Patent Application
20240102062
The present invention relates to the field of tagatose production, and specifically relates to an immobilized multi-enzyme system for producing tagatose and a method for preparing tagatose with the immobilized multi-enzymes.
Tagatose is a naturally occurring rare monosaccharide. It is a keto form of galactose and also an epimer of fructose. It has similar sweetness characteristics with sucrose, but only one-third of the calories of sucrose, and is thus called a low-calorie sweetener. It provides very fresh and pure sweetness, with a similar taste of fructose. Research shows that tagatose has important physiologically functional properties such as low calorie, low glycemic index, anti-caries effect, antioxidation, prebiotic actions, improvement of intestinal functions, immune regulation, and use as a drug precursor. It can be widely used in food, beverages, medicine, health care and other fields, having huge economic potential. (Oh D-K: Tagatose: properties, applications, and biotechnological processes. App. Microbiol. Biotechnol. 2007, 76:1-8). Currently, a mainstream method for producing tagatose comprises steps such as galactose isomerization, desalination, decolorization, separation, concentration, and crystallization to obtain pure tagatose. However, this method has several disadvantages. The galactose cannot be completely converted into tagatose, and thus the final product is a mixture of galactose and tagatose, resulting in a low conversion rate and the need of a complex tagatose separation process. While the cost for tagatose separation is high, the galactose as a raw material is not at a low price, both contributing to a high cost for tagatose production. (Rhimi M, Aghajari N, Juy M, Chouayekh H, Maguin E, Haser R, Bejar S: Rational design of Bacillus stearothermophilus US1001-arabinose isomerase: Potential applications for d-tagatose production. Biochim. 2009, 91:650-653. Oh H-J, Kim H-J, Oh D-K: Increase in d-tagatose production rate by site-directed mutagenesis of 1-arabinose isomerase from Geobacillus thermodenitrificans. Biotechnol. Lett. 2006, 28:145-149. Bosshart A, Hee C S, Bechtold M, Schirmer T, Panke S: Directed divergent evolution of a thermostable D-tagatose epimerase towards improved activity for two hexose substrates. ChemBioChem 2015, 16:592-601.) Both the CJ corporation in Korea and the Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences have developed a new route for producing tagatose with multiple enzymes in vitro (WO2018004310A1, CN109790524A, CN107988286A, and CN109666620A). With this route, starch, maltodextrin, or sucrose can be used as a raw material to produce tagatose through enzyme-based catalytic reactions in multiple steps. It represents a fundamental change from the existing isomerization-based tagatose production process.
However, this new route involves multiple enzyme molecules which require a plurality of steps for extraction and purification before they can be used in the catalytic reactions, resulting in a high cost for enzyme preparation. Moreover, the biological enzymes are water-soluble molecules and thus can hardly be recovered and reused after the completion of the catalytic reactions, resulting in a waste of enzymes. These factors lead to the high production cost for the new route of tagatose production. Therefore, there is an urgent need to develop a method for immobilizing multiple enzymes, which can enable the immobilization of the multiple enzymes in the new route of tagatose production, so that the enzymes can be recycled and the production cost can be lowered.
A method for preparing a porous magnetic microsphere and a carrier for immobilizing the enzyme was proposed (CN105039297A). The method had a simple preparation process with mild and green conditions. It achieved relatively high enzyme loading and relative enzyme activity by applying a porous magnetic microsphere to the immobilization of alcohol dehydrogenase. However, this method is limited to the immobilization of a single enzyme molecule, and there is no description about the effect of recycling of the immobilized enzyme. Thus, it needs further study and exploration on whether the method can be used to co-immobilize the multiple enzyme molecules in the above new route for tagatose production. Moreover, there are reports on production of tagatose using an immobilization technology. For example, Oh et al. used calcium alginate to immobilize the L-arabinose isomerase derived from E. coli to obtain 30 g/L tagatose within 24 h, with a conversion rate of 30%. The immobilized enzyme maintained a basically stable activity in conversion in 8 batches (Oh D K, Kim H J, Kim P, et al. Development of an immobilization method of L-arabinose isomerase for industrial production of tagatose. Biotechnology Letters, 2001, 23, 1859-1862). As another example, a method for immobilizing L-arabinose isomerase with spores of Bacillus subtilis was reported in 2018, wherein the immobilized enzyme can retain a residual activity of more than 85% after 20 times of recycling (CN 107937454 B). However, these reports are limited to the immobilization of L-arabinose isomerase only, and there are few reports on the immobilization of the multiple enzymes involved in the tagatose production route.
An object of the present invention is to provide a method for preparing an immobilized multi-enzyme system, an immobilized multi-enzyme system obtained therefrom, and a method for producing tagatose with the immobilized multi-enzyme system. The method for producing tagatose comprises immobilizing the multi-enzymes for enzyme-based catalytic conversion for tagatose production with a porous microsphere to obtain immobilized multi-enzymes, and producing tagatose with the immobilized multi-enzymes through catalysis. With the present invention, the enzymes can be recycled and thus the amounts of enzymes required to be added to produce tagatose in the new route can be greatly reduced, thereby reducing the production cost.
Specifically, the present invention provides a method for preparing an immobilized multi-enzyme system for producing tagatose. The immobilized multi-enzyme system is obtained by uniformly mixing a porous dopamine microsphere with a multi-enzyme mixture which is used for producing tagatose. In the present invention, a porous dopamine microsphere is used as a carrier for multi-enzyme immobilization. In terms of the structure of the carrier for multi-enzyme immobilization, a larger specific surface area of the material can provide more adsorption sites for enzyme molecules to achieve a higher loading capacity per unit mass of carrier. A higher porosity is conducive to rapid substrate transport, and thus reduction of the steric hindrance effect of the enzyme immobilized carrier on the mass transfer in reactions, ensuring the enzymes' activities in catalysis. In terms of components of the material, dopamine can provide appropriate hydrophilicity and hydrophobicity to enhance the stabilities of the immobilized multi-enzymes. In terms of recycling of enzymes, the microsphere has a relatively large particle size, and the immobilized multi-enzymes can be recycled simply by centrifugation. Thus, the amounts of the enzymes used for multi-enzyme catalysis for tagatose preparation and thus the production cost can be reduced.
The multi-enzyme mixture includes the enzymes for tagatose production, specifically, glucan phosphorylase (aGP), phosphoglucomutase (PGM), phosphoglucose isomerase (PGI), tagatose 6-phosphate 4-epimerase (T6PE) and tagatose 6-phosphate phosphatase (T6PP).
The porous dopamine microsphere can be prepared by a method comprising the following steps:
The present invention has optimized the method for preparing a porous dopamine microsphere, in which the structure, the composition and the enzyme binding force of the microsphere are modified to improve the catalysis and recycling performance of the immobilized multi-enzyme system.
In step (1) of the preparation of the porous dopamine microsphere, the concentrations of the raw materials and the stirring speed determine the basic structure and shape of the microsphere. In the present invention, the sodium carbonate aqueous solution and the calcium chloride aqueous solution preferably have the same concentration, and more preferably a concentration of 0.1-1 M. In actual operations, the sodium carbonate aqueous solution is preferably poured into an equal volume of calcium chloride aqueous solution quickly. The stirring speed in step (1) has a substantial impact on the size, shape, uniformity and the like of the microsphere, thereby affecting the catalytic effects of the immobilized multi-enzymes. In the present invention, the stirring is preferably carried out at a speed of 700-1,500 rpm, and more preferably 700-1,000 rpm.
In step (2) of the preparation of the porous dopamine microsphere, dopamine is introduced which can provide appropriate hydrophilicity and hydrophobicity to enhance the stabilities of the immobilized multi-enzymes. The present invention found through a lot of practical activities that when the ratio of the mass of dopamine in the dopamine solution to the mass of the porous calcium carbonate microsphere was 1:(3-5), preferably 1:4, the immobilized multi-enzyme system showed an excellent catalytic activity. Especially, with such a ratio, the recycling rate of the immobilized multi-enzyme system was improved, ensuring a relatively high production concentration of tagatose after multiple times of recycling of the immobilized multi-enzyme system. In practical use of the present invention, in order to increase the efficiency of dopamine binding to the porous calcium carbonate microsphere, the dopamine solution is preferably prepared by dissolving dopamine in a 40-60 mM Tris-HCl buffer at pH 8-9.
As a preferred embodiment of the present invention, the porous dopamine microsphere is prepared by a method comprising the following steps:
The enzymes for tagatose production involved in the present invention comprise glucan phosphorylase (αGP), phosphoglucomutase (PGM), phosphoglucose isomerase (PGI), tagatose 6-phosphate 4-epimerase (T6PE) and tagatose 6-phosphate phosphatase (T6PP). When the above five enzymes are co-immobilized to the porous dopamine microsphere, the enzymes can be recycled with their catalytic efficiencies retained, thereby significantly reducing the cost of enzyme supply. According to the present invention, each gram of the porous dopamine microsphere is preferably attached with 1,500-2,500 U of glucan phosphorylase, 1,500-2,500 U of phosphoglucomutase, 1,500-2,500 U of phosphoglucose isomerase, 1,500-2,500 U of tagatose 6-phosphate 4-epimerase, and 1,500-2,500 U of tagatose 6-phosphate phosphatase; more preferably, each gram of the porous dopamine microsphere is attached with 2,000 U of glucan phosphorylase, 2,000 U of phosphoglucomutase, 2,000 U of phosphoglucose isomerase, 2,000 U of tagatose 6-phosphate 4-epimerase, and 2,000 U of tagatose 6-phosphate phosphatase, so as to ensure that a desired catalytic efficiency and recycling of the immobilized multi-enzymes can be realized when appropriate amounts of immobilized multi-enzymes are added in practical production of tagatose.
To ensure an even distribution of the multi-enzymes on the microsphere, the immobilized multi-enzymes can be obtained by mixing and stirring well the porous dopamine microsphere and a solution of multi-enzyme mixture which is used for producing tagatose. As a preferred embodiment of the present invention, the solution of multi-enzyme mixture contains 1.5-2.5 U/ml of glucan phosphorylase, 1.5-2.5 U/ml of phosphoglucomutase, 1.5-2.5 U/ml of phosphoglucose isomerase, 1.5-2.5 U/ml of tagatose 6-phosphate 4-epimerase and 1.5-2.5 U/ml of tagatose 6-phosphate phosphatase. Preferably, it contains 2 U/ml of glucan phosphorylase, 2 U/ml of phosphoglucomutase, 2 U/ml of phosphoglucose isomerase, 2 U/ml of tagatose 6-phosphate 4-epimerase and 2 U/ml of tagatose 6-phosphate phosphatase. The porous dopamine microsphere and the solution of multi-enzyme mixture are in a mass-to-volume ratio of 1 g:(1.5-2.5) L.
The present invention also provides an immobilized multi-enzyme system obtained by the above preparation method. The present invention further provides a method for producing tagatose with the immobilized multi-enzyme system, wherein the method comprises using starch or a starch derivative as a raw material, and carrying out enzyme-based catalytic conversion with the immobilized multi-enzyme system to produce tagatose.
As a preferred embodiment of the present invention, the method specifically comprises taking 50-150 g/L of starch or starch derivative, an 80-120 mM HEPES buffer at pH 6.0-7.0, 10-50 mM inorganic phosphate, 3-7 mM divalent magnesium ions, 0.3-0.7 mM zinc ions or manganese ions, 3-7 U/ml of debranching enzyme, and 3-7 mg immobilized multi-enzymes/ml reaction liquid; carrying out enzyme-based catalytic conversion reactions at 40-70° C.; and collecting tagatose. The starch derivative is, for example, maltodextrin, amylose, amylopectin, or oligosaccharide.
After the above reactions are completed, solid-liquid separation is carried out to collect the immobilized multi-enzymes, which can be recycled for the preparation of inositol. The immobilized multi-enzymes can maintain relatively high catalytic activities when they are recycled for 1-9 times, preferably 1-7 times.
Compared with the prior art, the immobilized multi-enzyme system for producing tagatose prepared by the present invention has excellent catalytic activities and stabilities, especially, the enzymes can be recovered simply by centrifugation. Use of the porous dopamine microsphere-immobilized multi-enzyme system in tagatose production enables a simple process with mild conditions and recycling of the enzymes. It can greatly reduce the amounts of enzymes required to be added to prepare tagatose in multiple cycles and thereby lower the production cost.
The present invention discloses a method for producing tagatose with an immobilized multi-enzyme system. A person skilled in the art can, referring to the contents of the present invention, appropriately modify the parameters for implementation of the processes. Noted that all similar replacements or modifications are apparent to a person skilled in the art and are considered within the scope of the present invention. The methods of the present invention have been described through preferred embodiments. It is apparent that relevant personnel can modify or appropriately change and combine the methods and applications described herein to implement or apply the technology of the present invention without departing from the content, spirit, and scope of the present invention.
In this example, a porous dopamine microsphere was prepared by the following method.
The porous dopamine microsphere prepared above was used to prepare an immobilized multi-enzyme system. Specifically, 200 ml of a solution of multi-enzyme mixture and 3.5 g of the porous dopamine microsphere were mixed and stirred at 300 rpm for 2.5 h to obtain the system. The solution of multi-enzyme mixture contained 2 U/ml of glucan phosphorylase, 2 U/ml of phosphoglucomutase, 2 U/ml of phosphoglucose isomerase, 2 U/ml of tagatose 6-phosphate 4-epimerase, and 2 U/ml of tagatose 6-phosphate phosphatase.
Compared with Example 1, this example was different only in that, in step (2), the dopamine had a mass concentration of 2 mg/ml instead of 6 mg/ml.
Compared with Example 1, this example was different only in that, in step (1), the sodium carbonate solution was quickly poured into an equal volume of the calcium chloride solution at 1,500 rpm.
This comparative example provided a non-immobilized multi-enzyme mixture which had the same composition as the solution of multi-enzyme mixture of Example 1.
This comparative example provided a mixture of immobilized single enzymes for tagatose production. Compared with Example 1, this example was different only in that glucan phosphorylase, phosphoglucomutase, phosphoglucose isomerase, tagatose 6-phosphate 4-epimerase, and tagatose 6-phosphate phosphatase were respectively immobilized on the porous dopamine microsphere, and then theses five immobilized single enzymes were mixed. The amount of each enzyme, the amount of the porous dopamine microsphere and the specific method for immobilization were the same as those in Example 1.
The immobilized multi-enzymes provided by Examples 1-3, the multi-enzyme mixture provided by Comparative Example 1 or the mixture of immobilized single enzymes provided by Comparative Example 2 were respectively used to prepare tagatose using the following method.
A hundred (100) g/L of starch, 100 mM HEPES buffer at pH 6.5, 40 mM inorganic phosphate, 5 mM divalent magnesium ions, 0.5 mM zinc ions or manganese ions, 5 U/ml of debranching enzyme, 5 U/ml of the immobilized multi-enzymes or the non-immobilized multi-enzyme mixture or the mixture of immobilized single enzymes provided by any of the above Examples or the Comparative Examples were taken. Reactions were carried out at 70° C. The concentration of tagatose was determined by high performance liquid chromatography (HPLC).
When the immobilized multi-enzymes provided by Example 1 were used for preparation, after 18 hours of reaction, 60.2 g/L of tagatose was produced with a conversion rate of 60.2%. The tagatose production concentration within 18 h was shown in
When the immobilized multi-enzymes provided by Example 2 was used for preparation, after 18 hours of reaction, 54.1 g/L of tagatose was produced with a conversion rate of 54.1%. Compared with the immobilized multi-enzymes provided by Example 1, the immobilized multi-enzymes provided by Example 2 resulted in a lower conversion rate in preparation. This may be due to that an excessively high concentration of dopamine would lead to a reduced porosity of the carrier for immobilization, thereby reducing the activities of immobilized multi-enzymes.
When the immobilized multi-enzymes provided by Example 3 was used for preparation, after 18 hours of reaction, 48.4 g/L of tagatose was produced with a conversion rate of 48.4%. Compared with the immobilized multi-enzymes provided by Example 1, the immobilized multi-enzymes provided by Example 3 resulted in a lower conversion rate in preparation. This may be due to that an increased stirring speed during preparation of a carrier for immobilization would reduce the particle size and porosity of the carrier for immobilization, thereby reducing the activities of the immobilized multi-enzymes.
When the multi-enzyme mixture provided by Comparative Example 1 was used for preparation, after 18 hours of reaction, 66.7 g/L of tagatose was produced with a conversion rate of 66.7%. The tagatose production concentration within 18 h was shown in
When the mixture of immobilized single enzymes provided by Comparative Example 2 was used for preparation, after 18 hours of reaction, 31.2 g/L of tagatose was produced with a conversion rate of 31.2%. The tagatose production concentration within 18 h was shown in
Tagatose was prepared with the method provided by Example 4, and then solid-liquid separation was carried out. The immobilized multi-enzymes were collected and recycled for the preparation of tagatose according to the same method. The tagatose production concentration in each cycle was determined by HPLC. The results were expressed as relative tagatose production concentration. The tagatose production concentration in the first cycle of reactions was set as 100%.
Preparation was carried out with the immobilized multi-enzyme system provided by Example 1. After 9 cycles, the recycled immobilized multi-enzymes resulted in a relative tagatose production concentration of 54.1%, as shown in
Preparation was carried out with the immobilized multi-enzyme system provided by Example 2. After 9 cycles, the recycled immobilized multi-enzymes resulted in a relative tagatose production concentration of 31.2%, as shown in
Preparation was carried out with the immobilized multi-enzyme system provided by Example 3. After 9 cycles, the recycled immobilized multi-enzymes resulted in a relative tagatose production concentration of 43.0%, as shown in
Preparation was carried out with the mixture of immobilized single enzymes provided by Comparative Example 2. After 9 cycles, the recycled immobilized multi-enzymes resulted in a relative tagatose production concentration of 15.1%.
Note that, since the non-immobilized multi-enzymes provided by Comparative Example 1 were hard to be recovered and reused after one cycle of reactions for preparation, recycling was not carried out with the enzymes of Comparative Example 1. This also explained the advantages of enzyme immobilization.
Although the present invention has been described in detail with general description, embodiments, and experiments above, it is obvious to a person skilled in the art that some modifications or improvements can be made based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention are within the claimed scope of the present invention.
| Number | Date | Country | Kind |
|---|---|---|---|
| 212110330594.2 | Mar 2021 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2022/076062 | 2/11/2022 | WO |
