Novel L-rhamnose isomerase

Abstract

An L-rhamnose isomerase that can be obtained from a microorganism belonging to the genus Erwinia, has a subunit molecular mass of about 48 kDa when measured by SDS-PAGE, and has the following substrate specificities (A) and (B). (A) Having an isomerase activity of reacting with an aldose by recognizing a CHO group at C1 and an OH group at C2 to thereby convert the CHO group at C1 into an OH group and the OH group at C2 into a CO group, or reacting with a ketose by recognizing an OH group at C1 and a CO group at C2 to thereby convert the OH group at C1 into a CHO group and the CO group at C2 into an OH group. (B) Having an activity of catalyzing isomerization reactions between L-rhamnose and L-rhamnulose, between L-lyxose and L-xylulose, between L-mannose and L-fructose, between D-ribose and D-ribulose, between L-talose and L-tagatose, and between D-allose and D-allulose.

Description

[Technical Field]

[0001] The present invention relates to a novel L-rhamnose isomerase and a method for producing the same, a microorganism that produces the same, DNA encoding the enzyme, a recombinant vector containing the same, and a transformed host cell, an L-rhamnose isomerase mutant, and a method for producing ketose or aldose using the L-rhamnose isomerase or mutant. [Background technology]

[0002] According to the definition of the International Rare Sugar Society, a rare sugar is "a sugar that is rarely found in nature," that is, a monosaccharide that exists in small amounts in nature. There are four types of four-carbon monosaccharides (tetroses): four aldoses, two ketoses, and three sugar alcohols. There are eight types of five-carbon monosaccharides (pentoses): eight aldoses, four ketoses, and four sugar alcohols. There are 34 types of six-carbon monosaccharides (hexoses): 16 aldoses, eight ketoses, and 10 sugar alcohols. There are 32, 16, and 16 types of seven-carbon monosaccharides (heptoses): aldoheptoses, ketoheptoses, and heptitols, respectively. For example, among hexoses, the six types of aldoses that generally exist in large quantities in nature are D-glucose, D-galactose, D-mannose, D-ribose, D-xylose, and L-arabinose, while other aldoses, such as D-allose, are defined as rare sugars. That is, aldoses that are considered rare sugars include L-allose, L-gulose, L-glucose, L-galactose, L-altrose, L-idose, L-mannose, L-talose, D-talose, D-idose, D-altrose, D-gulose, and D-allose. As for ketoses, D-fructose exists in large quantities in nature, while the other ketoses do not exist in large quantities in nature, and therefore can be considered rare sugars. Among ketoses, those considered to be rare sugars include D-allulose (also known as D-psicose), D-tagatose, D-sorbose, L-fructose, L-allulose (also known as L-psicose), L-tagatose, and L-sorbose.

[0003] Recently, mass production technology for D-allulose (also known as D-psicose), the core raw material for all rare sugar production, has been established, making it possible to produce rare sugars that were previously difficult to obtain. Furthermore, D-allulose is thought to be at the center of new rare sugar production, such as D-allose, through enzymatic reactions. D-allose is an aldose isomer of D-glucose, differing only in the orientation of the OH group at the 3-position carbon. It is a rare monosaccharide known as an isomer of the ketose D-allulose. D-allose is known as an active ingredient in pharmaceutical compositions for treating renal diseases selected from acute renal failure and uremia (Patent Document 1), drugs for delaying the onset or progression of movement disorders caused by amyotrophic lateral sclerosis (Patent Document 2), antihypertensive agents (Patent Document 3), agents characterized by their use in inhibiting angiogenesis (Patent Document 4), T-lymphocyte proliferation inhibitors (Patent Document 5), and peritoneal deterioration inhibitors used in peritoneal dialysis fluids (Patent Document 6). It is also known as an edible pesticide (Patent Document 7). Recently, patent applications have been filed for its antitumor effect upon uptake into renal cell carcinoma cells (Patent Application No. 2019-52195) and its potent antitumor effect against human urothelial carcinoma cells (Patent Application No. 2019-58477). Due to these characteristics, D-allose is attracting attention as a next-generation core material in the fields of medicine and agrochemicals, and there is a demand for the establishment of mass production technology for D-allose, following D-allulose.

[0004] As a method for producing D-allose using an enzyme produced by a microorganism, the present inventors have developed a technology for producing allose from allulose using L-rhamnose isomerase isolated from Pseudomonas stutzeri (Non-Patent Document 1). L-rhamnose isomerase is an enzyme that catalyzes the reversible isomerization reaction between L-rhamnose and L-rhamnurose. However, it has been revealed that the L-rhamnose isomerase derived from P. stutzeri has a wide substrate specificity, acting not only between L-rhamnose and L-rhamnurose, but also between L-lyxose and L-xylulose, L-mannose and L-fructose, D-gulose and D-sorbose, D-ribose and D-ribulose, D-allose and D-allulose, and L-talose and L-tagatose. Utilizing this broad substrate specificity, it has become possible to produce a variety of rare aldoses and ketoses on Izumoring, primarily through the conversion of D-allulose to D-allose. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Patent No. 5330976 [Patent Document 2] Patent No. 5317055 [Patent Document 3] Patent No. 5158779 [Patent Document 4] Patent No. 4943839 [Patent Document 5] Patent No. 4724824 [Patent Document 6] Japanese Patent Application Laid-Open No. 2009-269887 [Patent Document 7] Patent No. 5816871 [Patent Document 8] U.S. Patent No. 10,480,018 [Non-patent literature]

[0006] [Non-Patent Document 1] J.Ferment.Bioeng.(1997) Vol.84, p.319 Summary of the Invention [Problem to be solved by the invention]

[0007] Today, as the production of most rare sugars has become possible, new challenges have arisen one after another in rare sugar production. For example, the present inventors have faced a new challenge: shifting from laboratory-level mass production of the rare sugar D-allose, whose physiological activity has been revealed, to market-scale mass production leading to industrialization. That is, in order to enable efficient mass production of rare sugars, it was first considered essential to select enzymes with higher thermotolerance and a wide range of optimal pH.

[0008] As described above, in conventional techniques, the rare sugar D-allose is produced using a known L-rhamnose isomerase (EC 5.3.1.14) from pure D-allulose, a rare sugar, as a raw material. L-rhamnose isomerase is an enzyme that catalyzes the isomerization of L-rhamnose to L-rhamnurose, and can also catalyze the isomerization of L-rhamnurose to L-rhamnose. It is also known to act on the isomerization between D-allose and D-allulose. Isomerases are named based on the substrate for which they show the highest activity, and therefore, even among enzymes named L-rhamnose isomerase, their substrate specificities vary. Furthermore, some of these known enzymes also produce the aldose D-altrose as a by-product during D-allose production, and the presence of the by-product D-altrose causes a decrease in the yield of D-allose in the D-allose purification process. Therefore, the purity of the rare sugar D-allose produced in mass production has become an issue.

[0009] The objectives of the present invention are to provide a novel L-rhamnose isomerase that is derived from a microorganism that is approved for use in food production and is considered non-toxic, has high activity, and is capable of isomerizing D-allulose to D-allose in high yield; to provide a microorganism that possesses the enzyme; and to provide a production method using the enzyme. [Means for solving the problem]

[0010] The present inventors focused on bacterial species that are considered to have little toxicity and are on the list of bacteria approved for use in food not only in Japan but also in Europe and America, and collected soil samples from various locations, isolated microorganisms from the soil, and continued to search for microorganisms that have L-rhamnose isomerase activity. As a result, among the many isolated strains, we found a microorganism belonging to the genus Erwinia that produces a novel L-rhamnose isomerase. This Erwinia microorganism, Erwinia billingiae, produces a novel L-rhamnose isomerase that is highly active and thermostable.

[0011] This novel microbial L-rhamnose isomerase catalyzes the isomerization reaction between an aldose and the corresponding ketose, recognizing and reacting the CHO group at C1 with the OH group at C2 of the aldose, converting the CHO group at C1 to an OH group and the OH group at C2 to a CO group, or recognizing and reacting the OH group at C1 with the CO group at C2 of the ketose, converting the OH group at C1 to a CHO group and the CO group at C2 to an OH group. It has aldose-ketose isomerase activity. Furthermore, since it does not produce the by-product D-altrose when producing D-allose from D-allulose as a substrate, it is suitable for D-allose production.

[0012] That is, the present invention relates to L-rhamnose isomerases described in (1) to (4) below, and a microorganism described in (5) below. (1) L-rhamnose isomerase derived from a microorganism belonging to the genus Erwinia, which has a subunit molecular mass of 48 kDa as measured by SDS-PAGE and has the substrate specificity of the following (A) and (B): (A) It has an isomerase activity that recognizes and reacts with the CHO group of C1 and the OH group of C2 of aldoses, converting the CHO group of C1 to an OH group and the OH group of C2 to a CO group, or recognizes and reacts with the OH group of C1 and the CO group of C2 of ketose, converting the OH group of C1 to a CHO group and the CO group of C2 to an OH group. (B) It has the activity to catalyze the isomerization reactions between L-rhamnose and L-rhamnurose, between L-lyxose and L-xylulose, between L-mannose and L-fructose, between D-ribose and D-ribulose, between L-talose and L-tagatose, and between D-allose and D-allulose. (2) The L-rhamnose isomerase according to (1) above, which has the following physicochemical properties (C) and (D): (C) The optimum pH for the reaction is 9. (D) The optimum reaction temperature is 70°C. (3) The L-rhamnose isomerase according to (1) or (2) above, wherein the microorganism belonging to the genus Erwinia is Erwinia billingiae. (4) The L-rhamnose isomerase according to any one of (1) to (3) above, wherein the microorganism belonging to the genus Erwinia is Erwinia billingiae GuaL218-3, which is internationally deposited with the International Patent Microorganisms Depositary Center under Accession Number NITE BP-03142.

[0013] The present invention also relates to the proteins described in (5) to (7) below, the DNAs, recombinant vectors, or transformed host cells described in (8) to (11) below, or the microorganisms described in (12) below. (5) A protein comprising the amino acid sequence represented by SEQ ID NO: 1. (6) A protein that is an amino acid substitution mutant of a protein consisting of the amino acid sequence represented by SEQ ID NO: 1, which has 80% or more identity with the amino acid sequence represented by SEQ ID NO: 1 and is identical to K3, L4, I5, Y9, E10, L11, Y16, D18, V19, I21, V23, Q25, V26, M27, T28, G32, I33, R46, N52, and E54 of the amino acid sequence represented by SEQ ID NO: 1. , R68, H73, A77, I79, E80, K81, M83, A89, D102, T103, E106, D108, A109, E111, Q113, S116, H117, Q124, H125 , K126, S134, S148, D151, K152, G153, C162, I168, H171, P179, V186, L193, I195, L198, A199, E202, A205, S2 06, V211, F216, D217, A218, S219, C245, L246, A250, T257, T275, V280, L300, T310, A313, N315, K316, N319 , K320, A345, S357, D358, Q359, R361, K362, L365, E366, A371, L375, V387, A390, W391, L393, H395, V397, D4 A protein having an amino acid substitution at at least one site selected from the group consisting of 00, A401, S402, S405, E406, H409, Q412, Q413, T414, R416, L417, and 419, and having the L-rhamnose isomerase activity of (A) and (B) below, and having higher L-rhamnose isomerase activity at an optimal temperature than a protein consisting of the amino acid sequence represented by SEQ ID NO: 1. (A) It has an isomerase activity that recognizes and reacts with the CHO group of C1 and the OH group of C2 of aldoses, converting the CHO group of C1 to an OH group and the OH group of C2 to a CO group, or recognizes and reacts with the OH group of C1 and the CO group of C2 of ketose, converting the OH group of C1 to a CHO group and the CO group of C2 to an OH group. (B) It has the activity to catalyze the isomerization reactions between L-rhamnose and L-rhamnurose, between L-lyxose and L-xylulose, between L-mannose and L-fructose, between D-ribose and D-ribulose, between L-talose and L-tagatose, and between D-allose and D-allulose. (7) A protein that is an amino acid substitution mutant of a protein consisting of the amino acid sequence represented by SEQ ID NO: 1, having 78% or more identity with the amino acid sequence represented by SEQ ID NO: 1, and having K3, L4, I5, Y9, E10, L11, Y16, D18, V19, I21, V23, E24, Q25, V26, M27, T28, G32, I33, R46, N52, E54, R68, H73, A77, I79, E80, K81, A82, M83, S84, A89, K90, I97, D102, T103, E106, D108, A109, E111, Q113, S116, H117, E120, Q124, H125, K126, S134, P139, L140, S148, A150, D151, K152, G1 53, I154, C162, R167, I168, H171, P179, V186, L193, I195, L198, A199, E202, A205, S206, E210, V211, K215, F216, D217, A218, S219, C2 45, L246, A250, T254, T257, T275, V280, R282, L300, T310, A313, N315, K316, N319, K320, A345, S357, D358, Q359, R361, K362, L363, L3 65, E366, Y369, A371, A374, L375, S381, V387, A390, W391, L393, H395, V397, D400, A401, S402, S405, E406, H409, Q412, Q413, T414, R4 A protein having an amino acid substitution at at least one site selected from L16, L417, and 419, and having the L-rhamnose isomerase activity of (A) and (B) below, and having the amino acid sequence represented by SEQ ID NO: 1, which protein has a higher ratio of L-rhamnose isomerase activity at reaction temperatures of 70°C and 50°C (T70 / T50), a higher residual activity after incubation at 60°C for 1 hour, or a higher ratio of L-rhamnose isomerase activity at a reaction temperature of 80°C and the optimum temperature (T80 / optimum temperature). (A) It has an isomerase activity that recognizes and reacts with the CHO group of C1 and the OH group of C2 of aldoses, converting the CHO group of C1 to an OH group and the OH group of C2 to a CO group, or recognizes and reacts with the OH group of C1 and the CO group of C2 of ketose, converting the OH group of C1 to a CHO group and the CO group of C2 to an OH group. (B) It has the activity to catalyze the isomerization reactions between L-rhamnose and L-rhamnurose, between L-lyxose and L-xylulose, between L-mannose and L-fructose, between D-ribose and D-ribulose, between L-talose and L-tagatose, and between D-allose and D-allulose. (8) A DNA encoding the protein according to any one of (5) to (7) above. (9) DNA containing the base sequence represented by SEQ ID NO: 2. (10) A recombinant vector containing the DNA according to (8) or (9) above. (11) A transformed host cell transformed with the recombinant vector described in (10) above. (12) Erwinia billingiae GuaL218-3, which produces the L-rhamnose isomerase described in any one of (1) to (4) above and is internationally deposited with the Patent Microorganisms Depositary Center under accession number NITE BP-03142.

[0014] The present invention also relates to immobilized proteins described in (13) to (16) below. (13) An immobilized protein in which the L-rhamnose isomerase according to any one of (1) to (4) above or the protein according to any one of (5) to (7) above is immobilized on a carrier. (14) An immobilized protein, in which the L-rhamnose isomerase described in any one of (1) to (4) above is immobilized on a carrier in the form of a crude enzyme present in a bacterial cell lysate, or the protein described in any one of (5) to (7) above is immobilized on a carrier in the form of a crude protein present in a bacterial cell lysate of a transformed host cell. (15) The immobilized protein according to (13) or (14) above, wherein the carrier is an ion exchange resin or a synthetic adsorbent. (16) The immobilized protein according to (15) above, wherein the carrier is WA30, FPA54, or FPA95.

[0015] The present invention also relates to a method for producing L-rhamnose isomerase or a method for producing ketose or aldose as set forth in (17) to (19) below. (17) A method for producing L-rhamnose isomerase, which comprises culturing an Erwinia microorganism producing the L-rhamnose isomerase according to any one of (1) to (4) above or a transformed host cell according to (11) above in a medium to accumulate the L-rhamnose isomerase in the microbial cells, and then recovering the accumulated L-rhamnose isomerase. (18) A method for producing L-rhamnose isomerase according to (17) above, wherein the medium is an inorganic salt medium supplemented with L-rhamnose. (19) A method for producing ketose or aldose, comprising reacting a solution containing one or more aldoses or ketoses with L-rhamnose isomerase according to any one of (1) to (4), a protein according to any one of (5) to (7), or an immobilized protein according to any one of (13) to (16), to produce the corresponding ketose or aldose, and collecting the resulting product. [Effects of the Invention]

[0016] The L-rhamnose isomerase of the present invention is characterized by its particularly high thermostability and high activity compared to conventional L-rhamnose isomerases derived from microorganisms. For example, while the conventional L-rhamnose isomerase derived from Pseudomonas stutzeri has an optimum temperature of 60°C, the enzyme of the present invention has a high optimum temperature of 70°C, and furthermore, retains 80% or more of its activity after heat treatment at 60°C for 10 minutes, making it suitable for use in industrial production. The excellent thermostability of this enzyme can be further enhanced by creating amino acid substitution mutants of this enzyme. Furthermore, when the substrate is 100 mM D-allulose, the enzyme has a high D-allose conversion activity of 2.26 U per unit protein at 60°C, making it possible to mass-produce D-allose. In particular, when the substrate is D-allulose, the enzyme of the present invention converts it only to D-allose without producing the by-product D-altrose, resulting in a correspondingly higher yield. [Brief explanation of the drawings]

[0017] [Figure 1] FIG. 1 shows a comparison of the amounts of D-allose produced by the crude enzyme of the present invention (derived from the GuaL218-3 strain) and the control crude enzyme (derived from the AgM30 strain). [Figure 2] The results of SDS-PAGE to confirm the molecular mass of this enzyme are shown below. The numbers on the left in the figure are in kDa. [Figure 3] FIG. 1 shows the optimum pH of the present enzyme and a control enzyme. [Figure 4] FIG. 1 shows the pH stability of the present enzyme and a control enzyme after 24 hours. [Figure 5] FIG. 1 shows the optimum temperatures of the present enzyme and a control enzyme. [Figure 6] FIG. 1 shows the temperature stability of the present enzyme and a control enzyme after 10 minutes of incubation. [Figure 7] FIG. 1 shows the substrate specificity of the present enzyme and a control enzyme. [Figure 8] FIG. 1 shows the effects of metal ions on the present enzyme and a control enzyme. [Figure 9] The relative activity expression rate of the immobilized enzyme of the present enzyme on the immobilization carrier is shown. [Figure 10] The results of SDS-PAGE of the recombinant enzyme expressed in E. coli are shown. [Figure 11] 1 shows the results of HPLC analysis of the reaction solution obtained by the conversion reaction of D-allulose to D-allose using a recombinant enzyme. DETAILED DESCRIPTION OF THE INVENTION

[0018] The present invention relates to an L-rhamnose isomerase that can be isolated from microorganisms belonging to the genus Erwinia, and which has the distinctive properties of high activity and thermostability. The L-rhamnose isomerase of the present invention has the isomerase activity of recognizing and reacting with the CHO group at C1 and the OH group at C2 of an aldose, converting the CHO group at C1 to an OH group and the OH group at C2 to a CO group to produce a ketose, or recognizing and reacting with the OH group at C1 and the CO group at C2 of a ketose, converting the OH group at C1 to a CHO group and the CO group at C2 to an OH group to produce an aldose.

[0019] The term "ketose" as used herein refers to a hexose (ketohexose) or a pentose (ketopentose) having a ketose structure. Ketohexoses include allulose (also known as psicose), sorbose, tagatose, and fructose, while ketopentoses include ribulose and xylulose. In the present invention, aldose refers to aldohexose, a hexose sugar, or aldopentose, a pentose sugar, both of which have an aldose structure. Aldohexoses include glucose, allose, altrose, gulose, idose, talose, galactose, and mannose, while aldopentoses include ribose, arabinose, xylose, and lyxose. D- or L- refers to the D- and L-forms of these sugars. The L-rhamnose isomerase of the present invention has broad substrate specificity, acting on L-rhamnose, L-lyxose, L-mannose, D-ribose, L-talose, and D-allose, and can catalyze conversions between L-rhamnose and L-rhamnurose, between L-lyxose and L-xylose, between L-mannose and L-fructose, between D-ribose and D-ribulose, between L-talose and L-tagatose, and between D-allose and D-allulose.

[0020] The L-rhamnose isomerase of the present invention can be prepared by culturing a microorganism belonging to the genus Erwinia capable of producing L-rhamnose isomerase and isolating the L-rhamnose isomerase from the cells grown in the culture medium. Examples of microorganisms belonging to the genus Erwinia that can be advantageously used include Erwinia billingiae GuaL218-3 and mutant strains thereof. In filing this application, the GuaL218-3 strain was internationally deposited with the Patent Microorganisms Depositary of the National Institute of Technology and Evaluation, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan, on February 28, 2020, and received under accession number NITE ABP-03142. Thereafter, the GuaL218-3 strain was officially internationally deposited under the Budapest Treaty on February 28, 2020, under accession number NITE BP-03142.

[0021] The L-rhamnose isomerase of the present invention is prepared by aerating and culturing an L-rhamnose isomerase-producing bacterium of the genus Erwinia in an inorganic salt medium supplemented with L-rhamnose, followed by centrifugation to recover the cells from the culture medium. The recovered cells are washed with 10 mM Tris-HCl buffer (pH 7.5) and then suspended in 10 mL of 10 mM Tris-HCl buffer (pH 7.5). The cells are disrupted by enzymatic treatment using the lytic enzyme lysozyme, or by using an ultrasonic homogenizer while the cell suspension is cooled in ice water. The disrupted cells are then centrifuged, and the supernatant is used as a crude enzyme solution.

[0022] The activity of L-rhamnose isomerase in the crude enzyme solution before purification can be confirmed by measuring the amount of D-allose produced using D-allulose as a substrate. The enzyme activity for the reverse reaction, converting D-allose to D-allulose, is also measured under similar conditions. These conversion reactions are typically carried out under the following conditions: substrate concentration: 1 to 60% (w / v), preferably about 5 to 50% (w / v); reaction temperature: 30 to 80°C, preferably about 50 to 70°C; reaction pH: 6 to 11, preferably about 8 to 11; reaction time can be selected as appropriate, but in the case of a batch reaction, it is typically selected within the range of 4 to 20 hours.

[0023] The crude enzyme solution can be purified by ion exchange chromatography and hydrophobic chromatography to isolate the purified enzyme. To confirm the purity of the enzyme, SDS-PAGE (gel concentration 12.5%) is used to confirm the formation of a single band and the apparent molecular mass. The L-rhamnose isomerase of the present invention purified as described above is a metalloenzyme whose subunit molecular mass determined by SDS-PAGE is approximately 48 kDa and whose activity is regulated by metal ions. The reaction with the substrate can be carried out in the presence of a metal ion selected from the group consisting of manganese, cobalt, nickel, magnesium, iron, copper, zinc, and calcium at a concentration of 0.5 to 5 mM.

[0024] The L-rhamnose isomerase of the present invention has a specific amino acid sequence, and examples thereof include a protein having the amino acid sequence set forth in SEQ ID NO: 1, or a protein having an amino acid sequence homologous thereto and maintaining equivalent L-rhamnose isomerase activity. A homologous amino acid sequence refers to, for example, an amino acid sequence having 75% or more, 78% or more, 80% or more, preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more identity with the amino acid sequence of SEQ ID NO: 1.

[0025] The percent identity between two amino acid sequences or two nucleic acid sequences (base sequences) can be determined, for example, by the following procedure. First, the two sequences are aligned to enable optimal comparison. In this case, gaps may be introduced into the first sequence to optimize alignment with the second sequence. When a molecule (amino acid residue or nucleotide) at a specific position in the first sequence is the same as a molecule at the corresponding position in the second sequence, the molecules at that position are said to be identical. The identity between two sequences is a function of the number of identical positions shared by the two sequences (i.e., identity (%) = number of identical positions / total number of positions × 100), and preferably, the number and size of gaps required for optimal alignment are also taken into account.

[0026] Comparison of two sequences and determination of identity can also be achieved using a mathematical algorithm. A non-limiting example of a mathematical algorithm that can be used for sequence comparison is the algorithm described in Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-68 and modified in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-77. Such an algorithm is incorporated into the NBLAST program and XBLAST program (version 2.0) described in Altschul et al. (1990) J. Mol. Biol. 215:403-10. To obtain a nucleotide sequence equivalent to the nucleic acid molecule of the present invention, for example, a BLAST nucleotide search can be performed using the NBLAST program with a score of 100 and a word length of 12.

[0027] The DNA of the present invention is a gene that encodes the protein and has a predetermined nucleotide sequence. Examples include a DNA sequence that encodes the amino acid sequence represented by SEQ ID NO: 1, a nucleotide sequence represented by SEQ ID NO: 2, or a DNA sequence that has a nucleotide sequence homologous to the nucleotide sequence represented by SEQ ID NO: 2 and encodes a protein that maintains L-rhamnose isomerase activity equivalent to that of the protein represented by SEQ ID NO: 1. A homologous nucleotide sequence refers to one that has 75% or more, 78% or more, 80% or more, preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more identity in nucleotide sequence with the nucleotide sequence represented by SEQ ID NO: 2, for example.

[0028] The DNA of the present invention can also be inserted into an appropriate autonomously replicating vector to produce a recombinant vector. Recombinant vectors consist of DNA and an autonomously replicating vector, and can be prepared relatively easily using standard recombinant DNA techniques once DNA is available. An appropriate vector is selected depending on the intended use (cloning or protein expression) and the host cell. Examples of such vectors include plasmid vectors such as pBR322, pUC18, pUB110, pTZ4, pC194, pHV14, TRp7, YEp7, and pBS7, and phage vectors such as λgt·λC, λgt·λB, ρ11, φ1, and φ105.

[0029] The recombinant vector thus obtained can be introduced into appropriate host cells such as Escherichia coli, Bacillus subtilis, actinomycetes, and yeast. The introduction can be performed using known methods such as calcium phosphate coprecipitation, electroporation, lipofection, and microinjection. To obtain transformed host cells, colony hybridization or other methods can be used.

[0030] The method for producing the L-rhamnose isomerase of the present invention or a protein having L-rhamnose isomerase activity is not particularly limited, and known methods can be used. Specifically, the L-rhamnose isomerase of the present invention can be produced by culturing a microorganism capable of producing the L-rhamnose isomerase of the present invention, or a host cell transformed with DNA encoding a protein having L-rhamnose isomerase activity of the present invention, in a nutrient medium, and then collecting the protein having L-rhamnose isomerase activity from the culture. Known methods can be used for the culture, and for example, both liquid and solid culture can be used.

[0031] After culturing the bacterial cells in this manner, the enzymes and proteins of the present invention are purified and recovered. Purification and recovery methods for enzymes and proteins can be freely selected from known methods. For example, when recovering from a culture medium, the culture supernatant can be filtered, centrifuged, or otherwise treated to remove insoluble matter, followed by separation and purification using an appropriate combination of concentration using an ultrafiltration membrane, salting out using ammonium sulfate precipitation or other methods, dialysis, various types of chromatography using ion exchange resins, and other methods. When recovering from bacterial cells, the bacterial cells can be disrupted, for example, by treatment with a lytic enzyme or ultrasonic treatment, and then similar separation and purification can be carried out.

[0032] Furthermore, the L-rhamnose isomerase of the present invention, like other isomerases, can be used by immobilizing the enzyme, and highly active immobilized enzymes can be obtained by various immobilization methods. The use of immobilized enzymes enables continuous large-scale isomerization reactions, and the immobilized enzymes can be obtained by known immobilization methods, such as carrier binding, crosslinking, gel entrapment, and microencapsulation, and the carrier may be any known carrier.

[0033] One embodiment of the method for immobilizing L-rhamnose isomerase of the present invention is immobilization using a crude enzyme solution. The crude enzyme solution containing L-rhamnose is obtained by ultrasonicating a bacterial cell suspension, and is added to an ion exchange resin or the like, where the L-rhamnose isomerase can be immobilized by binding at low temperature. To extract L-rhamnose isomerase from the bacterial cells, the bacterial cell walls must be disrupted. As mentioned above, disruption can be achieved by ultrasonication or treatment with an enzyme such as lysozyme. It has been found that the crude enzyme solution obtained by ultrasonication contains a large amount of active L-rhamnose isomerase. Any known immobilization carrier can be used to immobilize the enzyme from the crude enzyme solution, but ion exchange resins, sodium alginate, synthetic adsorbents, etc. are convenient and commonly used.

[0034] Among ion exchange resins, for example, as basic anion exchange resins, either strong basic anion exchange resins or weak basic anion exchange resins can be used, for example, strong basic anion exchange resins such as SA20A and PA418 (manufactured by Mitsubishi Chemical Corporation) and weak basic anion exchange resins such as WA30 (manufactured by Mitsubishi Chemical Corporation), FPA54 and FPA95 (manufactured by Organo Corporation) etc. An example of a synthetic adsorbent is XAD7HP (manufactured by Organo Corporation). When an immobilized enzyme is produced using a weakly basic anion exchange resin as a carrier, the immobilized L-rhamnose isomerase can be easily eluted after the reaction, which makes it very easy to regenerate the immobilized enzyme and improves production efficiency.

[0035] The L-rhamnose isomerase of the present invention can be produced by culturing a microorganism capable of producing L-rhamnose isomerase of the present invention or a host cell transformed with DNA encoding a protein having L-rhamnose isomerase activity in a nutrient medium, and then isolating the protein having L-rhamnose isomerase activity from the culture obtained. Any known culture method can be used, including, for example, liquid culture or solid culture.

[0036] After culturing the bacterial cells in a medium, the L-rhamnose isomerase of the present invention is purified and recovered. Protein purification and recovery methods can be freely selected from known methods. For example, when recovering from a culture medium, the culture supernatant can be filtered, centrifuged, or otherwise treated to remove insoluble matter, followed by separation and purification using an appropriate combination of methods such as concentration using an ultrafiltration membrane, salting out using ammonium sulfate precipitation, dialysis, and various types of chromatography using ion exchange resins. When recovering from bacterial cells, the bacterial cells can be disrupted, for example, by treatment with a lytic enzyme or ultrasonication, and then similar separation and purification can be carried out.

[0037] The purified L-rhamnose isomerase or immobilized L-rhamnose isomerase of the present invention can be used to act on a solution containing one or more aldoses or ketoses that can serve as substrates to produce the corresponding ketoses or aldoses, thereby enabling the production of these.The L-rhamnose isomerase of the present invention has higher substrate specificity for D-allulose than conventional L-rhamnose isomerases, and therefore D-allose, a rare sugar, can be produced in large quantities in the presence of D-allulose, a substrate.

[0038] Furthermore, by introducing mutations into the L-rhamnose isomerase gene of the present invention and substituting the corresponding amino acid residues with other amino acid residues by site-directed mutagenesis to prepare mutants with various amino acid substitutions, it is possible to obtain enzymes with higher D-allulose isomerase activity at the optimum temperature or with higher thermostability than the wild-type enzyme without amino acid substitutions. Thermostability is evaluated using four indices: the optimum temperature, the ratio of D-allulose isomerase activity at reaction temperatures of 70°C and 50°C (T70 / T50), the residual activity after incubation at 60°C for 1 hour, and the ratio of D-allulose isomerase activity at the reaction temperature of 80°C and the optimum temperature (T80 / optimum temperature). Even if the L-rhamnose isomerase of the present invention is a mutant with an amino acid sequence identity of approximately 78%, it is possible to obtain a mutant that retains enzymatic activity and, compared to the wild-type enzyme of SEQ ID NO: 1, has a higher optimum temperature, a higher ratio of D-allulose isomerase activity at reaction temperatures of 70°C and 50°C (T70 / T50), a higher residual activity after incubation at 60°C for 1 hour, or a higher ratio of D-allulose isomerase activity at a reaction temperature of 80°C and the optimum temperature (T80 / optimum temperature).

[0039] The amino acid substitution mutants were designed with reference to the method for creating a phylogenetic tree described in Patent Document 8. All DNA sequences encoding enzymes or putative enzymes with activity similar to that of the L-rhamnose isomerase of the present invention represented by SEQ ID NO: 1 are extracted, and a phylogenetic tree derived from the DNA sequence of phylogenetic origin as a common ancestral DNA is constructed using the method of Patent Document 8. The amino acid sequences encoded by the obtained DNA sequences that are in the same lineage as SEQ ID NO: 1 of the present invention in the phylogenetic tree are then compared, and the amino acid sequences of the portions where the sequences differ are focused on. Predicting that the portions where the amino acid residues differ are unlikely to contribute much to enzyme activity, various L-rhamnose isomerase mutants are prepared by changing the amino acids, etc., corresponding to the portions of SEQ ID NO: 1 that differ by base substitution through site-directed mutagenesis. From these, amino acid substitution mutants with higher D-allulose isomerase activity at the optimum temperature or higher heat resistance than the wild-type enzyme are selected. Furthermore, the amino acid substitution mutants of the present invention exceptionally include a mutant in which one amino acid residue is added to the 418th amino acid at the C-terminus.

[0040] Site-directed mutagenesis can be carried out by any method, such as inverse PCR or annealing (Muramatsu et al., eds., "New Genetic Engineering Handbook, 4th Revised Edition," Yodosha, pp. 82-88). If necessary, various commercially available site-directed mutagenesis kits, such as Stratagene's QuickChange II Site-Directed Mutagenesis Kit and QuickChange Multi Site-Directed Mutagenesis Kit, can also be used. Site-directed mutagenesis is most commonly performed using a mutagenic primer containing the nucleotide mutation to be introduced. Such a mutagenic primer can be designed to anneal to a region of a gene containing a nucleotide sequence encoding the amino acid residue to be modified, and to contain a base sequence having a nucleotide sequence (codon) encoding the modified amino acid residue in place of the nucleotide sequence (codon) encoding the amino acid residue to be modified.

[0041] An amino acid substitution mutant refers to a mutant in which an amino acid in the original sequence is substituted with a different amino acid. The substitution may be conservative or non-conservative, and is not particularly limited, but in a preferred embodiment of the invention, the substitution is conservative. Conservative substitutions include substitutions between amino acids with the same properties (basic, acidic, or neutral) or polarity (hydrophilic or hydrophobic), such as basic for basic, acidic for acidic, or polar for polar, or between aromatic amino acids or aliphatic amino acids. Conservative substitutions are made within groups such as, for example, basic amino acids (Arg, Lys, His), acidic amino acids (Glu, Asp), neutral nonpolar amino acids (Gly, Ala, Val, Leu, Ile, Met), aliphatic amino acids (Ala, Val, Leu, Ile, Met), polar amino acids (Gln, Asn, Ser, Thr), and aromatic amino acids (Phe, Trp, Tyr).

[0042] On the other hand, non-conservative substitutions involve exchanging an amino acid with an amino acid from another group, for example, deleting Cys or substituting it with another amino acid to prevent it from folding into a protein in a tertiary structure. Alternatively, amino acids are substituted taking into account the hydropathic index of amino acids (J. Mol. Biol. (1982) Vol. 157, pp. 105-132), which is an index of hydrophobicity / hydrophilicity for amino acids, so as to maintain a balance between hydrophilicity and hydrophobicity or to increase hydrophilicity for easier synthesis. In addition, substitution may be made with an amino acid that is less sterically hindered than the original amino acid, or a charged amino acid may be substituted with an uncharged amino acid.

[0043] Substitutions may be conservative or non-conservative, for example, Ala to Ser or Thr, Arg to Gln, His or Lys, Asn to Glu, Gln, Lys, His or Asp, Asp to Asn, Glu or Gln, Cys to Ser or Ala, Gln to Asn, Glu, Lys, His, Asp or Arg, Glu to Asn, Gln, Lys or Asp, Gly to Pro, His to Asn, Lys, Gln, Arg or Tyr, Ile to Leu. Examples of substitutions include, but are not limited to, substitutions of Lys with Met, Val, or Phe, substitutions of Leu with Ile, Met, Val, or Phe, substitutions of Lys with Asn, Glu, Gln, His, or Arg, substitutions of Met with Ile, Leu, Val, or Phe, substitutions of Phe with Trp, Tyr, Met, Ile, or Leu, substitutions of Ser with Thr or Ala, substitutions of Thr with Ser or Ala, substitutions of Trp with Phe or Tyr, substitutions of Tyr with His, Phe, or Trp, and substitutions of Val with Met, Ile, or Leu. These classifications of substitutable residues for amino acid residue substitutions are merely examples, and the substitutable amino acid residues are not limited to these classifications.

[0044] A large number of multiple mutants were prepared from the wild-type enzyme having the amino acid sequence of SEQ ID NO: 1, ranging from mutants with a single amino acid substitution to mutants with 90 amino acid substitutions, and 58 mutants were selected by performing enzyme activity and heat resistance tests. Of these, 13 of the 58 mutants were confirmed to have higher D-allulose isomerase activity at the optimum temperature than the wild-type enzyme without amino acid substitutions. In addition, 52 out of 58 mutants were identified that had a higher ratio of D-allulose isomerase activity at reaction temperatures of 70°C and 50°C (T70 / T50) than the wild-type enzyme, 17 out of 58 mutants had higher residual activity after incubation at 60°C for 1 hour than the wild-type enzyme, and 56 out of 58 mutants had a higher ratio of D-allulose isomerase activity at a reaction temperature of 80°C and the optimal temperature (T80 / optimal temperature) than the wild-type enzyme. The L-rhamnose isomerase of the present invention is also advantageous in that a large number of mutants having higher thermostability than wild-type L-rhamnose isomerase can be obtained. [Example]

[0045] The present invention will be explained in detail below by way of experiments, but the present invention is not limited to the following examples. <Experiment 1: Strain origin and identification> The present inventors inoculated a large number of bacteria isolated by screening into a liquid medium containing L-rhamnose, and cultured them with shaking. Using L-rhamnose as a substrate, they measured the amount of L-rhamnurose produced, thereby measuring the activity of L-rhamnose isomerase. In this way, the microorganism strain GuaL218-3 was found to be the strain with the highest activity, and phylogenetic analysis based on the 16S rRNA gene base sequence homology revealed that the strain GuaL218-3 belongs to Erwinia.

[0046] Strain identification (1) 16S rRNA gene sequence homology The 1-500 bp region of the 16S rRNA gene was analyzed to identify the 500 bp base sequence. (2) Homology search The 16S rRNA gene sequence of this strain was subjected to a BLAST search (Japan DNA Data Bank) to check for homology with known species that are used as reference strains. Based on the strain name and homology (%) values, which showed a homology of 98% or more with the 500-bp base sequence identified above, the microorganism GuaL218-3 strain was determined to be Erwinia billingiae. The GuaL218-3 strain was internationally deposited at the Patent Microorganisms Depositary Center of the National Institute of Technology and Evaluation on February 28, 2020, under the accession number NITE BP-03142.

[0047] <Experiment 2: Cultivation of Erwinia billingiae GuaL218-3 (Accession No. NITE BP-03142)> Erwinia billingiae GuaL218-3 (accession no. 1% (v / v) of seed culture solution of NITE BP-03142 strain was added aseptically, and the mixture was cultured at 30°C for 24 hours with aeration and agitation. As a control strain for comparison with the strain of the present invention, Arthrobacter globiformis M30 (NITE BP-1111) strain (hereinafter referred to as "AgM30 strain") was cultured in the same medium and under the same culture conditions as those for the GuaL218-3 strain.

[0048] <Experiment 3: Preparation of crude enzyme> The cultures of the GuaL218-3 and AgM30 strains were harvested by centrifugation. The harvested cells were washed with 10 mM Glycine-NaOH buffer (pH 9.0) and then suspended in 10 mL of Glycine-NaOH buffer (pH 9.0). The cell suspension was then disrupted using an ultrasonic homogenizer (Emerson Japan Co., Ltd.) while cooled in ice water. The disrupted material was centrifuged at 12,000 rpm for 20 minutes, and the supernatant was used as the crude enzyme.

[0049] <Experiment 4: Production of D-allose using crude enzyme> Using the two crude enzyme solutions obtained, D-allulose was added as a substrate to each solution to a final concentration of 100 mM, and the enzyme reaction was carried out at 30°C for 4 hours. The amount of D-allose produced was measured and compared based on the HPLC area. Specifically, the enzyme reaction solution composition was 50 mM Glycine-NaOH buffer (pH 9.0), 100 mM D-allulose, crude enzyme solution, and 1 mM manganese chloride were reacted at 30°C for 4 hours, boiled for 2 minutes to stop the reaction, and then the composition of the reaction mixture was measured by HPLC. The area ratio of the D-allose produced by HPLC analysis is shown in Figure 1. When the crude enzyme was used per the same volume of culture medium, the amount of D-allulose converted to D-allose by the crude enzyme derived from the GuaL218-3 strain was approximately 23 times that of the control crude enzyme derived from the AgM30 strain.

[0050] <Experiment 5: Enzyme Purification> 1. Purification by ion exchange chromatography and hydrophobic chromatography The two crude enzyme solutions were purified by ion exchange chromatography. The column used was a HiTrapQ HP equilibrated with buffer (20 mM Tris-HCl pH 7.5). Using the AKTA system, the column was fractionated at a flow rate of 5 mL / min with a 1 M NaCl gradient from 0% to 100% in 5 mL fractions. Fractions in which enzyme activity was detected were collected, and the purified enzyme was obtained by ion exchange chromatography.

[0051] 2. The enzyme purified by ion exchange chromatography was further purified by hydrophobic chromatography. The column used was a HiTrap PHENYL. Ammonium sulfate was added to the enzyme solution to make it 2M, and the elution was performed at a flow rate of 5mL / min with 2M ammonium sulfate, decreasing the concentration from 100% to 0%. The eluate was fractionated in 5mL increments. The fractions in which enzyme activity was detected were dialyzed to remove the ammonium sulfate, and the purified enzyme separated by hydrophobic chromatography was obtained.

[0052] 3. Polyacrylamide Gel Electrophoresis The purity of the purified enzyme was confirmed by SDS-PAGE (gel concentration 12.5%) according to standard methods. In Figure 2, the standard protein is on the left, and the center and right lanes are the enzyme after purification using hydrophobic chromatography. The result showed a single band at approximately 48 kDa, which confirmed that the enzyme had been purified to a high purity. SDS-PAGE revealed that the subunit molecular mass of the purified enzyme was approximately 48 kDa.

[0053] <Experiment 6: Measurement of the physicochemical properties of enzymes> The activity of the purified enzyme L-rhamnose isomerase was measured by the following experiment: 5 mM L-rhamnose was used as the substrate, and the enzymatic reaction was carried out for 10 minutes under each condition. After the reaction, 50 μL of 10% trichloroacetic acid solution was added to the reaction mixture to stop the reaction, and the amount of L-rhamnulose produced by the cysteine ​​carbazole sulfuric acid method was measured.

[0054] 1.Optimum reaction pH For the measurement, the optimal pH for the reaction was determined by using 5 mM L-rhamnose as a substrate and reacting it with various buffer solutions of pH 3 to 11 at 30°C for 10 minutes, and measuring the amount of L-rhamnulose produced using the cysteine ​​carbazole sulfate method. The reaction conditions are shown in Table 1. The buffers used are shown in Table 2.

[0055] [Table 1] [Table 2] The results are shown in Figure 3. The optimum reaction pH for this enzyme is 9, while that of the control enzyme is 10, which is on the alkaline side. This enzyme has a wider optimum reaction pH range and exhibits higher activity under more acidic conditions.

[0056] 2.pH stability Next, using the four buffer solutions of pH 4 to 11 in Table 2, the residual activity after being kept in each buffer solution at 30° C. for 24 hours is shown in FIG. Both the present enzyme and the control enzyme were stable at pH 6 to 11. However, the pH stability of the control enzyme was highest at pH 6, whereas the present enzyme was most stable at pH 7.5 and 10.

[0057] 3. Optimum reaction temperature The pH was adjusted to 9 with glycine-NaOH buffer, and the reaction was carried out at various temperatures between 30 and 80°C to determine the optimum temperature. The reaction conditions are shown in Table 3. The temperature range from 40 to 80°C is suitable for this enzyme, and Figure 5, which shows the results of measuring reaction temperature and relative activity, reveals that the optimum temperature for this enzyme is 70°C. [Table 3] The optimum temperature for the control enzyme is 50°C. However, the relative activity (%) of this enzyme, with 70°C (where the highest activity was observed) taken as 100, was 40% at 80°C, 80% at 60°C, and 70% at 50°C. In contrast, the relative activity (%) of the control enzyme, with 50°C (where the highest activity was observed) taken as 100, was 65% at 60°C and 8% at 70°C. This means that this enzyme maintains its activity particularly at high temperatures and has high heat resistance.

[0058] 4.Thermal stability Figure 6 shows the residual activity of this enzyme and the control enzyme after incubation at each temperature for 10 minutes under the reaction conditions (10 minutes) for which the optimal temperature was determined in section 3 above, as shown in Table 3. Regarding temperature stability after incubation for 10 minutes, the decrease in relative activity of this enzyme at 60°C was less than 20%, which was significantly less than the 80% decrease of the control enzyme, indicating that this enzyme is more stable at high temperatures than the control enzyme.

[0059] 5. D-allose or D-allulose isomerase activity, The D-allose isomerase activity of this enzyme was measured in the same manner as in Experiment 6. Using 100 mM D-allose as the substrate, the enzyme reaction was carried out at 60°C for 60 minutes. After the reaction, the reaction solution was quenched by placing it in boiling water for 3 minutes, and the composition of the solution after the reaction was measured by HPLC. One unit (U) of enzyme activity is the amount of enzyme that is required to isomerize D-allose to produce 1 μmol of D-allulose per minute under the above conditions. The reaction conditions are shown in Table 4. D-allulose isomerase activity was measured under similar conditions. 100 mM D-allulose was used as a substrate and the enzyme reaction was carried out at 60°C for 60 minutes to produce D-allose. One unit (U) of enzyme activity is the amount of enzyme required to isomerize D-allulose and produce 1 μmol of D-allose per minute under the above conditions.

[0060] [Table 4] The results showed that the specific activity of this enzyme for D-allose was 5.21 U / mg, and that for D-allulose was 2.26 U / mg. Furthermore, when D-allulose was isomerized to D-allose by this enzyme, the by-product D-altrose was not produced.

[0061] 6. Substrate specificity of L-rhamnose isomerase The isomerization activity of this enzyme for L-rhamnose and five types of aldoses (D-allose, L-talose, L-lyxose, D-ribose, and L-mannose) was examined. The enzyme reaction composition was as shown in Table 5 below, with a final substrate concentration of 5 mM and an enzyme solution (final concentration: 50 mM phosphate buffer, pH 8.0). The reaction was carried out at 70°C for 10 minutes, and the amount of ketose isomerized from each aldose was measured by HPLC analysis. The isomerization activity for L-rhamnose is set to 100, and the activity for each aldose is shown as a relative activity. The relative activity using D-allose, L-mannose, L-talose, L-lyxose, and D-ribose as substrates is shown in Table 6 and Figure 7. [Table 5]

[0062] [Table 6] The activity was strongest for L-rhamnose, followed by L-lyxose, L-mannose, D-ribose, L-talose, and D-allose. On the other hand, the control enzyme reacted with L-rhamnose, L-lyxose, L-mannose, D-ribose, and D-allose in that order, but did not react with L-talose. This enzyme catalyzes the isomerization reactions between L-rhamnose and L-rhamnulose, between L-lyxose and L-xylulose, between L-mannose and L-fructose, between D-ribose and D-ribulose, between L-talose and L-tagatose, and between D-allose and D-allulose.

[0063] 7. Effects of metal ions Next, to examine the effect of metal ions on D-allose isomerase activity, the enzyme was partially dialyzed and its activity was measured. Dialysis was performed by placing the enzyme solution in a cellulose membrane, immersing it in a glycine-NaOH buffer solution (pH 9.0) containing 20 mM EDTA, and slowly stirring the buffer solution for 16 hours to remove the effects of other metal ions. The enzyme activity of the enzyme thus obtained was measured by the cysteine ​​carbazole method after reaction in the presence of 1 mM of various divalent metal ions (under the reaction conditions shown in Table 7). As a result, CoCl2 significantly increased the activity of this enzyme, indicating that the enzyme is metal-dependent (Fig. 7).

[0064] [Table 7]

[0065] <Experiment 7: Preparation of immobilized enzyme> Fungal cells of Erwinia billingiae GuaL218-3 strain, which were cultured and collected in the same manner as in Experiment 2, were used. 1. Obtaining crude enzyme by ultrasonic treatment of bacterial cells The cells from 1 L of culture were suspended in 40 mL of 50 mM Glycine-NaOH buffer (pH 9.0), and the suspension was homogenized using an ultrasonic homogenizer while cooling in ice water. The homogenized material was centrifuged at 15,000 × g for 30 minutes, and the supernatant was used as the crude enzyme solution.

[0066] 2. Immobilization of crude enzyme The crude enzyme solution obtained above was added to an ion exchange resin or synthetic adsorbent equilibrated with 50 mM Glycine-NaOH buffer (pH 9.0) after thorough washing with pure water to swell it, and the crude enzyme protein was bound by gently mixing at 4°C for 20 hours. The resin was then washed with 50 mM Glycine-NaOH buffer (pH 9.0) to obtain the immobilized enzyme. The immobilization carriers used were strong basic anion exchange resins (SA10A, SA11A, NSA100, SA20A, PA306S, PA308, PA312, PA316, PA408, PA412, PA418 manufactured by Mitsubishi Chemical Corporation, weak basic anion exchange resins (WA10, WA20, WA21J, WA30 manufactured by Mitsubishi Chemical Corporation and FPA54, FPA60CL, FPA95 manufactured by Organo Corporation), or synthetic adsorbents (XAD7HP, XAD118ON manufactured by Organo Corporation).

[0067] 3.Method for measuring the enzyme activity of immobilized enzymes The enzymatic activity of the immobilized enzyme was measured by measuring the amount of D-allose produced when the enzyme reacted with D-allulose as a substrate. First, 100 mg of immobilized enzyme resin, glycine-NaOH buffer (pH 9.0) (final concentration 50 mM), manganese chloride (final concentration 1 mM), and D-allulose (final concentration 100 mM) solution (500 μL) were used as the reaction solution composition. The reaction was carried out for 24 minutes in a 30 ° C thermostatic water bath, and then the reaction was immediately stopped by boiling at 100 ° C for 2 minutes. The reaction solution was cooled to room temperature and desalted with ion exchange resin (a mixed resin of 200CT and IRA67 (both manufactured by Organo Corporation)), and further filtered to obtain an analytical sample. Analysis was carried out by measuring the peak area of ​​the D-allulose produced using high-performance liquid chromatography (column: GL-C611 (Hitachi), temperature: 60 ° C, eluent: 0.1 mM NaOH, flow rate: 1.0 mL / min, detector: RID-20A (Shimadzu)).

[0068] 4. Comparison of enzyme activity depending on the carrier used for enzyme immobilization To confirm the influence of the carrier used for enzyme immobilization, the enzyme activity of the present enzyme immobilized on each of the carriers described in 2 above was compared. The relative value was calculated by setting the sample with the highest activity expression rate among the comparative samples as 100. FIG. 9 shows the relative activity expression rates of various immobilized enzymes.

[0069] <Experiment 8: Cloning of DNA encoding the enzyme and preparation of recombinant vectors and transformed host cells containing the DNA> DNA encoding a protein with D-allulose isomerase activity was isolated from Erwinia The enzyme was cloned from Erwinia billingiae strain GuaL218-3, and autonomously replicating recombinant DNA was constructed. The base sequence of the DNA encoding the enzyme was determined, and transformed microorganisms were prepared.

[0070] <Experiment 8-1: Determining the complete base sequence of chromosomal DNA> Unlike existing similar enzymes, the D-allose isomerase enzyme of this fungus could not be isolated using PCR amplification techniques or existing protein databases. Therefore, we determined the whole genome sequence of Erwinia billingiae strain GuaL218-3 and constructed a database of proteins encoded by all ORFs in the genome. We requested Macrogen Japan Co., Ltd. to perform next-generation sequencing analysis using PacBio-RSII / Sequel using cultured Erwinia billingiae strain GuaL218-3 as the test fungus. As a result, four contigs with base lengths of 4,301,131 bp, 1,024,754 bp, 280,568 bp, and 126,938 bp were obtained. These four contigs total approximately 5.7 Mb, and we believe that they cover the entire genome sequence of the Erwinia billingiae strain GuaL218-3.

[0071] <Experiment 8-2: Building a protein database> Based on the obtained DNA sequence of approximately 5.7 Mb, 5,357 ORFs were deduced using the Prokka program, and the amino acid sequences of each ORF were deduced. These 5,357 amino acid sequences were used as the protein database for the Erwinia billingiae strain GuaL218-3.

[0072] <Experiment 8-3: Identification of proteins with D-allulose isomerase activity> Using the above protein database, the protein was registered in MASCOTserver (Matrix Science, Inc.), a protein identification system, and proteins showing D-allulose isomerase activity were identified. The test sample used was the 48 kDa band in SDS-PAGE described in paragraph

[0045] . After reduction and alkylation, the trypsin-digested fragments were subjected to MALDI-TOF-MS analysis. As a result, 78% similarity was observed with the underlined amino acid sequence in the amino acid sequence of Sequence 1 (SEQ ID NO: 1 in the Sequence Listing) consisting of 418 amino acids below, strongly suggesting that this protein is L-rhamnose isomerase from the Erwinia billingiae GuaL218-3 strain.

[0073] [Array 1] M TKLIEQAYELAK QRYADVGIDVEQVMTQLDGIPVSMHCWQGDDVR GFENPNGELTGGIQATGNYPGRARNAHELR ADIEKAMSLIPGAK RLNLHAIYLESDTPVERDAIEPQHFSHWVEWAK QHK LGLDFNPSCFSHPLSADGFTLSHADK GIR QFWID HCK ASRR ISAHFGEQLGTPSVMNIWVPDGMKDLTIDR LAPRER LASALDEVISEKFDASHHIDAVESKLFGIGAESYTVG SNEFCLGYAASRQTALTLDAGHFHPTEVISDK ISTAMLYVPR LLLHVSRPVRWDSDHVVLLDDETQAIATEIAR NKLFNK VHIGLDFFDASINRIAAWVIGTR NAKKALLR ALLEPSDQLRKLELEGDYTARLALLEEQKSLPWQAVWEAWCLRHDVPAD ASWLSEVRHYEQQTLR LR (Sequence 1: Amino acid sequence identified using a protein database of Erwinia billingiae strain GuaL218-3. The underlined amino acid sequence matches the peak obtained in MALDI-TOF-MS analysis.)

[0074] <Experiment 8-4: Isolation of an enzyme gene with D-allulose isomerase activity> The DNA sequence of gene sequence 2 (SEQ ID NO: 2 in the Sequence Listing) identified from the amino acid sequence was synthesized, inserted into the pQE60 vector (Qiagen), and transformed into E. coli for expression. The induced enzyme was confirmed using the constructed E. coli expression system. As shown in Figure 10, SDS-PAGE confirmed the induced protein in the soluble fraction. Furthermore, this induced recombinant enzyme was reacted with 60% (w / v) D-allulose as a substrate at 30°C for 24 hours, and the D-allulose isomerase activity was confirmed by HPLC. The peak at retention time 22.48 minutes in this figure corresponds to D-allose, and the peak at retention time 29.67 minutes corresponds to D-allulose. This recombinant enzyme catalyzed the isomerization of D-allulose to D-allose without producing D-altrose, a potential by-product.

[0075] [Array 2]

[0076] <Experiment 9: Creation of amino acid substitution mutants of this enzyme and their enzymatic activity and heat resistance> A phylogenetic tree was created using the amino acid sequence of the L-rhamnose isomerase of the present invention represented by SEQ ID NO: 1 by the method described in Patent Document 8, and the amino acid sequence of SEQ ID NO: 1 was compared with the amino acid sequence encoded by a DNA sequence classified in the same phylogeny as the DNA sequence of SEQ ID NO: 2 of the present invention. Various L-rhamnose isomerase mutants were created by changing the amino acids corresponding to the positions different from SEQ ID NO: 1 by base substitution through site-directed mutagenesis. The amino acid substitution mutants of the present invention exceptionally include a mutant in which one amino acid residue (G) is added to the 418th amino acid at the C-terminus.

[0077] Single-amino acid substitution mutants were first generated by site-directed mutagenesis. PCR primers for both DNA strands were prepared, each containing a base sequence with one or two base pair substitutions that would result in an amino acid substitution at the target mutation site. PCR reactions were then performed using the original plasmid DNA and these primers. The resulting PCR fragment was transformed into host E. coli, and plasmid DNA was extracted from the resulting clone. The introduction of the desired mutation was confirmed by sequence analysis. Multiple-substitution mutants were generated by repeating the above procedure.

[0078] Recombinant E. coli expressing recombinant enzymes containing site-directed mutations was pre-cultured for 12 hours at 30°C and 200 rpm in a medium containing 3.5% polypeptone, 2.0% yeast extract, 1.0% sodium chloride, and 2 mM manganese chloride, with ampicillin added to a final concentration of 100 μg / ml. One-twentieth the volume of the pre-culture was inoculated into expression medium containing ampicillin added to a final concentration of 100 μg / ml, and main culture was performed at 30°C and 200 rpm for 2 hours. After main culture, IPTG was added to a final concentration of 0.1 mM, and expression of the recombinant enzyme was induced overnight at 30°C and 200 rpm. The composition of the recombinant E. coli expression medium is shown in Table 8. [Table 8]

[0079] The enzymatic activity of the wild-type enzyme and crude enzymes of the amino acid substitution mutants obtained by recombination was measured by reacting them for 10 minutes at various temperatures using a final concentration of 100 mM D-allulose as the substrate and a final concentration of 50 mM Tris-HCl buffer (pH 8.0). The sugar composition of the reaction solution was then measured using HPLC to determine the amount of D-allose produced. In addition, to measure the residual activity of these crude enzymes after incubation at 60°C for 1 hour, the enzyme solution was heat-treated at 60°C for 1 hour, and then the enzyme reaction was carried out at 60°C for 10 minutes using the reaction composition in Table 9, and the residual activity was measured. The composition of the reaction solution is shown in Table 9 below. [Table 9]

[0080] Using the above method, the optimum reaction temperature and relative activity of each mutant were measured, and amino acid substitution mutants with higher D-allulose isomerase activity than the wild-type enzyme at the optimum reaction temperature of each mutant were selected. The wild-type enzyme is a crude enzyme produced by recombinant means, and its optimum temperature for D-allose production is 60°C, which is lower than the optimum temperature of 70°C for L-rhamnose of the purified enzyme. However, there are also mutants in which the optimum temperature was restored by inserting a mutation. As a result, 13 mutants were obtained that had a higher optimum temperature or higher D-allulose isomerase activity at the optimum temperature than the wild-type enzyme (Table 10).

[0081] As indicators of heat resistance, the ratio of D-allulose isomerase activity at reaction temperatures of 70°C and 50°C (T70 / T50), the residual activity after incubation at 60°C for 1 hour, and the ratio of D-allulose isomerase activity at a reaction temperature of 80°C and the optimum temperature (T80 / optimum temperature) were measured in the same manner as in the measurements of the optimum reaction temperature and relative activity of each mutant described above. Mutants with higher indices than the wild-type enzyme were selected (Tables 11 to 13).

[0082] As a result, 56 mutants were obtained that had higher activity than the wild-type enzyme in any of the four indices. The amino acid sequences of these 56 mutants are shown in SEQ ID NOS: 3 to 8, 10 to 28, and 30 to 60 in the Sequence Listing. The mutants with the amino acid sequences of SEQ ID NOS: 9 and 29 had activity, but did not reach the wild-type enzyme in any of the four indices. The mutants having the amino acid sequences of SEQ ID NOs: 3 to 60 in the sequence listing are designated as "SEQ ID NOs: 3 to 60." The amino acid substitution sites (positions) and substituted amino acids from the wild-type enzyme (SEQ ID NO: 1) in SEQ ID NOs: 3 to 60 are shown below. In this case, if the amino acid substitution sites shown below differ from those shown in SEQ ID NOs: 3 to 60 in the sequence listing, the amino acid sequence shown in the sequence listing is the correct sequence. The number in parentheses at the end of each sequence indicates the number of amino acid substitutions.

[0083] Array 3 419G (1) 4 V280I (1) 5 V387I (1) 6 L198F (1) 7 S405E (1) 8 N52E (1) 9 Q124R (1) 10 D102T (1) 11 Q359L (1) 12 K152P (1) 13 D18A, F216L, T310S, L365Q (4) 14 D18A, F216L, L365Q (3) 15 Y9F, D18A, V19I, I21V, Q25K, T28A, H73D, A77S, I79V, T103K, E106D, Q113K, H117N, Q124T, P179A, I195V, A 199G, E202Q, F216L, D217N, A218P, S219A, A250S, L300I, N315H, N319D, S357T, L365S, A371G, L375M, L417Q (31) 16 Y9F, D18A, V19I, I21V, T28A, H73D, A77S, I79V, T103K, E106D, Q113K, H117N, Q124T, P179A, I195V, E202Q, F216L, D217N, A218P, S219A, A250S, L300I, N315H, N319D, S357T, L365N, A371G, L375M, L417Q (29) 17 D18A, V19I, I21V, T28A, H73D, A77S, I79V, T103K, E106D, Q113K, H117N, Q124T, P179A, I195V, E202Q, F216L, D217N, A218P, S219A, A250S, L300I, N315H, N319D, S357T, L365S, A371G, L375M (27) 18 D18A、V19I、I21V、T28A、I79V、Q113K、H117N、Q124P、I195V、E202Q、F2 16L、A218P、S219A、A250T、T310S、N315H、N319D、S357T、L365N、L417Q (20) 19 D18A, V19I, I21V, T28A, H73D, T103K, Q113K, H117N, Q124N, H125N, E202Q, F216L, D217N, A218P, S219Q, A250T, N315H, N319D, L365N (19) 20 D18A, V19I, I21V, T28A, H73D, T103K, Q113K, H117N, Q124N, H125N, I195V, E202Q, F216L, D217N, A218P, S219Q, A250T, N315H, N319D, S357T, L365N (21)

[0084] 21 Y9F、D18A、V19I、I21V、M27L、T28G、G32R、N52Q、E54A、H73D、I79L、K81Q、T103K、E106A、A109E、Q113E、H117N、Q124N、K126Q、S134T、H171Y、I195V、E202Q、V211I、F216L、D217N、A218P、S219Q、A250T、T275A、T310N、N315Q、K316N、N319D、K320R、S357T、D358A、K362Q、L365N、E406D (40) 22 D18A、V19I、I21V、M27L、T28G、G32R、N52Q、E54A、H73D、I79L、K81Q、T103K、E106A、A109E、Q113E、H117N、Q124N、K126Q、S134T、H171Y、I195V、E202Q、V211I、F216L、D217N、A218P、S219Q、A250T、T275A、T310N、N315Q、K316N、N319D、K320R、S357T、D358A、K362Q、L365N、E406D (39) 23 Y9F、D18A、V19I、I21V、T28G、N52Q、E54A、H73D、I79L、T103K、Q113E、H117N、Q124N、S134T、H171Y、I195V、E202Q、F216L、D217N、A218P、S219Q、A250T、T275A、T310N、N315Q、N319D、S357T、K362Q、L365N、E406D (30) 24 Y9F、D18A、V19I、I21V、T28A、N52Q、E54A、H73D、I79L、T103K、Q113E、H117N、Q124N、H125N、S134T、H171Y、I195V、E202Q、V211I、F216L、D217N、A218P、S219Q、A250T、T310N、N315Q、N319D、S357T、K362Q、L365N、E406D (31) 25 D18A, V19I, I21V, T28A, N52Q, E54A, H73D, I79L, T103K, Q113E, H117N, Q124K, S134T, H171Y, I195V, E202Q, V211I, F216L, D217N, A218P, S219Q, A250S, T310N, N315Q, N319D, S357T, L365N (27) 26 D18A, V19I, I21V, V26A, T28A, G32R, N52Q, E54A, H73D, I79L, E80D, T103K, Q113E, H117N, Q124K, S134T, H171Y, I195V, E202Q, A205V, F216L, D217N, A218P, S219Q, A250T, T310N, N315Q, N319D, S357T, K362Q, L365N (31) 27 L4Q、Y9F、V19I、I21V、V26A、M27I、T28R、G32R、N52Q、E54A、H73D、A77S、E80D、T103K 、Q113A、S116A、H117N、E120A、Q124E、L140M、A150S、G153T、I154V、H171Y、L193I、I 195V, E202Q, A205M, F216L, D217N, A218P, S219Q, T257C, T275A, T310S, A313V, N315H, K316Q, N319D, A345T, S357T, Q359R, K362Q, L363A, L365N, A374T, S405G, E406D (48) 28 V19I、I21V、V26A、M27I、T28R、G32R、N52Q、E54A、H73D、A77S、E80D、T103K、Q113A 、S116A、H117N、E120A、Q124E、L140M、A150S、G153T、I154V、H171Y、L193I、I195V 、E202Q、A205M、F216L、D217N、A218P、S219Q、T257C、T275A、T310S、A313V、N315H、K316Q、N319D、A345T、S357T、Q359R、K362Q、L363A、L365N、A374T、S405G、E406D (46) 29 D18E、V19N、I21V、E24D、Q25L、T28A、G32E、E54Q、H73D、M83I、K90M、D102S、T103Q、Q113K、H117N、E120A、Q124S、H125N、K126R、S134T、P139A、L140K、K152T、R167H、H171Y、L198F、E202Q、E210A、V211I、K215Q、F216L、D217N、A218P、S219Q、A250S、T254I、T310H、N315Q、N319D、Q359R、K362R、L365S、Y369F (43) 30 K3T、L4Q、I5L、Y9W、E10D、Y16F、D18A、I21V、Q25E、V26A、M27L、T28R、G32R、I33L、R46A、N52G、E54S、R68K、H73T、I79L、K81L、A82T、M83L、A89P、T103E、E106A、D108N、A109E、E111K、Q113E、S116K、H117N、Q124A、H125N、S148A、K152D、G153E、C162V、I168V、H171Y、V186I、L193I、I195V、E202Q、A205L、S206A、V211A、F216L、A218P、S219A、C245Y、L246M、T257C、T275A、T310S、A313I、N315H、K316D、N319D、K320R、A345M、S357T、D358A、Q359E、R361K、L365A、E366N、A390M、W391Y、L393Q、H395N、V397A、D400G、A401S、S402Q、S405D、E406N、H409A、Q412E、Q413D、T414V、R416S、L417Q、419G (84)

[0085] 31 K3T、L4Q、I5L、Y9W、E10D、Y16F、D18A、I21V、Q25E、V26A、M27L、T28R、G32R、I33L、R46A、N52G、E54S、R68K、H73T、I79L、K81L、A82T、M83L、A89P、T103E、E106A、D108N、A109E、E111K、Q113E、S116K、Q124A、H125N、S148A、K152D、G153E、C162V、I168V、H171Y、V186I、L193I、I195V、E202Q、A205L、S206A、V211A、F216L、A218P、S219A、C245Y、L246M、A250T、T257C、T275A、T310S、A313I、N315H、K316D、N319D、K320R、A345M、S357T、D358A、Q359E、R361K、L365A、E366N、A390M、W391Y、L393Q、H395N、V397A、D400G、A401S、S402Q、S405D、E406N、H409A、Q412E、Q413D、T414V、R416S、L417Q、419G (84) 32 K3T、L4Q、I5L、Y9W、E10D、Y16F、D18A、I21V、Q25E、V26A、M27L、T28R、G32R、I33L、R46A、N52G、E54S、R68K、H73T、I79L、K81L、M83L、A89P、T103E、E106A、D108N、A109E、E111K、Q113G、S116T、H117N、Q124A、H125N、S148A、K152D、G153E、C162V、I168V、H171Y、V186I、L193I、I195V、E202Q、A205L、S206A、V211A、F216L、A218P、S219A、C245Y、L246M、A250T、T257C、T275A、T310S、A313I、N315H、K316D、N319D、K320R、A345M、S357T、D358A、Q359E、R361K、L365A、E366N、A390M、W391Y、L393Q、H395N、V397A、D400G、A401S、S402Q、S405D、E406N、H409A、Q412K、Q413D、T414V、R416S、L417Q、419G (84) 33 K3T、L4Q、I5L、Y9W、E10D、D18A、I21V、E24D、Q25E、V26A、M27L、T28R、G32R、I33L、R46A、N52G、E54S、R68K、H73T、I79L、K81L、M83L、A89P、E106A、D108N、A109E、E111K、Q113A、S116K、H117N、E120A、Q124A、H125N、K126Q、S148A、D151N、K152D、G153D、C162V、I168V、H171Y、V186I、L193I、I195V、E202Q、A205L、S206A、V211A、F216L、D217N、A218P、S219A、C245Y、L246M、A250T、T257C、T275A、T310N、A313I、N315H、K316D、N319D、K320R、A345M、S357T、D358S、Q359A、R361K、K362Q、L365E、E366N、A374V、S381T、A390M、W391Y、L393Q、H395N、V397T、D400G、A401S、S402Q、S405D、E406N、H409M、Q412K、Q413D、T414V、R416S、L417Q、419G (90) 34 L11I、D18A、I21V、Q25E、V26A、M27L、T28R、G32R、I33L、R46A、N52E、E54S、R68K、H73G、I79L、K81Q、M83L、A89P、E106A、D108N、A109E、E111K、Q113E、S116K、H117N、Q124A、H125N、D151N、K152D、G153E、I168V、H171Y、V186I、L193I、I195V、L198F、E202Q、A205L、S206N、F216L、A218P、S219A、C245Y、L246M、A250T、T257C、T275A、V280I、T310S、A313I、N315H、K316N、N319D、K320R、A345M、S357T、D358E、K362Q、L365A、E366D (60) 35 D18A、I21V、Q25E、V26A、M27L、T28R、G32R、I33L、R46A、N52E、E54S、R68K、H73G、I79L、K81Q、M83L、A89P、I97L、E106A、D108N、A109E、E111K、Q113E、S116K、H117N、Q124T、H125N、D151N、K152N、G153E、I168V、H171Y、V186I、L193I、I195V、L198F、E202Q、A205L、S206N、F216L、A218P、S219A、C245Y、L246M、A250T、T257C、T275A、V280I、T310S、A313I、N315H、K316N、N319D、K320R、A345M、S357T、D358E、K362Q、L365A、E366D、S381C (61) 36 Y16F、D18A、I21V、Q25E、V26A、M27L、T28R、G32R、I33L、R46A、N52E、E54A、R68K、H73T、I79L、K81Q、M83L、A89P、E106A、D108N、A109E、E111K、Q113E、S116K、H117N、Q124A、H125N、D151N、K152D、G153E、I168V、H171Y、V186I、L193I、I195V、E202Q、A205L、S206D、F216L、A218P、S219A、C245Y、L246M、A250T、T257C、T275A、T310S、A313I、N315H、K316N、N319D、K320R、A345M、S357T、K362Q、L365A、E366D (57) 37 Y16F、D18A、I21V、Q25E、V26A、M27L、T28R、G32R、I33L、R46A、N52E、E54A、R68K、H73T、I79L、K81Q、M83L、A89P、E106A、D108N、A109E、E111K、Q113E、S116K、H117N、Q124A、H125N、D151N、K152D、G153E、C162V、I168V、H171Y、V186I、L193I、I195V、E202Q、A205L、S206D、F216L、D217N、A218P、S219A、C245Y、L246M、A250T、T257C、T275A、T310S、A313I、N315H、K316N、N319D、K320R、A345M、S357T、K362Q、L365A、E366D (59) 38 Y16F、D18A、I21V、Q25E、V26A、M27L、T28R、G32R、I33L、R46A、N52E、E54A、R68K、H73T、I79L、K81Q、M83L、A89P、E106A、D108N、A109E、E111K、Q113E、S116K、H117N、Q124A、H125N、D151N、K152N、G153E、I168V、H171Y、V186I、L193I、I195V、E202Q、A205L、S206A、F216L、A218P、S219A、C245Y、L246M、A250T、T257C、T275A、T310S、A313I、N315H、K316N、N319D、K320R、A345M、S357T、D358E、K362Q、L365A、E366D (58) 39 Y16F、D18A、I21V、Q25E、V26A、M27L、T28R、G32R、I33L、R46A、N52E、E54A、R68K、H73T、I79L、K81Q、 M83L、A89P、E106A、D108N、A109E、E111K、Q113E、S116K、H117N、Q124A、H125N、K152N、G153E、I168V , H171Y, V186I, L193I, I195V, E202Q, A205L, S206A, F216L, A218P, S219A, C245Y, L246M, A250T, T257C, T275A, T310S, A313I, N315H, K316N, N319D, K320R, A345M, S357T, D358E, K362Q, L365A, E366N (57) 40 Y16F、D18A、Q25E、V26A、M27L、T28R、G32R、I33L、R46A、N52E、E54S、R68K、H73T、I79L、K81Q、M83L 、A89P、E106S、A109Q、E111K、Q113E、S116K、H117N、Q124A、H125N、K126Q、K152N、G153K、I168V、H1 71Y, V186I, L193I, I195V, E202Q, A205L, S206D, F216L, A218P, S219A, C245Y, L246M, A250T, T257C, T275A, T310S, A313V, N315H, K316N, N319D, K320R, A345M, S357T, D358E, K362Q, L365A, E366S (56)

[0086] 41 Y16F、D18A、Q25E、V26A、M27L、T28R、G32R、I33L、R46A、N52E、E54S、R68K、H73T、I79L、K81Q、M83L 、A89P、E106S、A109Q、E111K、Q113E、S116K、H117N、Q124A、H125N、K126Q、K152N、G153K、I168V、H1 71Y, V186I, L193I, I195V, E202Q, A205L, S206A, F216L, A218P, S219A, C245Y, L246M, A250T, T257C, T275A, T310S, A313V, N315H, K316N, N319D, K320R, A345M, S357T, D358E, K362Q, L365A, E366S (56) 42 Y16F, D18A, I21V, V26A, M27L, T28P, G32R, I33L, R46A, N52Q, E54A, R68K, H73T, I79L, K81Q, A89P, D108N, E111K, Q113E, S116K, H117N, Q124E, K152N, G153D, H171Y, V186I, L 193I, I195V, E202Q, A205L, S206A, F216L, A218P, S219A, C245Y, L246M, A250T, T257C, T275A, T310S, A313I, N315H, K316N, N319D, K320R, A345M, S357T, K362Q, L365A, E366N (50) 43 Y16F、D18A、Q25E、V26A、M27L、T28R、G32R、I33L、R46S、N52E、E54S、R68K、H73S、I79L、K81Q、S84R、A 89P、E106S、A109Q、E111K、Q113E、S116K、H117N、Q124A、H125N、K126Q、K152N、G153S、I168V、H171Y 、V186I、L193I、I195V、E202Q、A205L、S206A、F216L、A218P、S219A、C245Y、L246M、A250T、T257C、T275A、R282Q、T310S、A313V、N315H、K316D、N319D、K320R、A345M、S357T、D358A、Q359E、L365A、E366A (57) 44 Y16F、D18A、Q25E、V26A、M27L、T28R、G32R、I33L、R46S、N52E、E54S、R68K、H73S、I79L、K81Q、S84R、A 89P、E106S、A109Q、E111K、Q113E、S116K、H117N、Q124A、H125N、K126Q、K152N、G153S、I168V、H171Y , V186I, L193I, I195V, E202Q, A205L, S206A, F216L, D217N, A218P, S219A, C245Y, A250T, T257C, T275A, R282Q, T310S, A313V, N315H, K316D, N319D, K320R, A345M, S357T, D358A, Q359E, L365A, E366A (57) 45 Y16F、D18A、Q25E、V26A、M27L、T28R、G32R、I33L、R46S、N52E、E54S、R68K、H73S、A77T、I79L、K81Q、S84 R、A89P、E106S、A109Q、E111K、Q113E、S116K、H117N、Q124A、H125N、K126Q、K152N、G153S、I168V、H171Y , V186I, L193I, I195V, E202Q, A205L, S206A, F216L, D217N, A218P, S219A, C245Y, L246M, A250T, T257C, T275A, R282Q, T310S, A313V, N315H, K316D, N319D, K320R, A345M, S357T, D358A, Q359E, L365A, E366A (59) 46 Y16F、D18A、I21V、Q25E、V26A、M27L、T28R、G32R、I33 L、R46A、N52G、E54A、R68K、H73T、I79L、K81L、M83L、A 89P、T103E、E106A、D108N、A109E、E111K、Q113E、S116K、H117N、Q124A、H125N、K152D、G153E、C162V、I168V 、H171Y、V186I、L193I、I195V、E202Q、A205L、S206A、F216L、D217N、A218P、S219A、C245Y、L246M、A250T、T257C、T275A、T310S、A313I、N315H、K316D、N319D、K320R、A345M、S357T、D358A、Q359E、K362Q、L365A、E366D (61) 47 Y16F、D18A、I21V、Q25E、V26A、M27L、T28R、G32R、I33L 、R46A、N52G、E54A、R68K、H73T、I79L、K81L、M83L、A89 P、T103E、E106A、D108N、A109E、E111K、Q113E、S116K、 H117N、Q124A、H125N、S148A、K152D、G153E、C162V、I1 68V, H171Y, V186I, L193I, I195V, E202Q, A205L, S206A, F216L, D217N, A218P, S219A, C245Y, L246M, A250T, T257C, T275A, T310S, A313I, N315H, K316D, N319D, K320R, A345M, S357T, D358A, Q359E, K362Q, L365A, E366D (62) 48 Y16F、D18A、I21V、Q25E、V26A、M27L、T28R、G32R、I33L 、R46A、N52G、E54A、R68K、H73T、I79L、K81L、M83L、A89 P、T103E、E106A、D108N、A109E、E111K、Q113E、S116K、 H117N、Q124A、H125N、S148A、K152N、G153E、C162V、I1 68V, H171Y, V186I, L193I, I195V, E202Q, A205L, S206A, F216L, D217N, A218P, S219A, C245Y, L246M, A250T, T257C, T275A, T310S, A313I, N315H, K316D, N319D, K320R, A345M, S357T, D358A, Q359E, K362Q, L365A, E366N (62) 49 E10D、Y16F、D18A、I21V、Q25E、V26A、M27L、T28R、G32R、I 33L、R46A、N52G、E54S、R68K、H73T、I79L、K81L、M83L、A8 9P、T103E、E106A、D108N、A109E、E111K、Q113E、S116K、H 117N、Q124A、H125N、S148A、K152D、G153E、C162V、I168V、 H171Y, V186I, L193I, I195V, E202Q, A205L, S206A, V211A, F216L, D217N, A218P, S219A, C245Y, L246M, A250T, T257C, T275A, T310S, A313I, N315H, K316D, N319D, K320R, A345M, S357T, D358A, Q359E, R361K, K362Q, L365A, E366N (65) 50 E10D、Y16F、D18A、I21V、Q25E、V26A、M27L、T28R、G32R、I33L、R46A、N52G、E54S、R68K、H73T、I79L、K81L、M83L、A89P、T103E、E106A、D108N、A109E、E111K、Q113E、S116K、H117N、Q124A、H125N、S148A、K152D、G153E、C162V、I168V、H171Y、V186I、L193I、I195V、E202Q、A205L、S206A、V211A、F216L、A218P、S219A、C245Y、L246M、A250T、T257C、T275A、T310S、A313I、N315H、K316D、N319D、K320R、A345M、S357T、D358A、Q359E、R361K、K362Q、L365A、E366N (64)

[0087] 51 Y16F、D18A、I21V、Q25E、V26A、M27L、T28R、G32R、I33L、R46A、N52G、E54S、R68K、H73T、I79L、K81L、M83L、A89P、T103E、E106A、D108N、A109E、E111K、Q113E、S116K、H117N、Q124A、H125N、S148A、K152N、G153E、C162V、I168V、H171Y、V186I、L193I、I195V、E202Q、A205L、S206A、V211A、F216L、A218P、S219A、C245Y、L246M、A250T、T257C、T275A、T310S、A313I、N315H、K316D、N319D、K320R、A345M、S357T、D358A、Q359E、R361K、K362Q、L365A、E366N (63) 52 E10D、Y16F、D18A、I21V、Q25E、V26A M27L、T28R、G32R、I33L、R46A、N52G、E54S、R68K、H73T、I79L、K81L、M83L、A89P、T103E、E106A、D108N、A109E、E111K、Q113E、S116K、H117N、Q124A、H125N、K126R、S148A、D151N、K152D、G153E、C162V、I168V、H171Y、V186I、L193I、I195V、E202Q、A205L、S206A、V211A、F216L、A218P、S219A、C245Y、L246M、A250T、T257C、T275A、T310S、A313I、N315H、K316D、N319D、K320R、A345M、S357V、D358A、Q359A、R361K、K362Q、L365E、E366N (66) 53 Y16F、D18A、I21V、Q25E、V26A、M27L、T28R、G32R、I33L、R46A、N52G、E54S、R68K、H73T、I79L、K81L、M83L、A89P、T103E、E106A、D108N、A109E、E111K、Q113E、S116K、H117N、Q124A、H125N、K126R、S148A、D151N、K152N、G153E、C162V、I168V、H171Y、V186I、L193I、I195V、E202Q、A205L、S206A、V211A、F216L、A218P、S219A、C245Y、L246M、A250T、T257C、T275A、T310S、A313I、N315H、K316D、N319D、K320R、A345M、S357V、D358A、Q359A、R361K、K362Q、L365E、E366N (65) 54 E10D、D18A、I21V、E24D、Q25E、V26A、M27L、T28R、G32R、I33L、R46A、N52G、E54S、R68K、H73T、I79L、K81L、M83L、A89P、E106A、D108N、A109E、E111K、Q113A、S116K、H117N、E120A、Q124A、H125N、K126Q、S148A、D151N、K152D、G153D、C162V、I168V、H171Y、V186I、L193I、I195V、E202Q、A205L、S206A、V211A、F216L、D217N、A218P、S219A、C245Y、L246M、A250T、T257C、T275A、T310S、A313I、N315H、K316D、N319D、K320R、A345M、S357T、D358S、Q359A、R361K、K362Q、L365E、E366N、A374V、S381T (69) 55 K3T、L4Q、I5L、Y9W、L11I、D18A、V19I、I21V、V23A、Q25E、V26A、M27L、T28R、G32R、I33L、R46A、N52E、E54S、R68K、H73Q、I79L、K81Q、M83L、A89P、D102T、E106A、D108N、A109E、E111K、Q113E、S116K、Q124R、K126Q、D151N、K152P、G153E、H171Y、L193I、I195V、L198F、E202Q、A205L、S206N、F216L、A218P、S219A、C245Y、L246M、T257C、T275A、V280I、T310S、A313I、N315H、K316N、N319D、K320R、A345M、S357T、D358A、Q359L、K362Q、L365N、E366D、V387I、A390M、W391Y、L393Q、V397T、D400G、A401S、S402Q、S405E、E406N、H409T、Q412K、Q413D、T414V、R416S、L417Q、419G (81) 56 K3T、L4Q、I5L、Y9W、D18A、V19I、I21V、Q25E、V26A、M27L、T28R、G32R、I33L、R46A、N52D、E54S、R68K、I79L、K81Q、M83L、A89P、E106A、D108N、A109E、E111K、Q113E、S116K、H117N、Q124P、K126Q、D151N、K152E、G153E、H171Y、L193I、I195V、E202Q、A205L、S206N、F216L、A218P、S219A、C245Y、L246M、A250T、T257C、T275A、T310S、A313I、N315H、K316N、N319D、K320R、A345M、S357T、D358A、K362Q、L365N、E366D、A390M、W391Y、L393Q、V397T、D400G、A401S、S402Q、S405D、E406N、H409T、Q412K、Q413D、T414V、R416S、L417Q、419G (75) 57 K3T、L4Q、I5L、Y9W、D18A、V19I、I21V、Q25E、V26A、M27L、T28R、G32R、I33L、R46A、N52E、E54T、R68K、H73N、I79L、K81Q、M83L、A89P、E106A、D108N、A109E、E111K、Q113E、S116K、H117N、Q124G、K126Q、D151N、K152E、G153E、H171Y、L193I、I195V、E202Q、A205L、S206N、F216L、A218P、S219A、C245Y、L246M、T257C、T275A、T310S、A313I、N315H、K316N、N319D、K320R、A345M、S357T、D358A、Q359L、K362Q、L365N、E366D、A390M、W391Y、L393Q、V397T、D400G、A401S、S402Q、S405D、E406N、H409T、Q412K (71) 58 Y9W, D18A, V19I, I21V, Q25E, V26A, M27L, T28R, G32R, I33L, R46A, N52E, E54T, R68K, H73N, I79L, K81Q, M83L, A89P, E1 06A、D108N、A109E、E111K、Q113E、S116K、H117N、Q124G、K1 26Q、D151N、K152E、G153E、H171Y、L193I、I195V、E202Q、A20 5L, S206N, F216L, A218P, S219A, C245Y, L246M, T257C, T275A, T310S, A313I, N315H, K316N, N319D, K320R, A345M, S357 T、D358A、Q359L、K362Q、L365N、E366D、A390M、W391Y、L393 Q、V397T、D400G、A401S、S402Q、S405D、E406N、H409T、Q412K (68) 59 Y9W, D18A, V19I, I21V, Q25E, V26A, M27L, T28R, G32R, I33L, R46A, N52E, E54T, R68K, H73N, I79L, K81Q, M83L, A89P, E 106A、D108N、A109E、E111K、Q113E、S116K、H117N、Q124G、 K126Q、D151N、K152E、G153E、H171Y、L193I、I195V、E202Q、 A205L, S206N, F216L, A218P, S219A, C245Y, L246M, T257C, T275A, T310S, A313I, N315H, K316N, N319D, K320R, A345M 、S357T、D358A、Q359L、K362Q、L365N、E366D、A390M、W391Y、L393Q、V397T、D400G、A401S、S402Q、S405D、E406N、H409T (67) 60 K3T, L4Q, I5L, Y9W, Y16F, D18A, Q25E, V26A, M27L, T28R, G32R, I33L, R46S, N52E, E54S, R68K, H73S, I79L, K81Q, S84R, A89P, E106S, A10 9Q, E111K, Q113E, S116K, H117N, Q124A, H125N, K126Q, K152D, G153S, I168V, H171Y, V186I, L193I, I195V, E202Q, A205L, S206A, F216L , D217N, A218P, S219A, C245Y, L246M, A250T, T257C, T275A, R282Q, T310S, A313V, N315H, K316D, N319D, K320R, A345M, S357T, D358A, Q 359E, L365A, E366P, A390M, W391Y, L393Q, V397T, D400G, A401S, S402E, S405E, E406S, H409A, Q412K, Q413E, T414I, R416S, L417R, 419G (78)

[0088] In summary, the amino acid substitution mutants of sequences 3 to 60 have specific amino acid substitutions at the following 122 sites: K3T, L4Q, I5L, Y9F / W, E10D, L11I, Y16F, D18A / E, V19I / N, I21V, V23 A, E24D, Q25K / L / E, V26A, M27L / I, T28A / G / R / P, G32E / R, I33L, R46A / S、N52D / E / G / Q、E54A / Q / S / T、R68K、H73D / G / N / S / T / Q、A77S / T、I79L / V、E80D、K81Q / L、A82T、M83I / L、S84R、A89P、K90M、I97L、D102S / T、T 103E / K / Q、E106A / D / S、D108N、A109E / Q、E111K、Q113A / E / K / G、S116 A / K / T、H117N、E120A、Q124A / E / G / K / N / R / P / S / T、H125N、K126R / Q、S1 34T, P139A, L140K / M, S148A, A150S, D151N, K152D / E / N / P / T, G153D / E / K / S / T、I154V、C162V、R167H、I168V、H171Y、P179A、V186I、L193I I195V, L198F, A199G, E202Q, A205L / M / V, S206A / D / N, E210A, V2 A / I, K215Q, F216L, D217N, A218P, S219A / Q, C245Y, L246M, A250S / T. T254I、T257C、T275A、V280I、R282Q、L300I、T310N / H / S、A313I / V、N 315H / Q、K316D / N / Q、N319D、K320R、A345M / T、S357T / V、D358A / E / SQ 359A / E / L / R、R361K、K362Q / R、L363A、L365A / E / N / S / Q、E366A / D / N / P / S Y369F A371G A374T / V L375M S381C / T V387I A390M W391Y L393Q, H395N, V397A / T, D400G, A401S, S402Q / E, S405D / E / G, E406D / N / S、H409A / T / M、Q412E / K、Q413D / E、T414V / I、R416S、L417Q / R、419G

[0089] It is a range of 3~60 ranges of 122 Check out the range of 1000 sq. m. K3, L4, I5, Y9, E10, L11, Y16, D18, V19, I21, V23, E24, Q25, V26, M27, T28, G32, I33, R46 , N52, E54, R68, H73, A77, I79, E80, K81, A82, M83, S84, A89, K90, I97, D102, T103, E106 , D108, A109, E111, Q113, S116, H117, E120, Q124, H125, K126, S134, P139, L140, S148, A150, D151, K152, G153, I154, C162, R167, I168, H171, P179, V186, L193, I195, L198, A1 99, E202, A205, S206, E210, V211, K215, F216, D217, A218, S219, C245, L246, A250, T25 4, T257, T275, V280, R282, L300, T310, A313, N315, K316, N319, K320, A345, S357, D358, Q359, R361, K362, L363, L365, E366, Y369, A371, A374, L375, S381, V387, A390, W391, L 393, H395, V397, D400, A401, S402, S405, E406, H409, Q412, Q413, T414, R416, L417, 419

[0090] Among sequences 3 to 60, sequence 33 has the most amino acid substitutions, at 90, and the amino acid sequence identity with the wild-type enzyme of sequence 1 is 78%. Furthermore, among the 13 sequences shown in Table 10 below that have higher D-allulose isomerase activity at the optimum temperature than the wild-type enzyme of sequence 1, sequence 32 has the highest number of amino acid substitutions, at 84, and has 80% identity with the amino acid sequence of sequence 1. [Table 10]

[0091] Table 11 shows the numbers of 52 sequences that have a higher D-allulose isomerase activity ratio (T70 / T50) at reaction temperatures of 70°C and 50°C than the wild-type enzyme of Sequence 1. [Table 11]

[0092] Table 12 shows the numbers of 17 sequences that have higher residual activity than the wild-type enzyme of Sequence 1 after incubation at 60°C for 1 hour. [Table 12]

[0093] Table 13 shows the numbers of 56 sequences that have a higher ratio of D-allulose isomerase activity at a reaction temperature of 80°C to the optimum temperature (T80 / optimum temperature) than the wild-type enzyme of Sequence 1. [Table 13] [Industrial Applicability]

[0094] The L-rhamnose isomerase of the present invention is characterized by its particularly high thermostability and high activity compared to conventional microbial L-rhamnose isomerases. For example, while the optimum temperature of conventional L-rhamnose isomerase derived from Pseudomonas stutzeri is 60°C, the enzyme of the present invention has a high optimum temperature of 70°C, and furthermore, retains 80% or more of its activity after heat treatment at 60°C for 10 minutes, making it suitable for use in industrial production. Furthermore, when the substrate is D-allulose, it has a high D-allose conversion activity of 2.26 U per mg protein at 60°C, paving the way for the mass production of D-allose according to the present invention. Furthermore, the L-rhamnose isomerase of the present invention can be immobilized using various immobilization methods to obtain a highly active immobilized enzyme, which enables continuous large-scale isomerization reactions. Industrial immobilization allows for mass production of the target aldose. Furthermore, the amino acid substitution mutants of the L-rhamnose isomerase of the present invention include many that have higher enzymatic activity or higher thermostability than the enzyme before mutation. Therefore, the establishment of the L-rhamnose isomerase and its variants, as well as the method for producing them, of the present invention is of great industrial significance not only to the sugar industry, but also to the related food, cosmetics, pharmaceutical and agricultural chemical industries.

Claims

1. L-rhamnose isomerase derived from Erwinia billingiae GuaL218-3 strain, which has been internationally deposited with the International Patent Microorganisms Depositary Center under accession number NITE BP-03142, and which has a subunit molecular mass of 48 kDa as measured by SDS-PAGE, and has the substrate specificities (A) and (B) below and the physicochemical properties (C) and (D) below. (A) It has an isomerase activity of recognizing and reacting with the CHO group at C1 and the OH group at C2 of an aldose, converting the CHO group at C1 to an OH group and the OH group at C2 to a CO group, or recognizing and reacting with the OH group at C1 and the CO group at C2 of a ketose, converting the OH group at C1 to a CHO group and the CO group at C2 to an OH group. (B) It has the activity of catalyzing the isomerization reactions between L-rhamnose and L-rhamnurose, between L-lyxose and L-xylulose, between L-mannose and L-fructose, between D-ribose and D-ribulose, between L-talose and L-tagatose, and between D-allose and D-allulose. (C) The optimum pH for the reaction is 9. (D) The optimum reaction temperature is 70°C.

2. A protein comprising the amino acid sequence represented by SEQ ID NO:

1.

3. The protein is an amino acid substitution mutant of a protein consisting of the amino acid sequence represented by SEQ ID NO: 1, and has the amino acid sequence represented by SEQ ID NO: 6, 13, 15, 17, 20-22, 26-28, 30, 32, 34, 36, 37, 40-42, 46, 47, 49, 51, 55, or 56, and has the L-rhamnose isomerase activity of (A) and (B) below, and has higher L-rhamnose isomerase activity at an optimal temperature or higher residual activity after incubation at 60°C for 1 hour than a protein consisting of the amino acid sequence represented by SEQ ID NO: 1: (A) It has an isomerase activity of recognizing and reacting with the CHO group at C1 and the OH group at C2 of an aldose, converting the CHO group at C1 to an OH group and the OH group at C2 to a CO group, or recognizing and reacting with the OH group at C1 and the CO group at C2 of a ketose, converting the OH group at C1 to a CHO group and the CO group at C2 to an OH group. (B) It has the activity of catalyzing the isomerization reactions between L-rhamnose and L-rhamnurose, between L-lyxose and L-xylulose, between L-mannose and L-fructose, between D-ribose and D-ribulose, between L-talose and L-tagatose, and between D-allose and D-allulose.

4. The protein is an amino acid substitution mutant of a protein consisting of the amino acid sequence represented by SEQ ID NO: 1, and has the amino acid sequence represented by SEQ ID NO: 8, 10, 13, 16, 17, 19, 20-23, 26-28, 30-33, 36-53, 55, 56, 58, or 59, and has the L-rhamnose isomerase activity of the following (A) and (B), and has a higher ratio of L-rhamnose isomerase activity at reaction temperatures of 70°C and 50°C (T70 / T50) or a higher ratio of L-rhamnose isomerase activity at a reaction temperature of 80°C and the optimum temperature (T80 / optimum temperature) than the protein consisting of the amino acid sequence represented by SEQ ID NO: 1: (A) It has an isomerase activity of recognizing and reacting with the CHO group at C1 and the OH group at C2 of an aldose, converting the CHO group at C1 to an OH group and the OH group at C2 to a CO group, or recognizing and reacting with the OH group at C1 and the CO group at C2 of a ketose, converting the OH group at C1 to a CHO group and the CO group at C2 to an OH group. (B) It has the activity of catalyzing the isomerization reactions between L-rhamnose and L-rhamnurose, between L-lyxose and L-xylulose, between L-mannose and L-fructose, between D-ribose and D-ribulose, between L-talose and L-tagatose, and between D-allose and D-allulose.

5. A DNA encoding the protein according to any one of claims 2 to 4.

6. A DNA containing the base sequence represented by SEQ ID NO: 2 or its complementary sequence.

7. A recombinant vector containing the DNA of claim 5 or 6.

8. A transformed host cell transformed with the recombinant vector according to claim 7.

9. 2. Erwinia billingiae GuaL218-3, which produces the L-rhamnose isomerase of claim 1 and is internationally deposited with the Patent Microorganisms Depositary Center under accession number NITE BP-03142.

10. An immobilized protein, in which the L-rhamnose isomerase according to claim 1 or the protein according to any one of claims 2 to 4 is immobilized on a carrier.

11. An immobilized protein, wherein the L-rhamnose isomerase according to claim 1 is immobilized on a carrier in the form of a crude enzyme present in a bacterial cell lysate, or the protein according to any one of claims 2 to 4 is immobilized on a carrier in the form of a crude protein present in a bacterial cell lysate of a transformed host cell.

12. 12. The immobilized protein according to claim 10 or 11, wherein the carrier is an ion exchange resin or a synthetic adsorbent.

13. The immobilized protein of claim 12, wherein the carrier is DAIAION® WA30, AMBERLITE® FPA54, or AMBERLITE® FPA95.

14. A method for producing L-rhamnose isomerase, comprising culturing an Erwinia microorganism that produces the L-rhamnose isomerase according to claim 1 or a transformed host cell according to claim 8 in a medium, allowing the L-rhamnose isomerase to accumulate in the microbial cells, and then collecting the accumulated L-rhamnose isomerase.

15. The method for producing L-rhamnose isomerase according to claim 14, wherein the medium is an inorganic salt medium supplemented with L-rhamnose.

16. A method for producing a ketose or aldose, comprising: allowing the L-rhamnose isomerase of claim 1, the protein of any one of claims 2 to 4, or the immobilized protein of any one of claims 10 to 13 to act on a solution containing one or more aldoses or ketoses to produce the corresponding ketose or aldose, and collecting the corresponding ketose or aldose.

Images