WO2004007738A1

WO2004007738A1 - Optimization method for preparation of tagatose by thermostable isomerase isomerase

Info

Publication number: WO2004007738A1
Application number: PCT/KR2002/001321
Authority: WO
Inventors: Pil Kim; Jin-Hwan Choi; Sang-Hyun Yoon; Hoe-Jin Roh
Original assignee: Tongyang Confectionery Co.
Priority date: 2002-07-12
Filing date: 2002-07-12
Publication date: 2004-01-22
Also published as: AU2002315938A1

Abstract

An optimized method for the production of tagatose is disclosed. The method can greatly increase the conversion yield of tagatose from galactose using a thermostable galactose isomerase in the presence of specifice types of metal ions and concentrations thereof under a relatively high temperature and appropriately controlled pH conditions.

Description

OPTIMIZATION METHOD FOR PREPARATION OF TAGATOSE BY THERMOSTABLE ISOMERASE

Technical Field The present invention relates to an optimized method for the production of tagatose, and more particularly to a biological method for converting galactose to tagatose at high yield using a thermostable galactose isomerase in the presence of specific types of divalent metal ions at an optimum high reaction temperature.

Background Art

Tagatose, an isomer of galactose, is not commonly found in nature, and is a low calorie carbohydrate sweetener which is not metabolized by the body, but has almost the same sweetness as fructose. In general, sugar alcohols which are most commonly used as sugar substitutes, have a laxative effect when a person consumes more than a certain amount. However, tagatose has no side effects such as the laxative effect of sugar alcohols. Further, since tagatose undergoes browning like sugar but unlike sugar alcohols, it has a beneficial effect of giving appropriate flavors to foods during food processing. Due to these advantages, tagatose has drawn attention as a sugar substitute (Zehener, 1988, EP 257626; Marzur, 1989, EP 0341062A2). Major progress in the production of D-tagatose has been achieved using

chemical methods, microbial methods, methods using free enzymes or immobilized enzymes, etc. Among these methods, U. S. Pat. No. 4,273,922, published on June 16, 1981, discloses a process for converting aldose sugars to ketose sugars at high yield. According to the process, when a ketose is formed by adding boric acid to an aldose sugar in the presence of a tertiary or quaternary amine, the ketose and boric acid form a complex, thereby effectively driving the reaction equilibrium toward the ketose to produce tagatose. Another current chemical method for producing D-tagatose can be achieved by isomerizing aqueous galactose into a metal hydroxide under the conditions of a pH of 10 or higher and a temperature of -15~40°C in the presence of a soluble alkali metal salt or alkaline earth metal salt catalyst until an insoluble metal hydroxide-tagatose complex is precipitated (see, U.S. Pat. No. 5,002,612). However, these conventional chemical methods cannot to be applied for mass production of tagatose. While some chemical methods have been found to be effective in economic efficiency and yield, they have complicated and ineffective processes which can be performed only under specific conditions, and generate large amounts of industrial wastes.

For these reasons, there is a need for biological methods to achieve the environmentally friendly and economical production of tagatose. Particularly, considering environmental costs, many studies on biological processes for efficiently producing tagatose from low-priced carbohydrates obtainable from biological wastes materials using microbes are currently ongoing.

Kamori et. al. suggested a method for producing D-tagatose from dulicitol by culturing a bacterial strain belonging to Arthrobacter genus in an aqueous solution containing dulicitol under aerobic conditions at 20-35 °C for 1-15 days (see, JP

60248196, published on December 7, 1985). However, this conversion method has disadvantages that dulicitol is not available in large quantities and is expensive. Moreover, the enzyme galactitol dehydrogenase requires NAD (nicotinimide-adenine dimicleotide), an expensive co-enzyme which makes the conversion of dulcitol to D-tagatose more costly.

On the other hand, microbial methods have disadvantages that they require pre-incubation of microbes, and desired metabolic products require the process to separate and purify them from generated by-products with considerable costs. Further, since wastewater generated through the microbial methods has a high BOD value, additional air-feeding means for supplying oxygen into the generated wastewater must be installed.

On the contrary, enzymatic reactions have advantages that the reaction mixture is simple to formulate and thus optimum conditions for the reaction are easy to set. In addition, products of the enzymatic reactions can be produced at high yield.

Furthermore, since the reaction can be carried out under mild conditions, it requires less energy consumption.

The process for the converting aldoses or derivatives thereof to corresponding ketoses or derivatives thereof using the enzymatic methods is well known in the art. The enzymatic conversion of glucose to fructose, for example, is widely practiced on a commercial scale. Enzymatic methods for converting D-galactose to D-tagatose, however, have not been developed until recently.

U.K. Patent Specification No. 1,497,888 alleges that the conversion of D-galactose to D-tagatose using L-arabinose isomerase is known as producing L-ribulose from L-arabinose. However, no description of the conditions for such

conversion is provided.

Like glucose isomerase, arabinose isomerase exhibits different activities in vivo and in vitro. That is, arabinose isomerase converts arabinose to ribulose in vivo, but it converts galactose to tagatose in vitro and further changes the equilibrium of the isomerization between galactose (an aldose) and tagatose (a ketose), depending on reaction temperature, thereby driving the equilibrium toward the ketose with increasing

temperature.

The present inventors suggested a method for producing tagatose from

galactose using an E. co/z-derived arabinose isomerase (Korean Patent Application No. 99-16118; PCT WO Patent Pending PCT/KR99/00661). The method has disadvantages in terms of low thermal stability and conversion yield. Thus, the present inventors have earnestly tried to find a novel thermostable galactose isomerase capable of driving the equilibrium of the overall reaction toward tagatose while maintaining thermal stability and enzymatic activity even at high temperature, and as a result, have cloned a novel type of thermostable enzyme from natural sources having an arabinose isomerase activity capable of increasing thermal stability and converting

galactose to tagatose. In addition, the base sequence of the cloned enzyme gene was determined, and compared with base sequence of a known arabinose isomerase gene and the amino acid sequence of the arabinose isomerase. As a result, the cloned enzyme was found to have little homology with known arabinose isomerase. That is,

the cloned enzyme shares 9.5% of homology in the base sequence (Sequence No. 3) and 20.0% in the amino acid sequence with E. coli, 61.6% in the base sequence

(sequence No. 4) and 55.4% in the amino acid sequence with Bacillus subtilis, and 58.5 % in the base sequence and 54.3% in the amino acid sequence with Salmonella.

Accordingly, the cloned enzyme was identified to be a novel thermostable enzyme different from known arabinose isomerase and capable of converting galactose to

tagatose, and was finally named "galactose isomerase" (Korean Patent No.

2000-78833). In addition, mutagenesis of the cloned enzyme was conducted through an error-prone PCR method to produce a mutant enzyme having an activity of 11 times higher than intact galactose isomerase (Korean Patent No. 2001-21552, claiming the

Korean Patent No. 2000-78833 as a priority). No studies on optimum reaction conditions, such as types and concentrations of metal ions used, for producing tagatose using the arabinose isomerase or the thermostable galactose isomerase have hitherto been reported.

Disclosure of the Invention

In order to increase the conversion yield of tagatose from galactose using the thermostable galactose isomerase first cloned from natural sources, the present

inventors have examined optimum conditions such as reaction temperature, pH, cofactors, etc., and found that when some divalent metal ions are provided as cofactors of enzymatic reaction at appropriate concentration ranges, the reaction yield can be considerably increased and the stability to temperature and pH can be improved, and as

a result, they accomplished the present invention.

Therefore, the present invention has been made in view of the above problems,

and it is an object of the present invention to provide an optimized method for the production of tagatose from galactose using a novel thermostable enzyme.

Brief Description of the Drawings

The above and other objects, features and other advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings in which:

Fig. 1 is a graph showing the effects of various metal ions on the conversion yields of tagatose by a thermostable galactose isomerase;

Fig. 2 is a curve showing the conversion yields of tagatose at various

concentrations of Mn²⁺, Mg²⁺, Ba²⁺ and Fe²⁺, respectively;

Fig. 3 is a curve showing the conversion yields of tagatose by a thermostable galactose isomerase in the absence of Mn ⁺, with increasing temperature;

Fig. 4 is a curve showing the conversion yields of tagatose by a thermostable galactose isomerase in the presence of Mn²⁺, with increasing temperature;

Fig. 5 is a curve showing the conversion yields of tagatose by a thermostable galactose isomerase in the absence of Mn²⁺, plotted versus pH; and Fig. 6 is a curve showing the conversion yields of tagatose by a thermostable galactose isomerase in the presence of Mn , plotted versus pH.

Best Mode for Carrying Out the Invention

The features and other advantages of the present invention will become more apparent from the following detailed description.

In accordance with the present invention, the above and other objects can be accomplished by examining the conversion yield of tagatose using a thermostable galactose isomerase (Sequence No. 1) in accordance with various metal ion concentrations, pH, etc., at high temperatures. As the thermostable galactose isomerase used in the present invention, the

thermostable galactose isomerase encoded by Sequence No, 1 was used, The gene having Sequence No. 1 is a gene encoding the thermostable galactose isomerase first cloned by the present inventors. The thermostable galactose isomerase has an excellent effect capable of stably producing tagatose from galactose at high yield even at a temperature as high as 40-80 °C . However, no disclosure was made of optimum reaction conditions for producing tagatose from galactose.

In order to find cofactors capable of enhancing catalytic activity of the thermostable enzyme, the present inventors examined the conversion yields of tagatose at various concentrations of typical metal ions (K⁺, Na⁺, Mg²⁺, Ca²⁺) and transition metal ions (Fe²⁺, Cu²⁺, Mn²⁺, Zn²⁺, Co²⁺, Mn²⁺, Ni²⁺, etc.) having more reactivity (Fig. 1). In addition, they examined the conversion yield of tagatose at various temperatures and pH value within optimum concentration ranges of each metal ion. As a result, it was observed that the catalytic activity was considerably decreased in the presence of Zn²⁺, Ni²⁺, Cu²⁺, EDTA, etc., whereas the catalytic activity was highly increased in the presence of divalent metal ions such as Mn , Mg , Ba , Fe , Ca , etc. It was also observed that stability to temperature and pH was increased within the ranges showing maximum catalytic activity of each metal ion. Furthermore, it was observed that the conversion yields of tagatose in the presence of relatively low concentrations of Mn , Mg , Ba , Fe were proportional to concentrations of the metal ions, but the conversion yields at concentrations higher than 0.1 mM were maintained at equilibrium (Fig. 2).

In particular, the reaction yield of the thermostable galactose isomerase in a group containing manganese ion (Mn²⁺) was 2.6 times higher than in a control group containing no metal ions, and the absence or presence of Mn²⁺ influenced the

maximum activity of the enzyme against temperature and pH. That is, in the absence of Mn²⁺, the optimum temperature for activity was within the range of 5 -67 ^°C . Out of this range, the catalytic activity sharply decreased (Fig. 3). On the contrary, in the presence of Mn²⁺, the catalytic activity was very stable even at relatively high temperatures and thus the conversion yields of tagatose were increased over the control

(Fig. 4). In addition, in the absence of Mn²⁺, optimum pH for activity was within the

range of 7.0-8.8 (Fig. 5). On the contrary, in the presence of Mn²⁺, the range of the optimum pH range for activity was broadened (pH 7.0-10) to show very high activity even under basic conditions (Fig. 6).

In order to identify other examples of effects of metal ions in the production of

tagatose, the present inventors examined the conversion yield of tagatose using a

non-thermostable arabinose isomerase produced by a recombinant strain E. coli JMlC/pTClOl (KCTC-0606BP). The recombinant strain is a transformed strain containing a recombinant vector pTClOl of the gene αrαl encoding arabinose isomerase (L-arabinose isomerase; EC 5.3.1.4), which is cloned from E. coli by the present inventors. It was found that the arabinose isomerase produced by the recombinant strain can convert galactose to tagatose, and optimum conditions for the

conversion of tagatose were a temperature of 27-35 ^°C and a pH of 7.5-8.5. The effect of metal ions on the arabinose isomerase was similar to that of the thermostable

galactose isomerase. That is, the catalytic activity m the groups containing Fe , Mn²⁺, Mg²⁺, Ba²⁺, Ca²⁺, etc., amounted to 1.45-2.35 times that of the control group containing no metal ions. However, the catalytic activity in the groups containing Ni²⁺, Cu²⁺, Zn²⁺, etc., was considerably decreased (see, Table 1). In particular, as with other thermostable enzymes, Fe²⁺ as well as Mn²⁺ greatly increased the enzymatic

activity of the arabinose isomerase in the conversion of galactose to tagatose.

As described above, it can be seen that metal ions influence the production

yield of converting galactose to tagatose by the arabinose isomerase or the thermostable galactose isomerase cloned from natural sources. The optimized biological method for converting galactose to tagatose according to the present invention can be catalyzed by not only the thermostable isomerase but also the

thermostable isomerase encoded by the gene encoding an amino acid sequence having the same functions as the gene encoding the thermostable isomerase, as identified by codon degeneracy. In addition, the method according to the present invention can use other thermostable galactose isomerases derived from different sources or other thermostable arabinose isomerases producing tagatose from galactose. Furthermore, the metal ions can be added either alone or in admixture to increase the conversion yield of tagatose. Likewise, a thermostable galactose isomerase protein having an amino acid sequence encoded by Sequence No. 1 or thermostable protein derivatives having an amino acid sequences substituted with other amino acids exhibiting identical and similar functions to the galactose isomerase protein are included within the spirit and scope of the present invention. The thermostable galactose isomerase protein and thermostable protein derivatives can optimize the conversion yield of tagatose from galactose in the presence of various types of metal ions and at various metal ion concentrations, within the temperature range of 25~100^°C . For example, the

thermostable galactose isomerases and arabinose isomerases which have the same enzymatic activity as the galactose isomerase used in the present invention, or have identical or similar amino acid sequence as the galactose isomerase used in the present invention, may be derived from various microbes, such as E. coli, Bacillus sp.,

Salmonella sp., Enterobacter sp., Lactobacillus sp., Pseudornonas sp., Acetobacter sp., Zymornonas sp., Gluconobacter sp., Rhizobium sp., Rhodobacter sp., Agrobacterium

sp., etc. Using these isomerases, the conversion yield of tagatose from galactose can be optimized in the presence of various types of metal ions and at concentrations thereof. In particular, manganese ion (Mn²⁺) plays an important role as a promoter of the enzymatic activity to increase the conversion yield of tagatose.

The optimized method for producing tagatose using the thermostable enzymes according to the present invention is advantageous in terms of reaction conditions, solvents, reaction specificity, yield, etc., over chemical methods currently used.

Further, since the method according to the present invention is performed at a relatively higher temperature than conventional methods using arabinose isomerases, the reaction equilibrium shifts toward tagatose with increasing temperature and microbial contamination can be considerably reduced.

The thermostable galactose isomerase used to produce tagatose from galactose in the present invention may be in a free state or may be immobilized to an appropriate carrier under optimized reaction conditions such as types of metal ions and concentrations thereof, pH and temperature. Tagatose produced at high yield by the method according to the present invention can be used as a sweetener for low calorie

foods, a filler, an intermediate for synthesizing optically active compounds, and an additive of detergents, cosmetics, and pharmaceutical formulations.

The present invention is illustrated in greater detail below with reference to Examples. These Examples are provided only for illustrative purposes, but are not to be construed as limiting the scope of the present invention.

Example 1: Effect of metal ions on conversion yield of tagatose from galactose by thermostable galactose isomerase

First, E. coli JM105/pL151MO comprising a thermostable galactose isomerase

gene having the base sequence of Sequence No. 1 was cultured at 60 ^°C . The thermostable galactose isomerase was isolated and purified from the culture and used as an enzyme source for subsequent experiments. The amount of tagatose produced was quantified by the cystein-carbazole method. At this time, one unit of enzyme was defined as the amount of enzyme producing 1 μg of tagatose per minute.

(1) Experimental Example 1: Effect of metal ions on conversion yields of tagatose from galactose by thermostable galactose isomerase

After 3 units of the thermostable galactose isomerase were added to the mixtures of 1 g/1 galactose and 0.5mM Mn²⁺, Mg²⁺, Ba²⁺, Fe²⁺, Ca²⁺, Zn²⁺, Ni²⁺, Cu²⁺, etc., respectively, each reaction mixture was isomerized at 60 °C . After 12 hours, the amount of tagatose produced in the reaction mixtures was quantified (Fig. 1).

As shown in Fig. 1, In the presence of Zn²⁺, Ni²⁺, Cu²⁺, etc., the catalytic activity of the thermostable galactose isomerase was considerably decreased, compared to the control. On the contrary, in the presence of divalent metal ions such as Mn²⁺, Mg , Ba , Fe , Ca , etc., in particular, manganese ion (Mn ), the catalytic activity of the thermostable galactose isomerase was highly increased, thereby greatly increasing the conversion yield of tagatose compared to the control group containing no metal ions.

(2) Experimental Example 2: Change in conversion yields of tagatose

according to various concentrations of Mn²⁺, Mg²⁺, Ba²⁺ and Fe²⁺

The effect of Mn , Mg , Ba and Fe which were found to promote catalytic activity of the enzyme in Experimental Example 1, on the conversion yield of tagatose from galactose was evaluated at various concentrations of these metal ions (Fig. 2). As shown in Fig. 2, it was seen that the amount of tagatose produced at relatively low concentrations of the metal ions was proportional to concentrations, but

reaction rate reached equilibrium at concentrations higher than 0.1 mM (Fig. 2).

(3) Experimental Example 3: Change in conversion yield of tagatose over temperature

In order to examine the conversion yield of tagatose in the presence of manganese ion (Mn²⁺) with temperature, a control group containing no manganese ion and a group containing manganese ion (0.5mM) were used (Figs. 3 and 4). As a result, it was seen that the catalytic activity in the two groups largely depended on temperature. That is, the optimum temperature for activity in the control group containing no Mn ⁺ was within the range of 55-67 °C . When temperature was out of this range, the amount of tagatose produced was sharply decreased (Fig. 3). On the contrary, the catalytic activity in the group containing Mn²⁺ (0.5mM) was very stable even at high temperatures and thus the conversion yield of tagatose was much

higher than that in the control group (Fig. 4).

(4) Experimental Example 4: Change in conversion yield of tagatose over pH In order to examine the conversion yield of tagatose in the presence of manganese ion (Mn²⁺) with pH, a control group containing no manganese ion and a

group containing manganese ion (0.5mM) were used (Figs. 5 and 6).

As a result, it was seen that the optimum pH for activity in the control group

containing no Mn²⁺ was within the range of 7.0-8.8. Out of this range, the amount of tagatose produced sharply decreased (Fig. 5). On the contrary, the optimum pH range for activity in the group containing Mn²⁺ (0.5mM) was broadened to 7.0-10.0 to show very high activity even under basic conditions (Fig. 6).

Example 2: Effect of metal ions on conversion yield of tagatose from galactose by arabinose isomerase

After an arabinose isomerase was isolated and purified from the culture of the recombinant E. coli JMlO/pTClOl (KCTC 0603BP), the arabinose isomerase was EDTA-dialyzed to remove metal ions, and then Tris-HCL buffer (pH 8.0), 50mM galactose and each metal ion (0.5mM) were added thereto. Subsequently, the mixtures were reacted at 30^°C for 1 hour. The amount of tagatose produced was measured in each mixture. The results were shown in Table 1 below.

Table 1

As apparent from Table 1, groups containing Fe²⁺, Mn²⁺, Mg²⁺, Ba²⁺, Ca²⁺, etc.,

had 1.45-2.35 times higher conversion yield of tagatose than the control group containing no metal ions. However, groups containing Ni²⁺, Cu²⁺, Zn²⁺, etc., showed markedly decreased catalytic activity. It can be seen that the catalytic activity of the arabinose isomerase, like the thermostable galactose isomerase, was influenced

by similar types of metal ions.

Industrial Applicability

As described above, the method for converting galactose to tagatose using the thermostable galactose isomerase according to the present invention can greatly increase the conversion yield in the presence of specific types of metal ions and concentrations thereof under a relatively high temperature and appropriately controlled pH conditions.

Although the preferred embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that various

modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims.

Claims

Claims:

1. A method for producing tagatose from galactose using an arabinose isomerase or a thermostable galactose isomerase having a catalytic activity capable of converting galactose to tagatose in the presence of at least one metal ion selected from the group consisting of Mn²⁺, Mg²⁺, Ba²⁺, Fe²⁺ and Ca²⁺ as a promoter of the enzymatic activity at 25-100 °C

2. The method as set forth in claim 1, wherein the thermostable galactose isomerase is an enzyme encoded by Sequence No. 1 showing catalytic activity at 40-80 ^°C , or a thermostable isomerase having the same functions as the enzyme encoded by Sequence No. 1.

3. The method as set forth in claim 1, wherein the arabinose isomerase is derived from E. coli JMlC/pTClOl (KCTC-0606BP) and shows an optimum

catalytic activity at 25-35 ^°C .

4. The method as set forth in claim 1 or 2, wherein the metal ion is provided at a concentration of 0.1~5mM.

5. The method as set forth in claim 4, wherein the metal ion is manganese ion

(Mn²⁺).