JP7553238B2 - Enzyme capable of producing galactooligosaccharides and method for producing galactooligosaccharides - Google Patents

Description

The present invention relates to an enzyme capable of producing galactooligosaccharides. The present invention also relates to DNA encoding this enzyme, a method for producing this enzyme, and a method for producing galactooligosaccharides using this enzyme.

Galactooligosaccharides are valued as a nutrient source for useful bacteria such as bifidobacteria that inhabit the human intestinal flora. Various methods have been proposed for producing galactooligosaccharides (e.g., Patent Documents 1 to 6). The enzyme used in these methods is lactase, and the microorganisms used in these methods are lactase-producing microorganisms. Lactase (β-galactosidase) hydrolyzes lactose into β-galactose and glucose. Depending on the lactase, a reaction (sugar transfer reaction) can occur in which the β-galactosyl group obtained by decomposing lactose molecules is transferred to another lactose molecule, etc., and galactooligosaccharides can be produced by this reaction.

Currently, lactases used commercially for the production of galactooligosaccharides are mainly derived from Bacillus circulans. Lactase derived from Bacillus circulans has the advantage of being relatively efficient at producing galactooligosaccharides. However, this lactase has the disadvantage that it is more likely to produce galactooligosaccharides with 4 or more sugars. Galactooligosaccharides with 4 or more sugars have a lower yield from lactose than galactooligosaccharides with 3 sugars.

In addition, there are two methods for producing galactooligosaccharides: one using lactase-producing microorganisms and the other using lactase purified from the microorganisms. The method using lactase-producing microorganisms has the advantage that the enzyme purification step can be omitted compared to the method using purified lactase, and that the by-product monosaccharides such as glucose can be removed by assimilating them with the microorganisms. However, the study of this method is still insufficient.
Furthermore, the genus Bacillus is well known as a galactooligosaccharide-producing microorganism, but many species of the genus Bacillus, such as Bacillus anthracis and Bacillus cereus, are known to be pathogenic, and safer galactooligosaccharide-producing bacteria are needed.

In light of this background, the present inventors have developed an enzyme derived from a microorganism other than Bacillus circulans that has high selectivity for the production of trisaccharide galactooligosaccharides and can produce galactooligosaccharides with high efficiency, a method for producing galactooligosaccharides using the enzyme, and a method for producing galactooligosaccharides using a highly safe enzyme or microorganism (Patent Document 7).

Patent No. 2652049 Patent No. 2739335 Patent No. 5643756 Japanese Patent Application Publication No. 5-236981 Special Publication No. 2011-517553 Special table 2013-501504 publication WO2019/194062 publication

Z. Mozaffar, K. Nakanishi, R. Matsuno and T. Kamikubo, Purification and Properties of β-Galactosidases from Bacillus circulans, Agric. biol. chem., 48, 3053-3061 (1986) Sotoya et al., Identification of genes involved in galactooligosaccharide utilization in Bifidobacterium breve strain YIT 4014T., Microbiology, 163, 1420-1428. (2017) Van Leeuwen, S. S., 1H NMR analysis of the lactose/β-galactosidase-derived galacto-oligosaccharide components of Vivinal(R) GOS up to DP5., Carbohydrates. Res., 400, 59-73. (2014) T. Moriya, N. Nagahata, R. Odaka, H. Nakamura, J. Yoshikawa, K. Kurashima, T. Saito, Synthesis of an allergy inducing tetrasaccharide "4P-X"., Carbohydrates. Res., 439, 44- 49. (2017)

Based on the above background, the present invention aims to provide an enzyme capable of producing galactooligosaccharides with improved properties, which has high selectivity for the production of trisaccharide galactooligosaccharides and can produce galactooligosaccharides safely and efficiently, and a method for producing galactooligosaccharides with improved productivity.

As a result of intensive research into the above-mentioned objective, the inventors have succeeded in improving the heat resistance and/or glycosyl transfer ability of lactase, thereby completing the present invention.

That is, according to the present invention,
[1] (a) the amino acid sequence shown in SEQ ID NO:1;
(b) an amino acid sequence in which 1 to 10 amino acids have been substituted, deleted or inserted in the amino acid sequence shown in SEQ ID NO:1; or (c) an amino acid sequence having a homology of 70% or more but less than 100% to the amino acid sequence shown in SEQ ID NO:1,
An enzyme having an amino acid sequence having one or more mutations selected from the group consisting of N437H, T297A, R77A, Y296F, F414L, F104L, F104E, F104N, F104Q, F104S, F104T, F104V, and F104Y in the amino acid sequence shown in SEQ ID NO:1, and mutations corresponding thereto, and having galactooligosaccharide-producing activity;
[2] the enzyme according to [1] above, which has an amino acid sequence having N437H in the amino acid sequence shown in SEQ ID NO:1 or a mutation corresponding thereto;
[3] the enzyme according to [2] above, having an amino acid sequence having one or more mutations selected from the group consisting of T297A, F414L, F104L, F104E, F104N, F104Q, F104S, F104T, F104V, and F104Y in the amino acid sequence shown in SEQ ID NO: 1, and mutations corresponding thereto;
[4] the enzyme according to any one of [1] to [3] above, which has improved galactooligosaccharide-producing activity compared to the enzyme consisting of the amino acid sequence shown in SEQ ID NO: 1;
[5] A DNA encoding the enzyme according to any one of [1] to [4] above;
[6] a recombinant vector comprising the DNA according to [5] above;
[7] A transformant having the DNA according to [5] or the recombinant vector according to [6].
[8] A method for producing an enzyme having galactooligosaccharide-producing activity, comprising the steps of culturing the transformant according to [7] above, and recovering the enzyme having galactooligosaccharide-producing activity from the cultured transformant or a culture supernatant;
[9] An enzyme-containing composition comprising the enzyme according to any one of [1] to [4] above and having galactooligosaccharide-producing activity;
[10] There is provided a method for producing galactooligosaccharides, comprising contacting lactose with the enzyme according to any one of [1] to [4] above, the transformant according to [7] above, or the enzyme-containing composition according to [9] above.

The present invention provides an enzyme that is derived from Paenibacillus pabuli and has galactooligosaccharide-producing activity, and is capable of producing galactooligosaccharides with high efficiency and high selectivity in the production of trisaccharide galactooligosaccharides, as well as DNA encoding the enzyme, a method for producing the enzyme, and a method for producing galactooligosaccharides with improved productivity.

In some embodiments, the enzyme of the present invention has improved heat resistance, and is capable of performing an enzymatic reaction at higher temperatures with lactose at higher concentrations as a substrate. Since lactose becomes more soluble as the temperature is increased, it is desirable to have a higher reaction temperature when carrying out a galactooligosaccharide production reaction. Furthermore, according to the present invention, oligosaccharides can be produced at higher temperatures, which can prevent contamination by bacteria.

In another aspect, the enzyme of the present invention has improved glycosyl transfer ability, and can improve the production efficiency of galactooligosaccharides even under the same temperature conditions as before. Furthermore, by using the enzyme of the present invention with improved glycosyl transfer ability and heat resistance, it becomes possible to produce galactooligosaccharides with improved productivity even under the same reaction conditions as before.

Therefore, by using the enzyme and the method for producing galactooligosaccharides of the present invention, the productivity of galactooligosaccharide production is improved.

FIG. 1 is a graph comparing the residual activity at 60° C. between the wild-type enzyme (WT) and the enzyme of the present invention (mutant N437H). FIG. 1 shows a comparison of the galactooligosaccharide production amounts at 70° C. between the wild-type enzyme (WT) and the enzyme of the present invention (mutant N437H).

1. Definitions of Terms Used in the Present Description and Explanation of Methods In the present invention, the term "oligosaccharide" refers to a polysaccharide compound having 3 to 10 sugars.

In the present invention, galactooligosaccharides (also referred to as GOS) mainly refer to oligosaccharides represented by the following general formula:
(Gal)n-Gal-Glc
Here, Gal represents a galactose residue, Glc represents a glucose residue, and n is an integer from 1 to 8, particularly an integer from 1 to 3. The bond type between sugars is not particularly limited, but is typically a β-1,4-glycosidic bond. The bond type between sugars (particularly between Gal-Gal) may be a β-1,3-glycosidic bond.

Lactase is an enzyme that can hydrolyze lactose into β-galactose and glucose.

In the present invention, the lactase activity of an enzyme is defined in one of the following two ways:

The enzyme to be measured is added to a lactose solution (lactose concentration 10%), and the amount of glucose produced is measured and quantified under conditions of pH 6.5 and 40°C. The enzyme activity that produces 1 μmol of glucose per minute is defined as 1 LU.

The enzyme to be measured is added to an o-nitrophenyl-β-galactopyranoside (ONPG) solution (ONPG concentration 1.65 mM), and the amount of o-nitrophenyl produced is measured and quantified under conditions of pH 6.5 and 40°C. The enzyme activity that produces 1 μmol of o-nitrophenyl per minute is defined as 1 OU.

If the activity can be measured by at least one of these two methods, the enzyme may be determined to be lactase. More specifically, if the activity is 0.2 LU/mL or more or 0.5 OU/mL or more, the enzyme may be determined to be lactase.

When lactose is broken down, lactase can transfer the β-galactosyl group to other molecules. If it is transferred to water, β-galactose is produced, but if it is transferred to lactose (Gal-Glc), a trisaccharide (where n is 1 in the above formula (Gal)n-Gal-Glc) called galactooligosaccharide (Gal-Gal-Glc) is produced. Therefore, even if an enzyme has lactase activity, it does not necessarily have galactooligosaccharide production activity (also referred to as GOS production activity). The galactooligosaccharide production activity of an enzyme is evaluated by the method described below.

The enzyme to be measured is added to a lactose solution (lactose concentration 60%) and left to stand or shake for a specified time at pH 6.5 and 50°C. If the enzyme has galactooligosaccharide-producing activity, it will produce galactooligosaccharides from lactose during this time. After the specified time has passed, the reaction solution is analyzed by HPLC.

Columns used in HPLC include Transgenomic's CARBOSep CHO-620 6.5φ×300mm. Analysis is performed under the following conditions: mobile phase is water, flow rate is 0.4mL/min, temperature is 85°C, and detection is by RI.

If the ratio of the total HPLC peak area of galactooligosaccharides to the total peak area of monosaccharides, disaccharides, and galactooligosaccharides (three or more sugars, mainly three to five sugars) is equal to or greater than a reference value, it may be determined that there is galactooligosaccharide production activity. This reference value is usually 1%. Naturally, the higher this ratio, the higher the galactooligosaccharide production activity of the enzyme. If one wishes to obtain an enzyme with high galactooligosaccharide production activity, a high reference value may be set. Thus, the reference value may be set to 1%, 3%, 5%, 10%, 20%, 25%, 30%, 35%, or 40%.

When the above-mentioned transfer reaction of the β-galactosyl group occurs on a trisaccharide (Gal-Gal-Glc) (the above formula (Gal) _n -Gal-Glc, n is 1), a tetrasaccharide ((Gal) ₂ -Gal-Glc) (the above formula, n is 2) galactooligosaccharide is produced. Similarly, when the above-mentioned transfer reaction of the β-galactosyl group occurs on a tetrasaccharide (the above formula, n is 2), a pentasaccharide ((Gal) ₃ -Gal-Glc) (the above formula, n is 3) galactooligosaccharide is produced.

In the present invention, the yield of galactooligosaccharides means the percentage obtained by dividing the "amount of galactooligosaccharides produced" by the "amount of lactose consumed".
Yield (%)=Amount of galactooligosaccharides produced (mass)/Amount of lactose consumed (mass)×100
The amount of galactooligosaccharide produced and the amount of lactose consumed can be determined by HPLC analysis as described above.

When producing trisaccharide galactooligosaccharides, two molecules of lactose produce one molecule of glucose and one molecule of trisaccharide galactooligosaccharide. The theoretical yield of galactooligosaccharides in this case is approximately 74%.

When producing 4-saccharide galactooligosaccharides, 3 molecules of lactose produce 2 molecules of glucose and 1 molecule of 4-saccharide galactooligosaccharide. The theoretical yield of galactooligosaccharides in this case is about 65%.

Therefore, when producing galactooligosaccharides from lactose, it is most efficient in terms of yield to produce only trisaccharide galactooligosaccharides.

The production selectivity (%) of trisaccharide galactooligosaccharides means the percentage of trisaccharide galactooligosaccharides produced relative to the total amount of galactooligosaccharides produced. The higher this value, the more trisaccharide galactooligosaccharides are mainly produced. The total amount of galactooligosaccharides produced and the amount of trisaccharide galactooligosaccharides produced can be determined by HPLC analysis as described above. The production selectivity of trisaccharide galactooligosaccharides is preferably 60% or more, more preferably 70% or more, even more preferably 75% or more, and particularly preferably 80% or more.

2. Enzymes of the Invention According to the present invention, a novel enzyme having galactooligosaccharide-producing activity and DNA encoding the enzyme are provided. The enzymes of the present invention can be designed and/or produced by introducing specific mutations into the base amino acid sequence described below or the DNA encoding the same. The enzymes of the present invention may be isolated from naturally occurring microorganisms or may be produced synthetically.

2-1. Amino acid sequence that can be used as the basis for designing the enzyme of the present invention The amino acid sequence that is the basis for designing the enzyme of the present invention is the amino acid sequence of SEQ ID NO: 1 and the amino acid sequence of its family enzymes. The enzyme consisting of the amino acid sequence shown in SEQ ID NO: 1 is an enzyme derived from Paenibacillus publi (Patent Document 7). The molecular weight of the enzyme consisting of the amino acid sequence shown in SEQ ID NO: 1 is about 51 kDa. This enzyme functions as a lactase, and it has been found that it can be used to produce galactooligosaccharides using lactose as a raw material. It has about 8% homology to BgaD-D. BgaD-D is a lactase derived from Bacillus circulans, and is one of the four enzymes with different molecular weights that constitute BgaD, which is the lactase currently most commercially used for producing galactooligosaccharides (Patent Document 3), and is known to have the highest galactooligosaccharide production activity among them. In the method for producing galactooligosaccharides of the present invention described below, when BgaD-D is used as the lactase for producing galactooligosaccharides, the yield of galactooligosaccharides is about 60 to 70%, and the production selectivity of trisaccharide galactooligosaccharides is about 60% at maximum. When galactooligosaccharides are produced using BgaD-D, a large amount of galactooligosaccharides having tetrasaccharides or more is also produced.

In the galactooligosaccharide production method of the present invention described below, when an enzyme having the amino acid sequence shown in SEQ ID NO:1 is used as the lactase for producing galactooligosaccharides, the yield of galactooligosaccharides is about 60 to about 70%. However, the production selectivity of trisaccharide galactooligosaccharides among the galactooligosaccharides produced is about 80% or more.

The enzyme consisting of the amino acid sequence shown in SEQ ID NO:1 highly selectively transfers β-galactosyl groups (β-galactose residues) to sugars (e.g., lactose) to form β-1,3-glycosidic bonds. In other words, most GOS produced by the enzyme consisting of the amino acid sequence shown in SEQ ID NO:1 are trisaccharide GOS in which β-galactosyl groups are linked by β-1,3-glycosidic bonds. Among general intestinal bacteria, only bifidobacteria can degrade β-1,3-glycosidic bonds. Therefore, when such sugar-rich GOS is ingested, it is assumed that bifidobacteria will preferentially assimilate and grow in the intestine. In many conventional GOS, galactosyl groups are linked to lactose by forming β-1,4-glycosidic or β-1,6-glycosidic bonds.

In this way, the enzyme consisting of the amino acid sequence shown in SEQ ID NO:1 realizes a high galactooligosaccharide yield while also achieving high enzymatic properties and high selectivity for trisaccharide galactooligosaccharides. The use of this enzyme can reduce the generation of galactooligosaccharides with 4 or more sugars, which leads to a decrease in yield, more than ever before.

Naturally, it is inferred that the family of enzymes consisting of the amino acid sequence shown in SEQ ID NO:1 will have the same effect.

Of course, the enzyme may have an additional region as long as the function is not impaired. Examples of such additional regions include a tag domain. These may be added to either the N-terminus, the C-terminus, or both.

When an enzyme E1 is said to be a member of the family (or homologue) of another enzyme E2, it means that the homology (or identity or similarity) based on the amino acid sequences of E1 and E2 is 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. There is no upper limit, but it is less than 100%. In other words, there is a substitution, deletion, or insertion of at least one amino acid residue. Amino acid sequence homology can be easily calculated using known software using the BLAST algorithm and the BLAST 2.0 algorithm.

An enzyme E1 being a member of the family (or homologue) of another enzyme E2 means, inter alia, that the amino acid sequence of enzyme E1 can be achieved by substitution, deletion or insertion of 1 to 10 amino acids relative to the amino acid sequence of enzyme E2. Substitution means that an amino acid at a specific position has been replaced with another amino acid. Deletion means that an amino acid at a specific position has been deleted. Insertion means that an amino acid that was not present has been added to the amino acid sequence.

In addition, when an enzyme E1 and another enzyme E2 are said to be in the same family (or homologue), it means that the enzymes E1 and E2 can exert the same function. There is no particular restriction on the activity ratio in this function. For example, when the more active enzyme is E1 and its activity is taken as 100%, the activity of the less active enzyme E2 is 1% or more, 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more.

The family enzyme of the enzyme consisting of the amino acid sequence shown in SEQ ID NO: 1 means an enzyme having 70% or more but less than 100% homology to the amino acid sequence shown in SEQ ID NO: 1 and having galactooligosaccharide production activity. Alternatively, the family enzyme of the enzyme consisting of the amino acid sequence shown in SEQ ID NO: 1 means an enzyme consisting of an amino acid sequence in which 1 to 10 amino acids are substituted, deleted, or inserted in the amino acid sequence shown in SEQ ID NO: 1 and having galactooligosaccharide production activity. The family enzyme of the enzyme consisting of the amino acid sequence shown in SEQ ID NO: 1 may have a lower limit of homology to the amino acid sequence shown in SEQ ID NO: 1 of 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. Even in these cases, the upper limit of homology is less than 100%.

More specifically, the amino acid sequences on which the enzyme of the present invention is designed are the following amino acid sequences (a) to (c):
(a) the amino acid sequence shown in SEQ ID NO: 1; (b) the amino acid sequence of an enzyme having galactooligosaccharide-producing activity, which has an amino acid sequence in which 1 to 10 amino acids have been substituted, deleted or inserted in the amino acid sequence shown in SEQ ID NO: 1; (c) an amino acid sequence having an amino acid sequence that is 70% or more but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 1 and has any of the amino acid sequences of an enzyme having galactooligosaccharide-producing activity, or an amino acid sequence consisting of any of the amino acid sequences (a) to (c).

The enzyme having the amino acid sequence shown in SEQ ID NO:1 was isolated from Paenibacillus publi. However, the above family enzymes can also be obtained from the producing bacteria isolated from soil or the like, or from other bacteria of the genus Paenibacillus that are deposited in public institutions and are easily available to those skilled in the art, such as
Paenibacillus themophilus (e.g., accession number DSM 24746)
Paenibacillus popilliae (e.g., accession number DSM 22700)
Paenibacillus thiaminolyticus (e.g., accession number NBRC 15656)
Paenibacillus pabuli (e.g., accession number NBRC 13638)
Paenibacillus alvei (e.g., accession number NBRC 3343)
Paenibacillus alginolyticus (e.g., accession number NBRC 15375)
Paenibacillus chibensis (e.g., accession number NBRC 15958)
Paenibacillus chitinolyticus (e.g., accession number NBRC 15660)
Paenibacillus chondroitinus (e.g., accession number NBRC 15376)
Paenibacillus glucanolyticus (e.g., accession number NBRC 15330)
Paenibacillus lautus (e.g., accession number NBRC 15380)
Paenibacillus macerans (e.g., accession number NBRC 15307)
Paenibacillus peoriae (e.g., accession number NBRC 15541)
Paenibacillus polymyxa (e.g., accession number NBRC 15309 and accession number JCM 2507)
Paenibacillus validus (e.g., accession number NBRC 15382)
Paenibacillus apiarius (e.g., accession number DSM 5581)
Paenibacillus jamilae (e.g., accession number DSM 13815)
Paenibacillus kribbensis (e.g., accession number JCM 11465)
Paenibacillus terrae (e.g., accession number JCM 11466)
The family enzymes isolated from natural strains or mutant strains of these Paenibacillus bacteria, and recombinant family enzymes produced from them by genetic recombination techniques, can also be used in the present invention.

2-2. Amino acid sequence of the enzyme of the present invention The enzyme of the present invention has an amino acid sequence containing specific mutations in the amino acid sequence of the base enzyme as described above. Specifically, the specific mutations are any one or more of N437H, T297A, R77A, Y296F, F414L, F104L, F104E, F104N, F104Q, F104S, F104T, F104V, and F104Y in the amino acid sequence of SEQ ID NO: 1, and mutations equivalent thereto.

As described above, the amino acid sequence of the base enzyme may be the amino acid sequence of a family enzyme of an enzyme having the amino acid sequence shown in SEQ ID NO: 1. That is, the family enzyme of an enzyme consisting of the amino acid sequence shown in SEQ ID NO: 1 means an enzyme having 70% or more but less than 100% homology to the amino acid sequence shown in SEQ ID NO: 1 and having galactooligosaccharide production activity. The family enzyme of an enzyme consisting of the amino acid sequence shown in SEQ ID NO: 1 may have a lower limit of homology to the amino acid sequence shown in SEQ ID NO: 1 of 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. Even in these cases, the upper limit of homology is less than 100%. Alternatively, the family enzyme of an enzyme consisting of the amino acid sequence shown in SEQ ID NO: 1 means an enzyme consisting of an amino acid sequence in which 1 to 10 amino acids are substituted, deleted, or inserted in the amino acid sequence shown in SEQ ID NO: 1 and having galactooligosaccharide production activity.

In this case, the specific mutations may be any mutations that correspond to the respective mutations in the amino acid sequence shown in SEQ ID NO: 1. The amino acid sequence shown in SEQ ID NO: 1 consists of 447 amino acid residues, and it is believed that the amino acid sequences of the family enzymes are either roughly the same length or have identical or highly compatible regions that span a certain length (several tens to several hundred residues). Therefore, by using sequence comparison software known to those skilled in the art as described above, it is easy to find amino acids that correspond to the respective mutations in the amino acid sequences of the family enzymes.

Therefore, the enzyme of the present invention
(a) the amino acid sequence shown in SEQ ID NO:1;
(b) an amino acid sequence in which 1 to 10 amino acids have been substituted, deleted or inserted in the amino acid sequence shown in SEQ ID NO:1; or (c) an amino acid sequence having a homology of 70% or more but less than 100% to the amino acid sequence shown in SEQ ID NO:1,
The enzyme has an amino acid sequence having one or more mutations selected from the group consisting of N437H, T297A, R77A, Y296F, F414L, F104L, F104E, F104N, F104Q, F104S, F104T, F104V, and F104Y of the amino acid sequence shown in SEQ ID NO: 1, and mutations corresponding thereto, and has galactooligosaccharide-producing activity. As long as the enzyme has the above amino acid sequence, it may contain an additional portion in addition to the above amino acid sequence, or it may consist of only the above amino acid sequence.

The enzyme of the present invention may have multiple mutations. In this case, it is desirable to have N437H or an equivalent mutation in the amino acid sequence shown in SEQ ID NO: 1, or in addition to that, one or more mutations selected from the group consisting of T297A, F414L, F104L, F104E, F104N, F104Q, F104S, F104T, F104V, and F104Y in the amino acid sequence shown in SEQ ID NO: 1, and mutations equivalent thereto. As long as it has galactooligosaccharide production activity, it may contain any other mutations. Preferably, the enzyme of the present invention has improved galactooligosaccharide production activity, particularly heat resistance and/or yield, compared to the enzyme consisting of the amino acid sequence shown in SEQ ID NO: 1.

The enzyme of the present invention can be easily obtained by introducing desired mutations into DNA encoding the basic amino acid sequence such as those described above in (a) to (c), thereby preparing DNA encoding the enzyme of the present invention, and expressing this in an appropriate host. Alternatively, the enzyme may be isolated from naturally occurring microorganisms, such as bacteria of the genus Paenibacillus, particularly Paenibacillus publii, or may be produced synthetically.

3. DNA of the present invention
The present invention also provides DNA encoding the enzymes described above. Any DNA consisting of any base sequence is within the scope of the present invention as long as it encodes the enzymes described above. In particular, the DNA consisting of the base sequence described in detail below is a typical example of such a DNA.

The DNA encoding the enzyme of the present invention can be prepared from DNA encoding the basic amino acid sequence such as those described above in (a) to (c).
DNA consisting of the base sequence shown in SEQ ID NO:2 encodes an enzyme consisting of the amino acid sequence shown in SEQ ID NO:1 (including a stop codon at the 3' end). As described above, the enzyme consisting of the amino acid sequence shown in SEQ ID NO:1 has good galactooligosaccharide-producing activity. Therefore, enzymes encoded by DNA consisting of base sequences highly homologous to these base sequences are highly likely to have galactooligosaccharide-producing activity.

The present invention also includes DNAs consisting of base sequences with conservative base substitutions in the above DNAs. Conservative base substitutions in a base sequence refer to base substitutions in a base sequence that do not change the amino acid sequence of the enzyme they encode. In other words, this means that two or more DNAs have different base sequences but encode enzymes with the same amino acid sequence.

Of course, the above DNA may have additional regions as long as the function of the DNA itself or the enzyme it encodes is not impaired. Examples of such additional regions include repeat structures of the DNA itself, regions corresponding to stop codons, operator sequences, and tag peptides. These may be added to either the 5' end, the 3' end, or both.

Therefore, the DNA of the present invention is a DNA encoding the enzyme of the present invention, specifically,
A DNA encoding an enzyme having galactooligosaccharide producing activity,
(1) a base sequence shown in SEQ ID NO:2;
(2) a nucleotide sequence having high homology to the nucleotide sequence shown in SEQ ID NO: 2;
(3) a base sequence having a conservative base substitution with respect to any of the base sequences (1) to (2); or (4) a base sequence in which the base sequence of (1) to (2), a stop codon, an operator sequence, or the like has been added to any of the base sequences (1) to (2), the base sequence being an amino acid sequence having one or more mutations selected from the group consisting of N437H, T297A, R77A, Y296F, F414L, F104L, F104E, F104N, F104Q, F104S, F104T, F104V, and F104Y in the amino acid sequence encoded by the base sequence shown in SEQ ID NO: 2, and mutations corresponding thereto, or a DNA having a base sequence encoding an amino acid sequence including the same.

DNA consisting of a base sequence having high homology (or identity or similarity) to the base sequence of a certain DNA means DNA consisting of a base sequence that has a homology of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more to the base sequence of a certain DNA. There is no upper limit, and it is less than 100%. DNA base sequence homology can be easily calculated using known software using the BLAST algorithm and the BLAST 2.0 algorithm.

Alternatively/in addition, DNA consisting of a base sequence that has high homology to the base sequence of a certain DNA means DNA that hybridizes or is capable of hybridizing under stringent conditions to DNA consisting of a base sequence complementary to the base sequence of the certain DNA.

Here, "stringent conditions" refers to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. Hybridization stringency is determined mainly by temperature, ionic strength, and denaturing conditions. Examples of stringent conditions include washing at a salt concentration and temperature equivalent to 60°C, 1xSSC, and 0.1% SDS. Washing conditions may also be at a salt concentration and temperature equivalent to 60°C, 0.1xSSC, and 0.1% SDS, or at a salt concentration and temperature equivalent to 68°C, 0.1xSSC, and 0.1% SDS.

More specifically, the DNA of the present invention is
A DNA encoding an enzyme having galactooligosaccharide producing activity,
(1) a base sequence shown in SEQ ID NO:2;
(2) a nucleotide sequence having a homology of 70% or more but less than 100% to the nucleotide sequence shown in SEQ ID NO: 2;
(3) a nucleotide sequence that hybridizes under stringent conditions with a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO:2;
(4) a base sequence having conservative base substitutions with respect to any of the base sequences (1) to (3) above; or (5) a base sequence in which any of the repeat structures, stop codons, operator sequences, or the like of any of the base sequences (1) to (3) above has been added to any of the base sequences (1) to (3) above, the base sequence encoding an amino acid sequence having one or more mutations selected from the group consisting of N437H, T297A, R77A, Y296F, F414L, F104L, F104E, F104N, F104Q, F104S, F104T, F104V, and F104Y in the amino acid sequence encoded by the base sequence shown in SEQ ID NO:2, and mutations corresponding thereto.

In other words, the DNA of the present invention is
A DNA encoding an enzyme having galactooligosaccharide producing activity,
(A) a base sequence shown in SEQ ID NO: 2; or (B) a base sequence having a homology of 70% or more but less than 100% to the base sequence shown in SEQ ID NO: 2;
(C) a base sequence that hybridizes under stringent conditions with a base sequence complementary to the base sequence shown in SEQ ID NO: 2; or (D) a DNA comprising or consisting of any of the base sequences having conservative base substitutions with respect to any of the base sequences of (A) to (C), the encoded amino acid sequence of which is an amino acid sequence having one or more mutations selected from the group consisting of N437H, T297A, R77A, Y296F, F414L, F104L, F104E, F104N, F104Q, F104S, F104T, F104V, and F104Y in the amino acid sequence encoded by the base sequence shown in SEQ ID NO: 2, and mutations equivalent thereto.

The DNA that serves as the base for the DNA of the present invention can be synthesized, but it can also be obtained, for example, by recovering genomic DNA from bacteria of the genus Paenibacillus (particularly Paenibacillus publi) and then performing PCR using this as a template.

A primer pair for amplifying a structural gene encoding an enzyme consisting of the amino acid sequence shown in SEQ ID NO:1 or its homologue can be appropriately designed and synthesized.

In this way, a structural gene (e.g., DNA consisting of the base sequence shown in SEQ ID NO:2) encoding an enzyme consisting of the amino acid sequence shown in SEQ ID NO:1 or its homologue can be obtained.

The DNA of the present invention can be produced by introducing specific mutations into the DNA encoding the amino acid sequence of the base enzyme obtained in this manner, using a method known to those skilled in the art. Methods for introducing desired mutations into DNA encoding a specific amino acid sequence are known to those skilled in the art, and any method may be used to obtain DNA encoding the amino acid sequence of the enzyme of the present invention. The DNA of the present invention may itself be produced by synthesis, or may be isolated and amplified from a natural microorganism.

4. Vector of the Present Invention According to the present invention, there is provided a recombinant vector containing the DNA of the present invention. This recombinant vector enables the production of transformants and the mass expression of the enzyme.

There are no particular limitations on the vector, and any commonly used vector can be used. Examples include plasmids, bacteriophages, cosmids, and phagemids.

Examples of plasmids include pK4, pRK401, pRF31, pBR322, pBR325, pUC118, pUC119, pUC18, pBIC, pUB110, pTP5, YEp13, YEp24, and YCp50.

Examples of phages include λ phages (λgt10, λgt11, λZAP, etc.). In addition, animal viruses such as retroviruses or vaccinia viruses, and insect virus vectors such as baculoviruses can also be used.

Cis elements such as promoters and enhancers, splicing signals, polyA addition signals, selection markers, ribosome binding sequences (SD sequences), start codons, stop codons, etc. can be linked to the vector. A tag sequence can also be linked to facilitate purification of the enzyme produced. Known tags such as His tags, GST tags, and MBP tags can be used as tags. The vector may also contain an antibiotic resistance gene for selection.

There are no particular limitations on the method for inserting DNA into a vector, and any commonly used method can be used. This is usually done by the following method. In general, purified DNA can be cleaved with an appropriate restriction enzyme and inserted into the restriction enzyme site or multicloning site of the vector DNA to link it to the vector.

5. Transformants According to the present invention, there is provided a transformant which contains (or has been introduced into) the DNA and/or recombinant vector of the present invention.

There are no particular limitations on the host organism for the transformant, and any commonly used organism can be used. Any prokaryote, archaea, or eukaryote can be used, including, for example, eubacteria (such as Escherichia coli), yeast, plant cells, animal cells (such as COS cells and CHO cells), and insect cells.

Hosts include, in particular, microorganisms of the genera Bacillus (e.g., Bacillus subtilis), Paenibacillus, Brevibacillus (e.g., Brevibacillus chosinensis), Escherichia (e.g., Escherichia coli), Corynebacterium, Saccharomyces, Shizosaccharomyces, Kluyveromyces, Pichia, Aspergillus, Penicillium, and Trichoderma. Also include microorganisms that are commonly consumed as food, such as lactic acid bacteria and acetic acid bacteria.

There are no particular limitations on the method for introducing DNA and/or recombinant vectors into these hosts. Examples include electroporation, spheroplast, lithium acetate, calcium phosphate, and lipofection. They may also be inserted or added into the genome of the host using homologous recombination, etc.

In addition, any method can be used to confirm whether or not the DNA or recombinant vector has been introduced into the host. For example, PCR, Southern hybridization, Northern hybridization, etc. can be used.

6. Method for Producing the Enzyme The enzyme of the present invention or a composition containing the enzyme can be prepared by a conventionally known method using the transformant of the present invention. For example, the enzyme can be prepared by the method described below.

The transformant into which the target gene encoding the enzyme of the present invention has been introduced is mass-cultured in a liquid medium. After mass-culture, a specific inducer may be administered to the transformant to induce expression of the target gene. For example, when the vector uses the Lac operon, IPTG can be administered to induce expression of the target gene.

After large-scale expression, the culture supernatant or the disrupted liquid obtained by disrupting the cells with ultrasound or a cell wall-dissolving enzyme is collected. The supernatant or disrupted liquid contains a large amount of the enzyme of the present invention. The supernatant or disrupted liquid may be used as an enzyme-containing composition as is, or the liquid composition may be purified and collected as a purified enzyme.

This purification process can be carried out, for example, by salting out, membrane separation, or column chromatography. These procedures can be performed alone or in combination. There are no limitations on the type of column chromatography, but a column containing an antibody that specifically acts on these enzymes may be used, or a column that can interact with a tag domain, such as a His tag, that has been previously attached to the enzyme may be used.

By using such a method, it is possible to prepare the enzymes of the present invention in large quantities. These enzymes can be used to produce galactooligosaccharides.

7. Method for Producing Galactooligosaccharides Since the enzyme of the present invention has galactooligosaccharide-producing activity, it can be used to produce galactooligosaccharides from lactose. Therefore, the present invention provides a method for producing galactooligosaccharides. The method and conditions for producing galactooligosaccharides are not limited. An example will be described below, but the present invention is not limited thereto.

The method for producing galactooligosaccharides of the present invention includes a step of contacting lactose with the enzyme, transformant, or enzyme-containing composition of the present invention. This contact produces galactooligosaccharides. Optionally, the method may include additional steps such as isolating and/or purifying the produced galactooligosaccharides.

In the method for producing galactooligosaccharides of the present invention, the enzyme to be contacted with lactose may be any enzyme containing the enzyme of the present invention, and may be in a purified state (purified enzyme) from a microorganism that produces the enzyme. Alternatively, the transformant itself may be contacted with lactose, or lactose may be contacted with a mixture or crude product (enzyme-containing composition) containing other components in addition to the enzyme of the present invention, such as a crushed product or extract of the transformant. The enzyme-containing composition may be solid or liquid. When the transformant secretes the enzyme outside the cells, the medium from which the cells have been removed can also be used as the enzyme-containing composition. In terms of preventing side reactions, it is preferable to contact the purified enzyme with lactose.

Purified enzyme means an enzyme solution or enzyme solid obtained through a specific purification process. It means that it contains essentially no enzymes other than the enzyme in question, or the amount of other enzymes is reduced.

A reaction system (a mixture containing a solvent, lactose, and the enzyme, transformant, or enzyme-containing composition of the present invention) for producing galactooligosaccharides is formed by mixing an enzyme or the like with a solution containing lactose, or by mixing lactose with a solution containing an enzyme or the like. The solvent is arbitrary, but is usually water or an aqueous solvent mainly composed of water.

In the present invention, there is no particular restriction on the state of the lactose used as a raw material. It may be a lactose solution dissolved in any solvent. In addition, whey, which is the supernatant of curdled milk, and mixtures containing lactose such as its concentrate and skim milk powder may be used as the lactose raw material.

The lactose content of the reaction system is not particularly limited and can be set as desired. It may be, for example, 1-80% by mass, 2-70% by mass, 4-60% by mass, 5-60% by mass, or 30-60% by mass based on the total amount of the reaction system. Lactose may be appropriately replenished as lactose is consumed.

In some embodiments, the lactose content may be 4 to 50% by mass, 4 to 40% by mass, 4 to 30% by mass, 4 to 20% by mass, or 4 to 10% by mass. In such regions of relatively low substrate concentrations, the enzymes of the present invention are preferably used. These have high affinity for the substrate lactose, excellent Km values, and sufficient activity even at low substrate concentrations.

In another embodiment, the lactose content may be 5-60% by mass, 10-60% by mass, 20-60% by mass, 30-60% by mass, or 40-60% by mass. The enzymes of the present invention are preferably used even in such regions of relatively high substrate concentrations. These enzymes have both good galactooligosaccharide production activity and trisaccharide galactooligosaccharide selection activity, are less inhibited by products such as galactose, and can produce large amounts of trisaccharide galactooligosaccharides in a short period of time.

The content of the enzyme of the present invention in the reaction system is not particularly limited and can be set arbitrarily. For example, the content in the reaction system may be 0.1 to 10 LU/mL or 0.1 to 10 OU/mL.

The galactooligosaccharide-producing activity varies depending on the reaction system. The content of the enzyme of the present invention may be determined as follows.
In the first step, the amount of galactooligosaccharides produced relative to the content of the enzyme of the present invention in a specific reaction system (lactose content, content of the enzyme of the present invention, reaction pH, reaction temperature, reaction time, etc.) is confirmed.
In the second step, the lactase activity (at least one of LU/mL and OU/mL) of the enzyme of the present invention contained in the above reaction system is measured.
In the third step, the amount of the enzyme of the present invention contained in the reaction system is adjusted (the content of the enzyme of the present invention is increased or decreased) using the lactase activity measured in the second step as an indicator.
In the fourth step, the enzyme of the present invention is added to the reaction system so that the content of the enzyme is adjusted to a desired value.
The above first and second steps may be performed in reverse order.

The pH of the reaction system is not particularly limited and can be set arbitrarily. For example, it may be 3 to 9, 5 to 8, 6 to 8, 6 to 7.5, 6 to 7, 4 to 8, or 5 to 7. Naturally, the pH may be constant or may vary.

The temperature of the reaction system is not particularly limited and can be set arbitrarily. For example, it may be 20 to 75°C, 20 to 60°C, 30 to 50°C, 30 to 75°C, 40 to 75°C, 50 to 70°C, or 60 to 70°C. Naturally, the temperature may be constant or variable. It is preferable to select a temperature at which the lactose contained in the reaction system dissolves. The enzyme with excellent heat resistance of the present invention can be reacted particularly advantageously at 60 to 70°C.

The reaction time is not particularly limited and can be set as desired. For example, it may be 1 to 100 hours, 1 to 50 hours, 5 to 30 hours, 12 to 60 hours, 12 to 24 hours, or 24 to 48 hours.

These series of methods for producing galactooligosaccharides may be either batch or continuous. In the batch method, a predetermined amount of enzyme, transformant or enzyme-containing composition and raw lactose are added to a reactor, and galactooligosaccharides are produced for a predetermined period of time, after which a mixture containing galactooligosaccharides is recovered from the reactor. In the continuous method, the enzyme, transformant or enzyme-containing composition, and/or raw lactose are continuously or intermittently added to a reactor. Then, while galactooligosaccharides are produced, a mixture containing galactooligosaccharides is recovered from the reactor.

At the end of the above-mentioned steps, the reaction system contains a mixture of monosaccharides, disaccharides, and galactooligosaccharides. The galactooligosaccharides may be used as is as a solution, or may be purified from the solution. There are no particular limitations on the method for purifying the galactooligosaccharides, and any method may be used. For example, the galactooligosaccharides can be separated into their respective fractions by column chromatography using activated carbon, a cation exchange resin coordinated with a metal ion, or a gel filtration resin.

In column chromatography, conditions such as column size, solvent type, and solvent flow rate can also be adjusted as desired.

The production of galactooligosaccharides by contacting the transformant of the present invention with lactose is basically the same as described above. In a typical embodiment using the transformant,
an optional step of culturing the transformant of the present invention (culturing step);
Contacting the transformant of the present invention with raw lactose,
A step of producing galactooligosaccharides from raw lactose to obtain a galactooligosaccharide mixture (contact step);
an optional step of separating and/or purifying galactooligosaccharides from the galactooligosaccharide mixture (galactooligosaccharide purification step);
Such a process is carried out.

Usually, the transformant of the present invention is cultured before contacting it with lactose. There is no limitation on the culture conditions of the transformant of the present invention, and it can be any. For example, it can be cultured in a liquid medium at about 30°C for about 3 days under anaerobic or aerobic conditions. There is no limitation on the type of liquid medium, and any type can be used. Examples include LB medium, soybean casein digest (SCD) medium, and brain heart infusion (BHI) medium.

The transformant of the present invention obtained by culturing can be advantageously used as a culture or a processed product thereof, regardless of whether it is live or dead. The culture may be concentrated. The liquid medium and the cells can be separated by centrifugation, and wet cells can be obtained by washing with physiological saline, distilled water, etc. Other processed products of the culture include products treated with ultrasonic waves, lytic enzymes, surfactants, mechanical grinding, etc. These can be suspended in an appropriate solvent to obtain a fluid containing the cell components and/or enzymes of the transformant of the present invention (these are also transformants or enzyme-containing compositions).

The content of the transformant of the present invention in the reaction system is not particularly limited and can be set appropriately. For example, it may be 10 to 200 g/L, 30 to 150 g/L, or 50 to 100 g/L in wet cells. When enzymes are added using enzyme-containing mixtures such as disrupted material, extracts, and culture media, the content may be adjusted so that the original strain falls within the above range.

The ratio (kg/kg) of the content of the transformant of the present invention to the lactose content of the reaction system is not particularly limited and can be set appropriately. For example, the ratio of wet cells to the amount of lactose may be 1/30 to 1/1, 1/20 to 1/2, or 1/10 to 1/5. When enzymes are added using enzyme-containing mixtures such as disrupted materials, extracts, and culture media, the content may be adjusted so that the original strain falls within the above range.

In order to efficiently produce galactooligosaccharides, the reaction system may contain inorganic salts, etc. The content of inorganic salts, etc. is, for example, 0.00001 to 10% by mass, or 0.0001 to 1% by mass, based on the total amount of the reaction system. A liquid medium such as LB medium may be used as a solvent that contains inorganic salts.

The galactooligosaccharide mixture may be used as it is as a galactooligosaccharide solution containing bacteria, or may be used as a galactooligosaccharide solution after removing the bacteria, or the galactooligosaccharides may be purified from it. Any method may be used to remove the bacteria from the galactooligosaccharide solution containing bacteria. Examples include sterilization treatment by centrifugation, filtering, heating, etc. There are no particular limitations on the method for purifying the galactooligosaccharides, and any method may be used. The galactooligosaccharides can be separated into each fraction by column chromatography using activated carbon, a cation exchange resin coordinated with a metal ion, or a gel filtration resin.

The present invention will be specifically explained below with reference to examples, but the present invention is not limited to these examples.

1. Production of mutant enzymes using E. coli
1-1. Construction of expression plasmid The vector was amplified by PCR using the commercially available vector pET28a (Novagen, catalog number 69864-3CN) as a template. At this time, BamH I and Hind III recognition sequences were added to the amplification primers.

Similarly, the lactase gene having the base sequence shown in SEQ ID NO:2 (hereinafter also referred to as wild type) was amplified by PCR using the genome as a template. BamH I and Hind III recognition sequences were also added to the amplification primers. The primer pair used is shown below.

(for vector amplification)
5'-TCGACAAGCTTGCGGCCGCACTC-3' (SEQ ID NO: 3)
5'-GCTGCGGATCCTATATTCTCCTTCTTAAAG-3' (SEQ ID NO: 4)
(For amplifying the lactase gene shown in SEQ ID NO:2)
5'-AAAAAGGATCCATGACCATTTTTCAATTTC-3' (SEQ ID NO:5)
5'-TTTTTAAGCTTTTAACGGATTTCCAGCC-3' (SEQ ID NO: 6)

PCR was performed using "PrimeSTAR™ GXL DNA Polymerase" (Takara Bio, product code R050A) in the following 50 μL system. 3 ng/μL template DNA 3.0 μL, 2.5 mM dNTP Mixture 4.0 μL, 5× Prime STAR GXL Buffer 10 μL, 5 pmol/μL primers 2.5 μL, 1.25 U/μL Prime STAR GXL Polymerase 1.0 μL, and ultrapure water 27 μL were mixed. PCR was performed by repeating 30 cycles of (98°C, 10 seconds; 55°C, 15 seconds; 68°C, 1 minute/kb).
The resulting PCR products were treated with restriction enzymes BamH I and Hind III, and the lactase gene shown in SEQ ID NO:2 was inserted into the vector to construct an expression plasmid.

1-2 Introduction of site-specific mutations Candidates for mutations to be introduced into the enzyme having the amino acid sequence of SEQ ID NO: 1 were selected. A total of about 50 or more mutations were selected.

To introduce site-specific mutations, the PrimeSTAR™ Mutagenesis Basal Kit (Takara Bio, product code R046A) was used. The main primers used are shown below.

(For introducing N437H mutation)
5'-ACCAGCACGTAATCAAAAACAATTGG-3' (SEQ ID NO:7)
5'-ATTACGTGCTGGTACCAGTAGTAGCT-3' (SEQ ID NO: 8)
(For introducing T297A mutation)
5'-TATTACGCCATGGGCATCAATCGGTAT-3' (SEQ ID NO: 9)
5'-GCCCATGGCGTAATAATTAATGCCCAG-3' (SEQ ID NO: 10)
(For introducing F414L mutation)
5'-ATGCGTTTGGGGCTCATCCATGTGGAT-3' (SEQ ID NO:11)
5'-GAGCCCCAAACGCATGCGATACCCTTC-3' (SEQ ID NO: 12)
(For introducing F104L mutation)
5'-CATCGGCTCGTCGATAAATTGCTTGAA-3' (SEQ ID NO: 13)
5'-ATCGACGAGCCGATGGTAGAAGTCCAG-3' (SEQ ID NO: 14)
(For introducing F104E mutation)
5'-CATCGGGAAGTCGATAAATTGCTTGAA-3' (SEQ ID NO: 15)
5'-ATCGACTTCCCGATGGTAGAAGTCCAG-3' (SEQ ID NO: 16)
(For introducing F104N mutation)
5'-CATCGGAACGTCGATAAATTGCTTGAA-3' (SEQ ID NO: 17)
5'-ATCGACGTTCCGATGGTAGAAGTCCAG-3' (SEQ ID NO: 18)
(For introducing F104Q mutation)
5'-CATCGGCAAGTCGATAAATTGCTTGAA-3' (SEQ ID NO: 19)
5'-ATCGACTTGCCGATGGTAGAAGTCCAG-3' (SEQ ID NO: 20)
(For introducing F104S mutation)
5'-CATCGGAGTGTCGATAAATTGCTTGAA-3' (SEQ ID NO:21)
5'-ATCGACACTCCGATGGTAGAAGTCCAG-3' (SEQ ID NO:22)
(For introducing F104T mutation)
5'-CATCGGACAGTCGATAAATTGCTTGAA-3' (SEQ ID NO:23)
5'-ATCGACTGTCCGATGGTAGAAGTCCAG-3' (SEQ ID NO:24)
(For introducing F104V mutation)
5'-CATCGGGTTGTCGATAAATTGCTTGAA-3' (SEQ ID NO:25)
5'-ATCGACAACCCGATGGTAGAAGTCCAG-3' (SEQ ID NO:26)
(For introducing F104Y mutation)
5'-CATCGGTACGTCGATAAATTGCTTGAA-3' (SEQ ID NO:27)
5'-ATCGACGTACCGATGGTAGAAGTCCAG-3' (SEQ ID NO:28)
(For introducing R77A mutation)
5'-ACGTATGCATTCTCCATCGCATGGCCG-3' (SEQ ID NO:29)
5'-GGAGAATGCATACGTATTGATGCCCAG-3' (SEQ ID NO: 30)
(For introducing Y296F mutation)
5'-AATTATTTCACCATGGGCATCAATCGG-3' (SEQ ID NO:31)
5'-CATGGTGAAATAATTAATGCCCAGCAG-3' (SEQ ID NO: 32)

PCR was performed in the following 25 μL system: 1.5 μL of 100 pg/μL pET28a-nonhisBGL8, 12.5 μL of PrimeStar max Premix (2x), 1.0 μL of 5 μM each primer, and 9.0 μL of ultrapure water were mixed. PCR was performed by repeating the cycle of (98°C, 10 seconds; 59°C, 15 seconds; 72°C, 45 seconds) 35 times.

10 μL of the PCR product was used to transform E. coli DH5α, which was then cultured at 30°C for 2 days on LB agar medium containing Km (LB Km). Plasmids were extracted from each of the resulting transformants and purified using "MagExtractor™-Plasmid-" (Toyobo, code No. NPK-301). These procedures were repeated for the double mutants.

1-3 Production of recombinant enzyme using E. coli and preparation of cell-free extract The host used was E. coli BL21(DE3)lac-1 strain, in which E. coli-derived β-galactosidase was deleted by UV irradiation. The above-mentioned mutant and wild-type enzyme plasmids were transformed into the BL21(DE3)lac-1 strain, spread on LB Km agar medium and cultured at 30°C overnight. Each of the obtained transformants was inoculated into 3mL of LB Km medium and cultured at 37°C for 16 hours. 500μL of the culture solution was inoculated into 100mL of the same medium and cultured with shaking at 37°C. When _OD660 reached 0.5, IPTG was added to a final concentration of 0.1mM to induce the production of the recombinant protein. The induction culture was carried out at 30°C for 24 hours.

Each culture solution was centrifuged (8,000 rpm, 10 min, 4°C), and the collected cells were stored at -80°C. The frozen pellet was suspended in 1/10 volume of 100 mM sodium phosphate buffer (pH 6.5), and the cells were disrupted by sonication (UCD-200T, Bioruptor; power High, 30 min). The supernatant obtained by centrifugation (13,000 rpm, 5 min, 4°C) was used as a cell-free extract (crude enzyme solution).

2. Temperature stability (thermostability) test of mutant enzymes
2-1 Comparison of lactase activity at 60°C The ONPG decomposition activity at 60°C of the wild-type enzyme and each crude enzyme solution was measured. Each crude enzyme solution was diluted with 100 mM sodium phosphate buffer (pH 6.5) to 500-600 OU/mL. The crude enzyme solution to be measured prepared as described above was added to an o-nitrophenyl-β-galactopyranoside (ONPG) solution (ONPG concentration 1.65 mM), and the mixture was kept at pH 6.5 and 60°C for 0, 10, 30, and 60 minutes, after which the ONPG decomposition activity (o-nitrophenyl production amount) was measured and the remaining activity of each enzyme was compared. The results are shown in Figure 1.

The mutant N437H, which was assumed to contribute to thermal stability based on its three-dimensional structure, was expressed in Escherichia coli BL21(DE3)lac-1 strain as a host, and the residual activity at 60°C was compared with that of the wild-type enzyme. As a result, a significant improvement in heat resistance was observed in N437H.

2-2 GOS production test of mutant enzyme N437H at each temperature The lactose decomposition activity of the crude enzyme solution of wild-type enzyme and N437H was measured. This was diluted with 100 mM sodium phosphate buffer (pH 6.5) to 5, 10, 30, 50, and 100 LU/mL. 100 μL of each crude enzyme solution was added to 900 μL of 66% lactose solution dissolved in 100 mM sodium phosphate buffer (pH 6.5) to prepare 1 mL of reaction solution. This was carried out in a 1.5 mL tube. After keeping at 50, 55, 60, 65, 70, 75, and 80 ° C for 24 hours, the reaction was stopped by adding 20% sulfosalicylic acid. The reaction solution was diluted 100 times with water, and 5 μL was subjected to sugar analysis.

HPLC was performed on each sample to analyze the amount of GOS produced. The column used in the HPLC was a Transgenomic CARBOSep CHO-620 6.5φ×300 mm. The mobile phase was water, the flow rate was 0.4 mL/min, the temperature was 85°C, and detection was by RI. The percentage of the total HPLC peak area of GOS to the total peak areas of monosaccharides, disaccharides, and GOS (three or more sugars, mainly three to five sugars) was calculated.

The results of the evaluation at 70°C are shown in Figure 2. In Figure 2, "G3," "G4," and "G5" represent trisaccharides, tetrasaccharides, and pentasaccharides, respectively. The values represent the percentage of the HPLC area of GOS relative to the total HPLC area of GOS (pentasaccharides, tetrasaccharides, and trisaccharides), disaccharides (unreacted lactose and disaccharide transfers), and monosaccharides (glucose and galactose).

N437H was stable up to 70°C, and its heat resistance was improved by approximately 10°C compared to the wild-type enzyme. Since N437H is located near loops 441-447 and 47-50, which are involved in the formation of the complex, it was thought that the substitution with His strengthened the bonds between the subunits, contributing to stabilization.

3. Evaluation of heat resistance and glycosyltransferase activity of each mutant enzyme The crude enzyme solution of the wild type enzyme and each mutant was measured for lactose decomposition activity. This was diluted with 100 mM sodium phosphate buffer (pH 6.5) to 10, 15, 30 and 50 LU/mL. 100 μL of each crude enzyme solution was added to 900 μL of 11%, 33% and 66% lactose solution dissolved in 100 mM sodium phosphate buffer (pH 6.5) to prepare 1 mL of reaction solution. This was carried out in a 1.5 mL tube. After keeping at 30, 55 and 65 °C for 24 hours, the reaction was stopped by adding 20% sulfosalicylic acid. The reaction solution was diluted 100 times with water, and 5 μL was subjected to sugar analysis by HPLC as described above.
The results are shown in Table 1.

As shown in Table 1, it was confirmed that the N437H mutation contributes to improved heat resistance but does not affect GOS production ability.

For T297A, a GOS production reaction was carried out using 60% lactose as a substrate at 3 LU/mL (final concentration) at 55°C for 24 hours. As a result, the amount of GOS produced with three or more sugars was 44%, which was higher than the 41% produced by the wild-type enzyme.
In addition, amino acids were optimized for T297, but only the alanine substitution was found to improve metastatic ability. Since T297 is located on the surface of the active pocket, it is believed that the substitution of threonine with alanine widened the active pocket, making the product less susceptible to physical inhibition, which led to an increase in the amount of GOS produced.

In R77A and Y296F, improved transfer activity was observed when 10% or 30% lactose was used as a substrate at 30°C (1 or 5 LU/mL added). On the other hand, in the test groups where the reaction temperature was 55°C or higher, both enzymes showed lower GOS production and yield than the wild-type enzyme (data not shown).
From these findings, it was believed that R77A and Y296F contribute to the improvement of GOS productivity under the same conditions as conventional conditions or under conditions of lower temperature and lower substrate concentration. R77 and Y296 are highly conserved among family enzymes, and are presumed to be important amino acid residues for activity. In other words, in this case, it was believed that the transfer activity became dominant due to a specific decrease in hydrolysis activity, and the increase in GOS production was observed. In addition, since the GOS production and yield were lower than those of the wild-type enzyme in the test group where the reaction temperature was 55°C or higher, it was believed that the stability of the mutant was decreased and some of the enzyme was inactivated.

When a GOS production reaction was carried out for the double mutant combining T297A and the heat-resistant mutation N437H under conditions of 60% lactose, 5 LU/mL, 65°C, and 24 hours, the amount of GOS produced was greatly improved, reaching a maximum of 46%. In other words, the functionality of this double mutant was improved in terms of both heat resistance and glycosyltransferase activity.

When a GOS production reaction was carried out under conditions of 60% lactose at 55°C for 24 hours, the amount of GOS produced was 46% when 3 LU/mL was added for F414L, and 42% when 5 LU/mL was added for F104L, both of which exceeded those of the wild-type enzyme.
Furthermore, when amino acid optimization was performed for both mutation points, the transfer ability was improved in F104L with E, N, Q, S, T, V and Y substitutions. For F414L, only the leucine substitution was found to improve the transfer ability among the amino acids tested. In addition, F414L had a lower stability than the wild-type enzyme, but it was found that by combining it with the heat-resistant mutation N437H, it was possible to perform a GOS production reaction at 55°C similar to the wild-type enzyme. F414 is highly conserved among family enzymes and is presumed to be an amino acid residue important for activity. That is, in this case, as with R77A and Y296F, it was thought that the transfer activity became dominant due to a specific decrease in hydrolysis activity, and the increase in the amount of GOS produced was observed.

Claims (9)

(a) the amino acid sequence shown in SEQ ID NO:1;
(b) an amino acid sequence in which 1 to 10 amino acids have been substituted, deleted or inserted in the amino acid sequence shown in SEQ ID NO:1; or (c) an amino acid sequence having an identity of 90 % or more to the amino acid sequence shown in SEQ ID NO:1,
An enzyme having an amino acid sequence having one or more mutations selected from the group consisting of N437H, T297A, R77A, Y296F, F414L, F104L, F104E, F104N, F104Q, F104S, F104T, F104V, and F104Y in the amino acid sequence shown in SEQ ID NO:1, and having improved galactooligosaccharide-producing activity compared to an enzyme consisting of the amino acid sequence shown in SEQ ID NO:1 . The enzyme according to claim 1, having an amino acid sequence containing N437 H of the amino acid sequence shown in SEQ ID NO:1. The enzyme according to claim 2, having an amino acid sequence having one or more mutations selected from the group consisting of T297A, F414L, F104L, F104E, F104N, F104Q, F104S, F104T, F104V, and F104Y in the amino acid sequence shown in SEQ ID NO:1. A DNA encoding the enzyme according to any one of claims 1 to 3 . A recombinant vector comprising the DNA of claim 4 . A transformant comprising the DNA according to claim 4 or the recombinant vector according to claim 5 . A method for producing an enzyme having galactooligosaccharide-producing activity, comprising the steps of: culturing the transformant described in claim 6 ; and recovering the enzyme having galactooligosaccharide-producing activity from the cultured transformant or the culture supernatant. An enzyme-containing composition comprising the enzyme according to any one of claims 1 to 3 and having galactooligosaccharide producing activity. A method for producing galactooligosaccharides, comprising contacting lactose with the enzyme according to any one of claims 1 to 3 , the transformant according to claim 6, or the enzyme-containing composition according to claim 8 .

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