AMP (adenosine monophosphate) sulfating enzyme mutant for producing adenosine phosphoryl sulfuric acid and application of AMP sulfating enzyme mutant

Abstract

The invention discloses an AMP sulfating enzyme mutant for producing adenosine phosphoryl sulfuric acid and application, and belongs to the technical field of biological engineering. According to the invention, AMP is used as a raw material, and AMP sulfating enzymes BtaAPSST M2 and PcAPSK are used for biosynthesis of PAPS. Under the catalysis of 10 g / L of BtaAPSST M2 wet thallus and 20 g / L of PcAPSK wet thallus, the conversion rate of PAPS is 70.59%. Compared with the existing ATP sulfating enzyme capable of catalyzing ATP to generate APS, the ATP sulfating enzyme disclosed by the invention is lower in cost and equivalent in conversion rate. Wherein the PAPS yield of the BtaAPSST M2 is two times that of a wild enzyme, and the industrial process of producing the PAPS by a microbial synthesis method is accelerated.

Description

Technical Field

[0001] The invention relates to an AMP sulfurylase mutant for producing adenosine phosphosulfate and application thereof, belonging to the technical field of bioengineering. Background Art

[0002] Sulfated compounds are widely present in the cytoplasm, cell surfaces, and extracellular matrix, playing irreplaceable roles in various life processes, including cellular 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 sulfurylase to form adenosine phosphosulfate (APS). APS is then catalyzed by APS kinase to form PAPS, completing the conversion of inorganic sulfur to organic sulfur. PAPS, catalyzed by various reductases, forms sulfites and sulfides, which contribute to the synthesis and modification of compounds such as polysaccharides, proteins, hormones, and flavonoids. Research on the synthesis of PAPS is of great significance both for the understanding of sulfur metabolic pathways in vivo and for the synthesis and application of sulfonic acid compounds in vitro.

[0003] Currently, PAPS synthesis methods primarily include chemical and biological methods. However, chemical PAPS synthesis is energy-intensive and highly polluting, while biological PAPS 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. Therefore, this invention aims to obtain an engineered enzyme with high expression and activity that can catalyze the production of the PAPS precursor APS using the inexpensive substrate AMP. Through mechanistic analysis and protein engineering, the optimal synthase is obtained, increasing the supply of APS and achieving efficient PAPS production. Although research on APS synthetases started relatively late in China, significant progress has been made in recent years with the development of gene editing and synthetic biology technologies. While some progress has been made in gene cloning, expression, and enzymatic characterization of APS synthetases, there is still a gap in structural elucidation and engineering compared to earlier-stage countries such as the United States, Japan, and Europe. The modification and application of APS synthetases holds great promise, with potential breakthroughs in multiple fields in the future. Through gene mining, protein engineering, and metabolic engineering, it is hoped that their functions will be further optimized, promoting their development in related fields such as agriculture, environmental protection, industry, and medicine. Summary of the Invention

[0004] The present invention provides an AMP sulfurylase, which is a bis(5'-adenosine)-triphosphatase, whose English name is bis(5'-adenosyl)-triphosphatase, EC 3.6.1.29, and whose amino acid sequence is shown in SEQ ID NO.1 and nucleotide sequence is shown in SEQ ID NO.2.

[0005] SEQ ID NO.1

[0006] MSFRFGQHLIKPSVVFLKTELSFALVNRKPVVPGHVLVCPLRPVERFRDMSPEEVADLFQAAQRVGTVVEKHFQGTSLTFSMQDGPEAGQTVKHVHVHILPRKAGDFHRNDSIYDALEKHDREDKDSPALWRSEEEMAAEAAALRVYFQ

[0007] SEQ ID NO.2

[0008] ATGTCATTTAGGTTCGGACAACACCTAATAAAGCCGAGCGTTGTCTTTCTGAAAACCGAACTGAGCTTCGCCTTGGTTAACCGCAAGCCGGTTGTTCCGGGTCATGTGCTGGTGTGCCCGCTGAGACCTGTTGAACGTTTTCGCGACATGAGCCCGGAGGAGGTTGCAGACTTGTTTCAAGCGGCGCAGCGCGTGGGCACGGTGGTCGAGAAGCACTTCCAAG GTACTTCGCTGACCTTTTCCATGCAGGATGGTCCAGAGGCCGGCCAGACCGTAAAACACGTGCACGTTCATATTCTGCCGCGTAAAGCGGGCGACTTCCATCGTAATGATTCTATCTATGATGCATTGGAAAAGCACGATCGTGAAGACAAAGACTCCCCGGCACTGTGGCGTAGCGAAGAGGAGATGGCTGCTGAAGCGGCGGCGTTACGTGTGTACTTCCAA

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

[0010] Or the mutant is obtained by mutating the histidine at position 8 of the AMP sulfurylase shown in the amino acid sequence of SEQ ID NO.1 to methionine, and is named BtaAPSST H8M ; The amino acid is shown in SEQ ID NO.3, and the nucleotide is shown in SEQ ID NO.4;

[0011] Alternatively, the mutant is obtained by mutating the 83rd glutamine of the AMP sulfurylase shown in the amino acid sequence of SEQ ID NO. 1 to arginine, and is named Q83R;

[0012] Or the mutant is obtained by mutating the leucine at position 117 of the AMP sulfurylase shown in the amino acid sequence of SEQ ID NO.1 to aspartic acid and the histidine at position 8 to methionine, and is named BtaAPSST H8M / L117D or BtaAPSST M2 ; The amino acid is shown in SEQ ID NO.5, and the nucleotide is shown in SEQ ID NO.6;

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

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

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

[0016] The present invention also provides a gene encoding the AMP sulfurylase mutant or a recombinant vector carrying the gene of the 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 a microbial cell expressing the AMP sulfurylase mutant or the 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 cell is a host cell of Escherichia coli, Bacillus subtilis or yeast.

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

[0023] The present invention also provides a method for increasing the enzymatic activity of AMP sulfurylase or increasing the conversion rate of AMP sulfurylase to substrate AMP, wherein the method comprises mutating the leucine at position 117 of the AMP sulfurylase whose amino acid sequence is shown in SEQ ID NO.1 to aspartic acid or histidine;

[0024] Alternatively, the histidine at position 8 of the AMP sulfurylase whose amino acid sequence is shown in SEQ ID NO. 1 is mutated to methionine.

[0025] or mutating the glutamine at position 83 of the AMP sulfurylase whose amino acid sequence is shown in SEQ ID NO.1 to arginine;

[0026] Alternatively, the leucine at position 117 of the AMP sulfurylase shown in the amino acid sequence of SEQ ID NO. 1 is mutated to aspartic acid, and the histidine at position 8 is mutated to methionine.

[0027] Alternatively, the leucine at position 117 of the AMP sulfurylase shown in the amino acid sequence of SEQ ID NO. 1 is mutated to histidine, and the histidine at position 8 is mutated to methionine;

[0028] Alternatively, the leucine at position 117 of the AMP sulfurylase shown in the amino acid sequence of SEQ ID NO. 1 is mutated to histidine, and the histidine at position 8 is mutated to arginine;

[0029] Alternatively, the histidine at position 8 of the AMP sulfurylase shown in the amino acid sequence of SEQ ID NO. 1 is mutated to methionine, and the glutamine at position 83 is mutated to arginine.

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

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

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

[0033] In one embodiment of the present invention, the strain expressing adenosine 5'-phosphosulfate kinase uses Escherichia coli BL21 (DE3) as a 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 addition amount of the substrate AMP is: 5-100mM, the addition amount of the strain expressing the mutant according to claim 1 or the wet bacteria of the microbial cells according to claim 4 or 5 is: 2-50g / L of wet bacteria; the addition amount of the strain expressing adenosine 5'-phosphosulfate kinase is: 2-50g / L of wet bacteria, the addition amount of Na2SO4 is: 10-500mM, the addition amount of ATP is: 2-100mM, and the addition amount of MgCl2 is: 5-500mM.

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

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

[0038] The present invention also provides the use of the mutant, gene or recombinant vector, microbial cell or method in preparing 3'-adenosine phosphate-5'-phosphosulfate or a product containing 3'-adenosine phosphate-5'-phosphosulfate.

[0039] The present invention also provides a recombinant Escherichia coli, wherein the recombinant Escherichia coli expresses the AMP sulfurylase 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 sulfurylase BtaAPSST mutant, which comprises the following steps:

[0042] (1) The mutation site was determined based on the amino acid sequence of the AMP sulfurylase BtaAPSST in cattle (Bos taurus); saturation mutagenesis primers were designed and saturation mutagenesis was performed using a vector carrying the BtaAPSST gene as a template; and a plasmid vector containing the mutant was constructed;

[0043] (2) transforming the mutant plasmid into host cells;

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

[0045] The present invention provides a BtaAPSST containing M2 -pET-28a vector strain, the strain construction method is: constructing BtaAPSST M2 -pET-28a vector was introduced into E.coli BL21 (DE3) competent cells, and finally BtaAPSST was constructed M2 -pET-28a strain.

[0046] Beneficial effects

[0047] The present invention uses AMP as raw material and utilizes AMP sulfurylase BtaAPSST M2 and PcAPSK for the biosynthesis of PAPS. M2Under the catalysis of wet bacteria and 20g / L PcAPSK wet bacteria, 20mM AMP, 200mM Na2SO4, 4mM ATP and 20mM MgCl2 were incubated in 10mL Tris-HCl buffer (50mM, pH 7.0) at 37°C for 10h, and the conversion rate of PAPS was 70.59%. Compared with the current ATP sulfurylase that can catalyze ATP to APS, the present invention has lower cost and comparable conversion rate. Among them, BtaAPSST M2 The PAPS yield of the enzyme is twice that of the wild enzyme, which has accelerated the industrialization process of producing PAPS by microbial synthesis. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 : A method for synthesizing PAPS using AMP as catalyst.

[0049] Figure 2 : The results of purifying the mutants according to the steps in Example 4.

[0050] Figure 3 :BtaAPSST M2 HPLC (A) of the conversion of AMP to PAPS by PcAPSK and MS product identification of the catalytically generated APS (B) and PAPS (C). DETAILED DESCRIPTION

[0051] The pET-28a(+) plasmid used in the following examples was purchased from Novagen (Madison, WI, USA), and restriction enzymes, T4 DNA ligase, primeSTAR, etc. were purchased from TaKaRa (Dalian, China). The BtaAPSST mutants were all obtained by molecular engineering.

[0052] The culture medium involved in the following examples is as follows:

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

[0054] LB solid medium: Add 2% agar to LB liquid medium.

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

[0056] The detection method adopted in the following examples is as follows:

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

[0058] Preparation of BtaAPSST mutants:

[0059] After the mutant recombinant bacteria were inoculated into 96-deep-well plates for culture and induced expression, the APS reaction system was added to the bacterial solution for whole-cell catalytic reaction. The amount of APS produced was detected by HPLC. Mutants with APS production higher than the wild type were selected, and the plasmids were extracted for sequencing. The sequences were compared with the wild type to detect the mutation sites.

[0060] The relative enzyme activity detection method of BtaAPSST enzyme (AMP sulfurylase) is as follows:

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

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

[0063] The specific steps are as follows:

[0064] (1) The target protein sequence BtaAPSST from cattle (Bos taurus) was synthesized by Genwi (the sequence of wild-type AMP sulfurylase is shown in SEQ ID NO. 1) and, after codon optimization, ligated to the pET-28a vector via BamHI and HindIII. The recombinant expression plasmid pET-28a-BtaAPSST was obtained and transformed into E. coli BL21 (DE3). The positive engineered bacteria were 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) The E. coli BL21 (DE3) / pET-28a-BtaAPSST strain was inoculated from the glycerol tube into 3 mL of LB liquid medium and cultured at 37 °C for 12 h to obtain seed solution. Subsequently, the seed solution was transferred to 200 mL of TB liquid medium at an inoculum ratio of 1:100 and cultured at 200 rpm and 37 °C. When the OD 600 When the value was between 0.4 and 0.8, IPTG was added at a final concentration of 0.4 mM and cultured at 25 °C for 16 h. The wet cells were collected after centrifugation and stored in a freezer at -40 °C.

[0068] Example 2: Determining the rate-limiting enzyme in 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 shown in SEQ ID NO. 7) from Penicillium chrysogenum was codon-optimized in Escherichia coli and synthesized by GENEBANK. It was ligated to the pET-28a vector via BamHI and HindIII to obtain the recombinant expression plasmid pET28a-PcAPSK, which was then transformed into E. coli BL21 (DE3). The resulting positive engineered bacteria 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 the glycerol tube into 3 mL LB liquid medium and cultured at 37 ° C for 12 h to obtain seed solution. Subsequently, the seed solution was transferred to 200 mL TB liquid medium at an inoculum ratio of 1:100 and cultured at 200 rpm and 37 ° C. When the OD 600 When the value was between 0.4 and 0.8, IPTG was added at a final concentration of 0.4 mM and cultured at 25 °C for 16 h. The wet cells were collected after centrifugation and stored in a freezer at -40 °C.

[0072] 2. Determination of rate-limiting enzyme

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

[0074] The results showed that the AMP content in the reaction solution was 12.32 mM, APS did not accumulate, and the PAPS content was 4.57 mM. This indicates that the intermediate APS did not accumulate, but the product PAPS did. However, the substrate AMP consumption was only half of the pre-reaction level, indicating that the PcAPSK enzyme converts the intermediate APS to PAPS at a faster rate. Therefore, it was determined that the rate-limiting enzyme was BtaAPSST.

[0075] Example 3: Construction of single mutants

[0076] The specific steps are as follows:

[0077] (1) Using the whole plasmid PCR technique, the recombinant plasmid pET-28a-BtaAPSST was used as a template for site-directed saturation mutagenesis 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), L37 (histidine at position 36), and N27 (asparagine at position 27). The saturation mutation library includes the following: leucine at position 7), Q83 (glutamine at position 83), G89 (glycine at position 89), Q90 (glutamine at position 90), T91 (threonine at position 91), V92 (valine at position 92), H96 (histidine at position 96), H98 (histidine at position 98), L117 (leucine at position 117), and E118 (glutamic acid at position 118).

[0078] The PCR amplification system used the KOD system shown in Table 1, using the pET-28a-BtaAPSST plasmid as a template and the primers listed in Table 2 for PCR. The KOD system is shown in Table 2. PCR reaction conditions were: 1: 98°C for 5 min; 2: 94°C for 30 s; 3: 55°C for 30 s; 4: 72°C for 3 min 20 s; 30 cycles of steps 2-4; 5: 72°C for 10 min; 6: incubation at 12°C.

[0079] Table 1: KOD system table

[0080]

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

[0082] Table 2: Mutation primer sequences

[0083]

[0084]

[0085] Note: NNK / MNN is a degenerate codon 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 was: DpnI quick 0.3 μL, the above PCR product 8.7 μL, 10×T Buffer 1 μL). After the digestion was completed, the digestion product was obtained.

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

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

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

[0090] The specific steps are as follows:

[0091] (1) The recombinant bacteria containing mutants prepared in Example 3 (E. coli BL21 (DE3) / pET-28a-single mutant) were picked and placed in a 96-deep-well plate containing 0.05 mg / mL kanamycin-resistant LB liquid medium. After constant temperature cultivation at 220 rpm and 37°C for 12 h, seed liquid was prepared. The prepared seed liquid was added to TB liquid medium at an addition amount of 1:100 and cultured at 220 rpm and 37°C. After culturing for 2 to 3 h, IPTG was added with a final concentration of 0.4 mM, and the culture was induced at 25°C for 16 h. The cells were centrifuged at 10,000 rpm for 30 min, and the bacteria were collected.

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

[0093] The conversion rates of AMP to APS of the wild-type enzyme and the mutant enzyme were tested (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 following table:

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

[0095] BtaAPSST AMP→APS conversion rate 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 dominant mutants obtained by screening were: L117D, H8M, L117H, and Q83R.

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

[0098] 1) Preparation of mutants:

[0099] The beneficial mutant recombinant bacteria E.coli BL21(DE3) / pET-28a-BtaAPSST were selected and screened respectively. 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 cells were inoculated into 3 mL LB liquid medium containing 0.05 mg / mL kanamycin resistance and cultured at 37°C for 12 h to obtain seed solution. Subsequently, the seed solution was transferred to 200 mL TB liquid medium at an inoculum ratio of 1:100 and cultured at 200 rpm and 37°C. When the OD 600 When the value was between 0.4 and 0.8, IPTG was added at a final concentration of 0.4 mM, and the culture was induced at 25 °C for 16 h. The bacteria were collected after centrifugation.

[0100] 2) Purification of mutants:

[0101] Wash the collected cells twice with 20 mM Tris-HCl buffer, pH 8.0, and disrupt them by sonication on ice. Centrifuge at 12,000 rpm for 30 minutes at 4°C, collect the supernatant, and prepare the crude enzyme solution. Open the stored gravity column, allow the sealing liquid to drain naturally, then rinse the column with 2-3 column volumes of ultrapure water. Equilibrate the column with 1-2 column volumes of Buffer A. After equilibration, load the sample. The optimal loading volume is 1 mL of filler to 10 mL of protein, which can be adjusted based on protein concentration. After loading, allow the sample to settle naturally. Wash away contaminants with Buffer A (generally 4-5 column volumes, until the Coomassie Brilliant Blue color development solution turns blue). Elute with Buffer B (10-15 mL of eluent per 5 mL of filler), collect the solution immediately, and desalt using a desalting column of appropriate size at 4°C, 3700-4900 rpm. After desalting, collect the protein and determine its concentration. The wild-type crude enzyme solution was prepared according to the above method, and the relative enzyme activity was detected.

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

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

[0104] BtaAPSST Relative enzyme activity (U / mg) wild type 0.86 L117D 1.33 H8R 0.95 Q83K 1.13 Q83R 1.04

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

[0106] Example 5: Iterative combination of single mutants

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

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

[0109] Table 5: Mutation primer sequences

[0110]

[0111] According to the method of Example 3, recombinant bacteria: E. coli BL21 (DE3) / pET-28a-double mutant were prepared.

[0112] (2) According to the method of Example 4, the conversion rate of AMP→APS of the mutant obtained in step (1) was detected (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 of different mutants

[0115] BtaAPSST AMP→APS conversion rate 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 the screened BtaAPSST mutants

[0117] The mutants were purified and their relative enzyme activities were determined according to the steps in Example 4. The results of enzyme activity are shown in Table 1. Figure 2 The relative enzyme activity is shown in Table 7:

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

[0119] BtaAPSST Relative enzyme activity (U / mg) 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: 1U is defined as the amount of enzyme required to consume 1μM AMP in 1 min.

[0121] The combined mutant screening was performed according to the steps of Example 4 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 bacteria:

[0125] The mutant strain E. coli BL21 (DE3) / pET-28a-BtaAPSST obtained in Example 5 was sequenced correctly and the M2and the recombinant strain E. coli BL21(DE3) / pET-28a-BtaAPSST containing the wild-type enzyme were inoculated into LB seed medium and cultured at 220 rpm and 37°C for 8-12 h to prepare a seed solution;

[0126] The obtained seed solution was inoculated into the shake flask fermentation medium at an inoculum volume of 2% (v / v), and cultured at 220 rpm and 37°C until the OD 600 = 0.6 to 0.8, IPTG was added to a final concentration of 0.2 mM for induction at 220 rpm and 25°C for 16 h. After centrifugation, the cells were collected separately.

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

[0128] The reaction conditions are as follows: 10 g / L E. coli BL21 (DE3) / pET-28a-BtaAPSST M2 Wet cells or recombinant E. coli BL21 (DE3) / pET-28a-BtaAPSST containing the wild-type enzyme, and 20 g / L E. coli BL21 (DE3) / pET-28a-PcAPSK prepared in Example 2 were added to a reaction system of 10 mL Tris-HCl buffer (50 mM, pH 7.0), the reaction system also containing 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 minutes. The supernatant was filtered through a 0.22 μm microfiltration membrane and then measured using HPLC to determine the PAPS content and PAPS conversion rate in the reaction solution after the reaction was completed (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 M2 The PAPS content obtained from wet cells was 7.76 g / L, and the PAPS conversion rate was 70.59%.

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

[0133] It can be seen that the conversion rate of PAPS prepared by bacteria containing mutant enzyme is twice that of wild enzyme.

[0134] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. An AMP sulfurylase mutant, characterized in that The mutant is obtained by mutating the leucine at position 117 of the AMP sulfurylase shown in the amino acid sequence of SEQ ID NO.1 to aspartic acid or histidine; Or the mutant is obtained by mutating the histidine at position 8 of the AMP sulfurylase whose amino acid sequence is shown in SEQ ID NO.1 to methionine; Or the mutant is obtained by mutating glutamine at position 83 of the AMP sulfurylase with the amino acid sequence shown in SEQ ID NO.1 to arginine; Or the mutant is obtained by mutating the leucine at position 117 of the AMP sulfurylase as shown in SEQ ID NO. 1 to aspartic acid or histidine, and simultaneously mutating the histidine at position 8 to methionine; Or the mutant is obtained by mutating the leucine at position 117 of the AMP sulfurylase as shown in the amino acid sequence of SEQ ID NO.1 to histidine, and simultaneously mutating the histidine at position 8 to arginine; Alternatively, the mutant is obtained by mutating the histidine at position 8 of the AMP sulfurylase shown in the amino acid sequence of SEQ ID NO. 1 to methionine, and simultaneously mutating the glutamine at position 83 to arginine.

2. A gene encoding the AMP sulfurylase mutant according to claim 1 or a recombinant vector carrying the gene encoding the AMP sulfurylase mutant according to claim 1.

3. The recombinant vector according to claim 2, characterized in that The recombinant vector is an expression vector of the pET series; preferably, the expression vector is pET28a.

4. A microbial cell expressing the AMP sulfurylase mutant of claim 1 or carrying the gene or recombinant vector of claim 2.

5. The microbial cell according to claim 4, characterized in that The microbial cells are bacteria or fungi as host cells; preferably, the microbial cells are Escherichia coli, Bacillus subtilis or yeast as host cells.

6. A method for increasing the enzymatic activity of AMP sulfurylase or increasing the conversion rate of AMP sulfurylase to substrate AMP, characterized in that: The method comprises mutating the leucine at position 117 of the AMP sulfurylase whose amino acid sequence is shown in SEQ ID NO. 1 to aspartic acid or histidine; or mutating the histidine at position 8 of the AMP sulfurylase whose amino acid sequence is shown in SEQ ID NO.1 to methionine; or mutating the glutamine at position 83 of the AMP sulfurylase whose amino acid sequence is shown in SEQ ID NO.1 to arginine; Alternatively, the leucine at position 117 of the AMP sulfurylase sequence shown in SEQ ID NO. 1 is mutated to aspartic acid or histidine, and the histidine at position 8 is mutated to methionine; Alternatively, the leucine at position 117 of the AMP sulfurylase shown in the amino acid sequence of SEQ ID NO. 1 is mutated to histidine, and the histidine at position 8 is mutated to arginine; Alternatively, the histidine at position 8 of the AMP sulfurylase shown in the amino acid sequence of SEQ ID NO. 1 is mutated to methionine, and the glutamine at position 83 is mutated to arginine.

7. A method for preparing 3'-adenosine phosphate-5'-phosphosulfate, characterized in that: The method comprises adding the mutant according to claim 1 or a strain expressing the mutant according to claim 1 or the microbial cells according to claim 4 or 5, adenosine 5'-phosphosulfate kinase or a strain expressing adenosine 5'-phosphosulfate kinase to a reaction system containing substrate AMP to prepare 3'-phosphoadenosine-5'-phosphosulfate.

8. The method according to claim 7, characterized in that The adenosine 5'-phosphosulfate kinase is derived from Penicillium chrysogenum; Preferably, the strain expressing adenosine 5'-phosphosulfate kinase uses Escherichia coli, Bacillus subtilis or yeast as a host cell.

9. The method according to claim 7 or 8, characterized in that The reaction system also contains Na2SO4, ATP and MgCl2; Preferably, in the reaction system, the addition amount of the substrate AMP is 5-100 mM, the addition amount of the strain expressing the mutant according to claim 1 or the wet bacterial cell of the microorganism according to claim 4 or 5 is 2-50 g / L of wet bacterial cell; the addition amount of the strain expressing adenosine 5'-phosphosulfate kinase is 2-50 g / L of wet bacterial cell, the addition amount of Na2SO4 is 10-500 mM, the addition amount of ATP is 2-100 mM, and the addition amount of MgCl2 is 5-500 mM. Preferably, the reaction conditions are: reaction temperature of 16 to 42° C., and reaction time of 2 to 48 hours.

10. Use of the mutant according to claim 1, or the gene or recombinant vector according to claim 2, or the microbial cell according to claim 4 or 5, or the method according to any one of claims 7 to 9, in the preparation of 3'-phosphoadenosine-5'-phosphosulfate or a product containing 3'-phosphoadenosine-5'-phosphosulfate.

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