Expandase mutants and their use in the synthesis of g-7-adca

Abstract

The application provides an expandase mutant and application thereof in synthesis of G-7-ADCA. The application produces a series of enzymes with expandase activity through directional evolution and other technologies, and the enzymes can expand penicillin G into G-7-ADCA in one step. Therefore, the expandase mutants have important application value in industry.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and pharmaceutical preparation, specifically to a cyclase mutant and its application in the synthesis of G-7-ADCA. Background Technology

[0002] Cephalosporin antibiotics are a class of anti-infective drugs widely used in clinical practice, with advantages such as low toxicity and broad spectrum. 7-Amino-3-deacetoxycephalosporanic acid (7-ADCA) is one of the important parent nuclei in the synthesis of cephalosporin antibiotics, and can be used to synthesize cephalexin, cefadroxil, and cefadroxil, etc., with a large market demand.

[0003] Currently, the main industrial production methods for 7-ADCA include the chemical conversion of penicillin G (PenG) into G-7-ADCA. This process requires expanding the five-membered ring of penicillin into a six-membered ring of cephalosporin (e.g., US4003894), which is costly and environmentally harmful. In contrast, biocatalysis offers advantages such as mild reaction conditions and environmental friendliness. Ring-expanding enzymes are key enzymes in the catalytic ring-expanding process (the flow chart of the 7-ADCA reaction catalyzed by ring-expanding enzymes is shown in Figure 1). Figure 1 (As shown).

[0004] The widely used cycloexpanding enzyme is penicillin N cycloexpanding enzyme isolated from *Streptomyces clav.* Ligers. This enzyme's optimal substrate is penicillin N, but its activity against penicillin G (PenG) is low, making it unsuitable for industrial production. Therefore, it is necessary to modify the cycloexpanding enzyme using both irrational and rational methods to improve its catalytic activity against penicillin G. Hsu et al. (Applied and Environmental Microbiology, 2004, 70(10): 6257-6263) performed DNA shuffling on genes from eight different bacterial genera to obtain the FF8 mutant, which showed increased activity against the substrate penicillin G (k... cat / K MThe activity increased by 117.8 times, but the conversion rate to penicillin G was not determined. Ji et al. (Applied and Environmental Microbiology, 2012, 78(21):7809-7812) obtained combined mutants such as C155Y / Y184H / V275I / C281Y / I305M / S261M, C155Y / Y184H / V275I / C281Y / I305M / T213V / M73T and C155Y / Y184H / V275I / C281Y / I305M / T213V / S261M through iterative combination mutations, which increased the activity by 7-8 times and are the mutants with the highest reported activity to date. Although the activity of the thermoenzyme has increased, it still cannot meet the industrial demand. CN1446908A describes a cyclase. In this patent, it is mentioned that by replacing cysteine ​​at position 155 with tyrosine, tyrosine at position 184 with histidine, valine at position 275 with isoleucine, and cysteine ​​at position 281 with tyrosine, a mutant cyclase is produced by changing one or more of these amino acids. The report states that the enzyme activity is improved, but the improvement is still limited and cannot meet industrial needs.

[0005] Therefore, it is necessary to develop highly active cyclases based on penicillin G. Summary of the Invention

[0006] To address the shortcomings of the existing technology, the present invention provides a cyclase mutant for the preparation of G-7-ADCA.

[0007] This invention provides a mutant of a circulator obtained by mutating the circulator enzyme with the amino acid sequence shown in SEQ ID No. 1. The mutant is a mutant of scDAOCSH7 (a seven-mutant of the circulator enzyme from Streptomyces spp.), specifically a protein obtained by mutating positions 72, 73, 74, 94, 96, 97, 179, 242, 244, 301, 302 and / or 303 of scDAOCSH7.

[0008] More specifically, the scDAOCSH7 cyclase mutant is a protein obtained by modifying scDAOCSH7 using any one, two, three, four, five, six, seven, eight, nine, ten, eleven, or all of the following twelve methods:

[0009] X1. Mutate the threonine at position 72 of scDAOCSH7 to arginine, aspartic acid, glutamine, glycine, or lysine;

[0010] X2. Mutate the threonine at position 73 of scDAOCSH7 to aspartic acid, glutamine, and glycine;

[0011] X3. Mutate the arginine at position 74 of scDAOCSH7 to glutamic acid and lysine;

[0012] X4. Mutate the tyrosine residue at position 94 of scDAOCSH7 to serine;

[0013] X5. Mutate the aspartic acid at position 96 of scDAOCSH7 to glycine;

[0014] X6. Mutate the tyrosine at position 97 of scDAOCSH7 to lysine, glutamic acid, or aspartic acid;

[0015] X7. Mutate the arginine at position 179 of scDAOCSH7 to leucine, glutamic acid, glycine, or asparagine;

[0016] X8. Mutate the arginine at position 242 of scDAOCSH7 to serine;

[0017] X9. Mutate histidine at position 244 of scDAOCSH7 to arginine and lysine;

[0018] X10. Mutate the asparagine at position 301 of scDAOCSH7 to serine, histidine, and glycine;

[0019] X11. Mutate the tyrosine at position 302 of scDAOCSH7 to arginine, histidine, serine, or glutamic acid;

[0020] X12. Mutate valine at position 303 of scDAOCSH7 to glutamine, glutamic acid, and lysine;

[0021] In one embodiment, the scDAOCSH7 cyclase mutant is scDAOCSH7-T73D, scDAOCSH7-R74K, scDAOCSH7-R179L, or scDAOCSH7-V303K. Specifically, scDAOCSH7-T73D is a protein obtained by mutating the 73rd position of scDAOCSH7 to aspartic acid; scDAOCSH7-R74K is a protein obtained by mutating the 74th position of scDAOCSH7 to lysine; scDAOCSH7-R179L is a protein obtained by mutating the 179th position of scDAOCSH7 to leucine; and scDAOCSH7-V303K is a protein obtained by mutating the 303rd position of scDAOCSH7 to lysine.

[0022] In another embodiment, the scDAOCSH7 cyclase mutant is scDAOCSH7-T73D / R74K / R179L / V303K, which is a protein obtained by mutating positions 73, 74, 179, and 303 of scDAOCSH7 to aspartic acid, lysine, leucine, and lysine, respectively.

[0023] The present invention also provides a nucleic acid molecule encoding the above-mentioned protein. The nucleic acid molecule may be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA, hnRNA, or tRNA. Specifically, the nucleic acid molecule is a gene encoding the above-mentioned mutant.

[0024] The present invention further provides a recombinant vector, expression cassette, transgenic cell, or recombinant bacteria containing the above-mentioned nucleic acid molecules. The recombinant vector may be a recombinant expression vector or a recombinant cloning vector.

[0025] The recombinant expression vector can be constructed using existing expression vectors. The expression vector may also contain the 3' untranslated region of the foreign gene, i.e., the polyadenylated signal and any other DNA fragments involved in mRNA processing or gene expression.

[0026] The polyadenylated signal guides the addition of polyadenylated nucleotides to the 3' end of the mRNA precursor. When constructing a recombinant expression vector using the gene, any type of enhancing, constitutive, tissue-specific, or inducible promoter can be added before its transcription initiation nucleotide; these can be used alone or in combination with other promoters. Furthermore, when constructing a recombinant expression vector using the gene of this invention, enhancers, including translational enhancers or transcriptional enhancers, can also be used. These enhancer regions can be ATG start codons or adjacent region start codons, etc., but must be identical to the reading frame of the coding sequence to ensure correct translation of the entire sequence. The translation control signal and start codon are widely available and can be natural or synthetic. The translation initiation region can originate from the transcription initiation region or structural genes.

[0027] The expression cassette consists of a promoter capable of initiating the expression of the gene, the cyclase mutant gene, and a transcription termination sequence.

[0028] Preferably, the transgenic cells are immobilized cells; more specifically, the fermented cells are collected, centrifuged, and resuspended to achieve an OD of [missing value]. 600 For a concentration of 10 to 150, add 2-8% diatomaceous earth, 0.1-2% w / v flocculant, and 0.07-2% v / v crosslinking agent, and crosslink for 2-3 hours to obtain immobilized cells.

[0029] This invention further provides the application of the mutant as a penicillin ring expander. The application involves catalyzing the ring expansion of penicillin G to form G-7-ADCA, thereby generating penicillin G. Compared to the wild-type ring expander, it improves the conversion rate.

[0030] The use of the mutant, the nucleic acid molecule, the recombinant vector, expression cassette, transgenic cell line, or recombinant bacteria in any of the following is also within the scope of protection of this invention:

[0031] (a1) Prepare a product with penicillin cyclodextrin activity; (a2) Prepare G-7-ADCA.

[0032] This invention also protects a method for preparing G-7-ADCA, which involves contacting penicillin G with a cyclase mutant of this invention to produce G-7-ADCA. The cyclase mutant may be used in the form of a culture medium or a composition (containing purified free enzyme, enzyme immobilization, whole-cell cyclase mutant, or whole-cell immobilization). Preferably, the contact reaction between the cyclase mutant and penicillin G can be carried out in solution. Preferred concentrations of penicillin G are 1-500 mM, the amount of cyclase added is 0.1-100 U / mL, the reaction mixture is at pH 6 to 8, the reaction time is 0.1 to 24 h, and the reaction temperature is 4 to 40 °C. The G-7-ADCA prepared by the above enzyme can be separated and purified from the reaction mixture using conventional methods.

[0033] Furthermore, the invented cyclodextrin mutant can be contacted with penicillin G in vivo to produce G-7-ADCA. Specifically, G-7-ADCA can be produced through the following steps: introducing the cyclodextrin mutant encoding gene or its functionally equivalent derivative into microorganisms with cyclodextrin activity (such as Escherichia coli, Bacillus subtilis, and yeast); culturing the transformant under appropriate conditions in a suitable culture medium; and spontaneously biosynthesizing G-7-ADCA in the transformant with the cyclodextrin mutant.

[0034] This invention utilizes both rational and irrational design techniques to generate a series of enzymes with ring-expanding activity. These enzymes can expand the substrate penicillin G to G-7-ADCA in one step, and exhibit improved conversion rates compared to wild-type enzymes. Therefore, these ring-expanding enzymes have significant industrial applications. Attached Figure Description

[0035] Figure 1 This is a flowchart of the 7-ADCA reaction catalyzed by cyclase.

[0036] Figure 2 This is a schematic diagram showing the location of mutation sites related to cyclase.

[0037] Figure 3This is the liquid phase spectrum of the product of penicillin G substrate transformation by the mutant. Detailed Implementation

[0038] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0039] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0040] Example 1: Preparation of engineered strains with mutant scDAOCSH7 gene of circulatory expansion enzyme

[0041] Amino acid residues surrounding the active pocket of cyclases have a significant impact on substrate recognition and catalysis. Therefore, this invention rationally designs amino acids (T72, T73, R74, R75, Y94, D96, Y97, R242, H244, N301, Y302, V303) that may affect the binding pocket of PenG to the cyclase. Through computational design and screening, four cyclase mutant units were obtained. Using the cyclase scDAOCSH7 (SEQ ID NO.1) as a template, four combinatorial mutant libraries were constructed (Table 1). The positions of the mutation sites on the cyclase are shown below. Figure 2 As shown.

[0042] Table 1. Mutant library constructed based on the circulatory expander scDAOCSH7

[0043]

[0044] Using the selected primers, site combination mutations were performed, and the corresponding primers were designed as shown in Table 2.

[0045] Table 2 Primer sequence listing

[0046]

[0047]

[0048] The specific construction method is as follows:

[0049] 1. Construction of gene fragments

[0050] Using plasmid pET24a-scDAOCSH7 as a template, primers T72-F1, T72-F2, T72-F3, T72-F4, T73-F1, T73-F2, T73-F3, and T73-F4 from Table 2 were mixed in a ratio of 3:1:1:1:3:1:1:1. 2 μL of the mixture was used as the upstream primer, and 2 μL of T72-T73-R74-R from Table 2 was used as the downstream primer to amplify gene fragment L1. Using plasmid pET24a-scDAOCSH7 as a template, Y94-D95-Y97-F1, Y94-D95-Y97-F2, and Y94-D95-Y97-F3 from Table 2 were mixed in a 2:1:1 ratio. 2 μL of this mixture was used as the upstream primer, and 2 μL of Y94-D95-Y97-R from Table 2 was used as the downstream primer to amplify gene fragment L2. Using plasmid pET24a-scDAOCSH7 as a template, L179-F1, L179-F2, and L179-F3 from Table 2 were mixed in a 2:1:1 ratio. 2 μL of this mixture was used as the upstream primer, and 2 μL of R242-H244-R1 and R242-H244-R2 from Table 2 were mixed in a 2:1 ratio. 2 μL of this mixture was used as the downstream primer to amplify gene fragment L3. Using plasmid pET24a-scDAOCSH7 as a template, N301-F1, N301-F2, Y302-F1, Y302-F2, Y302-F3, V303-F1, and V303-F2 from Table 2 were mixed in a ratio of 2:2:6:6:3:6:2. 2 μL of this mixture was used as the upstream primer, and 2 μL of N301-Y302-V303-R from Table 2 was used as the downstream primer to amplify gene fragment L4. The first round of PCR program was: 94℃ for 2 min, (98℃ for 15 s, 55℃ for 30 s, 72℃ for 30 s) × 28 cycles, followed by incubation at 72℃ for 5 min.

[0051] 2. Second round of PCR

[0052] Using 2 μL of the first-round PCR products L1, L2, L3, and L4 as primers, and plasmid pET24a-scDAOCSH7 as a template, a second-round PCR was performed to construct ML1, ML2, ML3, and ML4 mutant libraries. The second-round PCR program was: 28 cycles of 94℃ for 2 min, (98℃ for 15 s, 60℃ for 30 s, and 72℃ for 5 min), followed by a final incubation at 72℃ for 10 min.

[0053] 3. Third round of PCR

[0054] The obtained second-round PCR product was processed as follows: 1 μL of Dpn I enzyme was added to the second-round PCR product to digest the plasmid template, and the mixture was treated at 37°C for 3 hours. 2 μL of the enzyme-digested second-round PCR product was electroporated into *E. coli* BL21(DE3). The electroporated *E. coli* BL21(DE3) bacterial culture was evenly spread on kanamycin-resistant (50 μg / mL) LB agar plates and cultured at 37°C for 14 hours. Single colonies grew, which were the engineered strain of the scDAOCSH7 gene mutant. Recombinant plasmids containing the target nucleotides of this strain with non-directional or directional mutations were used as expression vectors for expressing the scDAOCSH7 gene mutant.

[0055] Example 2: The circulatory expander scDAOCSH7 or its mutant catalyzes the production of G-7-ADCA from PenG.

[0056] Single colonies from the mutant library were inoculated into 96-well plates, and 300 μL of LB medium containing 50 μg / mL kanamycin was added. The plates were incubated at 37°C for 8–12 h. A negative control group (empty vector of pET-24a) and a positive control group (pET24a-scDAOCSH7) were set up. Then, 200 μL of seed culture was inoculated into 800 μL of TB medium containing 0.1 mM IPTG and 50 μg / mL kanamycin. The cells were expressed at 25℃ for 16 h, centrifuged to collect the cells, and 400 μL of 50 mM phosphate buffer was added. Then, 50 μg / mL FeSO4, 0.4 mM ascorbic acid, 6 mM α-ketoglutarate, and 5 mM Penicillin G (pH 7.4) were added to the phosphate buffer. The mixture was reacted at 30℃ for 2 h, centrifuged at 4000 rpm for 15 min, and 50 μL of the supernatant was collected. 50 μL of methanol was added, and the mixture was centrifuged at 12000 rpm for 1 min. The supernatant was then analyzed by high-performance liquid chromatography (HPLC).

[0057] The HPLC detection conditions were as follows: column: Agilent ZORBAX SB-C18 Stable Bond Analytical 4.6×250mm; mobile phase: sodium phosphate buffer (20mM, pH 3.0) to methanol in a ratio of 55:45; flow rate: 1mL / min; detection wavelength: 215nm.

[0058] The standard used was G-7-ADCA (Shandong Lukang Pharmaceutical Co., Ltd.), and a standard curve was prepared using G-7-ADCA. After liquid chromatography detection, the conversion rate was calculated based on the standard curve: Conversion rate = 100% × P / (5 × 10⁻⁶). -3 P represents the content of G-7-ADCA detected by liquid chromatography (mol / L).

[0059] Four mutants, scDAOCSH7-T73D, scDAOCSH7-R74K, scDAOCSH7-R179L, and scDAOCSH7-V303K, were obtained through screening. These mutants showed improved penicillin G substrate conversion rates compared to scDAOCSH7. Results are shown in Table 3, and liquid chromatography results are shown below. Figure 3 .

[0060] One enzyme activity unit: 1 mM of G-7-ADCA is produced in 1 min at 30°C.

[0061] Table 3 Mutant Transformation Rate

[0062]

[0063] In summary, the mutant constructed based on the scDAOCSH7 cyclase of this invention exhibits a 1.5-fold increase in enzyme activity and a 16% to 33% increase in conversion rate.

[0064] Example 3: Construction and enzyme activity assay of the scDAOCSH7-T73D / R74K / R179L / V303K mutant cyclase

[0065] 1. Four-site combined mutations were performed on T73D, R74K, R179L, and V303K.

[0066] Based on the four sites T73D, R74K, R179L, and V303K selected in Example 2, mutation site primers were designed, and combined mutations were constructed using the pET24a-scDAOCSH7 plasmid as a template. The primers used in the construction process are shown in Table 4.

[0067] Table 4 Primer List

[0068]

[0069] Using pET24a-scDAOCSH7 plasmid as a template, fragment P1 (73-179 fragment) was amplified using primer pairs T-73D / R74K-F and R179L-R, and fragment P2 (179-303 fragment) was amplified using primer pairs R179L-F and V303K-R. By overlapping PCR, a large fragment P was amplified using fragments P1 and P2 as templates. Then, MegaPrimer PCR was performed using the large fragment P as primers to construct the mutant.

[0070] The first round of PCR reaction system (50 μL) includes: 10 ng plasmid template, 10 pmol primer pair, 1 x KOD plus buffer, 0.2 mM dNTP, 1.5 mM MgSO4, and 5 units of KOD-plus DNA polymerase.

[0071] The first round of PCR reaction conditions were: 95℃ for 1 min; 98℃ for 10 s, 57℃ for 30 s, 68℃ for 1 min / kbp; 30 cycles; 68℃ for 10 min. Fragments P1 and P2 were recovered from the gel.

[0072] Using P1 and P2 as templates, and T-73D / R74K-F and V303K-R as primers, a second round of PCR was performed to obtain fragment P, which was then excised and recovered from the gel.

[0073] The second round of PCR reaction system (50 μL) includes: 50 ng each of gel-recovered fragments P1 / P2, 10 pmol primer pair, 1x KOD plus buffer, 0.2 mM dNTP, 1.5 mM MgSO4, and 5 units of KOD-plus DNA polymerase.

[0074] The conditions for the second round of PCR were: 95℃ for 3 min; 98℃ for 10 s, 60℃ for 30 s, 68℃ for 1 min / kbp; 25 cycles; 68℃ for 10 min.

[0075] The third round of PCR used fragment P as a large primer and KOD-plus DNA polymerase for MegaPrimer PCR.

[0076] The MegaPrimer PCR reaction system (50 μL) includes: 10 ng plasmid template, 250 ng fragment P, 1x KODplus buffer, 0.2 mM dNTP, 1.5 mM MgSO4, and 5 units of KOD-plus DNA polymerase.

[0077] The MegaPrimer PCR reaction conditions were: 94℃ for 5 min; 98℃ for 10 s, 60℃ for 30 s, 68℃ for 2 min / kbp, 25 cycles; 68℃ for 10 min. The plasmid template was digested with DpnI, electrotransformed into *E. coli* BL21(DE3), and single colonies were picked for sequencing identification. Sequencing confirmed that the constructed vector contained four mutation sites: T73D, R74K, R179L, and V303KL.

[0078] 2. The crude enzyme solution of the circulatory expansion enzyme scDAOCSH7-T73D / R74K / R179L / V303K mutant catalyzes the production of G-7-ADCA from PenG.

[0079] The plasmid containing pET24a-scDAOCSH7-T73D / R74K / R179L / V303K from point 1 was transformed into *E. coli* BL21(DE3). The transformed *E. coli* BL21(DE3) bacterial culture was evenly spread on LB agar plates containing kanamycin resistance (concentration of 50 μg / mL). After incubation at 37°C for 14 h, single colonies grew, which were the engineered strain of the scDAOCSH7-T73D / R74K / R179L / V303K gene mutant. A negative control group (empty vector of pET-24a) and a positive control group (pET24a-scDAOCSH7) were also set up. Single colonies were picked and inoculated into 5 mL LB medium containing 50 μg / mL kanamycin. The culture was incubated at 37°C for 6 h. Then, 2% of the colonies were inoculated into 200 mL TB medium containing 50 μg / mL kanamycin and incubated at 37°C for 3 h. Induction with 1 mM IPTG at 25°C for 13 h was then performed. The bacterial cells were collected, and phosphate buffer was added to a final concentration of 0.25 g / mL (mass of wet cells per mL). The cells were then sonicated to disrupt the lysis. The resulting lysate was centrifuged at 12000 rpm for 30 min, and the supernatant was collected.

[0080] Prepare a 1 mL reaction system: Add 100 μL crude enzyme solution, 50 μg / mL FeSO4, 0.4 mM ascorbic acid, 6 mM α-ketoglutarate, and 5 mM PenG. Add pH 7.4 phosphate buffer to 1 mL and react at 30°C for 2 h. Stop the reaction by adding 1 mL of methanol. Analyze the reacted sample for G-7-ADCA and PenG according to the method in Example 2. The results are shown in Table 5; the conversion rate reached 95% after 2 h.

[0081] Table 5 Mutant Transformation Rate

[0082]

[0083] 3. The scDAOCSH7-T73D / R74K / R179L / V303K mutant protein catalyzes the conversion of PenG to G-7-ADCA.

[0084] Culturing the cells as described in point 2, collecting the cells, and sonicating them are crucial for cell disruption. The lysate is then centrifuged at 12000 rpm for 30 min, and the supernatant is collected. The supernatant is then transferred to a pre-equilibrated Ni affinity column, and the mutant protein bound to the Ni column is eluted with phosphate buffer containing 400 mL of midazole, for a total elution of 20 mL. The eluted protein is dialyzed against phosphate buffer for 12 h, and the protein concentration is determined according to the manufacturer's instructions. G-7-ADCA is synthesized using the following PenG system:

[0085] Prepare a 1 mL reaction system containing: 0.1 mg / mL cyclase mutant, 50 μg / mL FeSO4, 0.4 mM ascorbic acid, 6 mM α-ketoglutarate, and 5 mM PenG. Add pH 7.4 phosphate buffer to 1 mL and react at 30°C for 2 h. Stop the reaction by adding 1 mL of methanol. Analyze the G-7-ADCA and PenG levels in the reacted sample according to the method in Example 2. The results are shown in Table 6; the conversion rate reached 95% after 2 h.

[0086] Table 6 Mutant Transformation Rate

[0087]

[0088] 4. Perform whole-cell reaction on bacteria containing the scDAOCSH7-T73D / R74K / R179L / V303K mutant.

[0089] Culture the bacteria as described in point 3 above, collect the bacteria, dissolve the bacteria in phosphate buffer, and adjust the OD of the bacteria. 600 To obtain a concentration of 20, take 100 mL of the above-mentioned bacterial cells and add 30 mM PenG, 45 mM α-ketoglutarate, 0.4 mM ascorbic acid, and 0.5 μg / mL ferrous sulfate, respectively. React at 30°C for 2 hours, then terminate the reaction. The G-7-ADCA and PenG assays were performed on the reacted sample according to the method in Example 2. The results are shown in Table 7; the conversion rate reached 79% after 2 hours.

[0090] Table 7 Mutant Transformation Rate

[0091]

[0092] For 30mM PenG, the conversion rate of the scDAOCSH7-T73D / R74K / R179L / V303K mutant was increased by 14% compared to scDAOCSH7.

[0093] Example 4: Immobilization of the scDAOCSH7-T73D / R74K / R179L / V303K mutant protein

[0094] The scDAOCSH7-T73D / R74K / R179L / V303K mutant protein was obtained according to Example 3, and the protein was quantified and immobilized onto an epoxy-based support.

[0095] Immobilized enzyme was obtained by adding buffer, carrier, and protein to a reactor, loading 50 mg of protein onto 1 g of wet carrier, and reacting at 70 rpm for 15 h, followed by 20 h of incubation. The immobilized enzyme was then subjected to the following reaction:

[0096] In a 1 mL reaction system, 5 mg of immobilized enzyme, 50 μg / mL FeSO4, 0.4 mM ascorbic acid, 6 mM α-ketoglutarate, and 5 mM PenG were added respectively. Phosphate buffer at pH 7.4 was added to 1 mL, and the reaction was stopped after 2 h. The G-7-ADCA and PenG of the reacted sample were detected according to the method in Example 2, and the conversion rate was calculated to be 90%.

[0097] Example 5: Immobilization of cells containing the scDAOCSH7-T73D / R74K / R179L / V303K mutant

[0098] The bacterial cells were cultured as described in Example 3, collected, and the OD was adjusted. 600 =100, add 1% diatomaceous earth to the suspension, stir well, then add 0.5% w / v polyethyleneimine with a molecular weight of 10000, flocculate the aqueous solution at room temperature, then add 2% v / v glutaraldehyde aqueous solution for crosslinking for 2 hours to obtain immobilized cells. The immobilized cells are then subjected to the following reaction:

[0099] In a 100 mL reaction system, add the final concentration of OD. 600 =20 immobilized cells, 50 μg / mL FeSO4, 0.4 mM ascorbic acid, 45 mM α-ketoglutarate, 30 mM PenG, and pH 7.4 phosphate buffer were added to 100 mL, and the reaction was carried out for 2 h. The reaction was then terminated. The G-7-ADCA and PenG were detected in the reaction sample according to the method in Example 2. The conversion rate was calculated to be up to 90%.

[0100] In summary, this invention constructed a cyclase mutant scDAOCSH7-T73D / R74K / R179L / V303K, which showed improved enzyme activity and conversion rate compared to the cyclase scDAOCSH7. Using the mutant scDAOCSH7-T73D / R74K / R179L / V303K as a catalyst, PenG conversion exceeded 90% in both whole-cell and immobilized cell processes at 30°C for 2 hours. Furthermore, this invention provides a method for converting PenG to G-7-ADCA, along with a corresponding immobilization technique, which has potential for industrial application. <110> Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences <120> Application of cyclases and their mutants in the synthesis of G-7-ADCA <160> 1 <210> 1 <211> 311 <212> PRT <213> Streptomyces clavuligerus <400> 1 MDTTVPTFSLAELQQGLHQDEFRRCLRDKGLFYLTDCGLTDTELKSAKDLVIDFFEHGSEAEKRAVTSPVPTTRRGFTGLESESTAQITNTGSYSDYSMCYSMGTADNLFPSGDFERIWTQYFDRQYTASRAVAREVLRATGTEPDGGVEAFLDYEPLLRFRYFPQVPEHRSAEEQPLRMAPHHDLSMVTLIQQTPCANGFVSLQAEVGGAFVDLPYRPDAVLVFCGAIATLVTGGQVKAPRHHVAAPRRDQIAGSSRTSSVFFLRPNADFTFSIPLAREYGFDVSLDGETATFQDWIGGNYVNMRRTSKA 311

Claims

1. A cyclase mutant, which is based on the cyclase scDAOCSH7 shown in SEQ ID No. 1, but with only the following mutations: The mutation at position 73 is aspartic acid; Alternatively, the 74th position of scDAOCSH7 could be mutated to lysine; or the 179th position of scDAOCSH7 could be mutated to leucine. Alternatively, the 303rd position of scDAOCSH7 could be mutated to lysine; Alternatively, threonine at position 73 of scDAOCSH7 may be mutated to aspartic acid, arginine at position 74 to lysine, arginine at position 179 to leucine, and valine at position 303 to lysine.

2. A nucleic acid molecule encoding the circulatory mutant of claim 1, wherein the nucleic acid molecule is DNA or RNA.

3. The nucleic acid molecule as described in claim 2, characterized in that, The DNA is cDNA, genomic DNA, or recombinant DNA; the RNA is mRNA, hnRNA, or tRNA.

4. A recombinant vector containing the nucleic acid molecule as described in claim 2 or 3.

5. The recombinant vector as described in claim 4, characterized in that, The recombinant vector is a recombinant expression vector or a recombinant cloning vector.

6. An expression cassette containing the nucleic acid molecule as described in claim 2 or 3.

7. The expression box as described in claim 6, characterized in that, The expression cassette consists of a promoter capable of initiating gene expression, the nucleic acid molecule, and a transcription termination sequence.

8. Transgenic cells containing the nucleic acid molecules as described in claim 2 or 3.

9. The transgenic cell as described in claim 8, characterized in that, The transgenic cells are immobilized cells.

10. The transgenic cell as described in claim 9, characterized in that, The fermented cells were collected, centrifuged, and resuspended to adjust their OD value. 600 For a concentration of 10 to 150, add 2-8% diatomaceous earth, 0.1-2% w / v flocculant, and 0.07-2% v / v crosslinking agent, and crosslink for 2-3 hours to obtain immobilized cells.

11. The use of the cyclase mutant of claim 1, or the nucleic acid molecule of claim 2 or 3, or a recombinant vector, expression cassette, transgenic cell line, or recombinant bacterium containing said nucleic acid molecule, in any of the following: (a1) Prepare a product with penicillin cyclase activity; (a2) Preparation of G-7-ADCA.

12. A method for preparing G-7-ADCA, comprising: Using PenG as a substrate, G-7-ADCA was obtained by catalytic reaction using the cyclase mutant of claim 1, or recombinant cells expressing the cyclase mutant, or lysates of the recombinant cells.

13. The method for preparing G-7-ADCA as described in claim 12, characterized in that, The process includes the following steps: preparing the cyclase mutant of claim 1, using the cyclase mutant as a penicillin cyclase to catalyze the cyclization of penicillin G to generate G-7-ADCA; wherein the cyclase mutant is used in the form of a culture medium.

14. The method as described in claim 13, characterized in that, The contact reaction between the cyclase mutant and penicillin G was carried out in solution with a penicillin G concentration of 1-500 mM, an added cyclase amount of 0.1-100 U / mL, a reaction mixture pH between 6 and 8, a reaction time of 0.1 to 24 h, and a reaction temperature of 4 to 40 °C.

15. The method as described in claim 14, characterized in that, The prepared G-7-ADCA was separated and purified from the reaction mixture.

16. The method for preparing G-7-ADCA as described in claim 12, characterized in that, The process includes the following steps: fermenting a microorganism containing the gene encoding the cyclase mutant of claim 1, so that the fermentation product catalyzes the cyclization of penicillin G to generate G-7-ADCA.

17. The method as described in claim 16, characterized in that, The method includes the following steps: cloning the gene encoding the cyclodextrin mutant of claim 1 into a penicillin G-producing bacterium to obtain a recombinant bacterium, fermenting the recombinant bacterium, and spontaneously catalyzing the biosynthesis of G-7-ADCA from penicillin G in the recombinant bacterium.

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