Construction of metal ion synergistic isoprene synthetase cascade reactor based on ZSM-5 substrate

Abstract

The invention provides a construction method of a ZSM-5 substrate-based metal ion synergistic isoprene synthase cascade reactor. The construction method comprises the steps of preparation of a ZSM-5 substrate with active metal ions and construction of the isoprene synthase cascade reactor. The ZSM-5 with the active metal ions is Mg-ZSM-5, and the molecular weight of the ZSM-5 with the active metal The cascade reactor is used for fixing five key enzymes at the downstream of a mevalonic acid pathway by using an adsorption method, namely mevalonic acid kinase (MK), phosphomevalonic acid kinase (PMK), diphosphate mevalonic acid decarboxylase (MVD), isopentenyl pyrophosphate isomerase (IDI) and isoprene synthetase (ispS). Meanwhile, the prepared cascade reactor is used for enzymolysis of mevalonic acid, and the performance of the reactor is evaluated through the yield of isoprene.

Description

Technical Field

[0001] The invention relates to the technical field of isoprene preparation, and in particular to a method for improving isoprene yield by constructing an enzyme cascade reactor with external aid. Background Art

[0002] Isoprene is one of the key ingredients in rubber manufacturing, and the market demand is huge. Currently, there are many methods for synthesizing isoprene. Among them, fermentation production has become a hot topic of research because of its mild conditions. Studies have shown that the core of fermentation production of isoprene is through the MVA pathway, that is, using mevalonate as the raw material and preparing it through the cascade reaction of five downstream enzymes. How to efficiently promote the cascade reaction of the five enzymes is the key to affecting the yield of isoprene. These five enzymes are mevalonate kinase (MK), phosphomevalonate kinase (PMK), diphosphomevalonate decarboxylase (MVD), isopentenyl pyrophosphate isomerase (IDI) and isoprene synthase (ispS). Studies have shown that by adding these five enzymes exogenously during the fermentation process, the yield of isoprene can be significantly increased. At the same time, studies have shown that these five enzymes are metal-activated enzymes. When magnesium ions are present in the fermentation system, the yield of isoprene can be significantly increased. However, the added magnesium ions and free enzymes cannot be recycled, making this method difficult to promote. Immobilizing the five enzymes and metal ions to prepare a cascade reactor is an effective way to solve the above problems. Summary of the invention

[0003] In view of this, the object of the present invention is to propose a method for constructing a metal ion-coordinated isoprene synthase cascade reactor based on a ZSM-5 substrate to solve the above problems.

[0004] The technical solution of the present invention is achieved in this way: A method for constructing a metal ion-coordinated isoprene synthase cascade reactor based on a ZSM-5 substrate, comprising constructing a ZSM-5 substrate with active metal ions, immobilizing mevalonate kinase (MK), phosphomevalonate kinase (PMK), diphosphomevalonate decarboxylase (MVD), isopentenyl pyrophosphate isomerase (IDI) and isoprene synthase (ispS) by an adsorption method, constructing an isoprene synthase cascade reactor, and enzymatically hydrolyzing mevalonate to prepare isoprene; The ZSM-5 substrate with active metal ions is prepared by a hydrothermal synthesis method to obtain a Mg-ZSM-5 substrate; The enzyme cascade reactor is a reactor in which MK, PMK, MVD, IDI and ispS are fixed on Mg-ZSM-5 by an adsorption method in a ratio of 1:1:1:2:16. DETAILED DESCRIPTION

[0005] In order to better understand the technical content of the present invention, specific embodiments are provided below to further illustrate the present invention.

[0006] Unless otherwise specified, the experimental methods used in the embodiments of the present invention are all conventional methods.

[0007] Unless otherwise specified, the materials, reagents, etc. used in the embodiments of the present invention can be obtained from commercial sources. Example

[0008] (1) ZSM-5 molecular sieve carrier powder was mixed with 10% magnesium chloride solution at a mass ratio of ZSM-5:MgCl2 of 1:1; the mixture was stirred at 50 °C for 4 h, the precipitate was filtered and rinsed three times.

[0009] (2) The precipitate was dried in an oven at 110 °C overnight and then treated in a muffle furnace at 550 °C for 4 h to obtain the carrier Mg-ZSM-5.

[0010] (3) using PBS buffer to adjust the concentrations of the five enzyme solutions of MK, PMK, MVD, IDI and ispS to a final concentration ratio of 1:1:1:2:16; (4) Accurately weigh 0.1 g of Mg-ZSM-5 molecular sieve material, take 100 μL of each of the five enzyme solutions and mix them, place Mg-ZSM-5 in the mixed enzyme solution to construct a multi-enzyme cascade reactor. Adjust the pH of the mixed solution to 7.0; (5) The mixture of Mg-ZSM-5 material and enzyme solution was placed in a shaker at 40°C at a speed of 150 r / min for 4 h for adsorption reaction; after the adsorption was completed, the mixture was taken out for filtration and washing to wash away the unfixed enzyme and other impurities; (6) The resulting product was dried in an oven at 40 °C to obtain a multi-enzyme cascade reactor Mg-ZSM-5@MVK-PMK-MVD-IDI-ISPS, which was then stored in the upper layer of a refrigerator at 4 °C.

[0011] Since mevalonic acid is very easy to undergo lactonization, it usually exists in the form of mevalonic acid lactone, so it needs to be further processed to obtain the product catalyzed by multiple enzymes in vitro. The specific operation process is as follows: 1) preparing a 1 mol / L KOH solution, and mixing mevalonate lactone and the KOH solution at a volume ratio of 1.05:1; 2) The mixture was subjected to saponification reaction on a shaker at 37°C for 1 h to obtain mevalonic acid.

[0012] According to the standard of 2 mL total reaction system volume, mevalonic acid and immobilized enzyme cascade reactor were added to 10 mL gas phase reaction bottle in sequence, and the reaction system was supplemented to 2 mL with PBS buffer. The reaction bottle was then placed in a constant temperature shaking incubator at 40°C for reaction, and 200 μL of air was taken from the top of the reaction bottle every 4 h using a gas phase microinjector for gas phase detection of isoprene content. In order to compare the catalytic activity of free enzyme and enzyme cascade reactor, it is necessary to control variables to ensure that the amount of free enzyme involved in the reaction is equal to the amount of enzyme in the enzyme cascade reactor, so it is necessary to ensure that the enzyme immobilization rate is above 90% when constructing the enzyme cascade reactor. When the experiment proceeds to the enzyme cascade reactor to catalyze the synthesis of isoprene in vitro, it is only necessary to use the enzyme cascade reactor to replace the free enzyme solution, and the system is still supplemented to 2 mL with PBS buffer. The reaction results showed that the in vitro catalytic synthesis of isoprene by the Mg-ZSM-5@MVK-PMK-MVD-IDI-ISPS multi-enzyme cascade reactor reached a yield of 495 mg / L, while the in vitro catalytic synthesis of isoprene by the free enzyme was only 196 mg / L, and the yield of isoprene was significantly improved. Example

[0013] The immobilized enzyme cascade reactor was recovered by filtration, and the experimental method of Example 1 was repeated 5 times, and the enzyme activity was still maintained above 93.85%.

[0014] The above description is only a preferred embodiment of the present invention and is not intended to limit the spirit and principle of the present invention. Any modifications, equivalent substitutions, improvements, etc. made should be included in the protection scope of the present invention.

Claims

1. A method for constructing a metal ion-coordinated isoprene synthase cascade reactor based on a ZSM-5 substrate, characterized in that: It includes the preparation of a ZSM-5 substrate with active metal ions and the construction of an isoprene synthase cascade reactor.

2. A method for constructing a metal ion-coordinated isoprene synthase cascade reactor based on a ZSM-5 substrate according to claim 1, characterized in that: The ZSM-5 having active metal ions is Mg-ZSM-5.

3. A method for constructing a metal ion-coordinated isoprene synthase cascade reactor based on a ZSM-5 substrate according to claim 1, characterized in that: The cascade reactor uses an adsorption method to immobilize five key enzymes in the downstream of the mevalonate pathway, namely mevalonate kinase (MK), phosphomevalonate kinase (PMK), diphosphomevalonate decarboxylase (MVD), isopentenyl pyrophosphate isomerase (IDI) and isoprene synthase (ispS), in a ratio of 1:1:1:2:16.