Method for catalyzing naphthalene to generate 1-naphthol and 2-naphthol by enzyme-linked system
By using an enzyme-linked system composed of P450 mutant enzyme and glucose-6-phosphate dehydrogenase, the problems of insufficient enzyme selectivity and activity and poor stability in the prior art were solved, and efficient catalytic naphthalene generation 1-naphthol and 2-naphthol reduces production costs and has significant industrial application potential.
Patent Information
- Application Number
- CN202411620322.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-14
- Publication Date
- 2025-02-25
AI Technical Summary
The prior art has problems such as insufficient enzyme selectivity and activity, poor stability, low coenzyme regeneration efficiency and insufficient reaction conditions when catalyzing the formation of 1-naphthol and 2-naphthol, which leads to increased the difficulty of isomers and the difficulty of separation and purification. Cost, and the decline in activity in industrial applications, limits the potential for industrial-scale applications.
The NADPH cycle is achieved by recombinantly expressing the P450 mutant enzyme and forming a stable enzyme-linked system with G6PD. Regeneration improves reactive activity and catalytic performance.
It significantly improves the activity and selectivity of catalytic naphthalene generation of 1-naphthol and 2-naphthol, reduces the consumption of NADPH, reduces production costs, improves the economic and sustainable reactions, and has good industrial application potential.
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Figure CN119506236A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of bioengineering, in particular to a method for generating 1-naphthol and 2-naphthol by catalyzing naphthalene with an enzyme-linked system. Background Art
[0002] Cytochrome P450 enzymes are a class of heme proteins that are widely present in organisms and are involved in the metabolism of a variety of endogenous and exogenous compounds. The typical reaction catalyzed by P450 enzymes is a monooxygenase reaction, in which the substrate is oxidized and molecular oxygen is reduced to water. This type of enzyme has always been an important tool in scientific research and drug development due to its important role in organisms and its potential in biotechnology applications.
[0003] Glucose-6-phosphate dehydrogenase (G6PD) is a key enzyme in the pentose phosphate pathway, which is an important branch of glucose metabolism. G6PD is involved in the metabolism of glucose and D-glucose-6-phosphate and NADP. + The reaction generates NADPH, which serves as an important reduced coenzyme and provides electrons in many biosynthetic reactions.
[0004] 1-Naphthol and 2-naphthol are important organic chemical raw materials and synthetic intermediates, widely used in dyes, medicines, pesticides, fragrances, rubber additives and other fields. Traditional methods for producing 1-naphthol and 2-naphthol, such as sulfonation-alkali fusion and gas-phase sulfonation, usually involve multi-step chemical reactions, use harmful chemicals, generate a large amount of waste, and have high energy consumption and low efficiency, causing serious pollution to the environment.
[0005] In the field of biocatalysis, although there have been studies exploring the use of related enzymes to convert naphthalene into 1-naphthol and 2-naphthol, there are many deficiencies and defects in the current related research, such as insufficient selectivity and activity of the enzyme: the existing enzymes may have low regioselectivity in the process of converting naphthalene into 1-naphthol and 2-naphthol, resulting in the generation of multiple isomers, which increases the difficulty and cost of subsequent separation and purification; poor enzyme stability and reusability: in industrial applications, enzyme stability and reusability are key factors. At present, the activity of some P450 enzymes decreases after long-term reaction or multiple cycles, limiting their potential for industrial-scale application; low coenzyme regeneration efficiency: in many P450 enzyme-catalyzed reactions, there is no regeneration of coenzymes such as NADPH or the regeneration efficiency is low, resulting in increased reaction costs and may limit the continued progress of the reaction; insufficient optimization of reaction conditions: existing studies may not fully optimize reaction conditions, such as temperature, pH value, substrate and enzyme concentration, to achieve the highest conversion rate and yield; for this purpose, a method for catalyzing naphthalene to generate 1-naphthol and 2-naphthol using an enzyme-linked system is provided. Summary of the invention
[0006] The purpose of the present invention is to provide a method for catalyzing naphthalene to produce 1-naphthol and 2-naphthol by an enzyme-linked system in view of the defects of the prior art, so as to solve the problems raised by the above-mentioned background technology.
[0007] To achieve the above-mentioned purpose, the present invention provides a P450 mutant enzyme or an enzyme-linked system composed of the mutant enzyme and glucose-6-phosphate dehydrogenase (G6PD), and an efficient biocatalytic method for catalyzing the production of 1-naphthol and 2-naphthol using naphthalene as a substrate. At the same time, the system realizes the cyclic regeneration of NADPH by recombinantly expressing the P450 mutant enzyme and forming a stable enzyme-linked system with G6PD, thereby reducing the consumption of NADPH in the reaction system and improving the reaction activity. Compared with the chemical synthesis method, the biocatalytic method of the present invention has the advantages of simple operation, environmental friendliness, high safety and significant cost-effectiveness, and is expected to replace the traditional chemical synthesis method in industrial production.
[0008] The present invention provides the following technical solution: an enzyme-linked system, which consists of glucose-6-phosphate dehydrogenase and P450 mutant enzyme.
[0009] A method for catalyzing naphthalene to produce 1-naphthol and 2-naphthol by an enzyme-linked system, the specific steps are as follows:
[0010] Step 1, mixing naphthalene, NADPH, P450 enzyme mutant, glucose-6-phosphate dehydrogenase, D-glucose-6-phosphate, and phosphate buffer for enzymatic reaction;
[0011] Step 2: The reaction is carried out at a temperature of 25-35° C. and a pH value of 7.0-9.0 for 2-8 hours to catalyze naphthalene to obtain 1-naphthol and 2-naphthol, and the molar ratio of the P450 mutant enzyme to naphthalene is 1:1-200.
[0012] As a preferred technical solution of the present invention, the concentration of NADPH is 0.1-2mM.
[0013] An application of a P450 mutant enzyme, wherein the P450 mutant enzyme is used to catalyze the conversion of naphthalene into 1-naphthol and 2-naphthol, and the specific steps are as follows:
[0014] a. mixing naphthalene, NADPH, P450 mutant enzyme, and phosphate buffer for enzymatic reaction;
[0015] b. The reaction is carried out at a temperature of 25-35° C. and a pH value of 7.0-9.0 for 2-8 hours to catalyze naphthalene to obtain 1-naphthol and 2-naphthol, and the molar ratio of the P450 mutant enzyme to naphthalene is 1:1-200.
[0016] As a preferred technical solution of the present invention, the amino acid sequence of the P450 mutant enzyme is selected from SEQ ID NO.1, and the alanine point mutation at position 82 is converted to phenylalanine.
[0017] As a preferred technical solution of the present invention, the method for preparing the P450 mutant enzyme comprises the following steps:
[0018] S1: construct a gene recombination vector containing SEQ ID NO.1 sequence and make a point mutation at position 82;
[0019] S2: The mutated recombinant vector is transferred into Escherichia coli and cultured to obtain a recombinant Escherichia coli bacterial liquid;
[0020] S3: Lysing the recombinant Escherichia coli bacterial solution to obtain a P450 mutant enzyme solution, wherein the mutant enzyme has improved hydroxylation activity and selectivity.
[0021] Compared with the prior art, the present invention has the following beneficial effects:
[0022] The present invention adopts a P450 mutant enzyme or an enzyme-linked system consisting of the mutant enzyme and glucose-6-phosphate dehydrogenase (G6PD) to catalyze naphthalene to generate 1-naphthol and 2-naphthol, and has the following specific advantages:
[0023] 1. The P450 mutant enzyme used in the present invention has its amino acid sequence optimized at a specific position (such as position 82), which significantly improves the hydroxylation activity and regioselectivity of naphthalene, thereby enhancing the catalytic performance.
[0024] 2. Through the action of glucose-6-phosphate dehydrogenase (G6PD) in the enzyme-linked system, the recycling regeneration of NADPH is achieved, which effectively reduces the consumption of NADPH during the reaction, reduces production costs, and improves the economy and sustainability of the reaction.
[0025] 3. The P450 mutant enzyme or the enzyme-linked system composed of the mutant enzyme and glucose-6-phosphate dehydrogenase (G6PD) in the present invention has good catalytic activity and good regional selectivity for para-hydroxylation, and has the significant advantages of small enzyme dosage, low enzyme preparation cost, short reaction time and good product yield, which can greatly reduce the production cost of 1-naphthol and 2-naphthol and has strong industrial application potential. BRIEF DESCRIPTION OF THE DRAWINGS
[0026] Figure 1 :HPLC diagram of 2-naphthol at 1mM concentration;
[0027] Figure 2 :HPLC diagram of 1-naphthol at 1mM concentration;
[0028] Figure 3:HPLC diagram of the production of 1-naphthol and 2-naphthol by naphthalene catalyzed by P450 mutant enzymes;
[0029] Figure 4 :HPLC diagram of the enzyme-linked system composed of P450 mutant enzyme and G6PD catalyzing naphthalene to produce 1-naphthol and 2-naphthol. DETAILED DESCRIPTION
[0030] The preferred embodiments of the present invention are described in detail below in conjunction with the accompanying drawings so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby making a clearer and more definite definition of the protection scope of the present invention.
[0031] The present invention provides an enzyme-linked system, which is composed of glucose-6-phosphate dehydrogenase and a P450 mutant enzyme. The enzyme-linked system is used to catalyze the conversion of naphthalene into 1-naphthol and 2-naphthol, and the glucose-6-phosphate dehydrogenase reacts with NADP to form 1-naphthol and 2-naphthol. + The reaction mechanism of generating NADPH realizes the cyclic regeneration of NADPH, reduces the consumption of NADPH in the reaction system and improves the reaction yield.
[0032] A method for catalyzing naphthalene to produce 1-naphthol and 2-naphthol by an enzyme-linked system, the specific steps are as follows:
[0033] Step 1, mixing naphthalene, NADPH, P450 enzyme mutant, glucose-6-phosphate dehydrogenase, D-glucose-6-phosphate, and phosphate buffer for enzymatic reaction;
[0034] Step 2, the reaction is carried out at a temperature of 25-35° C. and a pH value of 7.0-9.0 for 2-8 hours, catalyzing naphthalene to obtain 1-naphthol and 2-naphthol, the molar ratio of the P450 mutant enzyme to naphthalene is 1:1-200, the molar ratio of the glucose-6-phosphate dehydrogenase to the P450 mutant enzyme is 1:1-10, and the synergistic effect of the P450 mutant enzyme and the glucose-6-phosphate dehydrogenase in the enzyme-linked system improves the activity of the reaction; the concentration of NADPH is 0.1-2mM, and within this concentration range, the glucose-6-phosphate dehydrogenase can effectively realize the cyclic regeneration of NADPH.
[0035] An application of a P450 mutant enzyme, wherein the P450 mutant enzyme is used to catalyze the conversion of naphthalene into 1-naphthol and 2-naphthol, and the specific steps are as follows:
[0036] a. mixing naphthalene, NADPH, P450 mutant enzyme, and phosphate buffer for enzymatic reaction;
[0037] b. The reaction is carried out at a temperature of 25-35° C. and a pH value of 7.0-9.0 for 2-8 hours to catalyze naphthalene to obtain 1-naphthol and 2-naphthol, and the molar ratio of the P450 mutant enzyme to naphthalene is 1:1-200.
[0038] The amino acid sequence of the P450 mutant enzyme is selected from SEQ ID NO. 1, and the alanine at position 82 is mutated to phenylalanine. The mutant enzyme has improved hydroxylation activity and selectivity for naphthalene.
[0039] The preparation method of the P450 mutant enzyme comprises the following steps:
[0040] S1: construct a gene recombination vector containing SEQ ID NO.1 sequence and make a point mutation at position 82;
[0041] S2: The mutated recombinant vector is transferred into Escherichia coli and cultured to obtain a recombinant Escherichia coli bacterial liquid;
[0042] S3: Lysing the recombinant Escherichia coli bacterial solution to obtain a P450 mutant enzyme solution, wherein the mutant enzyme has improved hydroxylation activity and selectivity.
[0043] Example 1: P450 mutant enzyme catalyzes naphthalene to produce 1-naphthol and 2-naphthol;
[0044] P450 mutant enzyme reaction system: The reaction solution contains 5 mM naphthalene, 1 mM NADPH, 100 mM phosphate buffer (pH=8.0), 50 μM P450 mutant enzyme, and 5% DMSO.
[0045] Reaction conditions: 30°C, 220 rpm, in the dark for 6 h.
[0046] Product standard liquid phase diagram: 1mM concentration of 2-naphthol and 1mM concentration of 1-naphthol HPLC diagram is shown in Figure 1 and Figure 2 .
[0047] Sample treatment: After 6 hours of reaction, place on ice to terminate the reaction. Centrifuge the sample at 13000rpm / min for 30min, then filter with a 0.45μm filter head; store at 4℃ for testing. HPLC results are shown in Figure 3 , it was calculated that 0.02 mM of 1-naphthol and 0.18 mM of 2-naphthol were produced.
[0048] The reaction equation of the naphthalene reaction catalyzed by the P450 mutant enzyme is:
[0049]
[0050] Detection method:
[0051] The model of the high performance liquid chromatograph used is Shimadzu LC-20, Japan, and the chromatographic column selected is ZORBAX SB-C18 reverse phase column. Mobile phase A: water containing 0.1% formic acid. Mobile phase B: pure acetonitrile, the ratio of mobile phase A liquid to B liquid is 4:6, the flow rate is 1mL / min, the detection wavelength is 325nm, and the column temperature is 30℃.
[0052] Example 2: The enzyme-linked system consisting of a P450 mutant enzyme and glucose-6-phosphate dehydrogenase catalyzes naphthalene to produce 1-naphthol and 2-naphthol;
[0053] The enzyme-linked reaction system consisting of P450 mutant enzyme and glucose-6-phosphate dehydrogenase: the reaction solution contains 5mM naphthalene, 1mM NADPH, 100mM phosphate buffer (pH=8.0), 50μM P450 mutant enzyme, 5μM G6PD, 5μM D-glucose-6-phosphate, and 5% DMSO.
[0054] Reaction conditions: 30°C, 220 rpm, in the dark for 6 h.
[0055] Product standard liquid phase diagram: 1mM concentration of 2-naphthol and 1mM concentration of 1-naphthol HPLC diagram is shown in Figure 1 and Figure 2 .
[0056] Sample treatment: After 6 hours of reaction, place on ice to terminate the reaction. Centrifuge the sample at 13000rpm / min for 30min, then filter with a 0.45μm filter head; store at 4℃ for testing. HPLC results are shown in Figure 4 , it was calculated that 0.022mM 1-naphthol and 0.2mM 2-naphthol were generated. Compared with the P450 mutant single enzyme catalyzing naphthalene, the yield of 1-naphthol and 2-naphthol in the enzyme-linked system increased by about 10%.
[0057] Detection method: The model of the high performance liquid chromatograph used is Shimadzu LC-20, Japan, and the chromatographic column selected is ZORBAX SB-C18 reverse phase column. Mobile phase A: water containing 0.1% formic acid. Mobile phase B: pure acetonitrile, the ratio of mobile phase A liquid to B liquid is 40:60, the flow rate is 1mL / min, the detection wavelength is 325nm, and the column temperature is 30℃.
[0058] In summary, through the above two examples, it can be concluded that Example 1 uses a P450 mutant enzyme to catalyze naphthalene to generate 1-naphthol and 2-naphthol, which can produce 0.02mM 1-naphthol and 0.18mM 2-naphthol. Example 2 uses an enzyme-linked system composed of a P450 mutant enzyme and glucose-6-phosphate dehydrogenase to catalyze naphthalene to generate 1-naphthol and 2-naphthol, which can produce 0.022mM 1-naphthol and 0.2mM 2-naphthol, and Example 2 of the present invention has a higher yield, and the yield of 1-naphthol and 2-naphthol is increased by about 10%.
[0059] The above embodiments only express the implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention.
Claims
1. An enzyme-linked system, characterized in that: The enzyme-linked system consists of glucose-6-phosphate dehydrogenase and P450 mutant enzyme, and is used for catalyzing naphthalene to generate 1-naphthol and 2-naphthol.
2. A method for catalyzing naphthalene to produce 1-naphthol and 2-naphthol using an enzyme-linked system as claimed in claim 1, characterized in that: The specific steps are as follows: Step 1, mixing naphthalene, NADPH, P450 enzyme mutant, glucose-6-phosphate dehydrogenase, D-glucose-6-phosphate, and phosphate buffer for enzymatic reaction; Step 2: The reaction is carried out at a temperature of 25-35° C. and a pH value of 7.0-9.0 for 2-8 hours to catalyze naphthalene to obtain 1-naphthol and 2-naphthol, the molar ratio of the P450 mutant enzyme to naphthalene is 1:1-200, and the molar ratio of the glucose-6-phosphate dehydrogenase to the P450 mutant enzyme is 1:1-10.
3. The method for producing 1-naphthol and 2-naphthol by catalyzing naphthalene using an enzyme-linked system according to claim 2, characterized in that: The concentration of NADPH is 0.1-2 mM.
4. A use of the P450 mutant enzyme as claimed in claim 1, characterized in that: The P450 mutant enzyme is used to catalyze the conversion of naphthalene into 1-naphthol and 2-naphthol, and the specific steps are as follows: a. mixing naphthalene, NADPH, P450 mutant enzyme, and phosphate buffer for enzymatic reaction; b. The reaction is carried out at a temperature of 25-35° C. and a pH value of 7.0-9.0 for 2-8 hours to catalyze naphthalene to obtain 1-naphthol and 2-naphthol, and the molar ratio of the P450 mutant enzyme to naphthalene is 1:1-200.
5. The use of the P450 mutant enzyme according to claim 4, characterized in that: The amino acid sequence of the P450 mutant enzyme is selected from SEQ ID NO.1, and the alanine at position 82 is mutated to phenylalanine.
6. The use of the P450 mutant enzyme according to claim 4, characterized in that: The method for preparing the P450 mutant enzyme comprises the following steps: S1: construct a gene recombination vector containing SEQ ID NO.1 sequence and make a point mutation at position 82; S2: The mutated recombinant vector is transferred into Escherichia coli and cultured to obtain a recombinant Escherichia coli bacterial liquid; S3: Lysing the recombinant Escherichia coli bacterial solution to obtain a P450 mutant enzyme solution, wherein the mutant enzyme has improved hydroxylation activity and selectivity.