Complex enzyme, method for producing the same, and method for producing 1,3-propanediol and lactic acid

Abstract

The application provides a composite enzyme, a preparation method of the composite enzyme, and a method for preparing 1,3-propanediol and lactic acid. The composite enzyme comprises a biological enzyme of a glycerol-1,3-propanediol pathway and a biological enzyme of a glucose-lactic acid pathway, and is obtained by fermentation culture of Lactobacillus reuteri GDMCC NO: 64700. The method for preparing the composite enzyme can efficiently produce the composite enzyme for catalyzing the conversion of glycerol and sugar substances into 1,3-propanediol and lactic acid. The composite enzyme can not only catalyze the high production of 1,3-propanediol and lactic acid, but also has simpler components of a catalytic system, omits the sterilization treatment step, and simplifies the separation and purification process of target products.

Description

Technical Field

[0001] This application relates to the field of biology, specifically to a complex enzyme and its preparation method, and a method for preparing 1,3-propanediol and lactic acid. Background Technology

[0002] 1,3-Propanediol (1,3-PDO) and lactic acid are widely used in cosmetics, pharmaceuticals, food, and chemical industries as solvents, pH adjusters, and humectants, respectively. Currently, 1,3-propanediol and lactic acid are mainly prepared via microbial methods. 1,3-Propanediol primarily uses glycerol as a raw material. In the glycerol metabolic pathway, glycerol is first converted to 3-hydroxypropanal (3-HPA) by glycerol dehydratase (GDHt); then, 1,3-propanediol oxidoreductase (PDOR), under the action of the reducing coenzyme NADH, reduces 3-hydroxypropanal to 1,3-propanediol, releasing NAD+. + Lactic acid can be produced from carbohydrates. Taking glucose as an example, in the glucose metabolism pathway, 1 mol of glucose is broken down into 2 mol of lactate through glucokinase, 6-phosphofructokinase-1, and pyruvate kinase, respectively. This process can reduce NAD+ levels. + It is reduced to NADH, which forms a coenzyme cycle with the glycerol metabolic pathway.

[0003] However, since few microorganisms can completely separate the glycerol and glucose metabolic pathways, meaning that the two pathways do not compete with each other, it is difficult to obtain all the enzymes involved in the conversion of glycerol and glucose into 1,3-propanediol and lactic acid simultaneously. This makes it impossible to apply these enzymes to in vitro enzymatic reactions to co-produce 1,3-propanediol and lactic acid, and they can only be obtained through in vivo microbial fermentation, which greatly limits the industrial production and application of 1,3-propanediol and lactic acid. Summary of the Invention

[0004] This application aims to at least partially address the technical problems existing in the prior art. To this end, this application proposes a complex enzyme, its preparation method and application, and a method for preparing 1,3-propanediol and lactic acid. The method for preparing the complex enzyme using this application can efficiently produce a complex enzyme for catalyzing the conversion of glycerol and carbohydrates into 1,3-propanediol and lactic acid. This complex enzyme not only catalyzes high yields of 1,3-propanediol and lactic acid, but also has a simpler catalytic system, eliminating the need for sterilization and simplifying the separation and purification of the target products. Therefore, this method provides a green, low-cost, and efficient production process for the biological co-production of 1,3-propanediol and lactic acid.

[0005] In a first aspect of this application, a complex enzyme and a method for its preparation are disclosed, said complex enzyme comprising a bioenzyme of the glycerol-1,3-propanediol pathway and a bioenzyme of the glucose-lactic acid pathway. According to an embodiment of this application, the complex enzyme is obtained by fermentation culture of *Lactobacillus reuteri* GDMCC NO: 64700.

[0006] According to the method of the embodiments of this application, *Lactobacillus reuteri* GDMCC NO: 64700 can metabolize enzymes of the glycerol-1,3-propanediol pathway and enzymes of the glucose-lactic acid pathway, such as glycerol dehydratase, 1,3-propanediol oxidoreductase, glucokinase, 6-phosphofructokinase-1, pyruvate kinase, and NADH reducing coenzyme. Using the above-mentioned complex enzymes, glycerol and carbohydrates can be catalyzed in vitro to produce 1,3-propanediol and lactic acid.

[0007] According to an embodiment of this application, the method for preparing the complex enzyme includes: fermenting and culturing *Lactobacillus reuteri* GDMCC NO: 64700 to obtain the complex enzyme.

[0008] According to an embodiment of this application, the culture medium for fermentation includes carbohydrates;

[0009] The carbohydrates include at least one of glucose, starch, fructose, sucrose, and maltose;

[0010] And / or, the sugar content in the culture medium is 1.0 g / L to 50.0 g / L.

[0011] According to embodiments of this application, the culture medium further contains at least one of: peptone, beef extract, yeast extract, dipotassium hydrogen phosphate, sodium acetate, diammonium hydrogen citrate, magnesium sulfate, manganese sulfate, and Tween-80, and water.

[0012] And / or, the pH of the fermentation medium is 4 to 6.

[0013] According to an embodiment of this application, the fermentation culture temperature is 35℃~40℃ and the time is 24h~72h.

[0014] According to an embodiment of this application, the method further includes:

[0015] The fermentation broth obtained from the fermentation culture is subjected to a first centrifugation treatment, and the lower layer of bacterial precipitate is collected.

[0016] The bacterial cell precipitate was subjected to cell disruption treatment to obtain fragments;

[0017] The fragmented material is subjected to a second centrifugation process, and the supernatant is collected to obtain the complex enzyme.

[0018] According to an embodiment of this application, the rotation speed of the first centrifugation process is 8000 rpm to 12000 rpm, the time is 5 min to 20 min, and the temperature is 0℃ to 10℃.

[0019] And / or, before performing the cell disruption treatment, the lower bacterial cell precipitate is washed with buffer or physiological saline and resuspended, and the resulting bacterial suspension is subjected to the cell disruption treatment;

[0020] And / or, the cell disruption treatment includes ultrasonic cell disruption treatment, with a temperature of 0℃~10℃, a power of 200W~800W, and a time of 10min~60min;

[0021] And / or, the second centrifugation process is carried out at a speed of 8000 rpm to 12000 rpm, for a time of 5 min to 20 min, and at a temperature of 0℃ to 10℃.

[0022] In a second aspect of this application, the application of the complex enzyme described in the first aspect is proposed in the preparation of 1,3-propanediol and lactic acid.

[0023] In a third aspect of this application, a method for preparing 1,3-propanediol and lactic acid is provided. According to embodiments of this application, the method comprises reacting glycerol, a carbohydrate, the complex enzyme described in the first aspect, and water to obtain 1,3-propanediol and lactic acid.

[0024] According to embodiments of this application, the carbohydrate includes at least one of glucose, starch, fructose, sucrose, and maltose;

[0025] And / or, based on the total mass of the glycerol, carbohydrates and water, the glycerol accounts for 10% to 50% by mass, and / or, the carbohydrates account for 1% to 10% by mass;

[0026] And / or, the amount of the compound enzyme added is 0.05% to 1% of the total mass of the glycerol, carbohydrates and water;

[0027] And / or, the reaction temperature is 35℃~45℃, and the time is 30min~360min.

[0028] According to an embodiment of this application, the method further includes:

[0029] The reaction solution obtained from the reaction is concentrated and distilled to separate 1,3-propanediol and lactic acid;

[0030] The concentration is achieved by rotary evaporation at a temperature of 25℃ to 100℃.

[0031] The distillation process includes: first, controlling the distillation temperature to 122℃~125℃, collecting the fraction to obtain lactic acid; then, controlling the distillation temperature to 210℃~215℃, collecting the fraction to obtain 1,3-propanediol.

[0032] Additional aspects and advantages of this application will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of this application. Attached Figure Description

[0033] The above and / or additional aspects and advantages of this application will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0034] Figure 1 A metabolic diagram of 1,3-propanediol and lactic acid according to one embodiment of this application is shown. Detailed Implementation

[0035] The embodiments of the technical solution of this application are described in detail below. The following embodiments are only used to illustrate the technical solution of this application more clearly, and are therefore only examples, and should not be used to limit the scope of protection of this application.

[0036] In this document, the term "embodiment" means that a particular feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of this application. The appearance of this phrase in various places throughout the specification does not necessarily refer to the same embodiment, nor is it a separate or alternative embodiment mutually exclusive with other embodiments. It will be explicitly and implicitly understood by those skilled in the art that the embodiments described herein can be combined with other embodiments.

[0037] The "range" disclosed in this application is defined by a lower limit and an upper limit. A given range is defined by selecting a lower limit and an upper limit, which define the boundaries of a particular range. Ranges defined in this way can include endpoints and can be arbitrarily combined; that is, any lower limit can be combined with any upper limit to form a range. For example, if ranges of 60-120 and 80-110 are listed for a specific parameter, it is also expected that ranges of 60-110 and 80-120 are also included. Furthermore, if minimum range values ​​1 and 2 are listed, and if maximum range values ​​3, 4, and 5 are listed, then the following ranges are all expected: 1-3, 1-4, 1-5, 2-3, 2-4, and 2-5. In this application, unless otherwise stated, the numerical range "ab" represents a shortened representation of any combination of real numbers between a and b, where a and b are real numbers, and ranges defined in this way can include endpoints a and b. For example, the numerical range "0-5" indicates that all real numbers between "0-5" have been listed in this article; "0-5" is simply a shortened representation of these numerical combinations. Furthermore, when a parameter is stated as an integer ≥2, it is equivalent to disclosing that the parameter is, for example, an integer such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.

[0038] Unless otherwise specified, all embodiments and optional embodiments of this application can be combined to form new technical solutions.

[0039] Unless otherwise specified, all technical features and optional technical features of this application may be combined to form new technical solutions.

[0040] Unless otherwise specified, all steps in this application may be performed sequentially or randomly, preferably sequentially. For example, the method includes steps (a) and (b), indicating that the method may include steps (a) and (b) performed sequentially, or it may include steps (b) and (a) performed sequentially. For example, the mention that the method may also include step (c) indicates that step (c) may be added to the method in any order. For example, the method may include steps (a), (b), and (c), or it may include steps (a), (c), and (b), or it may include steps (c), (a), and (b), etc.

[0041] Therefore, in a first aspect of this application, a complex enzyme and a method for preparing the same are proposed, wherein the complex enzyme comprises a bioenzyme of the glycerol-1,3-propanediol pathway and a bioenzyme of the glucose-lactic acid pathway. According to an embodiment of this application, the complex enzyme is obtained by fermenting *Lactobacillus reuteri* GDMCC NO: 64700 in a fermentation medium containing glycerol and sugars.

[0042] like Figure 1 The metabolic diagram shown illustrates that *Limosilactobacillus reuteri* PDO / LA-3 (GDMCC NO: 64700, deposited at the Guangdong Provincial Microbial Culture Collection Center on May 29, 2024) possesses unique metabolic capabilities. It can metabolize enzymes required for 1,3-propanediol production, including glycerol dehydratase and 1,3-propanediol oxidoreductase; it can also metabolize enzymes required for lactate production, including glucokinase, 6-phosphofructokinase-1, and pyruvate kinase; and it can metabolize NADH-reduced coenzymes in the coenzyme cycle. However, strains commonly producing 1,3-propanediol, such as *Klebsiella*, *Escherichia coli*, *Lactobacillus*, or *Citrus*, rarely ferment all six enzymes simultaneously. For example, in *Klebsiella*, the enzymes in its metabolic pathway primarily catalyze the conversion of glycerol to 1,3-propanediol. Although it can convert a small amount of glycerol to lactate, it struggles to efficiently synthesize lactate from glucose, resulting in relatively low lactate yield and conversion rates.

[0043] Current processes for preparing 1,3-propanediol and lactic acid through fermentation result in a complex mixture containing live bacterial strains, protein nutrients, amino acids, vitamins, inorganic salts, and various metabolites. This necessitates sterilization of the product and significantly complicates the separation and purification of the target products. By separating a complex enzyme derived from the metabolism of *Lactobacillus reuteri* PDO / LA-3 and using it for in vitro catalysis of glycerol and sugars, high yields of 1,3-propanediol and lactic acid can be achieved. Furthermore, this catalytic system is simpler, eliminating the need for sterilization and simplifying the separation and purification of the target products. Therefore, this method provides a green, low-cost, and efficient biological co-production process for 1,3-propanediol and lactic acid.

[0044] According to embodiments of this application, the fermentation culture medium includes carbohydrates; the carbohydrates include at least one selected from glucose, starch, fructose, sucrose, and maltose. This provides energy and carbon sources for *Lactobacillus reuteri* PDO / LA-3, promoting its growth and metabolic activities, thereby increasing enzyme yield and activity.

[0045] According to embodiments of this application, the sugar content in the fermentation medium is 1.0 g / L to 50.0 g / L, for example, 1.0 g / L, 5.0 g / L, 10.0 g / L, 15.0 g / L, 20.0 g / L, 25.0 g / L, 30.0 g / L, 35.0 g / L, 40.0 g / L, 45.0 g / L, or 50.0 g / L. This provides sufficient energy and carbon sources for *Lactobacillus reuteri* PDO / LA-3, promoting its growth and metabolic activities, enhancing its ability to synthesize and secrete enzymes, thereby increasing enzyme yield and activity. It also avoids osmotic stress or metabolic burden caused by excessively high sugar concentrations, allowing the microorganism to produce enzymes efficiently under optimal growth conditions.

[0046] According to embodiments of this application, the culture medium further contains at least one of: peptone, beef extract, yeast extract, dipotassium hydrogen phosphate, sodium acetate, diammonium hydrogen citrate, magnesium sulfate, manganese sulfate, and Tween-80, and water. This provides *Lactobacillus reuteri* PDO / LA-3 with a nitrogen source, carbon source, vitamins, minerals, growth factors, and surfactants, enabling it to perform metabolic activities and synthesize enzymes under optimal conditions, enhancing its enzyme-synthesizing ability, thereby increasing enzyme yield and activity. In some embodiments, the culture medium further contains at least one of the following: 5.0–15.0 g / L peptone, 5.0–15.0 g / L beef extract, 1.0–8.0 g / L yeast extract, 1.0–5.0 g / L dipotassium hydrogen phosphate, 1.0–10.0 g / L sodium acetate, 1.0–5.0 g / L diammonium hydrogen citrate, 0.1–0.5 g / L magnesium sulfate, 0.01–1.0 g / L manganese sulfate, and 0.1–2.0 mL / L Tween-80, and water.

[0047] According to embodiments of this application, the pH of the fermentation medium is 4–6, for example, 4, 4.5, 5, 5.5, or 6. This facilitates the metabolic activities and enzyme synthesis of *Lactobacillus reuteri* PDO / LA-3 under optimal conditions, enhancing its enzyme synthesis capacity and thereby increasing enzyme yield and activity.

[0048] According to embodiments of this application, the fermentation culture temperature is 35℃~40℃, for example, 35℃, 36℃, 37℃, 38℃, 39℃, or 40℃, and the time is 24h~72h, for example, 24h, 30h, 36h, 42h, 48h, 54h, 60h, 66h, or 72h. This facilitates the metabolic activities and enzyme synthesis of *Lactobacillus reuteri* PDO / LA-3 under optimal conditions, enhancing its enzyme synthesis capacity and thus increasing enzyme yield and activity.

[0049] According to an embodiment of this application, the method further includes:

[0050] The fermentation broth obtained from the fermentation culture is subjected to a first centrifugation treatment, and the lower layer of bacterial precipitate is collected.

[0051] The bacterial cell precipitate was subjected to cell disruption treatment to obtain fragments;

[0052] The fragmented material is subjected to a second centrifugation process, and the supernatant is collected to obtain the complex enzyme.

[0053] Since the aforementioned six enzymes are all intracellular enzymes, centrifugation is required to separate and remove the upper fermentation supernatant. Then, the precipitated cells are disrupted to release the intracellular enzymes. After this process, a second centrifugation can yield the desired complex enzyme from the supernatant.

[0054] According to embodiments of this application, the rotation speed of the first centrifugation process is 8000 rpm to 12000 rpm, for example, 8000 rpm, 8500 rpm, 9000 rpm, 9500 rpm, 10000 rpm, 10500 rpm, 11000 rpm, 11500 rpm, or 12000 rpm; the time is 5 min to 20 min, for example, 5 min, 10 min, 15 min, or 20 min; and the temperature is 0℃ to 10℃, for example, 0℃, 2℃, 4℃, 6℃, 8℃, or 10℃. This allows for the separation and removal of extracellular substances and the culture medium.

[0055] According to an embodiment of this application, before performing the cell disruption treatment, the lower bacterial cell precipitate is washed with buffer or physiological saline and resuspended, and the resulting bacterial suspension is then subjected to the cell disruption treatment. This avoids residual extracellular substances and culture medium on the bacterial cells, which could affect the extraction of the complex enzyme.

[0056] According to embodiments of this application, the cell disruption treatment includes ultrasonic cell disruption, with a temperature of 0℃ to 10℃ (e.g., 0℃, 2℃, 4℃, 6℃, 8℃, 10℃), a power of 200W to 800W (e.g., 200W, 250W, 300W, 350W, 400W, 450W, 500W, 550W, 600W, 650W, 700W, 750W, 800W), and a time of 10min to 60min (e.g., 10min, 20min, 30min, 40min, 50min, 60min). This effectively disrupts the cell wall, releasing the complex enzymes within, while avoiding interference with the enzymes' activity.

[0057] According to embodiments of this application, the second centrifugation process is carried out at a speed of 8000 rpm to 12000 rpm, for example, 8000 rpm, 8500 rpm, 9000 rpm, 9500 rpm, 10000 rpm, 10500 rpm, 11000 rpm, 11500 rpm, or 12000 rpm; the time is 5 min to 20 min, for example, 5 min, 10 min, 15 min, or 20 min; and the temperature is 0℃ to 10℃, for example, 0℃, 2℃, 4℃, 6℃, 8℃, or 10℃. This facilitates the removal of cell wall residues, improves the yield and purity of the complex enzyme, and avoids affecting enzyme activity.

[0058] In a second aspect of this application, the application of the complex enzyme described in the first aspect in the preparation of 1,3-propanediol and lactic acid is proposed. The complex enzyme of this application can catalyze the production of 1,3-propanediol and lactic acid from glycerol and glucose in vitro, and its catalytic system has a simpler composition, eliminating the need for sterilization and simplifying the separation and purification of the target products. The features and advantages described for the complex enzyme above also apply to this application and will not be repeated here.

[0059] In a third aspect of this application, a method for preparing 1,3-propanediol and lactic acid is provided. According to embodiments of this application, the method comprises reacting glycerol, a carbohydrate, the complex enzyme described in the first aspect, and water to obtain 1,3-propanediol and lactic acid. The features and advantages described for the complex enzyme are equally applicable to this method for preparing 1,3-propanediol and lactic acid, and will not be repeated here.

[0060] For *Lactobacillus reuteri* PDO / LA-3, it is almost impossible to convert glycerol to 1,3-propanediol using only glycerol fermentation; that is, it requires the presence of glucose. When glycerol and glucose coexist, the complex enzyme can catalyze the conversion of glycerol and glucose to 1,3-propanediol and lactic acid.

[0061] When fermented with glycerol alone, *Lactobacillus reuteri* PDO / LA-3 is almost unable to convert glycerol to 1,3-propanediol using a complex enzyme, indicating that the conversion process depends on the presence of glucose. Only when glycerol and glucose coexist can the complex enzyme effectively catalyze the conversion of glycerol to 1,3-propanediol and glucose to lactic acid.

[0062] According to embodiments of this application, the carbohydrate includes at least one of glucose, starch, fructose, sucrose, and maltose. This facilitates the catalytic production of 1,3-propanediol and lactic acid by a complex enzyme.

[0063] According to embodiments of this application, based on the total mass of the glycerol, carbohydrates, and water, the mass percentage of the glycerol is 10% to 50%, for example, 10%, 11%, 12%, 13%, 14%, or 15%, and / or the mass percentage of the carbohydrates is 1% to 10%, for example, 1%, 2%, 4%, 5%, 6%, 8%, or 10%. This facilitates the effective binding of the enzyme's active site to the substrate molecule, avoids enzyme inactivation due to excessive substrate or limitation of the reaction rate due to insufficient substrate, ensures stable reaction, thereby improving conversion rate and yield, while reducing the occurrence of side reactions.

[0064] According to embodiments of this application, the reaction temperature is 35℃~45℃, for example, 35℃, 36℃, 37℃, 38℃, 39℃, 40℃, 41℃, 42℃, 43℃, 44℃, or 45℃; the reaction time is 30min~360min, for example, 30min, 60min, 90min, 120min, 150min, 180min, 210min, 240min, 270min, 300min, 330min, or 360min. Under these conditions, the activity of the complex enzyme can reach its optimal state, avoiding enzyme inactivation due to excessively high temperatures or excessively long reaction times, and also avoiding reduced reaction rates or incomplete enzymatic hydrolysis due to excessively low temperatures. This facilitates the complete conversion of glycerol and glucose into 1,3-propanediol and lactic acid, thereby achieving a highly efficient biocatalytic process and increasing the yield of 1,3-propanediol and lactic acid.

[0065] According to embodiments of this application, the method further includes: concentrating and distilling the reaction solution obtained from the reaction to separate 1,3-propanediol and lactic acid. This further improves the purity of 1,3-propanediol and lactic acid.

[0066] According to embodiments of this application, the concentration is achieved by rotary evaporation at a temperature of 25°C to 100°C, for example, 30°C, 40°C, 50°C, 60°C, 70°C, 80°C, 90°C, or 100°C. This facilitates the removal of low-boiling-point byproducts, such as ethanol, from the reaction solution, thereby enabling the concentration of 1,3-propanediol and lactic acid, and preventing side reactions between 1,3-propanediol and lactic acid caused by excessively high temperatures.

[0067] According to an embodiment of this application, the distillation includes: first controlling the distillation temperature to 122℃~125℃ (e.g., 122℃, 123℃, 124℃, 125℃), collecting the distillate to obtain lactic acid; then increasing the distillation temperature to 210℃~215℃ (e.g., 210℃, 211℃, 212℃, 213℃, 214℃, 215℃), collecting the distillate to obtain 1,3-propanediol. This facilitates the separation of 1,3-propanediol and lactic acid, and avoids side reactions between 1,3-propanediol and lactic acid caused by excessively high temperatures.

[0068] The following will explain the solution of this application with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of this application. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0069] Example 1

[0070] 1. Take *Lactobacillus reuteri* PDO / LA-3 (GDMCC NO: 64700) into MRS broth, adjust the pH to 5.5 with dilute hydrochloric acid and ammonia, and the total volume is 300 mL. Incubate anaerobically at 37℃ for 48 h, and collect the fermentation broth.

[0071] The MRS broth consisted of: 20.0 g / L glucose, 10.0 g / L peptone, 8.0 g / L beef extract powder, 4.0 g / L yeast extract powder, 2.0 g / L dipotassium hydrogen phosphate, 5.0 g / L sodium acetate, 2.0 g / L diammonium hydrogen citrate, 0.2 g / L magnesium sulfate (MgSO4·7H2O), 0.04 g / L manganese sulfate (MnSO4·4H2O), 1 mL / L Tween-80, and the remainder being water.

[0072] 2. Centrifuge the fermentation broth at 4℃ and 10000rpm for 10min to separate the supernatant and bacterial cells. Take the lower layer of bacterial cells (3.5g) and wash it 3 times with physiological saline.

[0073] 3. After rinsing the bacterial cells in the previous step, prepare a bacterial cell-physiological saline suspension with a solid content of 30%. Immerse the metal head of an ultrasonic cell disruptor into the suspension and perform ultrasonic cell disruption treatment in an ice-water bath at 4°C. The ultrasonic cell disruption conditions are: power 450W, time 45min. Measure the OD of the ultrasonically disrupted suspension at a wavelength of 600nm using a UV spectrophotometer. 600 When the rupture fluid OD 600 When the cell reaches 1 / 10 of its original size before ultrasonic disruption, it is considered to be completely disrupted.

[0074] The ultrasonically disrupted suspension was centrifuged at 4℃ and 10000rpm for 10min to separate the supernatant and bacterial fragments. The supernatant was collected to obtain the complex enzyme solution (complex enzyme concentration of 9w / w%).

[0075] 4. Add 20.0g of glycerol, 5.0g of glucose, and 0.1g of compound enzyme solution to an Erlenmeyer flask, add water to 100g, and react for 60min under nitrogen purging at 39℃ to obtain a reaction mixture.

[0076] 5. The reaction mixture was concentrated by rotary evaporation at 90℃ to remove low-boiling-point byproducts and water, and then purified by distillation at 122℃ to obtain lactic acid; further purification by distillation at 220℃ yielded 1,3-propanediol.

[0077] Example 2

[0078] The difference from Example 1 is that in step 1, the pH of the culture medium was adjusted to 4 using dilute hydrochloric acid and ammonia, and then anaerobic culture was carried out at 40°C for 24 hours. The cell mass in step 2, the concentration of the complex enzyme solution in step 3, and the mass of the complex enzyme therein are shown in Table 1.

[0079] Example 3

[0080] The difference from Example 1 is that in step 1, the pH of the culture medium was adjusted to 6 using dilute hydrochloric acid and ammonia, and then anaerobic culture was carried out at 35°C for 72 hours. The cell mass in step 2, the concentration of the complex enzyme solution in step 3, and the mass of the complex enzyme therein are shown in Table 1.

[0081] Example 4

[0082] The difference from Example 1 is that 10g of glycerol is added in step 4.

[0083] Example 5

[0084] The difference from Example 1 is that 50g of glycerol is added in step 4.

[0085] Example 6

[0086] The difference from Example 1 is that 1g of glucose is added in step 4.

[0087] Example 7

[0088] The difference from Example 1 is that 10g of glucose is added in step 4.

[0089] Example 8

[0090] The difference from Example 1 is that 0.05g of compound enzyme solution is added in step 4.

[0091] Example 9

[0092] The difference from Example 1 is that 0.5g of compound enzyme solution is added in step 4.

[0093] Example 10

[0094] The difference from Example 1 is that 1g of compound enzyme solution is added in step 4.

[0095] Example 11

[0096] The difference from Example 1 is that the temperature in step 4 is 45°C and the reaction time is 30 min.

[0097] Example 12

[0098] The difference from Example 1 is that the temperature in step 4 is 35°C and the reaction time is 360 min.

[0099] Example 13

[0100] The difference from Example 1 is that the temperature in step 4 is 39°C and the reaction time is 120 min.

[0101] Comparative Example 1

[0102] The difference from Example 1 is that *Lactobacillus reuteri* PDO / LA-3 was replaced with *Lactobacillus reuteri* CICC6118. The cell mass in step 2, the concentration of the complex enzyme solution in step 3, and the mass of the complex enzyme therein are shown in Table 1.

[0103] Comparative Example 2

[0104] The difference from Example 1 is that step 1 is as follows:

[0105] Klebsiella pneumoniae CGMCC1.6366 was placed in nutrient broth and cultured aerobically at 28°C for 48 hours. The fermentation broth was then collected.

[0106] The nutritional broth consists of 5.0 g / L peptone, 3.0 g / L beef extract powder, 5.0 g / L sodium chloride, and 0.5 g / L magnesium sulfate (MgSO4·7H2O). The remaining components are water.

[0107] The cell mass in step 2, the concentration of the complex enzyme solution in step 3, and the mass of the complex enzyme are shown in Table 1.

[0108] Comparative Example 3

[0109] The difference from Example 1 is that step 1 is as follows:

[0110] 1. Take Clostridium butyricum NCIMB 8082 into a Clostridium-enriched culture medium, adjust the pH to 7.0 with dilute hydrochloric acid and ammonia, and anaerobic culture at 30℃ for 48 h, then collect the fermentation broth.

[0111] The enhanced Clostridium culture medium consisted of 5.0 g / L peptone, 3.0 g / L beef extract, 5.0 g / L sodium chloride, 0.5 g / L magnesium sulfate (MgSO4·7H2O), with the remainder being water.

[0112] The cell mass in step 2, the concentration of the complex enzyme solution in step 3, and the mass of the complex enzyme are shown in Table 1.

[0113] The mass of the complex enzyme solutions obtained in Examples 1-13 and Comparative Examples 1-3, the consumption of glycerol and glucose, and the production of 1,3-propanediol and lactic acid are shown in Table 1.

[0114] Test case

[0115] 1. Detection of complex enzyme activity (glycerol pathway)

[0116] The enzyme activity of the composite enzyme solutions prepared in step 3 of Example 1 and Comparative Examples 1-3 was detected by measuring the glycerol pathway activity, and the specific methods are as follows:

[0117] The definition of complex enzyme activity (glycerol pathway): Under conditions of 39°C and pH 5.5, the amount of enzyme required to convert 1 μmol of glycerol in 1 minute is defined as one unit of enzyme activity (U), i.e., 1 U. 甘油 = 1 μmol glycerol / min.

[0118] Add 20.0 g of glycerol and 75.0 g of ultrapure water to an Erlenmeyer flask, adjust the pH to 5.5, preheat in a 39°C water bath for 10 min, add 100.0 mg of the complex enzyme solution, and react for 60 min under nitrogen purging at 39°C. Place the reaction solution in the Erlenmeyer flask in a boiling water bath for 10 min to inactivate the enzyme, thus terminating the reaction. Record the residual glycerol amount according to national standard GB / T 13216.6-91. The formula for calculating the specific activity of the glycerol pathway complex enzyme is as follows:

[0119]

[0120] Where: m 0甘油 m represents the initial mass (g) of added glycerol. t甘油 M represents the residual glycerol mass (g) after the reaction. 甘油 The molar mass of glycerol is 92.1 g / mol, V 复合酶液 The mass of the added compound enzyme solution is 100 mg, and t is the reaction time (min).

[0121] The test data are shown in Table 2.

[0122] 2. Detection of the conversion rate of glycerol to 1,3-propanediol by the complex enzyme

[0123] The conversion rate of the composite enzyme solution prepared in step 3 of Example 1 and Comparative Examples 1-3 to glycerol was tested respectively. The specific steps are as follows:

[0124] Add 20.0 g of glycerol and 75.0 g of ultrapure water to an Erlenmeyer flask, preheat in a 39°C water bath for 10 min, then add 100 mg of the complex enzyme solution and react for 60 min under nitrogen purging at 39°C. Place the reaction solution in the Erlenmeyer flask in a boiling water bath for 10 min to inactivate the enzyme, thus terminating the reaction. Calculate the conversion rate of glycerol to 1,3-propanediol promoted by the complex enzyme using the following formula:

[0125]

[0126] Where: m 1，3-丙二醇 m represents the mass (g) of 1,3-propanediol obtained after the reaction. 甘油 M represents the mass (g) of glycerol added before the reaction. 甘油 The molar mass of glycerol is 92.1 g / mol, M 1,3-丙二醇 The molar mass of 1,3-propanediol is 76.1 g / mol.

[0127] The test data are shown in Table 2.

[0128] 3. Detection of compound enzyme activity (glucose pathway)

[0129] The enzyme activity of the complex enzyme solutions prepared in step 3 of Example 1 and Comparative Examples 1-3 was detected by glucose pathway assay, and the specific methods are as follows:

[0130] The definition of complex enzyme activity (glucose pathway): Under conditions of 39℃ and pH 5.5, the amount of enzyme required to convert 1 μmol of glucose in 1 minute is defined as one unit of enzyme activity (U), i.e., 1U. 葡萄糖 = 1 μmol glucose / min;

[0131] Add 5.0 g of glucose and 75.0 g of ultrapure water to an Erlenmeyer flask, adjust the pH to 5.5, preheat in a 39°C water bath for 10 min, add 100.0 mg of the complex enzyme solution, and react for 60 min under nitrogen purging at 39°C. Place the reaction solution in the Erlenmeyer flask in a boiling water bath for 10 min to inactivate the enzyme, thus terminating the reaction. Record the residual glucose content according to national standard GB 5009.8-2016. The formula for calculating the specific activity of the glucose pathway complex enzyme is as follows:

[0132]

[0133] Where: m 0葡萄糖 m is the initial mass (g) of added glucose. t葡萄糖 M represents the mass (g) of glucose remaining after the reaction. 葡萄糖Given that the molar mass of glucose is 180.2 (g / mol), V 复合酶液 The mass of the added compound enzyme solution is 100 mg, and t is the reaction time (min).

[0134] The test data are shown in Table 3.

[0135] 4. Detection of glucose to lactate conversion rate by compound enzyme

[0136] The conversion rate of glucose to lactate by the composite enzyme solutions prepared in step 3 of Example 1 and Comparative Examples 1-3 was tested respectively. The specific steps are as follows:

[0137] Add 5.0 g of glucose and 75.0 g of ultrapure water to an Erlenmeyer flask, adjust the pH to 5.5 with dilute hydrochloric acid, preheat in a 39°C water bath for 10.0 min, add 100.0 mg of the compound enzyme solution, and react for 60 min under nitrogen purging at 39°C. Then, place the reaction solution in the Erlenmeyer flask in a boiling water bath for 10 min to inactivate the enzyme, thus terminating the reaction. Calculate the conversion rate of glucose to lactic acid promoted by the compound enzyme using the following formula:

[0138]

[0139] Where: m 乳酸 m represents the mass (g) of lactic acid obtained after the reaction. 葡萄糖 M represents the mass (g) of glucose added before the reaction. 葡萄糖 Given that the molar mass of glucose is 180.2 (g / mol), M 乳酸 The molar mass of lactic acid is 90.1 g / mol.

[0140] The test data are shown in Table 3.

[0141] The results are shown in Table 1. Compared with Comparative Examples 1-3, the complex enzymes generated by Lactobacillus reuteri PDO / LA-3 in Examples 1-13 have higher activity and can simultaneously and efficiently hydrolyze glycerol and glucose, thereby obtaining high yields of 1,3-propanediol and lactic acid.

[0142] The complex enzymes produced by *Lactobacillus reuteri* CICC 6118 (Comparative Example 1) and *Clostridium butyricum* NCIMB 8082 (Comparative Example 3) exhibited low enzyme activity in the glycerol pathway, resulting in poor catalytic ability to convert glycerol to 1,3-propanediol, leading to lower conversion rates and yields of 1,3-propanediol. Furthermore, the conversion rate and yield of lactic acid were also lower than those of *Lactobacillus reuteri* PDO / LA-3 (Example 1).

[0143] Comparative Example 2's *Klebsiella pneumoniae* CGMCC1.6366 primarily exhibits a metabolic pathway that converts glycerol to 1,3-propanediol. It also possesses a metabolic pathway to convert glycerol to lactic acid, and the extracted complex enzyme can catalyze this conversion to some extent, but the conversion efficiency and lactic acid yield are very low. Furthermore, the glycerol conversion rate and yield are lower than those of *Lactobacillus reuteri* PDO / LA-3 in Example 1.

[0144] Table 1

[0145]

[0146]

[0147] Table 2

[0148] Glycerylase activity (U / mg) Glycerin consumption (g) 1,3-Propanediol yield (g) 1,3-Propanediol conversion rate (%) Example 1 36.2 20 11.8 71.4 Comparative Example 1 10.9 6 2.8 16.9 Comparative Example 2 21.7 12 7.4 44.8 Comparative Example 3 18.1 10 5.1 30.9

[0149] Table 3

[0150] Glucosamine activity (U / mg) Glucose consumption (g) Lactic acid production (g) Lactic acid conversion rate (%) Example 1 4.6 5 1.8 72.0 Comparative Example 1 4.6 5 2.1 84.0 Comparative Example 2 0.5 0.5 0.1 4.0 Comparative Example 3 4.6 5 1.4 56.0

[0151] Although embodiments of this application have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting this application. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of this application.

Claims

1. A complex enzyme, characterized in that, The complex enzyme includes bioenzymes of the glycerol-1,3-propanediol pathway and bioenzymes of the glucose-lactic acid pathway, and is obtained by fermentation culture of Lactobacillus reuteri GDMCC NO: 64700.

2. A method for preparing the complex enzyme of claim 1, characterized in that, include, The complex enzyme was obtained by fermenting Lactobacillus reuteri GDMCC NO:64700.

3. The method according to claim 2, characterized in that, The culture medium for fermentation includes carbohydrates; The carbohydrates include at least one of glucose, starch, fructose, sucrose, and maltose; And / or, the sugar content in the culture medium is 1 g / L to 50 g / L.

4. The method according to claim 2, characterized in that, The culture medium further contains at least one of the following: peptone, beef extract, yeast extract, dipotassium hydrogen phosphate, sodium acetate, diammonium hydrogen citrate, magnesium sulfate, manganese sulfate, Tween-80, and water; And / or, the pH of the culture medium is 4 to 6; And / or, the fermentation culture temperature is 35℃~40℃, and the time is 24h~72h.

5. The method according to claim 2, characterized in that, Further includes: The fermentation broth obtained from the fermentation culture is subjected to a first centrifugation treatment, and the lower layer of bacterial precipitate is collected. The bacterial cell precipitate was subjected to cell disruption treatment to obtain fragments; The fragmented material is subjected to a second centrifugation process, and the supernatant is collected to obtain the complex enzyme.

6. The method according to claim 5, characterized in that, The first centrifugation process is carried out at a speed of 8000 rpm to 12000 rpm, a time of 5 min to 20 min, and a temperature of 0℃ to 10℃. And / or, before performing the cell disruption treatment, the lower bacterial cell precipitate is washed with buffer or physiological saline and resuspended, and the resulting bacterial suspension is subjected to the cell disruption treatment; And / or, the cell disruption treatment includes ultrasonic cell disruption treatment, with a temperature of 0℃~10℃, a power of 200W~800W, and a time of 10min~60min; And / or, the second centrifugation process is carried out at a speed of 8000 rpm to 12000 rpm, for a time of 5 min to 20 min, and at a temperature of 0℃ to 10℃.

7. The use of the complex enzyme of claim 1 in the preparation of 1,3-propanediol and lactic acid.

8. A method for preparing 1,3-propanediol and lactic acid, characterized in that, include: Glycerol, sugars, and the complex enzyme described in claim 1, along with water, are reacted to obtain 1,3-propanediol and lactic acid.

9. The method according to claim 8, characterized in that, The carbohydrates include at least one of glucose, starch, fructose, sucrose, and maltose; And / or, based on the total mass of the glycerol, carbohydrates and water, the mass percentage of the glycerol is 10% to 50%, and / or, the mass percentage of the carbohydrates is 1% to 10%. And / or, the amount of the compound enzyme added is 0.05% to 1% of the total mass of the glycerol, carbohydrates and water; And / or, the reaction temperature is 35℃~45℃, and the time is 30min~360min.

10. The method according to claim 9, characterized in that, Further includes: The reaction solution obtained from the reaction is concentrated and distilled to separate 1,3-propanediol and lactic acid; The concentration is achieved by rotary evaporation at a temperature of 25℃ to 100℃. The distillation process includes: first controlling the distillation temperature to 122℃~125℃, collecting the distillate, and obtaining lactic acid; The distillation temperature was then controlled at 210℃~215℃, and the fraction was collected to obtain 1,3-propanediol.

Images