An amp sulfurylase mutant for producing adenosine phosphosulfate and application thereof

Abstract

The application discloses an AMP sulfurylase mutant for producing adenosine phosphosulfate and application thereof, and belongs to the technical field of bioengineering. The application uses AMP as raw material, and uses BtaAPSST M2 and PcAPSK to carry out biosynthesis of PAPS. Under the catalysis of 10 g / L BtaAPSST M2 wet mycelium and 20 g / L PcAPSK wet mycelium, the conversion rate of PAPS is 70.59%. Compared with ATP sulfurylase which can catalyze ATP to generate APS, the cost of the application is lower, and the conversion rate is equivalent. The PAPS yield of BtaAPSST M2 is twice that of wild enzymes, and the industrialization process of the microbial synthesis method for producing PAPS is accelerated.

Description

Technical Field

[0001] This invention relates to an AMP sulfurylase mutant for the production of adenosine phosphoryl sulfate and its applications, belonging to the field of bioengineering technology. Background Technology

[0002] Sulfated compounds are widely distributed in the cytoplasm, cell surface, and extracellular matrix, playing an irreplaceable role in vital processes such as cell development, differentiation, immunity, detoxification, and signal transduction. 3'-phosphoadenosine-5'-phosphosulfate (PAPS) is the most commonly used sulfonic acid group donor in compound sulfation. In vivo, inorganic sulfuric acid is activated by adenosine triphosphate (ATP) via ATP-sulfatase to form adenosine phosphosulfate (APS). APS is then catalyzed by APS kinase to finally form PAPS, completing the conversion of inorganic sulfur to organic sulfur. PAPS is further catalyzed by various reductases to form sulfurous compounds (sulfites) and sulfur-containing compounds (sulfides), enabling the synthesis and modification of polysaccharides, proteins, hormones, and flavonoids. Research on PAPS synthesis is of great significance for both elucidating in vivo sulfur metabolism pathways and for the synthesis and application of sulfonic acid compounds in vitro.

[0003] Currently, PAPS synthesis methods mainly include chemical and biological methods. However, chemical synthesis of PAPS is energy-intensive and highly polluting; while biological synthesis is extremely costly and faces significant challenges in industrial application. Therefore, there is an urgent need for a method to efficiently synthesize PAPS using low-cost substrates. This invention aims to obtain an engineered enzyme with high expression, high enzyme activity, and the ability to catalyze the production of the PAPS precursor APS using the inexpensive substrate AMP. Through mechanism analysis and protein engineering, the optimal synthase will be obtained to increase the supply of APS and achieve efficient PAPS production. Research on APS synthases in China started relatively late, but significant progress has been made in recent years with the development of gene editing and synthetic biology technologies. Certain achievements have been made in the gene cloning, expression, and enzymatic properties of APS synthases, but there is still a gap in structural analysis and modification compared to the United States, Japan, and European countries, which started earlier. The modification and application prospects of APS synthases are broad, and breakthroughs may be achieved in multiple fields in the future. Through gene mining, protein engineering, and metabolic engineering, it is hoped that its function can be further optimized, promoting development in related fields such as agriculture, environmental protection, industry, and medicine. Summary of the Invention

[0004] This invention provides an AMP sulfurylase, which is a bis(5'-adenosyl)-triphosphatase, EC 3.6.1.29; its amino acid sequence is shown in SEQ ID NO.1. Its nucleotide sequence is shown in SEQ ID NO.2.

[0005] SEQ ID NO.1

[0006] MSFRFGQHLIKPSVVFLKTELSFALVNRKPVVPGHVLVCPLRPVERFRDMSPEEVADLFQAAQRVGTVVEKHFQGTSLTFSMQDGPEAGQTVKHVHVHILPRKAGDFHRNDSIYDALEKHDREDKDSPALWRSEEEMAAEAAALRVYFQ

[0007] SEQ ID NO.2

[0008] ATGTCATTTAGGTTCGGACAACACCTAATAAAGCCGAGCGTTGTCTTTCTGAAAACCGAACTGAGCTTCGCCTTGGTTAACCGCAAGCCGGTTGTTCCGGGTCATGTGCTGGTGTGCCCGCTGAGACCTGTTGAACGTTTTCGCGACATGAGCCCGGAGGAGGTTGCAGACTTGTTTCAAGCGGCGCAGCGCGTGGGCACGGTGGTCGAGAAGCACTTCCAAG GTACTTCGCTGACCTTTTCCATGCAGGATGTCCAGAGGCCGGCCAGACCGTAAAACACGTGCACGTTCATATTCTGCCGCGTAAAGCGGGCGACTTCCATCGTAATGATTCTATCTATGATGCATTGGAAAAGCACGATCGTGAAGACAAAGACTCCCCGGCACTGTGGCGTAGCGAAGAGGAGATGGCTGCTGAAGCGGCGGCGTTACGTGTGTACTTCCAA

[0009] The present invention provides an AMP sulfurylase mutant, which is obtained by mutating leucine at position 117 of the AMP sulfurylase as shown in SEQ ID NO.1 to aspartic acid or histidine, and is named L117D or L117H.

[0010] Alternatively, the mutant was obtained by mutating histidine at position 8 of the AMP sulfurylase, as shown in SEQ ID NO.1, to methionine, and was named BtaAPSST. H8M Amino acids are shown in SEQ ID NO.3, and nucleotides are shown in SEQ ID NO.4.

[0011] Alternatively, the mutant was obtained by mutating glutamine at position 83 of AMP sulfate enzyme, as shown in SEQ ID NO.1, to arginine, and was named Q83R;

[0012] Alternatively, the mutant was obtained by mutating leucine at position 117 of the AMP sulfate enzyme, as shown in SEQ ID NO.1, to aspartic acid, and simultaneously mutating histidine at position 8 to methionine, and was named BtaAPSST. H8M / L117D Or BtaAPSST M2 Amino acids are shown in SEQ ID NO.5, and nucleotides are shown in SEQ ID NO.6;

[0013] Alternatively, the mutant was obtained by mutating leucine at position 117 of the AMP sulfate enzyme, as shown in SEQ ID NO.1, to histidine, and simultaneously mutating histidine at position 8 to methionine, and was named H8M / L117H.

[0014] Alternatively, the mutant was obtained by mutating leucine at position 117 of the AMP sulfate enzyme, as shown in SEQ ID NO.1, to histidine, and simultaneously mutating histidine at position 8 to arginine, and was named Q83R / L117H.

[0015] Alternatively, the mutant was obtained by mutating histidine at position 8 of the AMP sulfurylase, as shown in SEQ ID NO.1, to methionine, and simultaneously mutating glutamine at position 83 to arginine, and was named H8M / Q83R.

[0016] The present invention also provides a gene encoding the above-mentioned AMP sulfurylase mutant or a recombinant vector carrying the above-mentioned AMP sulfurylase mutant.

[0017] In one embodiment of the present invention, the recombinant vector is a pET series expression vector.

[0018] In one embodiment of the present invention, the expression vector is pET28a.

[0019] The present invention also provides microbial cells expressing the above-mentioned AMP sulfurylase mutant or the above-mentioned gene or recombinant vector encoding the AMP sulfurylase mutant.

[0020] In one embodiment of the present invention, the microbial cell is a bacterial or fungal host cell.

[0021] In one embodiment of the present invention, the microbial cells are Escherichia coli, Bacillus subtilis, or yeast as host cells.

[0022] In one embodiment of the present invention, the microbial cell is Escherichia coli BL21(DE3) as the host cell.

[0023] The present invention also provides a method for improving the enzyme activity of AMP sulfate or improving the conversion rate of AMP sulfate to substrate AMP by AMP sulfate, wherein the method is to mutate the leucine at position 117 of the AMP sulfate enzyme as shown in SEQ ID NO.1 to aspartic acid or histidine.

[0024] Alternatively, the histidine at position 8 of the AMP sulfate enzyme, as shown in SEQ ID NO.1, may be mutated to methionine.

[0025] Alternatively, the glutamine at position 83 of the AMP sulfate enzyme, as shown in SEQ ID NO.1, could be mutated to arginine;

[0026] Alternatively, the leucine at position 117 of the AMP sulfate enzyme, as shown in SEQ ID NO.1, may be mutated to aspartic acid, and the histidine at position 8 may be mutated to methionine.

[0027] Alternatively, the leucine at position 117 of the AMP sulfate enzyme, as shown in SEQ ID NO.1, may be mutated to histidine, and the histidine at position 8 may be mutated to methionine.

[0028] Alternatively, the leucine at position 117 of the AMP sulfate enzyme, as shown in SEQ ID NO.1, may be mutated to histidine, and the histidine at position 8 may be mutated to arginine.

[0029] Alternatively, the histidine at position 8 of the AMP sulfate enzyme, as shown in SEQ ID NO.1, may be mutated to methionine, and the glutamine at position 83 may be mutated to arginine.

[0030] The present invention also provides a method for preparing 3'-adenosine-5'-phosphate sulfuric acid, wherein the method comprises adding the above-mentioned mutant or strain expressing the above-mentioned mutant or the above-mentioned microbial cells, adenosine 5'-phosphate kinase or strain expressing adenosine 5'-phosphate kinase to a reaction system containing substrate AMP to prepare 3'-adenosine-5'-phosphate sulfuric acid.

[0031] In one embodiment of the present invention, the adenosine 5'-phosphorylsulfate kinase is derived from Penicillium chrysogenum;

[0032] In one embodiment of the present invention, the strain expressing adenosine 5'-phosphoryl kinase uses Escherichia coli, Bacillus subtilis, or yeast as the host cell.

[0033] In one embodiment of the present invention, the strain expressing adenosine 5'-phosphorylsulfate kinase uses Escherichia coli BL21(DE3) as the host cell.

[0034] In one embodiment of the present invention, the reaction system further contains Na2SO4, ATP and MgCl2;

[0035] In one embodiment of the present invention, in the reaction system, the amount of substrate AMP added is 5-100 mM, the amount of wet bacterial cells expressing the mutant of claim 1 or the microbial cells of claim 4 or 5 added is 2-50 g / L of wet bacterial cells; the amount of strain expressing adenosine 5'-phosphoryl sulfate kinase added is 2-50 g / L of wet bacterial cells, the amount of Na2SO4 added is 10-500 mM, the amount of ATP added is 2-100 mM, and the amount of MgCl2 added is 5-500 mM.

[0036] In one embodiment of the present invention, the reaction conditions are: a reaction temperature of 16–42°C and a reaction time of 2–48 h.

[0037] In one embodiment of the present invention, the reaction is as follows: at 10 g / L BtaAPSST M2 Under the catalysis of wet bacterial cells and 20 g / L cAPSK wet bacterial cells, 20 mM AMP, 200 mM Na2SO4, 4 mM ATP and 20 mM MgCl2 were incubated in 10 mL Tris-HCl buffer (50 mM, pH 7.0) at 37 °C for 10 h.

[0038] The present invention also provides the use of the above-mentioned mutant, or the above-mentioned gene or recombinant vector, or the above-mentioned microbial cell, or the above-mentioned method in the preparation of 3'-adenosine-5'-phosphate sulfuric acid or products containing 3'-adenosine-5'-phosphate sulfuric acid.

[0039] The present invention also provides a recombinant Escherichia coli, wherein the recombinant Escherichia coli expresses the above-mentioned AMP sulfatase mutant BtaAPSST. M2 .

[0040] In one embodiment of the present invention, the recombinant Escherichia coli uses E. coli BL21(DE3) as the expression host.

[0041] The present invention also provides a method for obtaining the above-mentioned AMP sulfatase BtaAPSST mutant, the method comprising the following steps:

[0042] (1) Based on the amino acid sequence of AMP sulfate enzyme BtaAPSST in bovine taurus, the mutation site was determined; saturation mutation primers were designed, and saturation mutation was performed using a vector carrying the gene encoding BtaAPSST as a template; a plasmid vector containing the mutant was constructed.

[0043] (2) Transform the mutant plasmid into the host cell;

[0044] (3) Select positive clones for fermentation culture.

[0045] This invention provides a solution containing BtaAPSST M2 The strain constructed using the -pET-28a vector, wherein the strain is constructed by: [The text abruptly ends here, likely due to an incomplete sentence or a missing section.] M2 The pET-28a vector was introduced into E. coli BL21(DE3) competent cells, ultimately constructing BtaAPSST. M2 -pET-28a strain.

[0046] Beneficial effects

[0047] This invention uses AMP as a raw material and utilizes AMP sulfatase BtaAPSST M2 PAPS biosynthesis was performed using PcAPSK. At 10 g / L BtaAPSST M2Under the catalysis of wet bacterial cells and 20 g / L PcAPSK wet bacterial cells, 20 mM AMP, 200 mM Na2SO4, 4 mM ATP, and 20 mM MgCl2 were incubated in 10 mL Tris-HCl buffer (50 mM, pH 7.0) at 37°C for 10 h, achieving a PAPS conversion rate of 70.59%. Compared to ATP sulfatases currently used to catalyze the conversion of ATP to APS, this invention offers lower cost and comparable conversion rates. Among them, BtaAPSST... M2 The PAPS yield is twice that of wild enzymes, accelerating the industrialization of PAPS production via microbial synthesis. Attached Figure Description

[0048] Figure 1 A method for synthesizing PAPS using AMP catalysis.

[0049] Figure 2 The results of purifying the mutant using the steps in Example 4.

[0050] Figure 3 :BtaAPSST M2 HPLC (A) and MS identification of the catalytically generated APS (B) and PAPS (C) products from the conversion of AMP with PcAPSK to PAPS. Detailed Implementation

[0051] The pET-28a(+) plasmids used in the following examples were purchased from Novagen (Madison, WI, USA), and restriction endonucleases, T4 DNA ligases, primeSTAR, etc., were purchased from TaKaRa (Dalian, China). All BtaAPSST mutants were obtained through molecular modification.

[0052] The culture media involved in the following examples are as follows:

[0053] LB liquid medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, sterilized at 121°C for 20 min.

[0054] LB solid medium: LB liquid medium with 2% agar added.

[0055] TB liquid culture medium: KH2PO4 2.31g / L, K2HPO4·3H2O 16.42g / L, yeast extract 24g / L, peptone 12g / L, glycerol 4g / L.

[0056] The detection methods used in the following embodiments are as follows:

[0057] HPLC determination of AMP, APS, and PAPS: Analysis was performed using an HPLC system equipped with a polyamine column (YMC Pack polyamine II, 250 mm × 4.6 mm). 0.7 mM KH₂PO₄, after filtration and ultrasonic degassing, was used as the mobile phase at a flow rate of 0.6 mL / min. -1 Throughout the operation, the column temperature was maintained at 30°C, and the detection wavelength was 254 nm.

[0058] Preparation of BtaAPSST mutant:

[0059] After inoculating the mutant recombinant bacteria into 96-well plates and inducing expression, an APS reaction system was added to the bacterial culture to carry out a whole-cell catalytic reaction. The amount of APS generated was detected by HPLC. Mutants with APS production superior to wild-type were selected, and plasmids were extracted and sequenced. The sequences were compared with those of wild-type to detect mutation sites.

[0060] The method for detecting the relative enzyme activity of BtaAPSST enzyme (AMP sulfatase) is as follows:

[0061] Prepare a 10 mL reaction system containing 20 mM AMP, 200 mM Na₂SO₄, 4 mM ATP, 20 mM MgCl₂, and Tris-HCl buffer (50 mM, pH 7.0). Add 0.02 mM purified wild-type or mutant APSST enzyme and react at 37 °C for 2 h. After the reaction, the amount of APS produced is determined by HPLC. In this reaction system, the amount of enzyme required to consume 1 mM AMP per minute is defined as 1 enzyme activity unit (U).

[0062] Example 1: Construction and expression of BtaAPSST-pET28a-BL21(DE3) engineered bacteria

[0063] The specific steps are as follows:

[0064] (1) The target protein sequence BtaAPSST, derived from bovine taurus, was synthesized by Genewiz (the sequence of wild-type AMP sulfurylase is shown in SEQ ID NO.1). After codon optimization, it was ligated into the pET-28a vector using BamHI and HindIII. The recombinant expression plasmid pET-28a-BtaAPSST was obtained and transformed into E. coli BL21(DE3). The resulting positive engineered bacteria was named E. coli BL21(DE3) / pET-28a-BtaAPSST and stored in glycerol tubes at -80°C.

[0065] SEQ ID NO.1

[0066] MSFRFGQHLIKPSVVFLKTELSFALVNRKPVVPGHVLVCPLRPVERFRDMSPEEVADLFQAAQRVGTVVEKHFQGTSLTFSMQDGPEAGQTVKHVHVHILPRKAGDFHRNDSIYDALEKHDREDKDSPALWRSEEEMAAEAAALRVYFQ

[0067] (2) E. coli BL21(DE3) / pET-28a-BtaAPSST strain was inoculated from a glycerol tube into 3 mL of LB liquid medium and cultured at 37 °C for 12 h to obtain seed culture. Subsequently, the seed culture was transferred to 200 mL of TB liquid medium at an inoculation rate of 1:100 and cultured at 200 rpm and 37 °C. When OD 600 When the pH value is between 0.4 and 0.8, add IPTG to a final concentration of 0.4 mM, induce culture at 25°C for 16 h, collect wet cells after centrifugation, and store at -40°C.

[0068] Example 2: Determining the rate-limiting enzyme for the pathway

[0069] The specific steps are as follows:

[0070] 1. Preparation of PcAPSK wet cells

[0071] The target protein sequence PcAPSK (GENEBANK ID: 81454071, amino acid sequence as shown in SEQ ID NO.7), derived from Penicillium chrysogenum, was synthesized by Genewiz after codon optimization for E. coli. It was then ligated into the pET-28a vector using BamHI and HindIII to obtain the recombinant expression plasmid pET28a-PcAPSK, which was then transformed into E. coli BL21(DE3). The resulting positive engineered bacterium was named E. coli BL21(DE3) / pET-28a-PcAPSK and stored in glycerol tubes at -80°C. E. coli BL21(DE3) / pET-2PcAPSK was inoculated from a glycerol tube into 3 mL of LB liquid medium and cultured at 37 °C for 12 h to obtain a seed culture. Subsequently, the seed culture was transferred to 200 mL of TB liquid medium at a 1:100 inoculation rate and cultured at 37 °C and 200 rpm. When OD... 600 When the pH value is between 0.4 and 0.8, add IPTG to a final concentration of 0.4 mM, induce culture at 25°C for 16 h, collect wet cells after centrifugation, and store at -40°C.

[0072] 2. Determination of the rate-limiting enzyme

[0073] (1) The 10 g / L E. coli BL21(DE3) / pET-28a-BtaAPSST wet cells prepared in Example 1 and the 20 g / L E. coli BL21(DE3) / pET-28a-PcAPSK wet cells obtained in step 1 were added to a reaction system containing 10 mL Tris-HCl buffer (50 mM, pH 7.0). The reaction system also contained 20 mM AMP, 200 mM Na2SO4, 4 mM ATP and 20 mM MgCl2. The mixture was incubated at 37 °C for 10 h, and the contents of AMP, APS and PAPS were detected after the reaction.

[0074] The results showed that the AMP content in the reaction solution was 12.32 mM, APS did not accumulate, and PAPS content was 4.57 mM. This indicates that the intermediate APS did not accumulate, but the product PAPS did. Furthermore, the amount of substrate AMP consumed was only half of what it was before the reaction. This suggests that the PcAPSK enzyme converts the intermediate APS to PAPS more quickly. Therefore, the rate-limiting enzyme can be identified as BtaAPSST.

[0075] Example 3: Construction of a single mutant

[0076] The specific steps are as follows:

[0077] (1) Using whole plasmid PCR technology, site-directed saturation mutagenesis was performed using recombinant plasmid pET-28a-BtaAPSST as a template to obtain AMP sulfurylase mutants F5 (phenylalanine at position 5), H8 (histidine at position 8), I10 (isoleucine at position 10), N27 (asparagine at position 27), R28 (arginine at position 28), H35 (histidine at position 35), and L37 (isoleucine at position 36). A saturated mutant library of the following amino acids: leucine at position 7, glutamine at position 83, glycine at position 89, glutamine at position 90, threonine at position 91, valine at position 92, histidine at position 96, histidine at position 98, leucine at position 117, and glutamate at position 118.

[0078] The PCR amplification system used was the KOD system shown in Table 1. Using pET-28a-BtaAPSST plasmid as a template, PCR was performed using the primers shown in Table 2. The KOD system is shown in Table 2. The PCR reaction conditions were: 1: 98℃ for 5 min; 2: 94℃ for 30 s; 3: 55℃ for 30 s; 4: 72℃ for 3 min 20 s; repeat steps 2-4 30 times; 5: 72℃ for 10 min; 6: incubate at 12℃.

[0079] Table 1: KOD System Table

[0080]

[0081] The primer sequences involved are shown in Table 2.

[0082] Table 2: Mutant Primer Sequences

[0083]

[0084]

[0085] Note: NNK / MNN are degenerate codons that can encode twenty amino acids.

[0086] (2) The above PCR reaction system was incubated in a metal bath at 37°C for 30 min to digest the plasmid template (the digestion system consisted of: 0.3 μL of DpnI quick, 8.7 μL of the above PCR product, and 1 μL of 10×T Buffer). The digestion product was obtained after digestion.

[0087] (3) The digestion product was introduced into E. coli BL21(DE3) competent cells by heat shock. The specific steps were as follows: 10 μL of PCR product was introduced into 100 μL of E. coli BL21(DE3) competent cells; ice bath for 30 min; heat shock in a water bath at 42℃ for 90 s, and then immediately placed in ice for 3-5 min; 600 μL of antibiotic-free LB medium was added and mixed well, and cultured at 37℃ and 220 rpm for 1 h; centrifuged at 4000 rpm for 2 min; the supernatant was discarded, and the cells were mixed by pipetting and aspirating with the remaining 100-200 μL of LB medium and spread onto a plate containing 0.05 mg / mL kanamycin resistance, and cultured at 37℃ for about 12 h.

[0088] Recombinant bacteria were prepared as follows: E.coli BL21(DE3) / pET-28a- single mutant.

[0089] Example 4: Screening for the optimal single mutant recombinant bacteria

[0090] The specific steps are as follows:

[0091] (1) The recombinant bacteria containing the mutant (E.coli BL21(DE3) / pET-28a-single mutant) prepared in Example 3 were picked and placed in 96-well plates containing 0.05 mg / mL kanamycin-resistant LB liquid medium. After incubation at 220 rpm and 37 °C for 12 h, seed culture was prepared. The prepared seed culture was added to TB liquid medium at a ratio of 1:100 and incubated at 220 rpm and 37 °C. After incubation for 2-3 h, IPTG was added to a final concentration of 0.4 mM and induced to grow at 25 °C for 16 h. After centrifugation at 10,000 rpm for 30 min, the bacterial cells were collected.

[0092] (2) Add a final concentration of 20 mM AMP, 200 mM Na2SO4, 4 mM ATP, 20 mM MgCl2, and Tris-HCl buffer (50 mM, pH 7.0) to the bacterial cells obtained in the 96-well plate obtained in step (1), and react at 37°C for 2 h. After the reaction, the amount of APS generated was detected by HPLC. Three mutants superior to wild-type were selected from each well plate, and these mutants were then sent to Tianlin Biotechnology Co., Ltd. for sequencing.

[0093] The conversion rates of AMP to APS for wild-type and mutant enzymes were measured (the conversion rate of AMP to APS was calculated by dividing the amount of APS generated in the reaction solution by the amount of substrate AMP added). The results are shown in the table below:

[0094] Table 3: AMP→APS conversion rate of different mutants

[0095] wild type 36.59％ L117D 54.24％ H8M 41.02％ L117H 51.93％ Q83R 44.35％ N27T 19.02％ H8E 12.40％ Q90R 5.79％

[0096] The following mutants were selected: L117D, H8M, L117H, and Q83R.

[0097] (3) Determination of relative enzyme activity of screened BtaAPSST mutants

[0098] 1) Preparation of mutants:

[0099] Beneficial mutant recombinant bacteria E. coli BL21(DE3) / pET-28a-BtaAPSST were selected separately. L117D , E.coli BL21(DE3) / pET-28a-BtaAPSST L117H , E.coli BL21(DE3) / pET-28a-BtaAPSST H8M , E.coli BL21(DE3) / pET-28a-BtaAPSST Q83RThe seed culture was inoculated into 3 mL of LB liquid medium containing 0.05 mg / mL kanamycin resistance and cultured at 37°C for 12 h. Subsequently, the seed culture was transferred to 200 mL of TB liquid medium at a 1:100 inoculation ratio and cultured at 37°C and 200 rpm. When OD... 600 When the pH value is between 0.4 and 0.8, add IPTG to a final concentration of 0.4 mM, induce culture at 25°C for 16 h, and collect the bacterial cells after centrifugation.

[0100] 2) Purification of mutants:

[0101] The collected bacterial cells were washed twice with 20 mM Tris-HCl buffer (pH 8.0) and sonicated on ice. Centrifuged at 12,000 rpm for 30 min at 4°C, the supernatant was collected, and crude enzyme solution was prepared. The stored gravity column was opened, and after the sealing liquid had drained naturally, the column was washed with 2–3 column volumes of ultrapure water and equilibrated with 1–2 column volumes of Buffer A. After equilibration, the sample was loaded, with an optimal loading volume of 1 mL packing material to 10 mL protein, adjusted according to the protein concentration. After loading, the sample was allowed to settle naturally. Impurities were washed away with Buffer A (generally 4–5 column volumes, until the Coomassie Brilliant Blue reagent no longer turned blue). Elution was then performed with Buffer B (10–15 mL eluent in 5 mL packing material), collected immediately, and then desalted using a desalting column of appropriate size at 4°C and 3700–4900 rpm. After desalting, the protein was collected and its concentration was determined. Wild-type crude enzyme solution was prepared according to the above method, and the relative enzyme activity was tested.

[0102] The relative enzyme activities of the above mutants were detected respectively, and the results are shown in Table 4 below:

[0103] Table 4: Enzyme activity of BtaAPSST wild-type and mutant

[0104] wild type 0.86 L117D 1.33 H8R 0.95 Q83K 1.13 Q83R 1.04

[0105] Note: The amount of enzyme required to consume 1 μM AMP in 1 minute is defined as 1 U.

[0106] Example 5: Iterative Combinations of Single Mutants

[0107] The beneficial single mutants screened in Example 4 were iteratively combined, and the specific steps are as follows:

[0108] (1) Following the steps in Example 3, the four beneficial mutants BtaAPSST were screened. L117D BtaAPSST L117H BtaAPSST H8M BtaAPSST Q83RIterative mutations were performed. The primers involved are shown in Table 5.

[0109] Table 5: Mutant Primer Sequences

[0110]

[0111] Recombinant bacteria, E. coli BL21(DE3) / pET-28a- double mutant, were prepared according to the method in Example 3.

[0112] (2) The conversion rate of AMP→APS of the mutant obtained in step (1) was detected according to the method of Example 4 (the conversion rate of AMP→APS was calculated by dividing the amount of APS generated in the reaction solution by the amount of substrate AMP added).

[0113] The results are shown in Table 6 below:

[0114] Table 6: APS production levels of different mutants

[0115] wild type 36.59％ Q83R / L117D 36.01％ Q83R / L117H 57.22％ H8M / Q83R 46.54％ H8M / L117D 70.59％ H8M / L117H 59.33％

[0116] (3) Determination of relative enzyme activity of screened BtaAPSST mutants

[0117] The mutant was purified and its relative enzyme activity was measured according to the steps in Example 4. The enzyme activity results are as follows: Figure 2 As shown in Table 7, the relative enzyme activity was detected.

[0118] Table 7: Enzyme activity of BtaAPSST wild-type and mutant

[0119] wild type 0.86 Q83R / L117D 0.85 Q83R / L117H 1.35 H8M / Q83R 1.10 H8M / L117D 1.66 H8M / L117H 1.39

[0120] Note: The amount of enzyme required to consume 1 μM AMP in 1 minute is defined as 1 U.

[0121] Following the steps in Example 4, the combined mutants were screened to obtain the optimal mutant BtaAPSST. H8M / L117D Named BtaAPSST M2 .

[0122] Example 6: Preparation of PAPS

[0123] The specific steps are as follows:

[0124] (1) Preparation of bacterial cells:

[0125] The correctly sequenced mutant strain E. coli BL21(DE3) / pET-28a-BtaAPSST obtained in Example 5 was used respectively. M2The recombinant strain E. coli BL21(DE3) / pET-28a-BtaAPSST containing wild-type enzymes was inoculated into LB seed medium and cultured at 220 rpm and 37°C for 8–12 h to prepare seed liquid.

[0126] The obtained seed culture was inoculated into shake-flask fermentation medium at an inoculum rate of 2% (v / v) and cultured at 220 rpm and 37°C until OD reached. 600 =0.6-0.8, add IPTG to a final concentration of 0.2mM for induction, and incubate at 220rpm and 25℃ for 16h. After centrifugation, collect the bacterial cells separately.

[0127] (2) The PAPS production was determined by whole-cell reaction of the bacterial cells after induction of expression obtained in step (1).

[0128] The reaction conditions were as follows: 10 g / L E. coli BL21(DE3) / pET-28a-BtaAPSST was prepared. M2 Wet cells or recombinant strain E. coli BL21(DE3) / pET-28a-BtaAPSST containing wild-type enzymes, and 20 g / L E. coli BL21(DE3) / pET-28a-PcAPSK prepared in Example 2 were added to a reaction system containing 10 mL Tris-HCl buffer (50 mM, pH 7.0), which also contained 20 mM AMP, 200 mM Na2SO4, 4 mM ATP and 20 mM MgCl2, and incubated at 37 °C for 10 h.

[0129] After the reaction, a portion of the conversion solution was centrifuged at 10,000 rpm for 30 min. The supernatant was filtered through a 0.22 μm microfiltration membrane and analyzed by HPLC to determine the PAPS content and conversion rate in the reaction solution after the reaction (calculated by dividing the amount of PAPS generated in the reaction solution by the amount of substrate AMP added). The results are as follows. Figure 3 As shown, the formation of the product was further confirmed.

[0130] The results show:

[0131] E. coli BL21(DE3) / pET-28a-BtaAPSST was used. M2 The PAPS content obtained from the wet cell preparation was 7.76 g / L, and the PAPS conversion rate was 70.59%.

[0132] PAPS content prepared using recombinant strain E.coli BL21(DE3) / pET-28a-BtaAPSST containing wild-type enzymes was 3.86 g / L, and the PAPS conversion rate was 35.12%.

[0133] It is evident that the conversion rate of PAPS prepared from bacterial cells containing the mutant enzyme is twice that of the wild-type enzyme.

[0134] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. An AMP sulfurylase mutant, characterized in that, The mutant was obtained by mutating leucine at position 117 of AMP sulfate enzyme, as shown in SEQ ID NO. 1, to aspartic acid.

2. The gene encoding the AMP sulfurylase mutant of claim 1.

3. A recombinant vector carrying the gene encoding the AMP sulfurylase mutant of claim 1.

4. The recombinant vector according to claim 3, characterized in that, The recombinant vector is a pET series vector as the expression vector.

5. The recombinant vector according to claim 4, characterized in that, The expression vector is pET28a.

6. Microbial cells expressing the AMP sulfurylase mutant of claim 1.

7. A microbial cell carrying the gene of claim 2.

8. Microbial cells carrying the recombinant vector of claim 3.

9. The microbial cell according to any one of claims 6 to 8, characterized in that, The microbial cells are bacteria or fungi that serve as host cells.

10. The microbial cell according to claim 9, characterized in that, The microbial cells used are Escherichia coli, Bacillus subtilis, or yeast as host cells.

11. A method for increasing the activity of AMP sulfatase or increasing the conversion rate of AMP sulfatase to the substrate AMP, characterized in that, The method involves mutating leucine at position 117 of AMP sulfate enzyme, as shown in SEQ ID NO. 1, to aspartic acid.

12. A method for preparing 3'-adenosine-5'-phosphate sulfuric acid, characterized in that, The method involves adding any one of the following (1) to (4) to a reaction system containing the substrate AMP to prepare 3'-adenosine-5'-phosphate sulfate: (1) The AMP sulfate enzyme mutant and adenosine 5'-phosphorylsulfate kinase as described in claim 1; (2) The AMP sulfate enzyme mutant of claim 1 and the Escherichia coli strain expressing adenosine 5'-phosphorylsulfate kinase; (3) An Escherichia coli strain expressing the AMP sulfate mutant of claim 1 and adenosine 5'-phosphoryl sulfate kinase; (4) Escherichia coli strains expressing the AMP sulfate mutant of claim 1 and Escherichia coli strains expressing adenosine 5'-phosphorylsulfate kinase; The reaction system also contains Na2SO4, ATP, and MgCl2.

13. The method according to claim 12, characterized in that, The adenosine 5'-phosphosulfate kinase was derived from Penicillium chrysogenum (… Penicillium chrysogenum ).

14. The method according to claim 13, characterized in that, In the reaction system, the amount of substrate AMP added is 5-100 mM, the amount of Escherichia coli strain expressing the mutant of claim 1 added is 2-50 g / L of wet cells; the amount of Escherichia coli strain expressing adenosine 5'-phosphoryl sulfate kinase added is 2-50 g / L of wet cells, the amount of Na2SO4 added is 10-500 mM, the amount of ATP added is 2-100 mM, and the amount of MgCl2 added is 5-500 mM. The reaction conditions are: reaction temperature of 16~42℃ and reaction time of 2~48 h.

15. The use of the AMP sulfatase mutant and adenosine 5'-phosphate kinase according to claim 1 in the preparation of 3'-adenosine-5'-phosphate sulfate or products containing 3'-adenosine-5'-phosphate sulfate, characterized in that, The preparation of 3'-adenosine-5'-phosphate sulfuric acid or products containing 3'-adenosine-5'-phosphate sulfuric acid is carried out using AMP as a substrate, and the reaction system also contains Na2SO4, ATP and MgCl2.

Images