SELF-ASSEMBLY ENZYME SYSTEM SUPPLYING A-KETOGLUTARATE AND APPLICATION THEREOF IN CATALYTIC SYNTHESIS OF 4-HYDROXYISOLEUCINE
20250346934 ยท 2025-11-13
Inventors
Cpc classification
C12N9/0065
CHEMISTRY; METALLURGY
C12N9/0071
CHEMISTRY; METALLURGY
C12P13/06
CHEMISTRY; METALLURGY
C12N15/70
CHEMISTRY; METALLURGY
C12Y114/11
CHEMISTRY; METALLURGY
C12N9/0069
CHEMISTRY; METALLURGY
International classification
C12P13/06
CHEMISTRY; METALLURGY
Abstract
Disclosed are a self-assembly enzyme system supplying -ketoglutarate (-KG) and application thereof in catalytic synthesis of 4-hydroxyisoleucine. In the present disclosure, glutamate oxidase catalyzes glutamate to generate -KG, and catalase-peroxidase decomposes a byproduct H.sub.2O.sub.2. An interaction between RIAD and RIDD and a covalently linked combined state can mediate higher-order structures of various self-assembly enzymes. The LGOX/KatG self-assembly system is constructed through the affinity of short peptides in vitro to eliminate H.sub.2O.sub.2 in situ, thereby eliminating the inhibitory effect of H.sub.2O.sub.2 on Fe(II)/-KG DOs, and facilitating efficient and high-yield production of 4-HIL in a one-pot cascade reaction with IDO, with a highest yield up to 95% at a substrate concentration of 100 mM.
Claims
1. A self-assembly enzyme system, comprising a recombinant protein LGOX-RIAD and a recombinant protein KatG-RIDD; wherein the recombinant protein LGOX-RIAD is L-glutamate oxidase (LGOX) with a RIAD short peptide added to a C-terminal; the recombinant protein KatG-RIDD is catalase-peroxidase (KatG) with a RIDD short peptide added to a C-terminal; and the recombinant protein LGOX-RIAD and the recombinant protein KatG-RIDD are self-assembled through the RIAD short peptide and the RIDD short peptide.
2. The self-assembly enzyme system according to claim 1, wherein a stoichiometric ratio of the recombinant protein LGOX-RIAD to the recombinant protein KatG-RIDD is 1:2.
3. The self-assembly enzyme system according to claim 1, wherein the amino acid sequence of the LGOX is set forth in SEQ ID NO:4.
4. The self-assembly enzyme system according to claim 1, wherein the amino acid sequence of the KatG is set forth in SEQ ID NO:6.
5. The self-assembly enzyme system according to claim 1, wherein the amino acid sequence of the RIAD is set forth in SEQ ID NO:8.
6. The self-assembly enzyme system according to claim 1, wherein the amino acid sequence of the RIDD is set forth in SEQ ID NO:10.
7. The self-assembly enzyme system according to claim 1, wherein the LGOX is linked to the RIAD short peptide using a linker (GGGGS).sub.n, wherein n is 4, 5 or 6; and the KatG is linked to the RIDD short peptide using a linker (GGGGS).sub.n, wherein n is 4, 5 or 6.
8. The self-assembly enzyme system according to claim 1, wherein the LGOX is linked by (GGGGS).sub.4 and RIAD to obtain the recombinant protein LGOX-RIAD, and the KatG is linked by (GGGGS).sub.4 and RIDD to obtain the recombinant protein KatG-RIDD; or the LGOX is linked by (GGGGS).sub.6 and RIAD to obtain the recombinant protein LGOX-RIAD, and the KatG is linked by (GGGGS).sub.4 and RIDD to obtain the recombinant protein KatG-RIDD; or the LGOX is linked by (GGGGS).sub.4 and RIAD to obtain the recombinant protein LGOX-RIAD, and the KatG is linked by (GGGGS).sub.5 and RIDD to obtain the recombinant protein KatG-RIDD; or the LGOX is linked by (GGGGS).sub.4 and RIAD to obtain the recombinant protein LGOX-RIAD, and the KatG is linked by (GGGGS).sub.6 and RIDD to obtain the recombinant protein KatG-RIDD; or the LGOX is linked by(GGGGS).sub.5 and RIAD to obtain the recombinant protein LGOX-RIAD, and the KatG is linked by (GGGGS).sub.6 and RIDD to obtain the recombinant protein KatG-RIDD.
9. A method for multi-enzyme cascade conversion of (2S,3R,4S)-4-hydroxyisoleucine, wherein the method comprises using L-isoleucine and L-glutamic acid as starting substrates, and catalyzing the production of 4-HIL through a cascade reaction using the self-assembly enzyme system in claim 1 and L-isoleucine dioxygenase, employing the self-assembly enzyme system and L-isoleucine dioxygenase cascade catalysis to generate 4-HIL.
10. The method according to claim 9, wherein the multi-enzyme cascade adopts a conversion system for a two-step method or a one-pot method to convert and obtain (2S,3R,4S)-4-hydroxyisoleucine; wherein the two-step method comprises a first stage and a second stage, wherein in the first stage, monosodium L-glutamate is used as the substrate, and the self-assembly enzyme system is used as a catalyst to prepare and obtain a co-substrate -ketoglutarate (-KG); and in the second stage, the co-substrate -KG obtained in the first stage is used as a starting reaction solution, and L-isoleucine, FeSO.sub.4.Math.7H.sub.2O, L-ascorbic acid, and L-isoleucine dioxygenase are then added to prepare and obtain the (2S,3R,4S)-4-hydroxyisoleucine; and the one-pot method is to mix monosodium L-glutamate, L-isoleucine, FeSO.sub.4.Math.7H.sub.2O, L-ascorbic acid, L-isoleucine dioxygenase, and the self-assembly enzyme system together to prepare and obtain the (2S,3R,4S)-4-hydroxyisoleucine.
11. The method according to claim 10, wherein for the one-pot method, a concentration of the monosodium L-glutamate is 50-300 mM, a concentration of the L-isoleucine is 50-300 mM, a concentration of the FeSO.sub.4.Math.7H.sub.2O is 1-5 mM, a concentration of the L-ascorbic acid is 10-50 mM, and a concentration of the L-isoleucine dioxygenase is 0.1-1 mg mL.sup.1; and a concentration of the self-assembly enzyme system is 0.1-1 mg mL.sup.1.
12. The method according to claim 10, wherein for the one-pot method, a temperature is 25-35 C., a pH is 7.0-8.0, a conversion lasts for 2-9 hours, and a rotation speed is 200-400 rpm.
13. The method according to claim 10, wherein in the first stage of the two-step method, a concentration of the monosodium L-glutamate is 50-300 mM, and a concentration of the self-assembly enzyme system is 0.1-1 mg mL.sup.1; and in the second stage of the two-step method, a concentration of the L-isoleucine is 50-300 mM, a concentration of the FeSO.sub.4.Math.7H.sub.2O is 1-5 mM, a concentration of the L-ascorbic acid is 10-50 mM, and a concentration of the L-isoleucine dioxygenase is 0.1-1 mg.Math.mL.sup.1.
14. The method according to claim 10, wherein for the two-step method, a temperature is 25-35 C., and a pH is 7.0-8.0; and a conversion in the first stage lasts for 2-9 hours, a conversion in the second stage lasts for 2-9 hours, and a rotation speed is 200-400 rpm.
15. A product, comprising the self-assembly enzyme system of claim 1.
16. A preparation method for the self-assembly enzyme system of claim 1, comprising the following steps: (1) linking the gene encoding a recombinant protein LGOX-RIAD and the gene encoding a recombinant protein KatG-RIDD into expression vectors to obtain recombinant vectors, respectively; wherein the recombinant protein LGOX-RIAD is LGOX with a RIAD short peptide added to a C-terminal; and the recombinant protein KatG-RIDD is KatG with a RIDD short peptide added to a C-terminal; (2) converting the recombinant vectors obtained in the step (1) into Escherichia coli to obtain Escherichia coli expressing LGOX-RIAD and Escherichia coli expressing KatG-RIDD; respectively; (3) culturing, inducing expression, and purifying the Escherichia coli expressing LGOX-RIAD and the Escherichia coli expressing KatG-RIDD to obtain the recombinant protein LGOX-RIAD and the recombinant protein KatG-RIDD; and (4) mixing the recombinant protein LGOX-RIAD and the recombinant protein KatG-RIDD to obtain the self-assembly enzyme system.
17. The method according to claim 16, wherein in the step (1), the LGOX is linked to the RIAD short peptide using a linker (GGGGS).sub.n, wherein n is 4, 5 or 6; wherein the amino acid sequence of a basic unit (GGGGS) of the linker is set forth in SEQ ID NO: 12; and the KatG is linked to the RIDD short peptide using a linker (GGGGS).sub.n, wherein n is 4, 5 or 6; wherein the amino acid sequence of a basic unit (GGGGS) of the linker is set forth in SEQ ID NO:12.
18. The method according to claim 16, wherein the amino acid sequence of the LGOX is set forth in SEQ ID NO:4.
19. The method according to claim 16, wherein the amino acid sequence of the KatG is set forth in SEQ ID NO:6.
20. The method according to claim 16, wherein the amino acid sequence of the RIAD is set forth in SEQ ID NO:8, the amino acid sequence of the RIDD is set forth in SEQ ID NO:10, the expression vectors comprise pET28a, the Escherichia coli is Escherichia coli BL21(DE3); and wherein in the step (4), a stoichiometric ratio of the recombinant protein LGOX-RIAD to the recombinant protein KatG-RIDD is 1:2.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTIONS OF THE EMBODIMENTS
[0064] In order to make the objectives, technical solutions and advantages of the present disclosure clearer, the present disclosure will be further described in detail below in conjunction with the specific implementations with reference to the accompanying drawings.
[0065] The following examples detected various substances in the reaction liquid phase using high-performance liquid chromatography (HPLC), including the detection of amino acids and organic acids. The detection method is as follows:
[0066] Determination of amino acid content: a sample was subjected to derivatization reaction with a fluorenylmethoxycarbonyl chloride (Fmoc-Cl) reagent, a supernatant after centrifugation was diluted to an appropriate concentration, 250 L of the supernatant was taken, 250 L of 0.2 mM boric acid buffer (pH 9.2) was added to the supernatant, 500 L of 10 mM Fmoc-Cl was then added, and shaken well and reacted in a 25 C. metal bath for 10 minutes, 500 L of 40 mM 1-aminoadamantane was added to terminate the reaction, filtering was performed using a 0.22 m organic filter for liquid chromatography analysis. Detection conditions: a Diomansil C18 column was used, with a ultraviolet detection wavelength of 263 nm, a temperature of 25 C., a flow rate of 1 mL.Math.min.sup.1, and a sample injection volume of 10 L. Mobile Phase A: 50 mM NaAc-HAc buffer (pH 4.2)/acetonitrile at a volume ratio of 90:10; Mobile Phase B: 50 mM NaAc-HAc buffer (pH 4.2)/acetonitrile at a volume ratio of 20:80, and Mobile Phase A and B were used in a gradient elution program, with a flow rate of 1.0 mL/minute.
[0067] Determination of organic acid content: a supernatant was taken, centrifuged and diluted to an appropriate concentration, and then filtered through a 0.22 m aqueous filter for detection. A Waters T3 column was used, with an ultraviolet absorption wavelength of 210 nm, a column temperature of 40 C., a mobile phase of 20 mM KH.sub.2PO.sub.4 solution, pH 2.8, a flow rate of 0.8 mL.Math.min.sup.1, and an injection volume of 10 L.
[0068] Methods for enzyme activity assay in the following examples:
[0069] L-Isoleucine dioxygenase enzyme activity assay method: The activity of IDO was determined according to the amount of 4-HIL produced. An IDO enzyme activity assay solution (50 mM Tris-HCl, pH 7.0) containing 10 mM Ile, 0.5 mM FeSO.sub.4.Math.7H.sub.20, 10 mM ascorbic acid, and 10 mM -ketoglutaric acid with a final volume of 300 L (with an IDO concentration of 0.1 g L.sup.1) was incubated at 30 C. in an incubator (Eppendorf; Hamburg, Germany) at 800 rpm for 10 minutes, and 4-HIL content in the system was then measured. An amount of enzyme that catalyzed the generation of 1 mol of 4-HIL per minute was defined as one unit of IDO enzyme activity (U).
[0070] L-glutamate oxidase (LGOX) activity assay method: The activity of LGOX or LGOX-RIAD was determined according to the amount of -ketoglutarate (-KG) produced. An LGOX or LGOX-peptidase activity assay solution (50 mM Tris-HCl, pH 7.0) containing 10 mM L-Glu, and 1 mg L.sup.1 commercial catalase-peroxidase (KatG) with a final volume of 300 L (with an LGOX concentration of 0.1 g L.sup.1) was incubated at 30 C. at 800 rpm for 10 minutes, and a content of -ketoglutaric acid was then measured. The amount of enzyme used to release 1 mol of -KG per minute was defined as one unit of LGOX or LGOX-RIAD enzyme activity (U).
[0071] KatG activity assay method: The activity of KatG or KatG-RIDD was determined according to the consumption of H.sub.202. An enzyme activity assay solution 50 mM Tris-HCl, pH 7.0) containing 20 mM H.sub.2O.sub.2 with a final volume of 3 mL (with a KatG concentration of 0.1 g L.sup.1) was incubated at 30 C. at 200 rpm for 10 minutes, and an amount of reduction of H.sub.2O.sub.2 was detected using a UV spectrophotometer at 210 nm. The amount of enzyme used to decompose 1 mol of H.sub.2O.sub.2 per minute was defined as one unit of KatG or KatG-RIDD enzyme activity (U).
[0072] Strain Escherichia coli BL21(DE3) and plasmid pET28a were commercially available strains and plasmids.
[0073] The present disclosure will be further described below in conjunction with specific examples.
Example 1: This Example Illustrates the Expression and Purification Method of LGOX-RIAD and KatG-RIDD Fusion Proteins
1. Construction of Escherichia coli BL21(DE3)/pET-28-LGOX(pET-28a-LGOX-RIAD)
[0074] The LGOX and LGOX-linker-RIAD fusion protein fusion gene coding sequences containing enzyme restriction sites NcoI and XhoI were synthesized by a biotechnology company. Specifically, LGOX was the LGOX derived from Streptomyces ghanaensis (ATCC14672), and LGOX-RIAD was a fusion protein LGOX-linker-RIAD, that is, a linker was used to link the RIAD short peptide at a C-terminal of LGOX, and the linker was a flexible linker (GGGGS).sub.4-(GGGGS).sub.6 with different lengths. A nucleotide sequence of the gene encoding the LGOX was shown in SEQ ID NO:3, a nucleotide sequence of the gene encoding the short peptide RIAD was shown in SEQ ID NO:7, and a nucleotide sequence of the gene encoding a basic unit (GGGGS) of the linker was shown in SEQ ID NO:11. The synthesized gene fragments and the vector plasmid pET-28a were double-digested, and the synthesized gene fragments were ligated to a pET-28a plasmid using T4 ligase to obtain pET-28a-LGOX (pET-28a-LGOX-RIAD) expression plasmids with LGOX and linker lengths of (GGGGS).sub.4-(GGGGS).sub.6, respectively. The expression plasmids were then converted into an expression strain Escherichia coli BL21(DE3) to obtain recombinant Escherichia coli strains Escherichia coli BL21(DE3)/pET-28a-LGOX(pET-28a-LGOX-RIAD) expressing LGOX and LGOX-RIAD fusion proteins with different lengths of linker, that is, LGOX-RIAD (GGGGS).sub.3, LGOX-RIAD (GGGGS).sub.4, LGOX-RIAD (GGGGS).sub.5, and LGOX-RIAD (GGGGS).sub.6.
[0075] The free enzyme LGOX without connecting a short peptide and a linker was prepared in the same method using the nucleotide sequence of LGOX as shown in SEQ ID NO:3 as a control.
2. Construction of Escherichia coli BL21(DE3)/pET-28a-KatG(pET-28a-KatG-RIDD)
[0076] The KatG and KatG-linker-RIDD fusion protein fusion gene coding sequences containing enzyme restriction sites NcoI and XhoI were synthesized by a biotechnology company. Specifically, KatG was a catalase derived from Escherichia coli (W3110), and KatG-RIDD was a fusion protein KatG-linker-RIDD, that is, a linker was used to link the RIDD short peptide at a C-terminal of KatG, and the linker was a flexible linker of different lengths (GGGGS).sub.4-(GGGGS).sub.6. A nucleotide sequence of the gene encoding the KatG was shown in SEQ ID NO:5, a nucleotide sequence of the gene encoding the short peptide RIDD was shown in SEQ ID NO:9, and a nucleotide sequence of the gene encoding a basic unit (GGGGS) of the linker was shown in SEQ ID NO:11. The synthesized gene fragments and the vector plasmid pET-28a were double-digested, and the synthesized gene fragments were ligated to a pET-28a plasmid using T4 ligase to obtain pET-28a-KatG(pET-28a-KatG-RIDD) expression plasmids with KatG and linker lengths of (GGGGS).sub.4-(GGGGS).sub.6, respectively. The expression plasmids were then converted into an expression strain Escherichia coli BL21(DE3) to obtain recombinant Escherichia coli strains Escherichia coli BL21(DE3)/pET-28a-KatG(pET-28a-KatG-RIDD) expressing KatG and KatG-RIDD fusion proteins with different lengths of linker, that is, KatG-RIDD (GGGGS).sub.3, KatG-RIDD (GGGGS).sub.4, KatG-RIDD (GGGGS).sub.s, and KatG-RIDD (GGGGS).sub.6.
[0077] The free enzyme LGOX without connecting a short peptide and a linker was prepared in the same method using the nucleotide sequence of KatG as shown in SEQ ID NO:3 as a control.
3. Expression and Purification Method of LGOX-RIAD Fusion Protein
[0078] A method for preparing the recombinant pure protein enzyme was as follows:
[0079] (1) A single colony of Escherichia coli BL21(DE3)/pET-28a-LGOX-RIAD was picked from a plate with kanamycin and cultured continuously in 50 mL and 800 mL of LB medium (kanamycin 50 g.Math.mL.sup.1). When OD.sub.600 reached 0.6-0.8, isopropyl-o-D-thiogalactoside was added to a final concentration of 0.5 mM, and the protein expression was induced in a constant temperature shaker at 17 C. for 12-14 hours. A cultured bacterial solution was centrifuged at 4,600g for 10 minutes, a bacterial pellet was washed twice with 0.9% saline, and a wet bacterial pellet was then collected.
[0080] (2) The cell pellet was resuspended in a 20 mM Tris-HCl buffer (pH 7.5) containing 4 mM of 2-mercaptoethanol at a concentration of 0.1 mg L.sup.1, the resuspended cell pellet was then lysed by ultrasound in an ice-water bath, with working conditions as follows: power 40%, 2 seconds on, 3 seconds off, for 20 minutes. A supernatant was collected by centrifugation at 15,000g for 30 minutes, the supernatant was filtered through a 0.22 m filter and slowly injected into a His-trap nickel column (GE Healthcare, Little Chalfont, UK) using an AKTA AVANT 25 instrument for gradient elution to purify the target protein. An elution buffer for washing the impurity proteins contained 200 mM imidazole, 20 mM Tris, 300 mM NaCl, pH 7.5; and an elution buffer for washing the target protein contained 600 mM imidazole, 20 mM Tris, 300 mM NaCl, pH 7.5. The purified protein was then concentrated using an Amicon Ultra-15 centrifugal filter.
[0081] The purified protein was analyzed by SDS-PAGE, resulting bands were consistent with the theoretical molecular weight. The results are shown in
4. Expression and Purification Method of KatG-RIDD Fusion Protein
[0082] (1) A single colony of recombinant Escherichia coli BL21(DE3)/pET-28a-KatG-RIDD was picked from a plate with kanamycin and cultured continuously in 50 mL and 800 mL of LB medium (kanamycin 50 g.Math.mL.sup.1). When OD.sub.600 reached 0.6-0.8, isopropyl-o-D-thiogalactoside was added to a final concentration of 0.5 mM, and the protein expression was induced in a constant temperature shaker of 17-20 C. for 12-14 hours. A cultured bacterial solution was centrifuged at 4,600g for 10 minutes, a bacterial pellet was washed twice with 0.9% saline, and a wet bacterial pellet was then collected.
[0083] (2) The cell pellet was resuspended in a 20 mM Tris-HCl buffer (pH 7.5) containing 4 mM of 2-mercaptoethanol at a concentration of 0.1 mg L.sup.1, the resuspended cell pellet was then lysed by ultrasound in an ice-water bath, with working conditions as follows: power 40%, 2 seconds on, 3 seconds off, for 20 minutes. A supernatant was collected by centrifugation at 15,000g for 30 minutes, and the target protein was purified by gradient elution using a His-trap nickel column (GE Healthcare, Little Chalfont, UK) and AKTA AVANT 25. An elution buffer for washing the impurity proteins contained 80 mM imidazole, 20 mM Tris, 300 mM NaCl, pH 7.5; and an elution buffer for washing the target protein contained 300 mM imidazole, 20 mM Tris, 300 mM NaCl, pH 7.5. The purified protein was then concentrated using an Amicon Ultra-15 centrifugal filter. The purified protein was analyzed by SDS-PAGE, resulting bands were consistent with the theoretical molecular weight. The results are shown in
Example 2: This Example Illustrates the Expression and Purification Method of IDO Protein
1. Construction of Escherichia Coli BL21(DE3)/pET-28a-IDO
[0084] IDO fusion protein fusion gene coding sequences containing enzyme restriction sites NcoI and XhoI were synthesized by a biotechnology company. Specifically, an amino acid sequence of L-isoleucine dioxygenase (IDO) was shown in SEQ ID NO:2, a nucleotide sequence encoding the IDO was shown in SEQ ID NO:1, the synthesized gene fragments and the vector plasmid pET-28a were double-digested, and the synthesized gene fragments were ligated to a pET-28a plasmid using T4 ligase to obtain expression plasmids pET28a-IDO, the expression plasmids were then converted into an expression strain E. coli BL21(DE3) to recombinant Escherichia coli strains Escherichia coli BL21(DE3)/pET-28a-IDO.
2. Expression and Purification Method of IDO Protein
[0085] A method for preparing the recombinant pure protein enzyme was as follows:
[0086] The cell pellet was resuspended in a 20 mM Tris-HCl buffer (pH 7.5) at a concentration of 0.1 mg L.sup.1, the resuspended cell pellet was then lysed by ultrasound in an ice-water bath, with working conditions as follows: power 40%, 2 seconds on, 3 seconds off, for 20 minutes. A supernatant was collected by centrifugation at 15,000g for 30 minutes, and the target protein was purified by gradient elution using a His-trap nickel column (GE Healthcare, Little Chalfont, UK) and AKTA AVANT 25. An elution buffer for washing the impurity proteins contained 60 mM imidazole, 20 mM Tris, 280 mM NaCl, pH 7.5; and an elution buffer for washing the target protein contained 300 mM imidazole, 20 mM Tris, 300 mM NaCl, pH 7.5. The purified protein was then concentrated using an Amicon Ultra-15 centrifugal filter and to obtain IDO. The purified protein was analyzed by SDS-PAGE, resulting bands were consistent with the theoretical molecular weight. The results are shown in
[0087] Results of enzyme activity test of all enzymes in Example 1-2 are shown in Table 1.
TABLE-US-00001 TABLE 1 Enzyme activity results Enzyme activity Enzyme (U .Math. mg.sup.1) IDO 3.19 0.11 LGOX 1.90 0.09 LGOX-RIAD (GGGGS).sub.3 1.55 0.01 LGOX-RIAD (GGGGS).sub.4 1.78 0.10 LGOX-RIAD (GGGGS).sub.5 1.64 0.18 LGOX-RIAD (GGGGS).sub.6 1.56 0.07 KatG 5423.66 100.15 KatG-RIDD (GGGGS).sub.3 4646.84 670.48 KatG-RIDD (GGGGS).sub.4 4258.44 384.63 KatG-RIDD (GGGGS).sub.5 4866.78 223.85 KatG-RIDD (GGGGS).sub.6 4703.00 176.26
[0088] As shown in Table 1, the enzyme activities of LGOX and KatG after connecting short peptides reached more than 78% of the wild-type enzyme activity.
Example 3: Preparation of Self-Assembly Enzyme System
[0089] In this example, different self-assembly enzyme systems were prepared. In order to explore an optimal linker length, the assembly efficiency of LGOX-RIAD and KatG-RIDD fused with different linker lengths was analyzed.
[0090] The self-assembly enzyme systems were prepared as follows: The LGOX-RIAD and KatG-RIDD fusion enzymes with different linker lengths of fused short peptides prepared according to the method in Example 1 were mixed at a molar ratio of 1:2 to prepare a protein solution (that is, a multi-enzyme complex mixed solution), where a concentration of LGOX-RIAD was 1 mg.Math.mL.sup.1. Using the strong specific affinity between the RIAD-RIDD short peptides, the LGOX-RIAD and KatG-RIDD in the mixed solution achieved the self-assembly of LGOX and catalase, forming the self-assembly enzyme system.
[0091] As shown in Table 2, nine different combinations of the self-assembly enzyme systems were obtained, which was used as experimental groups. A mixed solution (LK) of free enzymes LGOX and KatG was used as a control group, and other conditions were the same as those of the experimental groups. The assembly status of enzyme complexes with different linker lengths of fused short peptides was analyzed using non-reducing SDS-PAGE (
TABLE-US-00002 TABLE 2 Different self-assembly enzyme systems obtained from nine combination methods LGOX-RIAD KatG-RIDD (GGGGS).sub.4 (GGGGS).sub.5 (GGGGS).sub.6 (GGGGS).sub.4 LK-1 LK-2 LK-3 (GGGGS).sub.5 LK-4 LK-5 LK-6 (GGGGS).sub.6 LK-7 LK-8 LK-9
Example 4 Application of Different Self-Assembly Enzyme Systems in Catalytic Generation of -KG
[0092] This example analyzes the reaction efficiency of assembled enzyme (that is, self-assembly enzyme system) of LGOX-RIAD and KatG-RIDD fused with different linker lengths.
[0093] In order to further explore the potential of each combination in producing-KG, the multi-enzyme complex mixed solutions from the nine combinations prepared in Example 3 were subjected to catalytic reaction for the production of -KG. The catalytic efficiency of the assembled enzymes was evaluated, and the mixed solution (LK) of free enzymes was used as a control. The results are shown in Table 3.
[0094] Specifically, a reaction system was 0.1 mg mL.sup.1 of LGOX (or LGOX-RIAD) and the corresponding two-fold molar amount of KatG (or KatG-RIDD), 50-300 mM (50, 100, 150, 200, 300 mM) of substrate monosodium L-glutamateand 50 mM of Tris-HCl buffer (pH 7.0), and the conversion was carried out at 200 rpm and 30 C. for 12 hours.
[0095] (1) The LK prepared in Example 3 was used to produce -KG. When a substrate concentration was 50 mM, an accumulated amount of -KG after 12 hours of conversion was 18.23 mM, with a molar yield of 36.47%; when a substrate concentration was 100 mM, an accumulated amount of -KG after 12 hours of conversion was 43.45 mM, with a molar yield of 43.45%; when a substrate concentration was 150 mM, an accumulated amount of -KG after 12 hours of conversion was 13.09 mM, with a molar yield of 8.72%; when a substrate concentration was 200 mM, an accumulated amount of -KG after 12 hours of conversion was 17.10 mM, with a molar yield of 8.55%; when a substrate concentration was 300 mM, an accumulated amount of -KG after 12 hours of conversion was 17.95 mM, with a molar yield of 5.99%;
[0096] (2) The LK-1 prepared in Example 3 was used to produce -KG. When a substrate concentration was 50 mM, an accumulated amount of -KG after 12 hours of conversion was 47.24 mM, with a molar yield of 94.48%; when a substrate concentration was 100 mM, an accumulated amount of -KG after 12 hours of conversion was 99.24 mM, with a molar yield of 99.24%; when a substrate concentration was 150 mM, an accumulated amount of -KG after 12 hours of conversion was 83.03 mM, with a molar yield of 55.35%; when a substrate concentration was 200 mM, an accumulated amount of -KG after 12 hours of conversion was 70.20 mM, with a molar yield of 35.10%; when a substrate concentration was 300 mM, an accumulated amount of -KG after 12 hours of conversion was 53.62 mM, with a molar yield of 17.87%;
[0097] (3) The LK-2 prepared in Example 3 was used to produce -KG. When a substrate concentration was 50 mM, an accumulated amount of -KG after 12 hours of conversion was 3.00 mM, with a molar yield of 6.00%; when a substrate concentration was 100 mM, an accumulated amount of -KG after 12 hours of conversion was 3.61 mM, with a molar yield of 36.07%; when a substrate concentration was 150 mM, an accumulated amount of -KG after 12 hours of conversion was 6.44 mM, with a molar yield of 4.30%; when a substrate concentration was 200 mM, an accumulated amount of -KG after 12 hours of conversion was 0.88 mM, with a molar yield of 0.44%; when a substrate concentration was 300 mM, an accumulated amount of -KG after 12 hours of conversion was 8.39 mM, with a molar yield of 2.80%;
[0098] (4) The LK-3 prepared in Example 3 was used to produce -KG. When a substrate concentration was 50 mM, an accumulated amount of -KG after 12 hours of conversion was 35.64 mM, with a molar yield of 48.62%; when a substrate concentration was 100 mM, an accumulated amount of -KG after 12 hours of conversion was 21.48 mM, with a molar yield of 21.48%; when a substrate concentration was 150 mM, an accumulated amount of -KG after 12 hours of conversion was 16.14 mM, with a molar yield of 10.76%; when a substrate concentration was 200 mM, an accumulated amount of -KG after 12 hours of conversion was 20.39 mM, with a molar yield of 10.20%; when a substrate concentration was 300 mM, an accumulated amount of -KG after 12 hours of conversion was 43.54 mM, with a molar yield of 13.51%;
[0099] (5) The LK-4 prepared in Example 3 was used to produce -KG. When a substrate concentration was 50 mM, an accumulated amount of -KG after 12 hours of conversion was 35.64 mM, with a molar yield of 71.28%; when a substrate concentration was 100 mM, an accumulated amount of -KG after 12 hours of conversion was 19.47 mM, with a molar yield of 19.47%; when a substrate concentration was 150 mM, an accumulated amount of -KG after 12 hours of conversion was 23.94 mM, with a molar yield of 15.96%; when a substrate concentration was 200 mM, an accumulated amount of -KG after 12 hours of conversion was 22.46 mM, with a molar yield of 11.23%; when a substrate concentration was 300 mM, an accumulated amount of -KG after 12 hours of conversion was 27.95 mM, with a molar yield of 9.32%;
[0100] (6) The LK-5 prepared in Example 3 was used to produce -KG. When a substrate concentration was 50 mM, an accumulated amount of -KG after 12 hours of conversion was 2.38 mM, with a molar yield of 4.77%; when a substrate concentration was 100 mM, an accumulated amount of -KG after 12 hours of conversion was 6.21 mM, with a molar yield of 6.21%; when a substrate concentration was 150 mM, an accumulated amount of -KG after 12 hours of conversion was 3.17 mM, with a molar yield of 2.11%; when a substrate concentration was 200 mM, an accumulated amount of -KG after 12 hours of conversion was 8.37 mM, with a molar yield of 4.19%; when a substrate concentration was 300 mM, an accumulated amount of -KG after 12 hours of conversion was 4.92 mM, with a molar yield of 1.64%;
[0101] (7) The LK-6 prepared in Example 3 was used to produce -KG. When a substrate concentration was 50 mM, an accumulated amount of -KG after 12 hours of conversion was 1.40 mM, with a molar yield of 2.81%; when a substrate concentration was 100 mM, an accumulated amount of -KG after 12 hours of conversion was 12.02 mM, with a molar yield of 12.02%; when a substrate concentration was 150 mM, an accumulated amount of -KG after 12 hours of conversion was 13.09 mM, with a molar yield of 8.73%; when a substrate concentration was 200 mM, an accumulated amount of -KG after 12 hours of conversion was 4.66 mM, with a molar yield of 2.33%; when a substrate concentration was 300 mM, an accumulated amount of -KG after 12 hours of conversion was 7.40 mM, with a molar yield of 2.47%;
[0102] (8) The LK-7 prepared in Example 3 was used to produce -KG. When a substrate concentration was 50 mM, an accumulated amount of -KG after 12 hours of conversion was 13.73 mM, with a molar yield of 27.36%; when a substrate concentration was 100 mM, an accumulated amount of -KG after 12 hours of conversion was 18.10 mM, with a molar yield of 18.10%; when a substrate concentration was 150 mM, an accumulated amount of -KG after 12 hours of conversion was 17.18 mM, with a molar yield of 11.45%; when a substrate concentration was 200 mM, an accumulated amount of -KG after 12 hours of conversion was 12.87 mM, with a molar yield of 6.44%; when a substrate concentration was 300 mM, an accumulated amount of -KG after 12 hours of conversion was 21.09 mM, with a molar yield of 7.03%;
[0103] (9) The LK-8 prepared in Example 3 was used to produce -KG. When a substrate concentration was 50 mM, an accumulated amount of -KG after 12 hours of conversion was 14.22 mM, with a molar yield of 28.45%; when a substrate concentration was 100 mM, an accumulated amount of -KG after 12 hours of conversion was 22.39 mM, with a molar yield of 22.39%; when a substrate concentration was 150 mM, an accumulated amount of -KG after 12 hours of conversion was 17.17 mM, with a molar yield of 11.45%; when a substrate concentration was 200 mM, an accumulated amount of -KG after 12 hours of conversion was 15.87 mM, with a molar yield of 7.94%; when a substrate concentration was 300 mM, an accumulated amount of -KG after 12 hours of conversion was 13.94 mM, with a molar yield of 4.65%;
[0104] (10) The LK-9 prepared in Example 3 was used to produce -KG. When a substrate concentration was 50 mM, an accumulated amount of -KG after 12 hours of conversion was 10.23 mM, with a molar yield of 2.46%; when a substrate concentration was 100 mM, an accumulated amount of -KG after 12 hours of conversion was 13.57 mM, with a molar yield of 13.57%; when a substrate concentration was 150 mM, an accumulated amount of -KG after 12 hours of conversion was 15.23 mM, with a molar yield of 10.15%; when a substrate concentration was 200 mM, an accumulated amount of -KG after 12 hours of conversion was 14.96 mM, with a molar yield of 7.48%; when a substrate concentration was 300 mM, an accumulated amount of ax-KG after 12 hours of conversion was 4.17 mM, with a molar yield of 1.390%; and
[0105] LK-1 exhibited strong catalytic activity at all substrate concentrations. When the substrate concentrations were 50 mM and 100 mM, LK-1 achieved a yield of >800 within 6 hours, and >90% after 12 hours. In addition, LK-3 and LK-4 showed superior catalytic efficiency over other combinations and the control group across all substrate concentration gradients. The catalytic efficiency of LK-7 and LK-8 had more obvious catalytic advantage compared with LK-2, LK-5, LK-6, and LK-9. Preferably, LK-1, LK-3, LK-4, LK-7 and LK-8 were used for in vitro coupling reaction with IDO.
TABLE-US-00003 TABLE 3 Molar yield of -KG at different enzymes and substrate concentrations Substrate concentration Enzyme 50 mM 100 mM 150 mM 200 mM 300 mM LK 36.47% 43.45% 8.72% 8.55% 5.99% LK-1 94.48% 99.24% 55.35% 35.10% 17.87% LK-2 6.00% 36.07% 4.30% 0.44% 2.80% LK-3 48.62% 21.48% 10.76% 10.20% 13.51% LK-4 71.28% 19.47% 15.96% 11.23 9.32% LK-5 4.77% 6.21% 2.11% 4.19% 1.64% LK-6 2.81% 12.02% 8.73% 2.33% 2.47% LK-7 27.36% 18.10% 11.45% 6.44% 7.03% LK-8 28.45% 22.39% 11.45% 7.94% 4.65% LK-9 20.46% 13.57% 10.15% 7.48% 1.39%
Example 5: Synthesis of 4-HILvia a Two-Step Method Using Different Self-Assembly Enzyme Systems
[0106] This example illustrates the two-step method for synthesizing 4-HIL, involving in vitro L-isoleucine dioxygenase and LGOX-KatG assembled enzyme. The results are shown in
[0107] The L-isoleucine dioxygenase obtained in Example 2 and the assembled enzyme of the multi-enzyme assembly combination screened in Example 4 were used to synthesize 4-HIL by the two-step method in vitro, and the mixed solution LK of the free enzyme was used as a control group. The results are shown in Table 4.
[0108] The conversion system as divided into two modules and then reacted in two steps: an a-KG generation module and a dioxygenase hydroxylation reaction module, where the -KG generation module included 100 mM L-Glu, 0.5 g L.sup.1 LGOX (or LGOX-RIAD), and a corresponding two-fold molar concentration of KatG (or KatG-RIDD), 50 mM Tris-HCl buffer (pH 8.0), which were converted at 200 rpm and 30 C. for 7 hours, and then boiled and inactivated for a next-step reaction; and the dioxygenase hydroxylation reaction module was as follows: 100 mM L-Ile, 5 mM FeSO.sub.4, 50 mM Vc, 1 g L.sup.1 IDO, with pH adjusted to 7.0, were added based on the -KG generation module (providing -KG as a co-substrate for the hydroxylation reaction), and then converted at 200 rpm and 30 C. for 7 hours.
[0109] (1) The L-isoleucine dioxygenase obtained in Example 2 and the free LGOX and KatG obtained in Example 1 were catalyzed at the same concentrations as that of the above-mentioned conversion system. After a two-step biocatalytic conversion for a total of 14 hours, a yield of the target product 4-HIL was 88%, with a space-time yield of 0.92 g L.sup.1 h.sup.1.
[0110] (2) The L-isoleucine dioxygenase obtained in Example 2 and the LK-1 screened in Example 4 were subjected to a two-step biocatalytic conversion for a total of 14 hours, resulting in a molar yield of 91.82% for the target product 4-HIL, with a space-time yield of 0.97 g L.sup.1 h.sup.1.
[0111] (3) The L-isoleucine dioxygenase obtained in Example 2 and the LK-3 screened in Example 4 were subjected to a two-step biocatalytic conversion for a total of 14 hours, resulting in a molar yield of 77.21% for the target product 4-HIL, with a space-time yield of 0.81 g L.sup.1 h.sup.1.
[0112] (4) The L-isoleucine dioxygenase obtained in Example 2 and the LK-4 screened in Example 4 were subjected to a two-step biocatalytic conversion for a total of 14 hours, resulting in a molar yield of 74.41% for the target product 4-HIL, with a space-time yield of 0.78 g L.sup.1 h.sup.1.
[0113] (5) The L-isoleucine dioxygenase obtained in Example 2 and the LK-7 screened in Example 4 were subjected to a two-step biocatalytic conversion for a total of 14 hours, resulting in a molar yield of 15.93% for the target product 4-HIL, with a space-time yield of 0.17 g L.sup.1 h.sup.1.
[0114] (6) The L-isoleucine dioxygenase obtained in Example 2 and the LK-8 screened in Example 4 were subjected to a two-step biocatalytic conversion for a total of 14 hours, resulting in a molar yield of 37.26% for the target product 4-HIL, with a space-time yield of 0.39 g L.sup.1 h.sup.1.
TABLE-US-00004 TABLE 4 Effect of different enzymes on the synthesis of 4-HILvia the two-step method Enzyme Molar yield Space-time yield LK 88% 0.92 g L.sup.1 h.sup.1 LK-1 91.82% 0.97 g L.sup.1 h.sup.1 LK-3 77.21% 0.81 g L.sup.1 h.sup.1 LK-4 74.41% 0.78 g L.sup.1 h.sup.1 LK-7 15.93% 0.17 g L.sup.1 h.sup.1 LK-8 37.26% 0.39 g L.sup.1 h.sup.1
Example 6 Synthesis of 4-HILvia a One-Pot Method Using Different Self-Assembly Enzyme Systems
[0115] This example illustrates the one-pot method for synthesizing 4-HIL involving in vitro L-isoleucine dioxygenase and LGOX-KatG assembled enzyme. The results are shown in
[0116] The L-isoleucine dioxygenase obtained in Example 2 and the assembled enzyme of the multi-enzyme assembly combination screened in Example 4 were used to synthesize 4-HIL by the one-pot method in vitro, and the mixed solution LK of the free enzyme was used as a control group. A conversion system was 100 mM L-Glu, 0.5 g L.sup.1 LGOX (or LGOX-RIAD) and a corresponding two-fold molar concentration of KatG (or LGOX-RIAD), 100 mM L-Ile, 5 mM FeSO.sub.4, 50 mM Vc and 1 g L.sup.1 IDO, and the conversion was carried out in 50 mM Tris-HCl buffer (pH 7.0) at 200 rpm and 30 C. for 7 hours.
[0117] (1) The L-isoleucine dioxygenase obtained in Example 2 and the free LGOX and KatG obtained in Example 1 were catalyzed at the same concentrations as that of the above-mentioned conversion system. After a biocatalytic conversion for a total of 7 hours, a yield of the target product 4-HIL was 4.14%, with a space-time yield of 0.09 g L.sup.1 h.sup.1.
[0118] (2) The L-isoleucine dioxygenase obtained in Example 2 and the LK-1 screened in Example 4 were subjected to a biocatalytic conversion for a total of 7 hours, resulting in a molar yield of 50.21% for the target product 4-HIL, with a space-time yield of 1.06 g L.sup.1 h.sup.1.
[0119] (3) The L-isoleucine dioxygenase obtained in Example 2 and the LK-3 screened in Example 4 were subjected to a biocatalytic conversion for a total of 7 hours, resulting in a molar yield of 50.69% for the target product 4-HIL, with a space-time yield of 1.07 g L.sup.1h.sup.1.
[0120] (4) The L-isoleucine dioxygenase obtained in Example 2 and the LK-4 screened in Example 4 were subjected to a biocatalytic conversion for a total of 7 hours, resulting in a molar yield of 65.25% for the target product 4-HIL, with a space-time yield of 1.38 g L.sup.1 h.sup.1.
[0121] (5) The L-isoleucine dioxygenase obtained in Example 2 and the LK-7 screened in Example 4 were subjected to a biocatalytic conversion for a total of 7 hours, resulting in a molar yield of 98.16% for the target product 4-HIL, with a space-time yield of 2.07 g L.sup.1 h.sup.1.
[0122] (6) The L-isoleucine dioxygenase obtained in Example 2 and the LK-8 screened in Example 4 were subjected to a biocatalytic conversion for a total of 7 hours, resulting in a molar yield of 92.57% for the target product 4-HIL, with a space-time yield of 1.95 g L.sup.1 h.sup.1.
[0123] The LGOX-KatG multi-enzyme self-assembly system mediated by the IAD-RlDD short peptides can efficiently produce ax-KG and simultaneously eliminate the byproduct H.sub.2O.sub.2 produced by LGOX catalysis in situ, thereby efficiently supplying the essential co-substrate -KG for Fe(II)/ai-KG-dependent dioxygenases in one pot, and promoting a C-H hydroxylation reaction catalyzed by dioxygenases.
TABLE-US-00005 TABLE 5 Effect of different enzymes on the synthesis of 4-HILvia the one-pot method Enzyme Molar yield Space-time yield LK 4.14% 0.09 g L.sup.1 h.sup.1 LK-1 50.21% 1.06 g L.sup.1 h.sup.1 LK-3 50.69% 1.07 g L.sup.1 h.sup.1 LK-4 65.25% 1.38 g L.sup.1 h.sup.1 LK-7 98.16% 2.07 g L.sup.1 h.sup.1 LK-8 92.57% 1.95 g L.sup.1 h.sup.1
Comparative Example 1
[0124] According to the method described in Example 1, the C-terminal of LGOX was linked to the short peptides RIAD and RIDD respectively through a (GGGGS).sub.3 linker, and the C-terminal of KatG was linked to the short peptides RIAD and RTDD respectively through a (GGGGS).sub.3 linker, and the crude enzyme activity was measured. The enzymatic activity of resulting crude enzymes was measured. Results are shown in Table 6. SDS-PAGE analysis was also performed, results are shown in
TABLE-US-00006 TABLE 6 Crude enzyme activity Crude enzyme activity Enzyme (mol .Math. mg.sup.1 .Math. min.sup.1) LGOX 30.41 1.84 LGOX-RIAD 25.5 2.59 LGOX-RIAD 6.87 1.50 KatG 286.15 11.65 KatG-RIAD 318.25 35.25 KatG-RIDD 323.95 34.35
[0125] As shown in Table 3 and
[0126] The above descriptions are merely preferred and feasible embodiments of the present disclosure and are not intended to limit the present disclosure, the present disclosure is not limited to the above examples, and variations, modifications, additions or substitutions made by those skilled in the art within the essential scope of the present disclosure are further within the protective scope of the present disclosure.