KR20020090282A - Optimization method for preparation of tagatose by thermostable isomerase - Google Patents

Abstract

PURPOSE: Provided is an optimization method for preparing tagatose by using thermostable isomerase under high temperature of reaction condition and within the appropriately adjusted pH range with differentiating kinds of and concentrations of metal ions, thereby dramatically increasing conversion rate of galactose to tagatose. CONSTITUTION: The method of tagatose from galactose is characterized by using arabinose isomerase or thermostable galactose isomerase, which is capable of converting galactose to tagatose, at 25-100 deg.C with providing a metal ion selected from the group consisting of Mn¬2+, Mg¬2+, Ba¬2+, Fe¬2+ and Ca¬2+ within the concentration range of 0.1-5mM.

Description

Optimization method for preparation of tagatose by thermostable isomerase

The present invention relates to a method for optimizing tagatose production, and more particularly, a bioconversion for converting galactose to tagatose in a high yield using heat-resistant galactose isomerase under high temperature reaction conditions optimized with a specific type of divalent cation. It is about process.

Tagatose is an isomer of galactose, which is rare in nature, and is a low-calorie substance that has almost similar sweetness than fructose and is hardly metabolized upon absorption by the body. Sugar alcohols, which are most commonly used as sugar substitute sweeteners, have an effect of causing diarrhea when consumed over a certain amount, whereas tagatose has no side effects, and unlike sugar alcohols, browning changes like sugar, resulting in food processing. Tagatose has attracted attention as a sugar substitute sweetener because of its ability to add timely flavors (Zehener, 1988, EP 257626; Marzur, 1989, EP 0341062A2).

Currently, D-tagatose is being manufactured or studied in chemical methods, methods using microorganisms, methods using free enzymes or immobilized enzymes. Chemically known method for preparing tagatose, in which boric acid is added to aldose sugar in the presence of tertiary or quaternary amines, forms complexes with boric acid and ketose when ketose is formed, and the equilibrium is effectively shifted toward tagatose. (US Pat. No. 4,273,922; June 6, 1981). Current chemical methods for producing D-tagatose are in the presence of soluble alkali metal salts or alkaline earth metal salt catalysts until an insoluble precipitate, consisting of a metal hydroxide-tagatose complex, is formed at a pH of 10 or higher and a temperature of -15 to 40 ° C. It is made by the process of isomerizing the galactose aqueous solution with a metal hydroxide (US Pat. No. 5,002,612). However, this conventional chemical method has a disadvantage that is not suitable for mass production of tagatose. Although the chemical method is sometimes excellent in terms of economics or yield, it is a chemical process, the process itself is complicated, the reaction proceeds only under certain conditions, there is a problem of generating an industrial waste.

Therefore, there is an increasing demand for tagatose manufacturing method using environmentally friendly and more economical biological methods. Above all, considering the environmental cost, biological processes using microorganisms from inexpensive carbohydrates obtained from waste biological resources are available. Research into a method that can produce tagatose in a more efficient manner is currently attempted.

As a bio-conversion process using microorganisms, Kamori et al. (JP60248196, 85.12.7) used Arthrobacter sp . In an aqueous solution containing dulicitol at 20-35 ° C. for 1-15 days under aerobic conditions. A method for producing D-tagatose from dulicitol by culturing has been disclosed, but doublyitol is limited in mass supply, expensive, and galactitol dehydrogenase which performs this reaction is expensive. There was a drawback of increasing the manufacturing cost of the process of converting dulisitol to D-tagatose by requiring NAD as a cofactor.

On the other hand, the microbial reaction requires a pre-cultivation process, and by-products are made in addition to the target metabolites after the end of the reaction, which requires considerable cost for separation and purification, high BOD of the wastewater, and increased aeration operation cost for oxygen supply. There is this.

In contrast, the enzyme reaction is very simple compared to the microbial reaction, it is easy to set the optimization conditions of the process, the concentration of the reaction product can be increased, and is usually energy-efficient because it is performed under mild conditions.

Bioconversion processes for converting aldose or aldose derivatives to ketose or ketose derivatives using enzymatic methods are known in the art. For example, the process of converting glucose to fructose using glucose isomerase is widely practiced on a commercial scale. However, the enzymatic method of converting galactose to tagatose has not been widely used until recently.

UK Patent Specification No. 1,497,888 discloses a method for isomerizing D-galactose to D-tagatose using L-arabinose isomerase known to produce L-ribulose from L-arabinose. However, there was no mention of reaction conditions that could increase the tagatose conversion yield in such bioconversions.

Like glucose isomerase, arabinose isomerase has different actions in vivo and in vitro. That is, in vivo, arabinose isomerized to ribulose, but in vitro, it converts galactose to tagatose, and like glucose isomerase, arabinose isomerase also acts as aldose, galactose and ketose, depending on the reaction temperature. Equilibrium between the tagatose is changed, the higher the temperature is characterized in that the equilibrium toward the ketose proceeds.

The present inventors have disclosed a method for preparing tagatose from galactose using E. coli-derived arabinose isomerase, but the thermal stability and conversion yield of the enzyme were low (domestic patent application No. 99-16118; PCT WO Patent Pending PCT / KR99 / 00661). For that reason, the present inventors have searched for a novel heat-resistant galactose isomerase that is stable at high temperatures and maintains enzymatic activity to shift the equilibrium of the overall reaction rate towards tagatose, resulting in increased thermal stability and conversion of galactose to tagatose. We cloned from nature a thermostable enzyme with a new type of arabinose isomerase activity, determined the isolated enzyme gene sequence and compared the homology between known arabinose isomerase gene sequences and amino acid sequences. E. coli and 9.5% (SEQ ID NO: 3), 20.0%, and Bacillus subtilis, respectively, 61.6% (SEQ ID NO: 4), 55.4%, Salmonella and 58.5% and 54.3%, respectively, known arabinose isomerase It is a new heat-resistant enzyme that can convert galactose into tagatose while being different from It was named "galactose isomerase" (Domestic Patent No. 2000-78833), and the enzyme gene was produced by an error-producing PCR method with an 11-fold improvement in enzyme activity. Korean Patent Priority Claim No. 2001-21552. However, the results of studies on the optimum reaction conditions such as varying the type and concentration of metal ions in tagatose production using arabinose isomerase or heat-resistant galactose isomerase have not been published.

The present inventors investigated the various reaction conditions such as temperature, pH, cofactors, etc. to increase the yield of tagatose conversion from galactose using the first heat-resistant galactose isomerase cloned from nature. The present invention was completed by observing that the reaction yield was significantly increased when the divalent cation was supplied at an appropriate concentration, as well as the stability to temperature and pH was further increased.

It is therefore an object of the present invention to provide an optimized method for producing tagatose from galactose using novel heat resistant enzymes.

Features and other advantages of the present invention will be more clearly understood by the following configuration.

1 is a diagram showing the effect of various metal ions on the tagatose conversion yield of heat-resistant galactose isomerase.

2 is a diagram showing the conversion yield of tagatose according to the concentration change of Mn ²⁺ , Mg ²⁺ , Ba ²⁺ , Fe ²⁺ .

3 is a diagram showing the tagatose conversion yield of the heat-resistant galactose isomerase with temperature in the absence of Mn ²⁺ .

4 is a diagram showing the tagatose conversion yield of the heat-resistant galactose isomerase with temperature in the state containing Mn ²⁺ .

5 is a diagram showing the tagatose conversion yield of the heat-resistant galactose isomerase according to pH in the state that Mn ²⁺ is not included.

6 is a diagram showing the tagatose conversion yield of heat-resistant galactose isomerase according to pH in the state containing Mn ²⁺ .

The above object of the present invention was achieved by using a heat resistant galactose isomerase (SEQ ID NO) to determine the tagatose conversion yield according to the concentration of metal ions, pH, and the like at a high temperature.

As an example of the heat resistant galactose isomerase used by the present inventors in the preparation of tagatose, a heat resistant galactose isomerase encoded from SEQ ID NO: 1 was used. The gene having SEQ ID NO: 1 is a heat-resistant galactose isomerase encoding gene cloned by the present inventors from nature in a high temperature condition. The heat-resistant galactose isomerase had a very excellent effect of producing tagatose from galactose with stable high yield even at high temperature of 40-80 ° C., but no other reaction conditions for optimizing tagatose production were disclosed. I never did.

In order to find such cofactors that can enhance the catalytic activity of the heat-resistant enzymes, the present inventors have a variety of transition metal ions (Fe ²⁺ ) that are abundant with typical metal ions (K ⁺ , Na ⁺ , Mg ²⁺ , Ca ²⁺ ). , Cu ²⁺ , Mn ²⁺ , Zn ²⁺ , Co ²⁺ , Mn ²⁺ , Ni ^2+, etc.) while varying the concentration of tagatose conversion for each metal ion concentration (Fig. 1), The yield of tagatose conversion at different temperatures and pHs in the optimized concentration range of the metal ions was investigated, indicating that catalytic activity was observed in the presence of metal ions such as Zn ²⁺ , Ni ²⁺ , Cu ²⁺ , or EDTA. On the other hand, it was found that the catalytic activity is greatly increased when divalent cations such as Mn ²⁺ , Mg ²⁺ , Ba ²⁺ , Fe ²⁺ and Ca ²⁺ are present. It was found that the stability to temperature and pH increases in the range where ions exhibit maximum activity. . Investigation of the catalytic activity of the Mn ²⁺ , Mg ²⁺ , Ba ²⁺ , Fe ²⁺ metal ion concentrations showed that the Tagatose conversion yield increased in proportion to the metal ion concentration in the low metal ion concentration range, but increased by 0.1. Above the mM concentration, it was found that the conversion yield almost reached equilibrium (FIG. 2).

In particular, among the metal ions that enhance the catalytic activity, manganese ions (Mn ²⁺ ) was found to increase the reaction yield of heat-resistant galactose isomerase by 2.6 times as compared to the control without the addition of metal ions, Mn ²⁺ It was observed that the maximum activity was different according to temperature and pH according to the presence or absence. That is, when Mn ²⁺ is not present, the optimum activity temperature is about 55-67 ℃, and when it is out of this range, activity decreases rapidly. B (FIG. 3), when the Mn ²⁺ ions were supplied, the catalytic activity was very stabilized even at a high temperature range, and the yield of tagatose conversion was significantly increased compared to the case where no additive was added (FIG. 4). In other words, when Mn ²⁺ is not present, the optimum active pH ranges from about 7.0-8.8 (FIG. 5), but when Mn ²⁺ ions are supplied, the reaction optimum active pH is further extended to alkaline conditions in the range 7.0-10. The activity was also very high at (Fig. 6).

In order to confirm this metal ion effect in tagatose production in another example, the present inventors determined the yield of tagatose conversion using non-heat-resistant arabinose isomerase produced by recombinant strain E. coli JM1C / pTC101 (KCTC 0606BP). Investigate. The strain is a transgenic strain comprising a vector pTC101 recombined with the a-rabinose isomerase (EC 5.3.1.4) gene ara I cloned from E. coli. The arabinose isomerase produced by the strain has the ability to convert galactose to tagatose, and the tagatose conversion activity was found to be optimal at a temperature of 27-35 ° C. and pH 7.5-8.5. As a result of investigating the catalytic activity effect of the metal ion on the enzyme, Fe ²⁺ , Mn ²⁺ , Mg ²⁺ , Ba ²⁺ , Ca ²⁺ , and the like compared to the control group without addition of the metal ion like the heat-resistant galactose isomerase It was found that the addition of divalent cations significantly increased the catalytic activity to 1.45-2.35 times, but the metal ions such as Ni ²⁺ , Cu ²⁺ , and Zn ²⁺ significantly reduced the catalytic activity (Table 1). In particular, as with heat-resistant enzymes, not only Mn ²⁺ but also Fe ²⁺ were found to greatly promote the catalytic activity of arabinose isomerase in tagatose conversion yield.

As described above, it can be seen that the metal ion has a very important effect on increasing the yield of tagatose of the arabinose isomerase or the heat-resistant galactose isomer cloned from nature. As such, the bioconversion process of optimizing the production of tagatose from galactose by varying the type and concentration of metal ions is not only the heat-resistant isomerase of the present invention, but also the amino acid sequence having the same possibility by the enzyme coding gene and codon degeneracy. The present invention can also be applied to heat-resistant isomerase encoded by a gene encoding, and also to other heat-resistant galactose isomerase or other heat-resistant arabinose isomerase that isomerizes tagatose from galactose. Furthermore, the tagatose conversion yield can be significantly increased by providing metal ions alone or in plurality. Likewise, heat-resistant galactose isomerase protein having an amino acid sequence encoded by SEQ ID NO: 1 or heat-resistant protein derivatives having an amino acid sequence substituted with another amino acid functionally identical or similar thereto are also within the spirit and scope of the present invention. The tagatose conversion yield can be optimized in the temperature range of 25-100 ° C by varying the type or concentration of ions. For example, the heat-resistant snow galactose isomerase or arabinose isomerase having the same activity as the galactose isomerase of the present invention or having the same similar amino acid sequence and converting the galactose to tagatose may be E. coli , Bacillus sp. Bacillus sp ), Salmonella sp. , Enterobacter sp. , Lactobacillus sp. , Pseudomas sp. , Acetobacter sp. ), jimo Pseudomonas sp (Zymomonas sp.), glucono bakteo sp (Gluconobactersp.), separation tank emptying sp (Rhizobium sp.), also bakteo sp (Rhodobacter sp.), Agrobacterium sp (Agrobacterium sp ) can be derived from a number of other microorganisms such as, and can be optimized as many tagatose conversion yield while varying the type or the concentration of metal ions and in particular manganese One will be able to significantly increase the conversion yield tagatose and (Mn ²⁺⁾ is an important catalyst activity promoter.

Tagatose manufacturing process using the heat-resistant enzyme of the present invention is not only superior to the reaction conditions, solvent, reaction specificity, product concentration, etc. than the tagatose manufacturing process by the current chemical method, but also using the conventional arabinose isomerase Otherwise, since the process is performed under high temperature reaction conditions, the reaction rate is increased to convert tagatose with temperature rise, and the risk of microbial contamination is significantly reduced.

Therefore, the heat resistant galactose isomerase of the present invention can be used to prepare tagatose from galactose in a free state or immobilized on a suitable carrier under optimized reaction conditions in which the type and concentration of metal ions, pH and temperature are properly controlled. Tagatose, prepared in high yield, can be used as low calorie food sweeteners and fillers, compound synthetic intermediates with optical activity, and as additives in detergents, cosmetics and pharmaceutical formulations.

Hereinafter, the configuration and operation of the present invention will be described in more detail with reference to Examples. However, the following examples are only for the purpose of illustrating the present invention and the scope of the present invention is not limited by the following examples.

Example 1 Investigation of the Effect of Metal Ions on the Tagatose Conversion Yield of Heat-Resistant Galactose Isomerase

In this Example, E. coli JM105 / pL151MO containing a novel heat resistant galactose isomerase gene having the nucleotide sequence of SEQ ID NO: 1 was incubated at 60 ° C., after separation and purification of the heat resistant galactose isomerase from the culture. Used as an enzyme source. The amount of tagatose produced was determined by cystein-carbazole method to confirm the degree of tagatose production. One unit of enzyme is defined as the amount of enzyme that produces 1 μg of tagatose per minute.

(1) Experimental Example 1: Effect of various metal ions on the tagatose conversion yield of heat-resistant galactose isomerase

0.5mM Mn ²⁺ , Mg ²⁺ , Ba ²⁺ , Fe ²⁺ , Ca ²⁺ , Zn ²⁺ , Ni ²⁺ , Cu ²⁺ and other metal ions and 1 g / l galactose added 3 units of heat-resistant galactose isomerase was added to the solution to undergo isomerization reaction at 60 ° C. for 12 hours, after which the reaction was terminated and the amount of tagatose produced was quantified (FIG. 1).

As shown in FIG. 1, metal ions such as Zn ²⁺ , Ni ²⁺ , Cu ²⁺ and the like have an effect of significantly reducing catalytic activity, while Mn ²⁺ , Mg ²⁺ , Ba ²⁺ , Fe ² Divalent cations such as ⁺ and Ca ²⁺ enhance catalytic activity and increase the tagatose conversion yield significantly compared to the metal ion-free control group. Among these metal ions, especially manganese ions (Mn ²⁺ ) are Toss conversion yield was found to have a very significant effect.

Experimental Example 2 Change in Tagatose Conversion Yield with Mn ²⁺ , Mg ²⁺ , Ba ²⁺ , Fe ²⁺ Concentration

In Experimental Example 1, the effect of the concentrations of these ions on the tagatose conversion yield was examined while varying the concentrations of Mn ²⁺ , Mg ²⁺ , Ba ²⁺ , Fe ²⁺ metal ions having catalyst promoting activity (FIG. 2).

As shown in FIG. 2, the production of tagatose increased primarily in the low metal ion concentration range, but isomerization by these metal ions reached almost equilibrium in the concentration range of 0.1 mM or more. .

(3) Experimental Example 3: change of tagatose conversion yield with temperature

Among the irradiated metal ions, the manganese ions (Mn ²⁺ ) having excellent catalytic activity were examined for the conversion of tagatose with temperature in the isomerization reaction in which the metal was not added or 0.5 mM was added (FIGS. 3 and 4).

As a result, it can be seen that the activity according to the temperature according to the presence or absence of Mn ²⁺ is a big difference. That is, when no addition of Mn ²⁺ optimum activity temperature is about 55-67 ℃ range and this range is out of tagatose production in the case of very reduced symptoms appear or nateu (Fig. 3), Mn ²⁺ was added to a temperature range In addition, it was found that the stabilization yield of the tagatose was significantly increased compared to the case where it was very stable (FIG. 4).

(4) Experimental Example 4: The change in tagatose conversion yield according to pH

In the isomerization reaction without adding manganese ions (Mn ²⁺ ) or 0.5 mM, the yield of tagatose conversion according to pH was investigated and the results are shown in FIGS. 5 and 6.

The experimental results, in the case of additive-free Mn ²⁺ optimum activity is about pH 7.0 to 8.8 range, and if outside this range eoteuna indicate aspects that tagatose production decreased (Fig. 5), in the case of the Mn ²⁺ activated addition reaction optimum pH is It was further extended to 7.0-10 range, and the yield of tagatose was almost similar, indicating that the activity was maintained very high even under alkaline conditions (FIG. 6).

Example 2 Investigation of the Effect of Metal Ions on the Tagatose Conversion Yield of Arabinos Isomerase

The purified arabinose isomerase from the culture of recombinant E. coli JM10 / pTC101 (KCTC 0603BP) was EDTA-dialyzed to remove metal ions and added Tris-HCL buffer (pH 8.0), 50 mM galactose, and 0.5 mM each metal ion. After reacting at 30 ° C. for 1 hour, the amount of tagatose produced by each metal ion added was investigated (Table 1).

As can be seen in Table 1, the divalent cations such as Fe ²⁺ , Mn ²⁺ , Mg ²⁺ , Ba ²⁺ , Ca ²⁺ and the like compared to the control group to which the metal ion was not added, yield the tagatose conversion yield. Although it is very effective to increase 1.45-2.35 times, it can be seen that metal ions such as Ni ²⁺ , Cu ²⁺ , and Zn ²⁺ decrease the catalytic activity significantly. The types of metal ions that promote or inhibit catalytic activity were very similar to those of heat-resistant galactose isomerase.

As described above, the isomerization reaction using heat-resistant galactose isomerase with varying types and concentrations of metal ions within a high temperature reaction condition and a properly controlled pH range may significantly increase the conversion yield of galactose to tagatose. It has a very excellent effect.

Claims (5)

Arabinose having an activity of converting galactose to tagatose by providing a metal activity promoter selected from the group consisting of Mn ²⁺ , Mg ²⁺ , Ba ²⁺ , Fe ²⁺ and Ca ²⁺ at 25-100 ° C. A method for producing tagatose from galactose using isomerase or heat resistant galactose isomerase. The method of claim 1, wherein the heat resistant galactose isomerase is an enzyme encoded by SEQ ID NO: 1 exhibiting catalytic activity at 40-80 ° C, or a heat resistant galactose isomerase having a functionally equivalent function. The method of claim 1, wherein the arabinose isomerase is an arabinose isomerase derived from E. coli JM1C / pTC101 (KCTC 0606BP) that exhibits optimum catalytic activity at 25-35 ° C. 3. The method according to claim 1 or 2, wherein tagatose is prepared from galactose by providing metal ions at a concentration in the range of 0.1-5 mM. The method of claim 4, wherein the metal ion is manganese ion (Mn ²⁺ ).