CN103627756A - Method for enzymatic synthesis of isoquercitrin by using continuous-flow tubular microreactor - Google Patents

Abstract

A method for enzymatically synthesizing isoquercitrin using a continuous-flow tubular microreactor, adopting a continuous-flow tubular microreactor with a micron-level inner diameter, using glycine-NaOH at pH 9 as a buffer to prepare aloe with a concentration of 0.01 to 5 g/L Ding mother liquor, with hesperidinase as a catalyst, distilled water for enzyme proportioning at a concentration of 0.001g/mL～0.1g/mL, adding ionic liquid [Bmim][BF ₄ ], rutin mother liquor, enzyme liquid and The ionic liquids were prepared in a volume ratio of 72:18:10, and the reaction solution was pumped into the microreactor at a flow rate of 0.1-100 μL/min by a micro-syringe pump to carry out the enzymatic reaction, and the temperature was controlled at 20-60 °C. This method is a time-saving and efficient biocatalytic (transformation) method, which is beneficial to the large-scale preparation of isoquercitrin.

Description

A method for enzymatically synthesizing isoquercitrin using a continuous-flow tubular microreactor

technical field

The invention relates to the field of biochemical pharmacy, in particular to a method for enzymatically synthesizing isoquercitrin by using a continuous flow microreactor.

Background technique

Isoquercitrin is a natural flavonoid compound with biological activities such as anti-tumor, anti-oxidation and hypoglycemia, and is a synthetic intermediate of food colorant (Enzymatically modified isoquercitrin) EMIQ (Food Chem. ):214-219). Isoquercitrin can be extracted from plants such as mulberry leaves and Tianjihuang. For example, CN102827220A discloses that isoquercitrin is separated from lotus leaves by column chromatography, but the content of isoquercitrin in natural products is extremely low. far from meeting market demand. In addition, isoquercitrin can be prepared by chemical methods. For example, CN1817876A discloses a method of heating and hydrolyzing rutin in a high-pressure reactor to prepare isoquercitrin. However, isoquercitrin and quercetin coexist in the product, and isoquercitrin The highest yield of glycosides is only 13%, most of which are quercetin. Therefore, considering the high cost of extraction and chemical methods, enzymatic selective hydrolysis of rutin to prepare isoquercitrin has been recognized as an effective way in recent years.

Isoquercitrin was obtained by selective enzymatic hydrolysis of rhamnoside bond with cheap rutin as substrate, which provided a new idea for the production of isoquercitrin. For example, Petr et al. used rhamnosidase to catalyze the hydrolysis of rutin to produce isoquercitrin, and the conversion rate could reach 93% after 25 hours of reaction (Bioresour.Technol., 2011, 115(0):222-227). However, the enzymatic cleavage efficiency of this hydrolysis process is low, the reaction time is long, the enzyme is expensive and the reusability is low, which all limit the production efficiency of isoquercitrin. In a novel ionic liquid co-solvent reaction system, Wang et al. used hesperidinase to hydrolyze rutin to produce isoquercitrin, and the conversion rate could reach 93.4% after 10 hours of reaction (Bioresour.Technol., 2013, 128, 156-163). Compared with the traditional method, the method greatly shortens the reaction time, but it still needs to be further improved. Therefore, it is urgent to find a more efficient new technology for preparing isoquercitrin based on this new catalytic system.

Since the rise of microreactor technology in the mid-1990s, it has rapidly become a research hotspot due to its unique features and advantages, and has been used more and more in the field of biocatalysis. Continuous-flow microreactor (Continuous-flow microreactor) is a continuous-flow tubular reactor, and the size of its reaction space is generally on the order of microns or even nanometers. Compared with conventional reactors, continuous flow tubular microreactors have the advantages of large surface area, high mass transfer and heat transfer efficiency, and easy scale-up (Lab Chip., 2012, 12, 4080-4084). Du et al studied the synthesis of sugar esters catalyzed by lipase TLIM in a microreactor, which immobilized the lipase in the pipe within 2 mm. Compared with traditional reactors, the yield of about 90% can be achieved within 30 minutes, and the by-products are less (RSC Adv., 2012, 2, 2663-2665). However, there is no method for enzymatically hydrolyzing rutin to synthesize isoquercitrin with a continuous flow tube microreactor.

Therefore, for the first time, the present invention uses a continuous flow tube microreactor to enzymatically hydrolyze rutin to synthesize isoquercitrin, which is beneficial to improve the efficiency of the enzymatic reaction, shorten the reaction time, reduce energy consumption, and is simple to operate and easy to industrialize the production of high-purity Isoquercitrin is of great significance to promote its use in cosmetics, pharmaceuticals, food and other industries.

Contents of the invention

Technical problem to be solved: The present invention provides a method for enzymatically synthesizing propyl caffeate using a continuous flow tube microreactor to solve the problems of low reaction rate, long time-consuming and difficult synthesis of isoquercitrin in the prior art. issues of continuous production.

Technical solution: The method of enzymatically synthesizing isoquercitrin by using a continuous-flow tubular microreactor, using a continuous-flow tubular microreactor with a micron-level inner diameter, and using glycine-NaOH with pH 9 as a buffer to prepare a concentration of 0.01-5g/ L of rutin mother liquor, using hesperidinase as a catalyst, and distilled water for the enzyme in a ratio of 0.001g/mL to 0.1g/mL, adding ionic liquid [Bmim][BF ₄ ], rutin mother liquor, Enzyme solution and ionic liquid are mixed according to the volume ratio of 72:18:10, and the reaction solution is pumped into the microreactor with a flow rate of 0.1-100μL/min by a micro-syringe pump to carry out the enzymatic reaction, and the temperature is controlled at 20-60℃ .

The tubular microreactor is provided with a sample inlet, a micropipe and a sample outlet connected in sequence, the sample inlet end of the reactor is connected with a micro-injection pump, the sample outlet end of the reactor is connected with a collector, and the micropipe is The serpentine channel is carved by thermal injection molding or etching. The inner diameter of the channel is 0.25m-2m long, 100-500μm wide, and 50μm deep. The microreactor is made of glass, PDMS (polydimethylsiloxane) or PMMA (Poly( methyl methacrylate)).

The concentration of rutin in the buffer solution is preferably 0.1 g/L.

The concentration of the enzyme solution is preferably 0.1 g/mL.

The flow rate controlled by the micro-syringe pump is preferably 2 μL/min.

Beneficial effect:

The present invention chooses to utilize the continuous flow tube type microreactor to enzymatically synthesize isoquercitrin, the traditional reaction time is reduced from 10h to 30min, the yield can reach 100%, the energy consumption is greatly reduced, and the reaction efficiency is greatly improved; the production process consists of Intermittent operation becomes continuous operation and continuous production is possible. In addition, the device adopted in the present invention has a simple structure, and the production capacity can be expanded in equal proportion by simply increasing the number, has good industrial application prospects, and can meet the needs of the rapidly developing pharmaceutical industry.

Description of drawings

Fig. 1 is the tubular microreactor device figure of enzymatic synthesis isoquercitrin reaction formula and continuous flow of the present invention, among the figure 1 micro injection pump, 2 micropipes, 3 collectors;

Figure 2 is an enlarged schematic diagram of the continuous flow tubular microreactor device pipeline, in which there are 4 sample inlets, 2 micropipes, and 5 sample outlets.

Detailed ways

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Example 1

Prepare 5 g/L rutin mother solution with pH of 9 with glycine-sodium hydroxide buffer solution, prepare hesperidin enzyme solution with concentration of 0.001 g/mL with distilled water, rutin mother solution, enzyme solution and ionic liquid [Bmin][ BF ₄ ] were prepared into a biotransformation reaction system at a volume ratio of 72:18:10. The prepared reaction system was pumped into a continuous flow reactor at a flow rate of 100 μL/min through a microsyringe pump for reaction. The tubular microreactor is provided with a sample inlet 4, a micropipe 2 and a sample outlet 5 connected in sequence, the sample inlet end of the reactor is connected with the micro-injection pump 1, and the sample outlet end of the reactor is connected with the collector 3, The reactor is made of PDMS-glass material through thermal injection molding to produce a serpentine pipe with a length of 0.25m, a width of 500μm, and a depth of 50μm. The temperature is controlled at 60°C, and the reaction liquid stays in the reactor for 36s. The export material was collected, and the yield of enzymatic synthesis of isoquercitrin detected by HPLC was 2.3%.

Example 2

Prepare 0.01g/L rutin mother solution with pH of 9 with glycine-sodium hydroxide buffer solution, prepare hesperidin enzyme solution with concentration of 0.1g/mL with distilled water, rutin mother solution, enzyme solution and ionic liquid [Bmin] [BF ₄ ] was prepared into a biotransformation reaction system at a volume ratio of 72:18:10. The prepared reaction system was pumped into a continuous flow reactor at a flow rate of 2 μL/min through a microsyringe pump for reaction. The structure of the device is the same as that of Example 1. The reactor is made of PMMA material by etching to carve a serpentine pipe with a length of 2 m, a width of 100 μm, and a depth of 50 μm. The temperature was controlled at 20°C, and the reaction solution stayed in the reactor for 30 minutes. The export material was collected, and the yield of enzymatic synthesis of isoquercitrin was detected by HPLC to be 5.7%.

Example 3

Prepare 0.1g/L rutin mother solution with pH of 9 with glycine-sodium hydroxide buffer solution, prepare hesperidin enzyme solution with concentration of 0.01g/mL with distilled water, rutin mother solution, enzyme solution and ionic liquid [Bmin] [BF ₄ ] was prepared into a biotransformation reaction system at a volume ratio of 72:18:10. The prepared reaction system was pumped into a continuous flow reactor at a flow rate of 20 μL/min through a micro-syringe pump for reaction. The structure of the device is the same as that of Example 1. The reactor is made of PDMS material and a serpentine pipe with a length of 1 m, a width of 100 μm and a depth of 50 μm is made by thermal injection molding. The temperature was controlled at 40°C, and the reaction liquid stayed in the reactor for 15 minutes. The export material was collected, and the yield of enzymatic synthesis of isoquercitrin detected by HPLC was 58.6%.

Example 4

Prepare 0.1g/L rutin mother solution with pH of 9 with glycine-sodium hydroxide buffer solution, prepare hesperidin enzyme solution with concentration of 0.05g/mL with distilled water, rutin mother solution, enzyme solution and ionic liquid [Bmin] [BF ₄ ] was prepared into a biotransformation reaction system at a volume ratio of 72:18:10. The prepared reaction system was pumped into a continuous flow reactor at a flow rate of 10 μL/min through a microsyringe pump for reaction. The structure of the device is the same as that of Example 1. The reactor is made of PMMA material by etching to carve a serpentine pipe with a length of 2 m, a width of 200 μm, and a depth of 50 μm. The temperature was controlled at 40°C, and the reaction liquid stayed in the reactor for 30 minutes. The export material was collected, and the yield of enzymatic synthesis of isoquercitrin detected by HPLC was 72.1%.

Example 5

Prepare 1 g/L rutin mother solution with a pH of 9 with glycine-sodium hydroxide buffer solution, prepare a 0.1 g/mL hesperidinase solution with distilled water, rutin mother solution, enzyme solution and ionic liquid [Bmin][ BF ₄ ] were prepared into a biotransformation reaction system at a volume ratio of 72:18:10. The prepared reaction system was pumped into a continuous flow reactor at a flow rate of 4 μL/min through a microsyringe pump for reaction. The structure of the device is the same as that of Example 1. The reactor is made of glass material by etching to carve a serpentine pipe with a length of 1 m, a width of 200 μm, and a depth of 50 μm. The temperature was controlled at 35°C, and the reaction liquid stayed in the reactor for 15 minutes. The export material was collected, and the yield of enzymatically synthesized isoquercitrin was detected by HPLC to be 89.1%.

Example 6

Prepare 0.5g/L rutin mother solution with pH of 9 with glycine-sodium hydroxide buffer solution, prepare hesperidin enzyme solution with concentration of 0.1g/mL with distilled water, rutin mother solution, enzyme solution and ionic liquid [Bmin] [BF ₄ ] was prepared into a biotransformation reaction system at a volume ratio of 72:18:10. The prepared reaction system was pumped into a continuous flow reactor at a flow rate of 2 μL/min through a microsyringe pump for reaction. The structure of the device is the same as that of Example 1. The reactor is made of PDMS material and carved a serpentine pipe with a length of 1 m, a width of 100 μm, and a depth of 50 μm by thermal injection molding. The temperature was controlled at 35°C, and the reaction liquid stayed in the reactor for 15 minutes. The export material was collected, and the yield of enzymatically synthesized isoquercitrin was detected by HPLC to be 93.5%.

Example 7

Prepare 0.1g/L rutin mother solution with pH of 9 with glycine-sodium hydroxide buffer solution, prepare hesperidin enzyme solution with concentration of 0.1g/mL with distilled water, rutin mother solution, enzyme solution and ionic liquid [Bmin] [BF ₄ ] was prepared into a biotransformation reaction system at a volume ratio of 72:18:10. The prepared reaction system was pumped into a continuous flow reactor at a flow rate of 2 μL/min through a microsyringe pump for reaction. The structure of the device is the same as that in Example 1. The reactor is made of PDMS-glass material and carved a serpentine pipe with a length of 2 m, a width of 200 μm, and a depth of 50 μm by hot injection molding. The temperature was controlled at 40°C, and the reaction liquid stayed in the reactor for 30 minutes. The export material was collected, and the yield of enzymatically synthesized isoquercitrin was detected by HPLC to be 99.9%.

Claims (5)

1. Utilize the method for enzymatically synthesizing isoquercitrin by a continuous-flow tubular microreactor, characterized in that: the continuous-flow tubular microreactor with a micron-level internal diameter is used as a buffer preparation concentration of 0.01 with the glycine-NaOH of pH9 ～5g/L rutin mother liquor, with hesperidinase as catalyst, distilled water for enzyme at a concentration of 0.001g/mL～0.1g/mL, add ionic liquid [Bmim][BF ₄ ] into the reaction system, reed The mother liquid, enzyme liquid, and ionic liquid were mixed in a volume ratio of 72:18:10, and the reaction liquid was pumped into the microreactor at a flow rate of 0.1-100 μL/min with a micro-syringe pump to carry out the enzymatic reaction, and the temperature was controlled at 20 -60°C. 2. The method for enzymatically synthesizing isoquercitrin using a continuous-flow tubular microreactor according to claim 1, characterized in that: the tubular microreactor is provided with a sequentially connected sample inlet (4), a micropipe (2) and the sample outlet (5), the inlet port of the reactor is connected to the micro-injection pump (1), the sample outlet end of the reactor is connected to the collector (3), the micropipe is a serpentine channel, through Carved by thermal injection molding or etching, the inner diameter of the channel is 0.25m-2m in length, 100-500μm in width, and 50μm in depth. The material of the microreactor is glass, PDMS or PMMA. 3. The method for enzymatically synthesizing isoquercitrin by using a continuous-flow tubular microreactor according to claim 1, characterized in that: the concentration of rutin in the buffer solution is preferably 0.1 g/L. 4. The method for enzymatically synthesizing isoquercitrin using a continuous-flow tubular microreactor according to claim 1, characterized in that: the concentration of the enzyme solution is preferably 0.1 g/mL. 5. The method for enzymatically synthesizing isoquercitrin using a continuous-flow tubular microreactor according to claim 1, characterized in that: the flow rate controlled by the micro-injection pump is preferably 2 μL/min.

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