Expanding ring enzyme mutant and application thereof in synthesis of G-7-ADCA

Abstract

The invention provides an expansible ring enzyme mutant and an application of the expansible ring enzyme mutant in synthesis of G-7-ADCA. A series of mutants with penicillin G ring expansion activity are obtained through ancestor enzyme sequence reconstruction, directed evolution and the like, and the mutants can directly expand the ring of the substrate penicillin G to generate G-7-ADCA. Therefore, the expansive ring enzyme mutants have important application value in industry.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology and the field of pharmaceutical preparation, and in particular to a mutant of ring-expanding enzyme and its application in synthesis of G-7-ADCA. BACKGROUND

[0002] Cephalosporins have been widely used in clinical due to their low toxicity, broad spectrum, resistance to lactamase and other characteristics. 7-ADCA (7-amino-3-deacetoxycephalosporanic acid) is an important mother nucleus of cephalosporins. It is usually synthesized by chemical enzymatic method in industry, but this method needs multiple steps such as oxidation, ring expansion, rearrangement, and has high cost and heavy pollution. The biological enzymatic method is to expand penicillin G into G-7-ADCA under the action of penicillin ring-expanding enzyme, and then remove the side chain under the action of penicillin acylase to produce 7-ADCA ( Figure 1 ). This method has simple steps, is environmentally friendly, and has good application prospect. However, the natural substrate of penicillin ring-expanding enzyme is penicillin N, and its catalytic activity on the cheaper substrate penicillin G is low, which cannot meet the industrial demand. Therefore, it is necessary to modify the ring-expanding enzyme to improve its catalytic activity on penicillin G. Ji et al. (Applied and Environmental Microbiology, 2012, 78(21): 7809-7812) obtained a dominant mutant C155Y / Y184H / V275I / C281Y / I305M / T213V / M73T of ring-expanding enzyme by directed evolution technology based on previous research. The activity is 7-8 times higher than that of the wild type, which is the highest mutant reported so far, but its optimum working temperature is 25℃, which still cannot meet the industrial production demand.

[0003] In addition to focusing on the activity of the enzyme, the enzyme often needs to go through high temperature, high substrate / product concentration, extreme pH and other conditions when it functions, which also prompts us to focus on the stability of the enzyme. Ancestral sequence reconstruction (ASR) technology can trace the evolution process of the enzyme based on the phylogeny of modern enzymes and other information. The novel enzyme protein sequence reconstructed by ASR technology often has the advantage of improved stability (Current Opinion in Structural Biology, 2016, 38, 37-43). Therefore, it is crucial to reconstruct the ancestral enzyme sequence of penicillin ring-expanding enzyme by ASR to improve the activity and thermal stability of the ring-expanding enzyme for the industrial production of cephalosporins. SUMMARY

[0004] ​In view of the deficiencies in the prior art, the present application produces a series of enzymes with ring expansion activity through ancestral enzyme sequence reconstruction, directed evolution and other technologies, and further obtains ring expansion enzyme mutants, which can be used for preparing G-7-ADCA.

[0005] The present application provides a PenG ring expansion enzyme, which is any one of the PenG ring expansion enzymes of AD 1 to AD 12 obtained by ancestral enzyme reconstruction, and the corresponding amino acid sequences are shown in SEQ ID NO: 1 to SEQ ID NO: 12, respectively.

[0006] Further, on the basis of the amino acid sequence of the AD6 PenG ring expansion enzyme, at least one amino acid at positions 71, 73, 150, 155, 170, 184, 275, 280, 280, 292, 300 or 305 is replaced by another amino acid, and the amino acid sequence of the AD6 is further modified as follows: any one, any two, any three, any four, any five or all of the following proteins are obtained, and have higher activity, higher conversion rate and / or better thermal stability relative to the starting PenG ring expansion enzyme:

[0007] X1, the phenylalanine at position 71 of AD6 is mutated to glutamine;

[0008] X2, the isoleucine at position 73 of AD6 is mutated to threonine;

[0009] X3, the aspartic acid at position 150 of AD6 is mutated to valine;

[0010] X4, the cysteine at position 155 of AD6 is mutated to tyrosine;

[0011] X5, the aspartic acid at position 170 of AD6 is mutated to alanine;

[0012] X6, the tyrosine at position 184 of AD6 is mutated to histidine;

[0013] X7, the valine at position 275 of AD6 is mutated to isoleucine;

[0014] X8, the glutamic acid at position 280 of AD6 is mutated to valine;

[0015] X9, the cysteine at position 281 of AD6 is mutated to tyrosine;

[0016] X10, the alanine at position 292 of AD6 is mutated to glutamic acid;

[0017] X11, the glycine at position 300 of AD6 is mutated to aspartic acid;

[0018] X12, the isoleucine at position 305 of AD6 is mutated to methionine;

[0019] Alternatively, on the basis of the AD10 amino acid sequence, at least one amino acid at positions 72, 74, 151, 156, 171, 185, 276, 281, 282, 293, 301 and 306 is replaced by another amino acid, further to the amino acid sequence of the AD10 is modified as follows any one, any two, any three, any four, any five or all of the following six proteins, and relative to the starting PenG expandase has higher activity, higher conversion rate and / or better thermal stability:

[0020] X1, the isoleucine at position 74 of AD10 is mutated to threonine;

[0021] X2, the cysteine at position 156 of AD10 is mutated to tyrosine;

[0022] X3, the tyrosine at position 185 of AD10 is mutated to histidine;

[0023] X4, the valine at position 276 of AD10 is mutated to isoleucine;

[0024] X5, the cysteine at position 282 of AD10 is mutated to tyrosine;

[0025] X6, the isoleucine at position 306 of AD10 is mutated to methionine;

[0026] Alternatively, on the basis of the AD11 amino acid sequence, at least one amino acid at positions 72, 74, 151, 156, 171, 185, 276, 281, 282, 293, 301 and 306 is replaced by another amino acid, further to the amino acid sequence of the AD11 is modified as follows any one, any two, any three, any four, any five or all of the following six proteins, and relative to the starting PenG expandase has higher activity, higher conversion rate and / or better thermal stability:

[0027] X1, the isoleucine at position 74 of AD11 is mutated to threonine;

[0028] X2, the cysteine at position 156 of AD10 is mutated to tyrosine;

[0029] X3, the tyrosine at position 185 of AD10 is mutated to histidine;

[0030] X4, the valine at position 276 of AD10 is mutated to isoleucine;

[0031] X5, the cysteine at position 282 of AD10 is mutated to tyrosine;

[0032] X6, mutating isoleucine at position 306 of AD10 to methionine;

[0033] The present application provides a coding gene comprising the said expandase mutant.

[0034] The present application provides a recombinant vector or a recombinant strain comprising the said coding gene.

[0035] Preferably, the starting strain is E. coli, more preferably, it is E. coli BL21(DE3).

[0036] The present application also provides the use of the said PenG expandase (or its mutant) or the said coding gene or the said recombinant vector in any of the following:

[0037] (a1) preparing a product having penicillin expandase activity;

[0038] (a2) preparing G-7-ADCA.

[0039] The present application further provides a method for preparing G-7-ADCA, comprising: using the said PenG expandase, or a recombinant cell expressing the same, or a lysate of the recombinant cell, to catalyze a reaction with PenG as a substrate, to obtain G-7-ADCA.

[0040] Specifically, the method comprises the following steps: preparing the said PenG expandase, and using the said PenG expandase as a penicillin expandase to catalyze the ring expansion of penicillin G to generate G-7-ADCA; wherein the expandase mutant is used in the form of a culture solution or a composition (containing purified free enzyme, immobilized enzyme, whole cell of expandase, immobilized whole cell); preferably, the contact reaction between the PenG expandase and penicillin G is carried out in a solution, more preferably, the concentration of penicillin G is 1-500 mM, the amount of added expandase is 0.1-100 U / mL, the pH of the reaction mixture is between 5 and 8, the reaction time is 0.1 to 24 h, the reaction temperature is 4 to 60℃, and optionally, the prepared G-7-ADCA is separated and purified from the reaction mixture.

[0041] Specifically, a microorganism containing a coding gene encoding the said PenG expandase is fermented to obtain a fermentation product, which is used to catalyze the ring expansion of penicillin G to generate G-7-ADCA; preferably, a recombinant bacterium containing a coding gene encoding the said PenG expandase is fermented, and in the said recombinant bacterium, penicillin G is spontaneously catalyzed to biosynthesize G-7-ADCA.

[0042] The application produces a series of enzymes with ring expansion activity through ancestral enzyme sequence reconstruction, directed evolution and other technologies, which can expand the substrate penicillin G into G-7-ADCA in one step, and has improved conversion rate compared with the control enzyme or wild type enzyme. Therefore, these ring expansion enzymes have important application value in industry. BRIEF DESCRIPTION OF DRAWINGS

[0043] Figure 1 Reaction flow chart of 7-ADCA catalyzed by ring expansion enzyme. DETAILED DESCRIPTION

[0044] The experimental methods used in the following examples are conventional methods unless otherwise specified.

[0045] The materials, reagents and the like used in the following examples can be obtained from commercial channels unless otherwise specified.

[0046] Example 1, ancestral enzyme sequence reconstruction of ring expansion enzyme and preparation of genetically engineered strain

[0047] The main process of ancestral enzyme sequence reconstruction of ring expansion enzyme includes six steps of local amino acid database construction, gBlock analysis of conserved amino acid region, iq-Tree amino acid substitution model analysis, iq-Tree ML method phylogenetic tree construction, database sequence length equalization, and CodeML reconstruction of ancestral enzyme (PaML). Through calculation design and screening, 12 ancestral enzyme AD 1 to AD 12 sequences (SEQ ID NO: 1 to SEQ ID NO: 12) of ring expansion enzyme were obtained by taking the wild type of Streptomyces clavuligerus ring expansion enzyme as a template. AD 1 to AD 12 were synthesized and connected to the pET 24a expression vector digested by NdeI and XhoI, to obtain the recombinant expression vector pET 24a-AD 1~12.

[0048] AD1 Amino Acid Sequence (SEQ ID NO: 1): MDTTVPTFSLAELQQGLHQDEFRRCLRDKGLFYLTDCGLTDTELKSAKDIVIDFFE HGSEAEKRAVTSPVPTMRRGFTGLESESTAQITNTGSYSDYSMCYSMGTADNLFPSGDFERIWTQYFDRQYTASRAVAREVLRATGTEPDG GVEAFLDCEPLLRFRYFPQVPEHRSAEEQPLRMAPHYDLSMVTLIQQTPCANGFVSLQAEVGGAFTDLPYRPDAVLVFCGAIATLVTGGQV KAPRHHVAAPRRDQIAGSSRTSSVFFLRPNADFTFSVPLARECGFDVSLDGETATFQDWIGGNYVNIRRTSKA.

[0049] AD2 Amino Acid Sequence (SEQ ID NO: 2): MMDTTVPTFSLAELQEGLHQEEFRRCLAEKGVFYLTDSGLSEADHKSAKDVAVDFF EHGTEEEKRAVTSPVPTIRRGFTGLESESTAQITNTGSYTDYSMCYSMGTADNLFPSADFERIWTHYFDRMYTASREVARQVLKATGTEPE GGVEAFLDCEPLLRFRYFPEVPEHRSAEEQPLRMAPHYDLSMVTLIQQTPCANGFVSLQAEVGGTFVDLPARPDAVLVFCGAVATLVTGGQ VKAPRHHVAAPGRDQIAGSSRTSSVFFLRPNADFTFSVPLARECGFDVSLDGETATFQDWIGGNYVNIRRTSKA.

[0050] AD3 amino acid sequence (SEQ ID NO: 3): MDTTVPTFSLAELQEGLHQEEFRRCLREKGVFYLTDSGLSEADHKSAKDVAVDFFEHGTEAEKRAVTSPVPTIRRGFTGLESESTAQITNTGSYSDYSMCYSMGTADNLFPTADFERIWTHYFDRMYDASREVARQVLKATGTEPEG GVEAFLDCEPLLRFRYFPEVPEHRSAEEQPLRMAPHYDLSMVTLIQQTPCANGFVSLQAEVGGTFVDLPARPDAVLVFCGAVATLVTGGQV KAPRHHVAAPGRDQIAGSSRTSSVFFLRPNADFTFSVPLARECGFDVSLDGETATFQDWIGGNYVNIRRTSKA.

[0051] AD4 amino acid sequence (SEQ ID NO: 4): MMDTTVPTFSLAELQEGLHQEEFRRCLAEKGVFYLTDSGLSEADHKSAKDVAVDFFEHGTEEEKRAVTSPVPTIRRGFTGLESESTAQITNTGSYSDYSMCYSMGTADNLFPTADFERIWTHYFDRMYAASREVARQVLKATGTEPD GGVEAFVDCEPLLRFRYFPEVPEHRSAEEEPLRMAPHYDLSMVTLIQQTPCPNGFVSLQAEVGGTFVDLPARPDAVLVFCGAVATLVTGGQ VKAPRHHVAAPGRDQIAGSSRTSSVFFLRPNADFTFSVPLARECGFDISLDGETATFQDWIGGNYVNIRRTSKA.

[0052] AD5 amino acid sequence (SEQ ID NO: 5): MTDTTVPTFSLAELQEGLHQEEFRRCLREKGVFYLTDSGLSEADHASAKDVAVDFF EH GTEEEKKAVTTPIPTIRRGFTGLESESTAQITNTGTYTDYSMSYSMGTADNLFPSADFERIWTDYFDRMYTASREVARQVLKATGTEPE GGVDAFLDCEPLLRFRYFPEVPEHRSAEEQPLRMAPHYDLSIVTLIHQTPCANGFVSLQAEVGGTFVDLPARPDAVLVFCGAVATLVTGGR VKAPRHHVAAPGADQRVGSSRTSSVFFLRPNADFSFSVPLARECGFDVSLDGETATFQDWIGGNYVNIRRTSAA.

[0053] AD6 amino acid sequence (SEQ ID NO: 6): MDTTVPTFSLAELQEGLHQEEFRRCLSEKGVFYLTDTGLTEADHASAREVAVDFFE HGTEEEKKAVTSPIPTIRRGFTGLESESTAQITNTGKYSDYSMCYSMGTADNLFPTADFERIWTDYFDRMYAASQEVARQVLKAVGAEPEE GVDAFLDCEPLLRFRYFPEVPEDRVAEEQPLRMAPHYDLSIVTLIHQTPCANGFVSLQVEVDGSFVDLPARPGAVLVFCGAVATLVTGGRI KAPKHHVAAPGRDQRVGSSRTSSVFFLRPNADFSFSVPLARECGFDVSIPGETATFNDWIGGNYVNIRRTSAAR.

[0054] AD7 Amino Acid Sequence (SEQ ID NO: 7): MMDTTVPTFSLAELQEGLHQEEFRRCLAEKGVFYLTDSGLSEADHKSAKDVAVDFF EHGTEEEKRAVTSPVPTIRRGFTGLESESTAQITNTGSYTDYSMCYSMGTADNLFPTADFERIWTHYFDRMYAASREVARQVLKATGTEPD GGVEAFVDCEPLLRFRYFPEVPEHRSAEEEPLRMAPHYDLSTVTLIQQTPCPNGFVSLQAEVGGTFVDLPARPDAVLVFCGAVATLVTGGQ VKAPRHHVAAPGRDQIAGSSRTSSVFFLRPNADFTFSVPLARECGFDISLDGETATFQDWIGGNYVNIRRTTKA.

[0055] AD8 Amino Acid Sequence (SEQ ID NO: 8): MTDTTVPTFSLAELQKGVHQEEFRRCLTEKGVFYLTDSGLSEADHASATDAAMDFF EHGTEEEKKAVTTPIPTIRRGFSALESESTAQITNTGTYTDYSMSYSMGISDNLFPSARFERIWTDYFDRLYTASREVARAVLKATGTEPD GDVDAFLDCDPLLRFRYFPEVPEHRSAEQEPRRMAPHYDLSIVTLIHQTPCANGFVSLQAEVGGRFVDLPARPDAVVVFCGAVAPLVTGGR VKAPRHHVASPGADQRVGSSRTSSVFFLRPNADFSFSVPLARACGLDVSLDGETATFGDWIGGNYVNMRATTAP.

[0056] AD9 amino acid sequence (SEQ ID NO: 9): MTDTTVPTFSLTELQEGLHQEEFRRCLTEKGVFYLTDSGLSEADHASARDVAVDFF EHGTEEEKRAVTTPIPTIRRGFTGLESESTAQITNTGKYTDYSMCYSMGTADNLFPTPDFERIWTDYFDRMYAASREVARQVLKATGTEPDGGIDAFLDCEPLLRLRYFPEVPEDRVAEEEPLRMAPHYDLSIVTLIHQTPCANGFVSLQAEVGGTFVDLPARPDAVLVFCGAVATLVTGGRIKAPRHHVAAPGADQRVGSSRTSSVFFLRPNADFSFSVPLARECGFDVSLDGETATFNDWIGGNYVNIRRTTAARAGAEVAAAAPVSTAAPIAT.

[0057] AD10 amino acid sequence (SEQ ID NO: 10): MTDTTVPTFSLTELQAGLHQEEFRRCLTEKGVFYLTDSGLSEADHASARDVAVD FFEHGTEEEKKAVTTPIPTIRRGFSGLESESTAQITNTGKYTDYSMCYSMGTADNLFPTPDFERIWTDYFDRMYAASREVARQVLKATGAE PDGGIDAFLDCEPLLRLRYFPEVPEDRVAEEEPLRMAPHYDLSIVTLIHQTPCANGFVSLQVEVGGTFVDLPARPDAVLVFCGAVATLVTG GRIKAPRHHVAAPGADQRVGSSRTSSVFFLRPNADFSFSVPLARECGFDVSLPGETATFNDWIGGNYVNIRRTTAA.

[0058] AD11 amino acid sequence (SEQ ID NO: 11): MTDTTVPTFSLTELQEGLHQEEFRRCLTEKGVFYLTDSGLAEADHASARDVAVD FFEHGTEEEKRAVTTPIPTIRRGFSGLESESTAQITNTGKYTDYSMCYSMGTADNLFPTPDFERIWTDYFDRMYAASREVARQVLKATGAE PDGGIDAFLDCEPLLRLRYFPEVPEHRVAEEQPLRMAPHYDLSIVTLIHQTPCANGFVSLQVEVGGTFVDLPARPGAVLVFCGAVATLVTGGRIKAPRHHVAAPGADQRVGSSRTSSVFFLRPNADFSFSVPLARECGFDVSLDGETATFNDWIGGNYVNIRRTTAA.

[0059] AD12 amino acid sequence (SEQ ID NO: 12): MTDTTVPIFSLTELQKGLHQEEFRRCLTEKGVFYLTDSGLSEADHASARDTAVD FFEHGTEEEKRAVTTPIPTIRRGFSGLESESTAQITNTGKYTDYSMCYSMGISDNLFPTPDFERIWTDYFDRMYAASREVARAVLEATGAEPDGDIDAFLDCDPLLRLRYFPEVPEDRVAEEEPLRMAPHYDLSIVTLIHQTPCANGFVSLQAEVGGEFVDLPARPDAVVVFCGAVATLVTGGRIKAPRHHVASPGADQRVGSSRTSSVFFLRPNADFSFSVPLARECGFDVSLDGETATFGDWIGGNYVNMRRTTAARAGAERAAAAPVSTAAPIAT.

[0060] The above-mentioned recombinant expression vector is transformed into a suitable microbial host. The host microorganism is various host microorganisms which are conventional in the art, as long as it can meet the above-mentioned stable self-replication of the recombinant expression vector and the effective expression of the expandase ancestral enzyme gene. In the present embodiment, the aforementioned recombinant expression plasmid is introduced into E. coli BL21 (DE3) competent cells by electroporation, and is inverted and cultured in a LB solid plate containing kanamycin resistance for 12-16 h. The positive transformants are selected for DNA sequencing verification, and the correct transformants are verified as the expandase ancestral enzyme gene engineering strain.

[0061] Example 2, determination of whole-cell catalytic activity and thermal stability of expandase ancestral enzyme

[0062] The 12 ring-expanding enzyme ancestor enzymes were streaked on LB solid plates (with the ring-expanding enzyme from Streptomyces clavuligerus as a positive control, designated WT), and single colonies were picked from the plates and transferred to 5 ml of LB broth containing 5 μL of kanamycin (final concentration of 50 μg / mL) in test tubes, and incubated at 37 °C, 220 rpm for 8-10 h. The seed liquid was then transferred to TB medium containing Kan at a 2% inoculation amount, and incubated at 37 °C, 220 rpm for 2 h, after which IPTG was added, and the mixture was induced at 25 °C, 220 rpm for 12-14 h. The bacterial liquid in the shake flask was transferred to a 50 ml centrifuge tube, and centrifuged at 4 °C, 4000 rpm for 10 min to collect the bacteria, which were then washed and resuspended in PBS buffer (50 mM, pH 7.4). The bacterial cells were then diluted with PBS buffer at a ratio of 0.1 g / mL.

[0063] Whole-cell catalytic activity assay: The reaction system (1 ml) was 50 mM KH2PO4 / K2HPO4buffer (pH 7.4), 0.1 g / mL whole cells, Pen G (final concentration of 5 mM), α-ketoglutaric acid (final concentration of 5 mM), ascorbic acid (final concentration of 0.4 mM), ferrous sulfate (final concentration of 50 μg / mL), and the reaction was carried out in a capped incubator at 30 °C, 1000 rpm for 2 h, after which pure methanol was added in equal proportions to terminate the reaction. The mixture was centrifuged at 12000 rpm for 15 min, and the supernatant was analyzed by HPLC. The HPLC detection conditions were as follows: chromatographic column Agilent ZORBAX SB-C18 Stable Bond Analytical 4.6 x 250 mm; mobile phase 0.1% TFA water and pure methanol at a ratio of 50:50; flow rate 1 mL / min; detection wavelength 215 nm. The standard used was G-7-ADCA (Shandong Lu Kang Pharmaceutical Co., Ltd.), and a standard curve was prepared using G-7-ADCA. After the liquid phase detection was completed, the conversion rate was calculated according to the standard curve, and the conversion rate = 100% x P / (5 x 10-3), where P is the content of G-7-ADCA detected by liquid phase detection (mol / L). The results are shown in Table 1.

[0064] Thermal stability assay: The thermal stability of the ring-expanding enzyme ancestor enzymes was detected by differential scanning calorimetry (DSC). The protein concentration of the pure enzyme sample was determined by the Bradford kit, and the protein was adjusted to a final concentration of 1 mg / mL with potassium phosphate buffer (50 mM, pH 7.4). The protein sample and PBS buffer were then centrifuged at 4 °C, 12000 rpm for 10 min, and degassed for 10 min before detection. The DSC scan was performed at a temperature range of 20-100 °C at a heating rate of 1 °C / min. The results are shown in Table 1.

[0065] By screening, AD6, AD10 and AD11 with relatively good thermal stability and high activity were selected as the object of modification to further improve the activity.

[0066] Table 1, whole cell catalytic activity determination and thermal stability determination of ancestral enzymes

[0067]

[0068]

[0069] Example 3, directed evolution of expandase ancestral enzymes

[0070] By homology modeling and pocket amino acid analysis, a series of amino acids around the pocket were obtained, which were mutated to amino acids conducive to the binding of the substrate and the pocket amino acid. Therefore, the mutated amino acids were introduced into the expandase ancestral enzymes AD6, AD10 and AD11 to obtain the first round of single-point mutants, and then whole cell catalytic activity determination and HPLC screening were performed to screen the highest activity single-point mutant as the starting strain for the next round of combination mutation. The remaining sites were superimposed on the dominant single mutant, and then whole cell catalytic activity determination and screening were performed, and the cycle was repeated until all six sites were iteratively mutated. The results are shown in Table 2.

[0071] Table 2, screening results of directed evolution mutants of ancestral enzymes

[0072]

[0073]

[0074]

[0075]

[0076] In order to construct the above mutants, the following cloning was performed:

[0077] PCR to obtain the cloned fragment, the system is 25 μl: DNA template 1 μl (10 ng / μl), forward primer 1 μl (10 ng / μl), reverse primer 1 μl (10 ng / μl), DNA polymerase 12.5 μl, ddH2O 9.5 μl. PCR program is 94℃, 2 min, (98℃, 15 s; 60℃, 30 s; 68℃, 5 min) 30 cycles, 72℃, 7 min. Take 2 μl of the amplified PCR product for agarose gel electrophoresis, if the band is as expected, add 1 μl of Dpn I restriction endonuclease to the remaining PCR product, mix well and digest at 37℃ for 1 h, and then electroporate into E. coli competent BL21 (DE3). After recovery at 37℃ for 1 h at 220 rpm, spread on LB solid plates containing kanamycin and incubate at 37℃ for 12 h. Pick single colonies for sequencing identification. After successful construction of the mutant, it can be used for HPLC screening. The primers used for directed evolution mutation are shown in Table 3.

[0078] Table 3, list of ancestor enzyme mutation primers

[0079]

[0080]

[0081]

[0082]

[0083] Example 4, detection of whole cell activity and thermal stability of the advantage mutant of the expandase ancestor enzyme

[0084] The whole cell activity determination is shown in Example 2.

[0085] The three with the highest conversion rate were selected for testing. Thermal stability determination method: thermal stability detection uses DSC thermal analysis method (Differential Scanning Calorimeter). The protein concentration of the pure enzyme sample of the expandase ancestor enzyme was determined by the Bradford kit, and the protein final concentration was adjusted to 1 mg / mL with potassium phosphate buffer (50 mM, pH 7.4). Then the protein sample and PBS buffer were centrifuged at 12000 rpm for 10 min at 4℃, and degassed for 10 min before detection. The temperature range is 20-100℃, and the DSC scanning is performed at a temperature rising rate of 1℃ / min.

[0086] Table 4, whole cell activity and thermal stability of the optimal mutant of the ancestor enzyme

[0087] Mutant Pen G conversion (%) Tm(°C) WT 10.0 47.69 AD6-73T / 275I / 184H / 281Y / 305M 65.3 62.12 AD10-74T / 185H / 276I / 282Y / 306M 66.4 63.52 AD11-156Y / 185H / 276I / 282Y / 306M 60.21 59.99

[0088] In summary, the obtained expandase mutant AD6-73T / 275I / 184H / 281Y / 305M, AD10-74T / 185H / 276I / 282Y / 306M and AD11-156Y / 185H / 276I / 282Y / 306M, using the mutant as a catalyst, through whole cell reaction at 30 DEG C for 2h, the whole cell catalytic activity and stability are improved to different degrees compared with WT. Meanwhile, the application provides a method for mining new expandase enzymes, which provides a new idea for the modification of expandase and has industrial application potential.

Claims

1. A PenG ring expansion enzyme, characterized in that, Any one of the PenG expandase of AD 1 to AD12 is obtained by reconstitution of ancestral enzymes, and the corresponding amino acid sequences are shown in SEQ ID NO: 1 to SEQ ID NO: 12, respectively.

2. The PenG expandase enzyme of claim 1, wherein, On the basis of the amino acid sequence of the PenG expandase of AD6, at least one amino acid at positions 71, 73, 150, 155, 170, 184, 275, 280, 280, 292, 300 or 305 is replaced by another amino acid, and further, the amino acid sequence of AD6 is modified by any one, any two, any three, any four, any five or all of the following six modifications to obtain a protein with higher activity, higher conversion rate and / or better thermal stability relative to the starting PenG expandase: X1, the phenylalanine at position 71 of AD6 is mutated to glutamine; X2, the isoleucine at position 73 of AD6 is mutated to threonine; X3, the aspartic acid at position 150 of AD6 is mutated to valine; X4, the cysteine at position 155 of AD6 is mutated to tyrosine; X5, the aspartic acid at position 170 of AD6 is mutated to alanine; X6, the tyrosine at position 184 of AD6 is mutated to histidine; X7, the valine at position 275 of AD6 is mutated to isoleucine; X8, the glutamic acid at position 280 of AD6 is mutated to valine; X9, the cysteine at position 281 of AD6 is mutated to tyrosine; X10, the alanine at position 292 of AD6 is mutated to glutamic acid; X11, the glycine at position 300 of AD6 is mutated to aspartic acid; X12, the isoleucine at position 305 of AD6 is mutated to methionine.

3. The PenG expandase enzyme of claim 1, wherein, On the basis of the amino acid sequence of the PenG expandase of AD10, at least one amino acid at positions 72, 74, 151, 156, 171, 185, 276, 281, 282, 293, 301 and 306 is replaced by another amino acid, and further, the amino acid sequence of AD10 is modified by any one, any two, any three, any four, any five or all of the following six modifications to obtain a protein with higher activity, higher conversion rate and / or better thermal stability relative to the starting PenG expandase: X1, the isoleucine at position 74 of AD10 is mutated to threonine; X2, the cysteine at position 156 of AD10 is mutated to tyrosine; X3, the tyrosine at position 185 of AD10 is mutated to histidine; X4, the valine at position 276 of AD10 is mutated to isoleucine; X5, the cysteine at position 282 of AD10 is mutated to tyrosine; X6, the isoleucine at position 306 of AD10 is mutated to methionine.

4. The PenG expandase enzyme of claim 1, wherein, On the basis of the amino acid sequence of the PenG expander enzyme of AD11, at least one of positions 72, 74, 151, 156, 171, 185, 276, 281, 282, 293, 301 and 306 is substituted by another amino acid, and the amino acid sequence of AD11 is further modified by any one, any two, any three, any four, any five or all of the following six modifications to obtain a protein with higher activity, higher conversion rate and / or better thermal stability relative to the starting PenG expander enzyme: X1, the isoleucine at position 74 of AD11 is mutated to threonine; X2, the cysteine at position 156 of AD10 is mutated to tyrosine; X3, the tyrosine at position 185 of AD10 is mutated to histidine; X4, the valine at position 276 of AD10 is mutated to isoleucine; X5, the cysteine at position 282 of AD10 is mutated to tyrosine; X6, the isoleucine at position 306 of AD10 is mutated to methionine.

5. A coding gene comprising the PenG expander enzyme of any one of claims 1 to 4.

6. A recombinant vector or recombinant strain containing the coding gene of claim 5.

7. The recombinant bacterial strain of claim 6, wherein The starting bacterium is E. coli, more preferably it is E. coli BL21(DE3).

8. Use of the expander enzyme mutant of any one of claims 1 to 4 or the coding gene of claim 4 or the recombinant vector of claim 5 in any one of the following: (a1) preparation of a product with penicillin expander enzyme activity; (a2) preparation of G-7-ADCA.

9. A process for the preparation of G-7-ADCA comprising: Using the PenG expander enzyme of any one of claims 1 to 4 or the recombinant cells expressing the same or the lysate of the recombinant cells as catalyst, a catalytic reaction is carried out with PenG as substrate to obtain G-7-ADCA.

10. The process for the preparation of G-7-ADCA as claimed in claim 9 wherein, The method comprises the following steps: preparing the PenG expander enzyme of any one of claims 1 to 4, and using the PenG expander enzyme as the expander enzyme to catalyze the ring expansion of penicillin G to produce G-7-ADCA; wherein the PenG expander enzyme is used in the form of a culture solution or a composition (containing purified free enzyme, immobilized enzyme, expander enzyme mutant whole cells, immobilized whole cells); preferably, the contact reaction between the PenG expander enzyme and penicillin G is carried out in a solution, more preferably the concentration of penicillin G is 1-500 mM, the amount of expander enzyme added is 0.1-100 U / mL, the reaction mixture is at pH 5 to 8, the reaction time is 0.1 to 24 h, the reaction temperature is 4 to 60 ℃, and optionally, the prepared G-7-ADCA is separated and purified from the reaction mixture; Specifically, a microorganism containing a coding gene encoding the PenG expander enzyme of any one of claims 1 to 4 is fermented to obtain a fermentation product that catalyzes the ring expansion of penicillin G to produce G-7-ADCA; preferably, a recombinant bacterium containing a coding gene encoding the PenG expander enzyme of any one of claims 1 to 4 is fermented, and in the recombinant bacterium, penicillin G is spontaneously catalyzed to biosynthesize G-7-ADCA.

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