Method for preparing (R)-citronellal through whole-cell catalysis

Abstract

The invention belongs to the technical field of biological catalysis, and particularly relates to a method for preparing (R)-citronellal through whole-cell catalysis. According to the preparation technology of the (R)-citronellal through whole-cell catalysis, escherichia coli is used as a fermentation strain, a selected induction substrate is added in the fermentation process, and expression of key enzymes synthesized by the interior of thalli and the (R)-citronellal is enhanced in microorganisms through induction and metabolic engineering means; and coenzyme factors are added in the thallus catalytic conversion process to synthesize (R)-citronellal, so that enzyme with high catalytic efficiency and specificity can be generated, and the substrate conversion rate, yield and optical purity ee value can be remarkably improved.

Description

Technical Field

[0001] The invention belongs to the technical field of biocatalysis, and particularly relates to a method for preparing (R)-citronellal by whole-cell catalysis. Background Art

[0002] Citronellal is the main component of citronella oil. It exists naturally in essential oils such as lemon eucalyptus oil and citronella oil. It has a strong, fresh, green citrus-like, slightly woody aroma. However, due to the overly strong aroma of citronellal and its unstable chemical properties, it is only used in small quantities in low-grade lilac, lily of the valley and other cosmetics and mosquito coils. At present, there are two main methods for producing citronellal: one is to vacuum distill the crude aldehyde from the essential oil and then purify it with sodium bisulfite; the other is to produce it by oxidation of citronellol.

[0003] Optically pure (R)-citronellal is an important flavor and pharmaceutical intermediate, which can be used for flavoring beverages, foods, perfumes, etc., and can also be used as a raw material for synthesizing L-menthol. It is mainly obtained by separation from natural essential oils in industry, and can also be obtained by hydrogenation of citral catalyzed by metal catalysts. However, the traditional chemical synthesis method of (R)-citronellal usually involves the use of heavy metal catalysts and harmful solvents, and there are problems such as harsh reaction conditions and low product purity, which not only burdens the environment, but also may affect the safety of the product.

[0004] At present, although the biocatalytic pathway is widely popular, it is still very challenging. This is mainly because citral is a mixture of two isomers (E-isomer geranial and or Z-isomer neral), and the isomers have a great influence on the enantioselectivity of OYEs. So far, it has not been reported that OYEs can achieve efficient conversion of citral. In addition, the preparation of (R)-citronellal by biocatalysis also has problems such as low substrate concentration and excessive reduction of aldehydes, and pure enzymes need to be used as catalysts, resulting in high prices. It can be seen that the biological synthesis of R-citronellal often has problems such as interference of isomers and low conversion rate, which limits its further application in this field. Therefore, the art expects to develop an efficient and environmentally friendly (R)-citronellal synthesis method, which has important application value for the application of this product. Summary of the invention

[0005] To this end, the technical problem to be solved by the present invention is to provide a method for preparing (R)-citronellal by whole-cell catalysis, which can significantly improve the substrate conversion rate and the yield and optical purity ee value of the product, has the advantages of high efficiency, high selectivity and environmental protection, and is suitable for large-scale production of (R)-citronellal.

[0006] In order to solve the above technical problems, the present invention provides a method for preparing (R)-citronellal by whole-cell catalysis, comprising the following steps:

[0007] (1) inoculating Escherichia coli into a fermentation medium for induction culture;

[0008] (2) adding an induction substrate to the fermentation medium and continuing the induction culture;

[0009] (3) collecting the fermentation broth and adding the conversion liquid containing E / Z citral to carry out catalytic reaction to obtain the product.

[0010] Specifically, in the method for preparing (R)-citronellal by whole-cell catalysis, in step (1), the fermentation strain includes Escherichia coli.

[0011] Specifically, in the method for preparing (R)-citronellal by whole-cell catalysis, in the step (1), the fermentation medium comprises the following components: 6-9 g / L yeast powder, 2-7 g / L casein peptone, 0.7-4 g / L YNB, 0.08-0.16 g / L inositol, 0.27-0.45 g / L riboflavin, 6-15 g / L magnesium sulfate, and 1-6 g / L ammonium sulfate hydrochloride.

[0012] Specifically, in the method for preparing (R)-citronellal by whole-cell catalysis, in step (1), the temperature of the induction culture step is 28-32° C., and the pH is regulated to be 6-8.

[0013] Specifically, in the method for preparing (R)-citronellal by whole-cell catalysis, in step (2), the induction substrate includes IPTG and / or geraniol.

[0014] Specifically, the method for preparing (R)-citronellal by whole-cell catalysis is as follows:

[0015] The IPTG is added at a concentration of 8-25 mM based on the fermentation medium; and / or,

[0016] The geraniol is added at a concentration of 5-12 mM based on the fermentation medium.

[0017] Specifically, in the method for preparing (R)-citronellal by whole-cell catalysis, in step (2):

[0018] When the wet weight of the bacteria reaches 8-12%, the induction substrate is added; and / or,

[0019] When the wet weight of the bacteria reaches 15-20%, the fermentation is stopped.

[0020] Specifically, in the method for preparing (R)-citronellal by whole-cell catalysis, in the step (3), the amount of the conversion liquid added is 1-3 wt % based on the mass of the fermentation medium.

[0021] Specifically, in the method for preparing (R)-citronellal by whole-cell catalysis, in step (3), the concentration of E / Z citral in the conversion solution is 1-3 mol / L.

[0022] Specifically, in the method for preparing (R)-citronellal by whole-cell catalysis, in the step (3), the conversion solution further comprises: a glucose concentration of 0.8-1.2 mol / L, a NADH concentration of 0.2-0.4 mM, and a NAD + The concentration is 0.1-0.3 mM and the isohexane concentration is 20-35 v / v%.

[0023] Specifically, in the method for preparing (R)-citronellal by whole-cell catalysis, in the step (3), the temperature of the catalytic reaction is 28-35° C., the pH is adjusted to 8.0-8.5 with sodium carbonate, the stirring speed is 180-220 rpm, and the reaction time is 20-24 h.

[0024] The whole-cell catalytic (R)-citronellal preparation process of the present invention uses Escherichia coli as a fermentation strain, adds a selected induction substrate during the fermentation process, uses IPTG and / or geraniol for induction, and enhances the expression of key enzymes for (R)-citronellal synthesis in the microorganism through induction and metabolic engineering means; and, in the process of bacterial catalytic conversion, uses E / Z citral as a substrate, and adds coenzyme factors to the conversion liquid to synthesize (R)-citronellal under certain conditions. The whole-cell catalytic (R)-citronellal preparation process of the present invention can produce enzymes with high catalytic efficiency and specificity through a unique process design, and can significantly improve the substrate conversion rate, yield and optical purity ee value. The method has the advantages of high efficiency, high selectivity and environmental protection, and is suitable for large-scale production of (R)-citronellal. DETAILED DESCRIPTION

[0025] The present invention provides a method for preparing (R)-citronellal by whole-cell catalysis, comprising the following steps:

[0026] (1) inoculating the fermentation strain into a fermentation medium for induction culture;

[0027] (2) adding an induction substrate to the fermentation medium and continuing the induction culture;

[0028] (3) collecting the fermentation broth and adding the conversion liquid containing E / Z citral to carry out catalytic reaction to obtain the product.

[0029] In some specific embodiments, the fermentation strain comprises Escherichia coli.

[0030] In some specific embodiments, the fermentation medium is prepared, and the medium composition is: yeast powder 6-9 g / L, casein peptone 2-7 g / L, YNB 0.7-4 g / L, inositol 0.08-0.16 g / L, riboflavin 0.27-0.45 g / L, magnesium sulfate 6-15 g / L, and ammonium sulfate hydrochloride 1-6 g / L.

[0031] In some specific embodiments, the fermentation medium is sterilized at 115-121° C. for 15-30 minutes.

[0032] In some specific embodiments, the fermentation strain is inoculated into the culture medium at a volume ratio of 3%-8%, the temperature is set at 28-32°C, the pH is 6-8, the rotation speed is 150-180 rpm, and the culture is carried out for 12-20 hours.

[0033] In some specific embodiments, during the fermentation process, when the wet weight of the bacteria reaches 8-12%, an aqueous solution containing 8-25mM IPTG and / or 5-12mM geraniol at a weight ratio of 1-3wt% is added for induction, thereby enhancing the expression of key enzymes related to the synthesis of (R)-citronellal in the cells through metabolic engineering.

[0034] In some specific embodiments, the fermentation is stopped when the wet weight of the microorganisms reaches 15-20%.

[0035] In some specific embodiments, in the conversion solution, the amount of E / Z citral added to the fermentation solution is 1-3 mol / L, the glucose concentration is 0.8-1.2 mol / L, the NADH concentration is 0.2-0.4 mM, and the NAD + The concentration is 0.1-0.3 Mm, and the isohexane concentration is 20-35% by volume, and the reaction is carried out.

[0036] In some specific embodiments, the conversion reaction conditions are a temperature of 28-35° C., preferably sodium bicarbonate is used to adjust the pH to 8.0-8.5, a stirring speed of 180-220 rpm, and a reaction time of 20-24 h.

[0037] In some specific embodiments, after the reaction is completed, samples are taken to detect the content and purity of (R)-citronellal, and the conversion rate and yield are calculated.

[0038] In the following examples of the present invention, Escherichia coli was used as the fermentation strain, and each component of the culture medium was commercially available chemicals.

[0039] In the following examples of the present invention, as an exemplary scheme, the fermentation strain selected was Escherichia coli purchased from the China Industrial Microbiological Culture Collection Center, with a collection number of CICC 23917.

[0040] Example 1

[0041] The preparation method of (R)-citronellal described in this embodiment comprises the following steps:

[0042] (1) preparing a fermentation medium according to the following composition: 6 g / L yeast powder, 2 g / L casein peptone, 0.7 g / L YNB, 0.08 g / L inositol, 0.27 g / L riboflavin, 6 g / L magnesium sulfate, and 1 g / L ammonium sulfate hydrochloride; sterilizing the prepared fermentation medium at 121° C. for 20 minutes; after the sterilized fermentation liquid returns to room temperature, inoculating the preserved Escherichia coli strain into the fermentation medium at a volume ratio of 3%, setting the temperature at 28° C., pH 6, and the rotation speed at 150 rpm for 12 hours;

[0043] (2) During the fermentation process, when the wet weight of the bacterial cells reaches 8%, an induction substrate accounting for 1 wt% of the fermentation medium is added and the fermentation is continued, wherein the induction substrate is an aqueous solution containing 8 mM IPTG and 5 mM geraniol; when the wet weight of the bacterial cells reaches 15%, the fermentation is stopped;

[0044] (3) collecting the fermentation broth, and adding the conversion solution according to the volume of the fermentation broth and mixing thoroughly, wherein the components of the conversion solution include: a final concentration of E / Z citral of 1 mol / L, a final concentration of glucose of 0.8 mol / L, a final concentration of NADH of 0.2 mM, and a final concentration of NAD + The final concentration of isohexane is 0.1 mM and 20 v / v% by volume; the reaction temperature is adjusted to 28°C, and sodium bicarbonate is added to adjust the pH to 8.0, the stirring speed is 180 rpm, and the reaction is carried out for 20 hours; after the reaction, samples are taken to detect the content and purity of (R)-citronellal, and the conversion rate and yield are calculated.

[0045] Example 2

[0046] The preparation method of (R)-citronellal described in this embodiment comprises the following steps:

[0047] (1) preparing a fermentation medium according to the following composition: 8 g / L yeast powder, 4 g / L casein peptone, 2 g / L YNB, 0.16 g / L inositol, 0.45 g / L riboflavin, 15 g / L magnesium sulfate, and 6 g / L ammonium sulfate hydrochloride; sterilizing the prepared fermentation medium at 121° C. for 20 minutes; after the sterilized fermentation liquid returns to room temperature, inoculating the preserved Escherichia coli strain into the fermentation medium at a volume ratio of 8%, setting the temperature at 32° C., pH 8, and the rotation speed at 150 rpm for 20 hours;

[0048] (2) During the fermentation process, when the wet weight of the bacterial cells reaches 12%, an induction substrate accounting for 2 wt% of the fermentation medium is added and the fermentation is continued, wherein the induction substrate is an aqueous solution containing 20 mM IPTG and 12 mM geraniol; when the wet weight of the bacterial cells reaches 18%, the fermentation is stopped;

[0049] (3) collecting the fermentation broth, and adding the conversion solution according to the volume of the fermentation broth and mixing thoroughly, wherein the components of the conversion solution include: a final concentration of E / Z citral of 3 mol / L, a final concentration of glucose of 1 mol / L, a final concentration of NADH of 0.3 mM, and a final concentration of NAD + The final concentration of isohexane is 0.2 mM and 35 v / v% by volume; the reaction conditions are adjusted to a temperature of 32°C, and sodium bicarbonate is added to adjust the pH to 8.4, with a stirring speed of 220 rpm, and the reaction is carried out for 23 hours; after the reaction is completed, samples are taken to detect the content and purity of (R)-citronellal, and the conversion rate and yield are calculated.

[0050] Example 3

[0051] The preparation method of (R)-citronellal described in this embodiment comprises the following steps:

[0052] (1) preparing a fermentation medium according to the following composition: 6 g / L yeast powder, 2 g / L casein peptone, 0.7 g / L YNB, 0.08 g / L inositol, 0.27 g / L riboflavin, 6 g / L magnesium sulfate, and 1 g / L ammonium sulfate hydrochloride; sterilizing the prepared fermentation medium at 121° C. for 20 minutes; after the sterilized fermentation liquid returns to room temperature, inoculating the preserved Escherichia coli strain into the fermentation medium at a volume ratio of 3%, setting the temperature at 28° C., pH 6, and the rotation speed at 150 rpm for 12 hours;

[0053] (2) During the fermentation process, when the wet weight of the bacterial cells reaches 8%, an induction substrate accounting for 1 wt% of the fermentation medium is added and the fermentation is continued, wherein the induction substrate is an aqueous solution containing 8 mM IPTG; when the wet weight of the bacterial cells reaches 15%, the fermentation is stopped;

[0054] (3) collecting the fermentation broth, and adding the conversion solution according to the volume of the fermentation broth and mixing thoroughly, wherein the components of the conversion solution include: a final concentration of E / Z citral of 1 mol / L, a final concentration of glucose of 0.8 mol / L, a final concentration of NADH of 0.2 mM, and a final concentration of NAD +The final concentration of isohexane is 0.1 mM and 20 v / v% by volume; the reaction temperature is adjusted to 28°C, and sodium bicarbonate is added to adjust the pH to 8.0, the stirring speed is 180 rpm, and the reaction is carried out for 20 hours; after the reaction, samples are taken to detect the content and purity of (R)-citronellal, and the conversion rate and yield are calculated.

[0055] Example 4

[0056] The preparation method of (R)-citronellal described in this embodiment comprises the following steps:

[0057] (1) preparing a fermentation medium according to the following composition: 6 g / L yeast powder, 2 g / L casein peptone, 0.7 g / L YNB, 0.08 g / L inositol, 0.27 g / L riboflavin, 6 g / L magnesium sulfate, and 1 g / L ammonium sulfate hydrochloride; sterilizing the prepared fermentation medium at 121° C. for 20 minutes; after the sterilized fermentation liquid returns to room temperature, inoculating the preserved Escherichia coli strain into the fermentation medium at a volume ratio of 3%, setting the temperature at 28° C., pH 6, and the rotation speed at 150 rpm for 12 hours;

[0058] (2) during the fermentation process, when the wet weight of the bacterial cells reaches 8%, an induction substrate accounting for 1 wt% of the fermentation medium is added and the fermentation is continued, wherein the induction substrate is an aqueous solution containing 5 mM geraniol; when the wet weight of the bacterial cells reaches 15%, the fermentation is stopped;

[0059] (3) collecting the fermentation broth, and adding the conversion solution according to the volume of the fermentation broth and mixing thoroughly, wherein the components of the conversion solution include: a final concentration of E / Z citral of 1 mol / L, a final concentration of glucose of 0.8 mol / L, a final concentration of NADH of 0.2 mM, and a final concentration of NAD + The final concentration of isohexane is 0.1 mM and 20 v / v% by volume; the reaction temperature is adjusted to 28°C, and sodium bicarbonate is added to adjust the pH to 8.0, the stirring speed is 180 rpm, and the reaction is carried out for 20 hours; after the reaction, samples are taken to detect the content and purity of (R)-citronellal, and the conversion rate and yield are calculated.

[0060] Example 5

[0061] The preparation method of (R)-citronellal described in this embodiment comprises the following steps:

[0062] (1) preparing a fermentation medium according to the following composition: 9 g / L yeast powder, 7 g / L casein peptone, 4 g / L YNB, 0.12 g / L inositol, 0.36 g / L riboflavin, 10 g / L magnesium sulfate, and 4 g / L ammonium sulfate hydrochloride; sterilizing the prepared fermentation medium at 121° C. for 20 minutes; after the sterilized fermentation liquid returns to room temperature, inoculating the preserved Escherichia coli strain into the fermentation medium at a volume ratio of 5%, setting the temperature to 30° C., pH 7, and the rotation speed to 180 rpm for 20 hours;

[0063] (2) During the fermentation process, when the wet weight of the bacterial cells reaches 10%, an induction substrate accounting for 3 wt% of the fermentation medium is added and the fermentation is continued, wherein the induction substrate is an aqueous solution containing 25 mM IPTG and 8 mM geraniol; when the wet weight of the bacterial cells reaches 20%, the fermentation is stopped;

[0064] (3) collecting the fermentation broth, and adding the conversion solution according to the volume of the fermentation broth and mixing thoroughly, wherein the components of the conversion solution include: a final concentration of E / Z citral of 2 mol / L, a final concentration of glucose of 1.2 mol / L, a final concentration of NADH of 0.4 mM, and a final concentration of NAD + The final concentration of isohexane is 0.3 mM and 30 v / v% by volume; the reaction conditions are adjusted to a temperature of 35°C, and sodium bicarbonate is added to adjust the pH to 8.5, with a stirring speed of 200 rpm, and the reaction is carried out for 24 hours; after the reaction is completed, samples are taken to detect the content and purity of (R)-citronellal, and the conversion rate and yield are calculated.

[0065] Comparative Example 1

[0066] The preparation method of (R)-citronellal in this comparative example is the same as that in Example 1, the only difference being that in step (2), when the wet weight of the bacteria reaches 8%, only sterile water accounting for 1 wt% of the fermentation medium is added without adding the inducing substrate.

[0067] Comparative Example 2

[0068] The preparation method of (R)-citronellal in this comparative example is the same as that in Example 1, except that in step (3), NADH and NAD are not added to the conversion solution. + , and replaced with an equal amount of water.

[0069] Comparative Example 3

[0070] The preparation method of (R)-citronellal in this comparative example is the same as that in Example 1, the only difference being that in step (3), isohexane is not added to the conversion liquid but is replaced by an equal amount of water.

[0071] Comparative Example 4

[0072] The preparation method of (R)-citronellal in this comparative example is the same as that in Example 1, the only difference is that in step (1), the culture strain is replaced by Saccharomyces cerevisiae, which is purchased from the China Industrial Microbiological Culture Collection Center, with a collection number of CICC1388.

[0073] Comparative Example 5

[0074] The preparation method of (R)-citronellal in this comparative example is the same as that in Example 1, the only difference is that in step (2), when the wet weight of the bacteria reaches 8%, the induction substrate is not added but replaced by an equal amount of methanol.

[0075] Comparative Example 6

[0076] The preparation method of (R)-citronellal in this comparative example is the same as that in Example 1, the only difference being that in step (3), NADH is not added to the conversion solution but is replaced by an equal amount of water.

[0077] Comparative Example 7

[0078] The preparation method of (R)-citronellal in this comparative example is the same as that in Example 1, except that in step (3), the NAD + , and replaced with an equal amount of water.

[0079] Experimental example

[0080] The products after catalytic conversion in the above Examples 1-5 and Comparative Examples 1-7 were respectively sampled to detect the content and purity of (R)-citronellal, and the conversion rate and yield were calculated; the specific testing method includes:

[0081] (1) Wet cell weight (unit: %): Weigh 1.00 kg of fermentation liquid, centrifuge at 8000 rpm for 10 min, add 1 L of water to obtain the cells, resuspend evenly, centrifuge again at 8000 rpm for 10 min, obtain wet cells, and weigh their mass to be m1 kg (accurate to 4 decimal places); calculation formula: wet cell weight = m1 / 1 × 100%;

[0082] (2) Citronellal content: Determined by method 1 (hydroxylamine method) of the aldehyde and ketone determination method (OT-7);

[0083] (3) Optical purity ee value: The sample size is 1.1 g and should be left to stand for 1 hour before titration; the equivalent factor (e) in the calculation is 77.13 and is determined using the non-polar column method in GT-10-4.

[0084] The test results of the products in the above Examples 1-5 and Comparative Examples 1-7 are shown in Table 1 below.

[0085] Table 1 (R)-Citronellal test results

[0086]

[0087]

[0088] It can be seen that the whole-cell catalytic preparation method of (R)-citronellal of the present invention can enhance the expression of key enzymes for (R)-citronellal synthesis inside the microorganism by induction and metabolic engineering means, and combine with the optimized system in the conversion reaction, so as to produce enzymes with high catalytic efficiency and specificity, and can significantly improve the substrate conversion rate, yield and optical purity ee value. This method has the advantages of high efficiency, high selectivity and environmental protection, and is suitable for large-scale production of (R)-citronellal.

[0089] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A method for preparing (R)-citronellal by whole-cell catalysis, characterized in that: The steps include: (1) inoculating the fermentation strain into a fermentation medium for induction culture; (2) adding an induction substrate to the fermentation medium and continuing the induction culture; (3) collecting the fermentation broth and adding the conversion liquid containing E / Z citral to carry out catalytic reaction to obtain the product.

2. The method for preparing (R)-citronellal by whole-cell catalysis according to claim 1, characterized in that: In the step (1), the fermentation strain includes Escherichia coli.

3. The method for preparing (R)-citronellal by whole-cell catalysis according to claim 1 or 2, characterized in that: In the step (1), the fermentation medium comprises the following components: 6-9 g / L yeast powder, 2-7 g / L casein peptone, 0.7-4 g / L YNB, 0.08-0.16 g / L inositol, 0.27-0.45 g / L riboflavin, 6-15 g / L magnesium sulfate, and 1-6 g / L ammonium sulfate hydrochloride.

4. The method for preparing (R)-citronellal by whole-cell catalysis according to any one of claims 1 to 3, characterized in that: In the step (1), the temperature of the induction culture step is 28-32°C, and the pH is regulated to 6-8.

5. The method for preparing (R)-citronellal by whole-cell catalysis according to any one of claims 1 to 4, characterized in that: In the step (2), the induction substrate includes IPTG and / or geraniol; Preferably, the IPTG is added at a concentration of 8-25 mM based on the fermentation medium; Preferably, the geraniol is added at a concentration of 5-12 mM based on the fermentation medium.

6. The method for preparing (R)-citronellal by whole-cell catalysis according to any one of claims 1 to 5, characterized in that: In the step (2): When the wet weight of the bacteria reaches 8-12%, the induction substrate is added; and / or, When the wet weight of the bacteria reaches 15-20%, the fermentation is stopped.

7. The method for preparing (R)-citronellal by whole-cell catalysis according to any one of claims 1 to 6, characterized in that: In the step (3), the amount of the conversion liquid added is 1-3 wt % based on the mass of the fermentation medium.

8. The method for preparing (R)-citronellal by whole-cell catalysis according to any one of claims 1 to 7, characterized in that: In the step (3), the concentration of E / Z citral in the conversion solution is 1-3 mol / L.

9. The method for preparing (R)-citronellal by whole-cell catalysis according to claim 8, characterized in that: In step (3), the conversion solution further comprises: glucose concentration of 0.8-1.2 mol / L, NADH concentration of 0.2-0.4 mM, NAD + The concentration is 0.1-0.3 mM and the isohexane concentration is 20-35 v / v%.

10. The method for preparing (R)-citronellal by whole-cell catalysis according to any one of claims 1 to 9, characterized in that: In the step (3), the temperature of the catalytic reaction is 28-35° C., the pH is adjusted to 8.0-8.5, the stirring speed is 180-220 rpm, and the reaction time is 20-24 h.

Images