control. The fluorescence intensity of the display strain stained with FITC was measured under the conditions of Amax=488nm; Emax=517nm ( Figure 6 The fluorescence intensity of the display strain stained with FITC was measured under the conditions of Amax = 488nm; Emax = 517nm). According to the flow cytometer test results, the positive sample EBY- Bs -PPK detected that the proportion of cells bound to FITC fluorescent secondary antibody was 88.00%, while the negative control sample EBY100-pYD1 detection result was 0.36%, indicating that the PPK protein was successfully expressed and anchored on the surface of EBY100 yeast.

[0075] SEQ ID NO: 3 is:

[0076] ATGAACGTTAATAAATATAAGATAGAAGGGAATAAGGAAATTAAGTTAAAAAACTACCCAACCTCCGAAGATCATGGCTTTTCTGAAGAGGAATTGTTGGACCATCATATCCCTGAGTCAATTTCAAAATTGAAAGATTTGCATTGGAAACTGCATGCAGATGAGAAAAAAGGGATAGTGGTCGTTCTGCAGGCGATGGACGCCGCCGGTAAGGACGAGGCGATTTCATATATCTTCTCAAACCTAAATGCACAGGGTTTGAAAACTACTAGCTTTCAAAAGCCTTCCGACACTGAAAGAAACCACGATTATTTGTGGCGTATTCGTGAAGGTTTACCTGAACGTGGCCAGGTAGGTATTTTAAATAGATCTCATTATGAAGATGTGATTGCTCCAAGGGTTCATGATTTGCTTGAAGAAGGAACTATTCCCGACGACAAGGACGAACACGAAGTTTGGAAGATGAGATACAGACAGATCAAAGATTTCGAAAGATATATGGTGGAAAACGGATTCGAAGTCGTCAAATTCTTTTTCAATATGAGCAAAGAAGAACAAAAAAACAGGTTGTTGGAAAGAATGAAAAATCCTAAAAAGAATTGGGAATTTAGTTTCTCTGACGTAGAAGAAAGGGAGCACTGGGATTCTTATCAGCATATCTTTGAGGATATGTTGAACAATACCAGCACCGAATATGCCCCATGGCACATATTGCCGGCCGACGACGAATGGTACAGCCGTTATATAATCACTGAGACCATGATCGAATGTCTAGAGAAAATCGATCCTCAATTTCCTGAGATATCTGAGGAAAACAGAAAAAAGTTGGACGAATATATTGAAAAATTGGAGAAAGAG。

[0077] Example 4 Recombinant Bacillus subtilis Polyphosphatase Bs -Determination of the Enzyme Activity and Enzymatic Properties of -PPK

[0078] Collect the induced Saccharomyces cerevisiae EBY100- Bs-PPK bacterial cells were washed twice with saline and the supernatant was discarded. The cells were freeze-dried at -45°C for 48 h to obtain the freeze-dried cells, which were the whole-cell catalysts and stored at 4°C for future use.

[0079] Prepare ATP / ADP standard solutions with concentrations of 0.05, 0.5, 1, 2, 5, and 6 mM, respectively, and detect them by HPLC. Plot the concentration of ATP / ADP as the horizontal axis and the corresponding peak area as the vertical axis to plot the ATP ( Figure 7 a)) / ADP( Figure 7 b) Standard curve.

[0080] Use HPLC to detect ATP and ADP in the system under the following conditions:

[0081] The mobile phase was methanol: water (pH 6.0) at a ratio of 6:94. The chromatographic column was Agilent ZORBAX Eclipse SB-C18 (4.6 mm × 250 mm, 5 µm). The column temperature was 25 °C, the flow rate was 0.8 mL / min, and the concentrations of ATP and ADP were determined by the external standard method at 260 nm using a UV detector.

[0082] 3 mg / mL Bs -PPK enzyme was added to a 5 mL reaction bottle, followed by 6 mM ADP, 9 mM sodium hexaphosphate (phosphate donor), and 5 mM MgCl2. The reaction was carried out at pH 7.0 and 30°C for 10 min. After centrifugation, the supernatant was filtered through a 0.22 μm filter membrane and then HPLC was used to determine whether a product was generated, and the molar production rate of ATP was calculated.

[0083] The molar generation rate is calculated as follows:

[0084] .

[0085] Bs -PPK whole cell enzyme preparation catalytic activity HPLC detection spectrum as shown Figure 8 As shown ( Figure 8 The reaction system is: 3 mg / mL whole cell enzyme preparation, ADP 6 mM, phosphate donor sodium hexaphosphate 9 mM and MgCl2 5 mM at 30℃ for 10 min. a) The figure shows the ATP standard; b) The figure shows the 0h reaction control; c) The figure shows the Bs-PPK reaction result for 10 min). 3 mg / mL of Bs -PPK enzyme converted 6 mM ADP into 5.54 mM ATP within 10 min, with a molar ATP production rate of 92.34%.

[0086] The reaction temperature gradient (20, 30, 35, 37, 40, 45, 60, 80 °C), pH gradient (5.5, 6.5, 7.0, 7.5, 8.0, 8.5, 9.5) and 5 mM metal ion (K + , Ca 2+ Mg 2+ 、Zn 2+ , Fe 3+ , Cu 2+ , Ba 2+ , Mn 2+ ), place the test group under different conditions for reaction, take samples after the reaction to measure the amount of ATP generated, and calculate the molar generation rate. Draw a bar graph of temperature and catalytic activity to analyze the optimal conditions for the enzyme.

[0087] The effect of temperature on Bs -PPK catalytic activity, the results are as follows Figure 9a The enzyme has high catalytic activity in a wide temperature range (30-45°C), and the ATP molar production rate is higher than 90%. The effect of pH on the activity of Bs -PPK catalytic activity, the results are as follows Figure 9b As shown, Bs -PPK has a relatively high catalytic activity in a wide pH range (pH5.5-8.5), and the molar generation rate of ATP is above 90%, which can meet the needs of practical applications; the effect of metal ions on the catalytic activity of enzymes, the results are as follows Fig.9c As shown in Figure 2, none of the above metal ions inhibited the activity of the enzyme. + Mg 2+ , Fe 3+ , Ba 2+ It has a certain promoting effect on catalytic activity, among which Mg 2+ Best results.

[0088] Example 5 Surface display of Saccharomyces cerevisiae Bs - Analysis of the reproducibility of PPK enzymes

[0089] An important function of enzymes is that they can be recycled, which is beneficial for their industrial application. Bs -PPK whole cell catalyst ( Bs -PPK whole cell enzyme preparation) was subjected to multiple cycles of reaction and the molar production rate of ATP was determined by HPLC. The reaction system was as shown in Example 4. After the reaction was completed, the reaction was separated by centrifugation. Bs-PPK whole cell catalyst was washed once with PBS and then put back into a new round of reaction, which was repeated 5 times. After each reaction, the supernatant was filtered through a 0.22 μm filter membrane and the molar production rate of ATP was determined by HPLC. The ATP molar production rate of the initial reaction was converted to 100% for plotting.

[0090] The results are as follows Fig.10 shown. Bs -PPK whole cell catalyst retained 73.24% of the original enzyme activity after recycling once. The rate of catalytic activity reduction slowed down from the 2nd to the 5th time. It still maintained 50% activity when recycled for the 5th time, which has good reusability. For the practical application of ordinary enzyme proteins, it is difficult to retain high enzyme activity after complex recycling procedures, and it is difficult to achieve enzyme recycling. By displaying enzyme proteins on the surface of living cells, it is close to enzyme immobilization to a certain extent. The cell particles of brewer's yeast engineered bacteria are 5-10 μm and can be quickly separated by centrifugation. Therefore, the yeast display whole cell catalyst can be recycled multiple times in a mild reaction environment.

[0091] In summary, the Bacillus subtilis disclosed in the present invention Bs -PPK can stably and efficiently convert ADP to ATP.

[0092] The above experimental results can further illustrate that the present invention can be safely and efficiently used in actual production, thereby effectively reducing the actual cost investment.

[0093] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A Bacillus subtilis polyphosphatase Bs -PPK, characterized in that The protein encoded by the gene fragment described in (a) or (b) below: (a) a gene fragment consisting of the base sequence shown in SEQ ID NO: 1; (b) The base sequence in (a) is codon optimized and adapted to obtain a gene fragment derived from (a) suitable for a Saccharomyces cerevisiae vector.

2. A Bacillus subtilis polyphosphatase as claimed in claim 1 Bs -PPK, characterized in that The amino acid sequence of the protein encoded by the gene fragment in (b) is shown in SEQ ID NO:

2.

3. Application of the Bacillus subtilis polyphosphatase Bs-PPK according to claim 1 or 2 in biosynthesis of ATP, wherein the Bacillus subtilis polyphosphatase Bs -PPK catalyzes the conversion of ADP and sodium hexaphosphate to produce ATP.

4. The use according to claim 3, characterized in that: The preparation method of the Bacillus subtilis polyphosphatase Bs-PPK is as follows: after culturing Bacillus subtilis in a Sc liquid medium containing glucose, transferring to a Sc liquid medium containing galactose for induction culture, and collecting bacterial cells by centrifugation to obtain Bacillus subtilis polyphosphatase Bs-PPK; the Bacillus subtilis is a Bacillus subtilis deposited in the Guangdong Provincial Microbiological Culture Collection Center on December 7, 2022 with a deposit number of GDMCC No: 1.3763; the taxonomic name of the Bacillus subtilis is: Bacillus subtilis.

5. The use according to claim 4, characterized in that: The glucose-containing Sc liquid culture medium comprises 1.5 wt %-2.5 wt % glucose, 0.67 wt %-1.34 wt % yeast-free amino nitrogen source and 2 wt %-2.5 wt % acid-hydrolyzed casein; the galactose-containing Sc liquid culture medium comprises 2 wt %-2.5 wt % galactose, 0.67 wt %-1.34 wt % yeast-free amino nitrogen source and 2 wt %-2.5 wt % acid-hydrolyzed casein.

6. The use according to claim 4, characterized in that: The culturing of Bacillus subtilis in the Sc liquid medium containing glucose is specifically: culturing in the Sc liquid medium containing glucose at 28-30° C. and 180-200 rpm for 18-24 hours; the induction culturing is specifically: induction culturing at 20-25° C. for 60-72 hours.

7. The use according to claim 4, characterized in that: The centrifugation is carried out at 5000-90000 rpm and 0-8° C. for 8-10 min.

8. The use according to claim 4, characterized in that: The centrifugation was performed at 9000 rpm and 4° C. for 10 min.

9. The use according to claim 3, characterized in that: Bacillus subtilis polyphosphatase Bs -PPK catalyzes ADP and sodium hexaphosphate to produce ATP within 10 minutes.

10. The Bacillus subtilis polyphosphatase according to claim 1 Bs -PPK yeast surface vector construction method, characterized in that, The steps include: constructing a recombinant plasmid pYD1- Bs -PPK, recombinant polyphosphatase via the surface display system of Saccharomyces cerevisiae EBY100 Bs -PPK was displayed on the surface of Saccharomyces cerevisiae cells to obtain a whole-cell enzyme catalyst.

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