Enzymatic reaction catalyst and application thereof in synthesis of 10-hydroxycamptothecin-glucoside

Abstract

The invention relates to an enzymatic reaction catalyst and application thereof in synthesis of 10-hydroxycamptothecin-glucoside, and relates to the technical field of biological catalysis. The enzymatic reaction catalyst comprises glycosyl transferase with an amino acid sequence as shown in SEQ ID NO: 1 and sucrose synthase with an amino acid sequence as shown in SEQ ID NO: 2. The high-efficiency and low-cost enzymatic synthesis of 10-hydroxycamptothecin glucoside is realized through the catalytic reaction of the enzymatic reaction catalyst.

Description

Technical Field

[0001] This invention relates to the field of biocatalysis technology, specifically to enzyme-catalyzed reaction catalysts and their application in the synthesis of 10-hydroxycamptothecin-glucoside. Background Technology

[0002] 10-hydroxycamptothecin (HCPT) is a natural product unique to Camptotheca acuminata Decne, which has anti-tumor activity (inhibits topoisomerase I) and is of great application value in cancer treatment.

[0003] However, its poor water solubility and high toxicity limit its clinical application. Therefore, the development of water-soluble HCPT derivatives has become a research hotspot in recent years. Glycosylation modification significantly improves the water solubility of HCPT. However, the content of 10-hydroxycamptothecin-glucoside in Camptothecin fruit is extremely low. Large-scale preparation of 10-hydroxycamptothecin-glucoside requires large-scale planting of Camptothecin, resulting in a serious waste of land resources and raw materials. In addition, the extraction process of 10-hydroxycamptothecin-glucoside is complex and inefficient, resulting in poor overall economic efficiency. The chemical synthesis of 10-hydroxycamptothecin-glucoside requires low temperature of -20 °C and cumbersome steps such as group protection and deprotection.

[0004] Currently, it has been reported that the glycosyltransferase oleD from Streptomyces antibioticus can catalyze the production of 10-hydroxycamptothecin from 10-hydroxycamptothecin. However, the yield of the product, 10-hydroxycamptothecin-glucoside, is only 13%. Summary of the Invention

[0005] This invention aims to overcome the above-mentioned defects and provide an enzymatic reaction catalyst and its application in the synthesis of 10-hydroxycamptothecin-glucoside, specifically, the combined use of a glycosyltransferase and a sucrose synthase (ATSUS1) to achieve high-efficiency and low-cost preparation of 10-hydroxycamptothecin-glucoside. The specific scheme is as follows:

[0006] This invention provides an enzyme-catalyzed reaction catalyst, characterized in that: the enzyme-catalyzed reaction catalyst comprises glycosyltransferase and sucrose synthase;

[0007] The amino acid sequence of the glycosyltransferase is shown in SEQ ID NO: 1;

[0008] The amino acid sequence of sucrose synthase is shown in SEQ ID NO: 2.

[0009] The present invention also provides a gene encoding an enzyme-catalyzed reaction catalyst, characterized in that: the gene encodes a glycosyltransferase and / or sucrose synthase in an enzyme-catalyzed reaction catalyst as claimed in claim 1;

[0010] The nucleotide sequence of the gene encoding glycosyltransferase is shown in SEQ ID NO: 3;

[0011] The nucleotide sequence of the gene encoding sucrose synthase is shown in SEQ ID NO: 4.

[0012] The present invention also provides a recombinant plasmid for an enzyme-catalyzed reaction catalyst, characterized in that: the recombinant plasmid includes the encoding gene of an enzyme-catalyzed reaction catalyst according to claim 2;

[0013] The backbone of the recombinant plasmid includes at least one of pET28a, pYES2, and pLysS.

[0014] The present invention also provides a transformant of an enzyme-catalyzed reaction catalyst, characterized in that: the transformant comprises a recombinant plasmid of an enzyme-catalyzed reaction catalyst of claim 3.

[0015] The present invention also provides the application of the above-mentioned enzymatic reaction catalyst in the preparation of 10-hydroxycamptothecin-glucoside.

[0016] In addition, the present invention also provides a method for preparing 10-hydroxycamptothecin-glucoside, characterized in that: using uridine diphosphate (UDP), sucrose, and 10-hydroxycamptothecin as raw materials, and using glycosyltransferase and sucrose synthase as catalysts, an enzymatic reaction is carried out to obtain the target 10-hydroxycamptothecin-glucoside.

[0017] The amino acid sequence of the glycosyltransferase is shown in SEQ ID NO: 1;

[0018] The amino acid sequence of sucrose synthase is shown in SEQ ID NO: 2.

[0019] Preferably, the molar ratio of UDP to 10-hydroxycamptothecin is 0.4-0.6:0.2-0.3.

[0020] Preferably, the ratio of sucrose synthase to sucrose is (150-200) μg: (300-400) mmol.

[0021] Preferably, the mass ratio of glycosyltransferase to sucrose synthase is 100-130:150-200.

[0022] Preferably, the temperature of the enzymatic reaction is 25℃-35℃, and the reaction time is 20 h-28 h.

[0023] The function and effects of this invention:

[0024] This invention utilizes glycosyltransferase and sucrase synthase (ATSUS1) as enzymatic catalysts to achieve the efficient and low-cost enzymatic synthesis of the natural product 10-hydroxycamptothecin-glucosinolate. In this reaction, glycosyltransferase glycosylates 10-hydroxycamptothecin; while sucrase synthase (ATSUS1) catalyzes the breakdown of sucrose into one molecule of fructose and one molecule of glucose. Simultaneously, glucose, under the action of sucrase synthase (ATSUS1), binds to UDP to generate UDPG, thereby providing activated glucose for the glycosylation reaction of 10-hydroxycamptothecin, thus promoting the glycosylation process and improving its production efficiency and yield. Attached Figure Description

[0025] Figure 1 This is a schematic diagram of the 10-hydroxycamptothecin enzymatic reaction catalyzed by glycosyltransferase combined with sucrose synthase (ATSUS1) in this embodiment;

[0026] Figure 2 This is an SDS-PAGE gel electrophoresis image of the glycosyltransferase and sucrose synthase (ATSUS1) in this embodiment;

[0027] Figure 3 This is a schematic diagram of the liquid chromatography detection results of 10-hydroxycamptothecin-glucoside in this embodiment;

[0028] Figure 4 This is a schematic diagram of the high-resolution mass spectrometry identification results of 10-hydroxycamptothecin-glucoside in this embodiment;

[0029] Figure 5 This is a schematic diagram of the mass-to-charge ratio analysis of the product obtained in this embodiment. Detailed Implementation

[0030] The principles and features of this invention are described below. The examples given are for illustrative purposes only and are not intended to limit the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they should be performed according to the techniques or conditions described in the literature in this field, or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0031] Example 1. Glycosyltransferase and sucrose synthase (ATSUS1)

[0032] 1.1. Construction of glycosyltransferase or sucrose synthase (ATSUS1) includes the following steps:

[0033] S1. Construction of expression strain: Select the base sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4, with NdeI at the 5' end and XhoI at the 3' end, and the target vector is pET28a, pYES2, or pLysS (in this experimental example, pET28a was selected, which was provided by Beijing Qingke Biotechnology Co., Ltd., and the gene synthesis was entrusted to Beijing Qingke Biotechnology Co., Ltd.); Add 4 μg of the synthesized plasmid to 100 μL of ddH2O, take out 3 μL of the plasmid and add it to 50 μL of E coli BL21(DE3) competent cells (purchased from Beijing TransGen Biotech Co., Ltd.), reset the EP tube to ice for 30 min, then heat shock it in a 42℃ water bath for 45 s, and then put it on ice for 2 min. Add 900 μL of LB medium (Wuhan Taisheng Biotechnology Co., Ltd.) to each tube and incubate at 37℃ and 180 rpm for 60 min on a shaker. Then centrifuge at 2000 g for 5 min, take 850 μL of supernatant, gently mix the precipitate, spread it on an LB plate containing 100 mg / L kanamycin, and incubate at 37℃ for 10-12 h.

[0034] SEQ ID NO: 3

[0035]

[0036] SEQ ID NO:4

[0037]

[0038] S2. Screening of positive clones: Single colonies grown on the plate were inoculated onto LB plates containing 50 mg / L kanamycin and incubated at 37°C for 5 h. Single colonies were picked and sequenced. The sequencing results showed that the length of the glycosyltransferase fragment was 1443 bp and the length of the sucrose synthase (ATSUS1) fragment was 2427 bp.

[0039] S3. Synthesis of glycosyltransferase or sucrose synthase (ATSUS1): Select the kanamycin-resistant strain from step S2 and expand it to 1 L of culture. When the cell density reaches OD200, continue culturing until the desired cell density is reached. 600 When the concentration of the bacterial culture medium was 0.8, 0.5 mM isopropyl thiogalactoside (IPTG, purchased from BioFroxx Biotechnology Co., Ltd., purity ≥99%) was added for induction culture. After 24 h, the bacterial cells were collected by centrifugation. The bacterial cell pellet was resuspended in 100 mL Tris-HCl buffer and homogenized (1000 bar, 5 min) until the solution became translucent. Then, the cells were centrifuged at 12000 rpm for 1 h at 4 °C, and the supernatant obtained after centrifugation was collected after the cells were broken up.

[0040] S4. Purified Glycosyltransferase or Sucrose Synthase (ATSUS1): Prepare a Ni-NTE column (Ni Sepharose™ Excel, purchased from GE Healthcare, 17-3712-01), wash 3 times with ultrapure water and 3 times with Lysis buffer. Pass the supernatant through the Ni-NTE column twice, collecting the unbound portion of the protein mixture after passing through the binding column. Wash the nickel column with Wash buffer to remove contaminating proteins, 3 mL each time, 3 times. Elute the target band with Eluntion buffer to obtain purified glycosyltransferase or sucrose synthase (ATSUS1), and then pass it through a molecular sieve SD75 (purchased from GE Healthcare) to obtain glycosyltransferase or sucrose synthase (ATSUS1) with higher purity.

[0041] The components and amounts of the buffer solution used for protein purification are as follows:

[0042] The Lysis buffer consists of: 20 mM Tris-HCl (pH = 8.0), 300 mM KCl, 10% glycerol, and 1 mM MPMSF.

[0043] The Wash Buffer consists of: 20 mM Tris-HCl (pH = 8.0), 300 mM KCl, 10 mM, 20 mM, 30 mM, 50 mM, 100 mM, and 200 mM imidazole, 10% glycerol, and 1 mM PMSF.

[0044] The Elution Buffer consists of: 20 mM Tris-HCl (pH = 8.0), 300 mM KCl, 500 mM imidazole, 10% glycerol, and 1 mM PMSF.

[0045] Molecular sieve buffer: 20 mM Tris-HCl (pH = 8.0), 10% glycerol.

[0046] S5. SDS-PAGE electrophoresis analysis: After passing the obtained glycosyltransferase or sucrose synthase (ATSUS1) through nickel column affinity chromatography, a portion of the collected sample was mixed with loading buffer (containing 0.25M Tris-HCl pH 6.8, 10% SDS, 50% glycerol, 0.5% bromophenol blue, and 25% β-mercaptoethanol) and analyzed by SDS-PAGE electrophoresis. The results are as follows: Figure 2 As shown, by Figure 2 It is known that glycosyltransferase or sucrose synthase (ATSUS1) was prepared. The amino acid sequence of glycosyltransferase SEQ ID NO: 1 or the amino acid sequence of sucrose synthase SEQ ID NO: 2 is shown below:

[0047] SEQ ID NO: 1

[0048] MEESKTPHVAIIPSPGMGHLIPLVEFAKRLVHLHGLTVTFVIAGEGPPSKAQRTVLDSLPSSISSVFLPPVDLTDLSSSTRIESRISLTVTRSNPELRKVFDSFVEGGRLPTALVVDLFGTDAFDVAVEFHVPPYIFYPTTANVLSFFLHLPKLDETVSCEFRELTEPLMLPGCVPVAGKDFLDPAQDRKDDAYKWLLHNTKRYKEAEGILVNTFFELEPNAIKALQEPGLDKPPVYPVGPLVNIGKQEAKQTEESECLKWLDNQPLGSVLYVSFGSGGTLTCEQLNELALGLADSEQRFLWVIRSPSGIANSSYFDSHSQTDPLTFLPPGFLERTKKRGFVIPFWAPQAQVLAHPSTGGFLTHCGWNSTLESVVSGIPLIAWPLYAEQKMNAVLLSEDIRAALRPRAGDDGLVRREEVARVVKGLMEGEEGKGVRNKMKELKEAACRVLKDDGTSTKALSLVALKWKAHKKELEQNGNH

[0049] SEQ ID NO:2

[0050] MANAERMITRVHSQRERLNETLVSERNEVLALLSRVEAKGKGILQQNQIIAEFEALPEQTQKKLEGGPFFDLLKSTQEAIVLPPWVALAVRPRPGVWEYLRVNLHALLVEELQPAEFLHFKEELVDGVKNGNFTLELDFEPFNASIPRPTLHKYIGNGVDFLNRHLSAKLFHDKESLLPLLKFLRLHSHQGKNLMLSEKIQNLNTLQHTLRKAEEYLAELKPETLYEEFEAKFEEIGLERGWGDNAERVLDMIRLLLDLLEAPDPCTLETFLGRVPMVFNVVILSPHGYFAQDNVLGYPDTGGQVVYILDQVRALEIEMLQRIKQQGLNIKPRILILTRLLPDAVGTTCGERLERVYDSEYCDILRVPFRTEKGIVRKWISRFEVWPYLETYTEDAAVELSKELDGKPDLIIGNYSDGNLVASLLAHKLGVTQQCTIAHALEKTKYPDSDIYWKKLDDKYHFSCQFTADIFAMNHTDFIITSTFQEIAGSKETVGQYESHTAFTLPGLYRVVHGIDVFDPKFNIVSPGADMSIYFPYTEEKRRLTKFHSEIEELLYSDVENKEHLCVLKDKKKPILFTMARLDRVKNLSGLVEWYGKNTRLRELANLVIVGGDRRKESKDNEEKAEMKKMYDLIEEYKLNGQFRWISSQMDRVRNGELYRYICDTKGAFVQPALYEAFGLTVVEAMTCGLPTFATCKGGPAEIIVHGKSGFHIDPYHGDQAADTLADFFTKCKEDPSHWDEISKGGLQRIEDEYTWQIYSQRLLTLTGVYGFWKHVSNLDRLEARRYLEMFYALKYRPLAVPLAQDD

[0051] 1.2. Determination of enzyme concentration

[0052] The concentrations of glycosyltransferase and sucrose synthase (ATSUS1) were determined by the BCA method:

[0053] 0 µL, 1 µL, 2 µL, 4 µL, 8 µL, 12 µL, 16 µL, and 20 µL of 2 mg / mL BSA solution (Wuhan Taisheng Biotechnology Co., Ltd.) were added to the first column of a 96-well plate, respectively. 20 µL, 19 µL, 18 µL, 16 µL, 12 µL, 8 µL, 4 µL, and 0 µL of PBS buffer were added to the corresponding wells, respectively. In the first row of the second, third, and fourth columns, 19 µL of PBS buffer and 1 µL of the unknown protein solution were added. 200 μL of a mixture of BCA reagent A and BCA reagent B (50:1 volume ratio) was added to each well. The plates were incubated at 37°C for 30 min using a microplate reader and a shaker. The 96-well plate was cooled to room temperature, and the absorbance of the samples at 562 nm was measured using a microplate reader. A standard curve y = ax + b (where y is absorbance and x is protein concentration) was plotted to calculate the concentration of the unknown protein.

[0054] Example 2. Enzymatic reaction

[0055] The synthesized glycosyltransferase, in conjunction with sucrose synthase (ATSUS1), catalyzes the enzymatic reaction of 10-hydroxycamptothecin, as follows: Figure 1 As shown, glycosyltransferases can glycosylate 10-hydroxycamptothecin; while sucrase synthase (ATSUS1) can catalyze the breakdown of sucrose into one molecule of fructose and one molecule of glucose. At the same time, glucose can combine with UDP under the action of sucrase synthase (ATSUS1) to generate UDPG, thereby providing activated glucose for the glycosylation reaction of 10-hydroxycamptothecin; thus, the activated glucose reacts with 10-hydroxycamptothecin, introducing glucose into 10-hydroxycamptothecin to achieve glycosylation.

[0056] 2.1. Methods of enzyme-catalyzed reactions:

[0057] The 1 mL enzymatic reaction system is as follows: 30-80 mM Tris-HCl buffer (pH = 8.0), UDP 0.4-0.6 mM (≥98%, Shanghai Yuanye Biotechnology Co., Ltd.), 10-hydroxycamptothecin 0.2-0.3 mM (95%, Shanghai Yuanye Biotechnology Co., Ltd.), sucrose 300-400 mM (AR, Sinopharm Chemical Reagent Co., Ltd.), 100-130 μg glycosyltransferase, and 150-200 μg sucrose synthase (ATSUS1). The reaction temperature is 25-35℃, and the reaction time is 20-28 h.

[0058] In a preferred embodiment, bovine serum albumin (BCA protein concentration assay kit (enhanced version), solution concentration 2 mg / mL, purchased from Yeasen Biotechnology Co., Ltd.) was used as a standard. The concentration of glycosyltransferase or sucrose synthase (ATSUS1) was determined using the BCA method, with 115 μg of glycosyltransferase and 161 μg of sucrose synthase (ATSUS1) added to a 1 mL reaction system. The specific preferred enzymatic reaction system is as follows: 50 mM Tris-HCl buffer (pH = 8.0), 0.5 mM UDP, 0.25 mM 10-hydroxycamptothecin, 350 mM sucrose, 115 μg of glycosyltransferase, and 161 μg of sucrose synthase (ATSUS1), reaction temperature 30℃, and reaction time 24 h.

[0059] 2.2 Results of the enzymatic reaction (using the preferred experimental example as an example):

[0060] like Figure 3 The liquid phase detection results shown indicate that the substrate in the enzymatic reaction has been completely converted into the product 10-hydroxycamptothecin-glucoside.

[0061] like Figure 4 As shown, the precise molecular weights of the obtained products were identified using high-resolution mass spectrometry (Thermo Fisher Q Exactive), with a mobile phase of methanol:0.1% formic acid water = 20:80. Figure 5 As shown, further mass-to-charge ratio analysis of the product obtained from the enzymatic reaction revealed that the precise molecular weight of the product was 526.29077, indicating the synthesis of 10-hydroxycamptothecin-glucoside in the system.

[0062] 10-Hydroxycamptothecin at concentrations of 0 mM, 0.01 mM, 0.03 mM, 0.0625 mM, 0.125 mM, 0.25 mM, 0.40 mM, and 0.50 mM was selected. High-resolution liquid chromatography (HPLC) was used for detection, and a standard curve for 10-hydroxycamptothecin was fitted based on the peak area: y = ax, where y represents the integrated peak area of ​​10-hydroxycamptothecin and x represents the concentration of quercetin. Substituting the 10-hydroxycamptothecin after the enzymatic reaction into the standard curve, detection showed that there was no residue of 10-hydroxycamptothecin in the enzymatic reaction system, indicating a conversion rate of over 90%.

[0063] Compared with the paper "Assessing the Regioselectivity of OleD-Catalyzed Glycosylation with a Diverse Set of Acceptors" by Zhou, M., Hamza, A., Zhan, C.-G. & Thorson, JS, Journal of Natural Products 76, 279–286 (2013), which showed that the yield of 10-hydroxycamptothecin as a substrate using the glycosyltransferase oleD was only 13% (1.1 mg), the conversion rate of the present invention is significantly higher than that of known techniques.

[0064] The function and effect of this embodiment:

[0065] This embodiment provides a glycosyltransferase and sucrose synthase (ATSUS1) to establish a 10-hydroxycamptothecin glycosylation reaction. This enables the enzymatic synthesis of the natural product 10-hydroxycamptothecin-glucoside; during this synthesis, the substrate loading of 10-hydroxycamptothecin can reach 91 mg / L, and the conversion rate is over 90%.

[0066] This embodiment utilizes glycosyltransferase and sucrose synthase (ATSUS1) as enzyme catalysts, and based on the functions of glycosyltransferase and sucrose synthase (ATSUS1), achieves the enzymatic synthesis of the natural product 10-hydroxycamptothecin-glucoside.

[0067] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. An enzyme-catalyzed reaction catalyst, characterized in that: The enzyme-catalyzed reaction catalyst includes glycosyltransferase and sucrose synthase; The amino acid sequence of the glycosyltransferase is as shown in SEQ ID NO: 1; The amino acid sequence of the sucrose synthase is shown in SEQ ID NO:

2.

2. A gene encoding an enzyme-catalyzed reaction catalyst, characterized in that: The encoding gene encodes a glycosyltransferase and / or sucrose synthase in an enzyme-catalyzed reaction catalyst as described in claim 1; The nucleotide sequence of the gene encoding the glycosyltransferase is shown in SEQ ID NO: 3; The nucleotide sequence of the gene encoding the sucrose synthase is shown in SEQ ID NO:

4.

3. A recombinant plasmid for an enzyme-catalyzed reaction catalyst, characterized in that: The recombinant plasmid includes the gene encoding an enzyme-catalyzed reaction catalyst as described in claim 2; The backbone of the recombinant plasmid includes at least one of pET28a, pYES2, and pLysS.

4. A converter of an enzyme-catalyzed reaction catalyst, characterized in that: The transformant comprises a recombinant plasmid of an enzyme-catalyzed reaction catalyst as described in claim 3.

5. The application of the enzymatic reaction catalyst as described in claim 1 in the preparation of 10-hydroxycamptothecin-glucoside.

6. A method for preparing 10-hydroxycamptothecin-glucoside, characterized in that: Using uridine diphosphate (UDP), sucrose, and 10-hydroxycamptothecin as raw materials, and glycosyltransferase and sucrose synthase as catalysts, an enzymatic reaction was carried out to obtain the target 10-hydroxycamptothecin-glucoside. The amino acid sequence of the glycosyltransferase is as shown in SEQ ID NO: 1; The amino acid sequence of the sucrose synthase is shown in SEQ ID NO:

2.

7. The method for preparing 10-hydroxycamptothecin-glucoside as described in claim 6, characterized in that: The molar ratio of UDP to 10-hydroxycamptothecin is 0.4-0.6:0.2-0.

3.

8. The method for preparing 10-hydroxycamptothecin-glucoside as described in claim 6, characterized in that: The ratio of sucrose synthase to sucrose is (150-200) μg: (300-400) mmol.

9. The method for preparing 10-hydroxycamptothecin-glucoside as described in claim 6, characterized in that: The mass ratio of the glycosyltransferase to the sucrose synthase is 100-130:150-200.

10. The method for preparing 10-hydroxycamptothecin-glucoside as described in claim 6, characterized in that: The temperature of the enzymatic reaction is 25℃-35℃, and the reaction time is 20 h-28 h.

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