KR101740098B1 - Biofuel cell comprising aldose sugar dehydrogenase - Google Patents

Abstract

The present invention relates to the use of Thermus thermophilus HJ6 thermophilus HJ6), and a method for producing electricity using the biofuel cell. BACKGROUND OF THE INVENTION 1. Field of the Invention [0002] The present invention relates to a biofuel cell. The biofuel cell comprising the anode electrode fixed with aldose dehydrogenase provided by the present invention can use an alcohol such as methanol or ethanol as a substrate as compared with the conventional biofuel cell and exhibits improved heat resistance In the environment where the temperature is increased, the electric productivity is increased, so that it can be widely used in the development of various biofuel cells.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention [0001] The present invention relates to a biofuel cell comprising an aldehyde dehydrogenase enzyme (biofuel cell comprising aldose sugar dehydrogenase)

The present invention relates to a biofuel cell including a sugar aldose dehydrogenase, more specifically, the present invention provides a brush Summers standing Russ HJ6 (Thermus thermophilus HJ6), and a method for producing electricity using the biofuel cell. BACKGROUND OF THE INVENTION 1. Field of the Invention [0002] The present invention relates to a biofuel cell.

Biofuel cell (BFC) refers to a device that converts the chemical energy of a substrate, which is an electron donor, into electrical energy by utilizing the functions of organisms such as enzymes and microorganisms. Specifically, it includes means for converting chemical energy generated in the process of decomposition of a substrate by enzymatic reaction or metabolism of a microorganism into electrical energy, and means for recovering the converted electrical energy. As a result, Means an apparatus that produces electricity from a battery. Typically, the biofuel cell comprises an anode electrode to which an oxidizing enzyme or a microorganism expressing the oxidizing enzyme is immobilized, a reducing enzyme or a cathode electrode to which the microorganism expressing the reducing enzyme is immobilized, And an anode electrode and a cathode electrode. When each of the reaction vessels is filled with a medium containing the respective substrates, the oxidizing enzyme or microorganisms immobilized on the anode electrode are decomposed to form decomposition products The generated electrons are transferred from the anode electrode to the cathode electrode and the reducing enzyme immobilized on the cathode electrode oxidizes the substrate while the dissolved oxygen in the medium is dissolved in the form of water So that the electric current continuously flows through the electric wire. The core of such a biofuel cell is an enzyme or microorganism immobilized on each electrode. As each enzyme or microorganism acts on the substrate and the generation and consumption efficiency of electrons increases, the production amount of electricity produced by the microbial fuel cell increases.

These biofuel cells generally use microorganisms rather than enzymes. Unlike enzymes, in which the substrate degrading activity is maintained for a certain period of time and then the substrate degrading activity is lowered, the microorganism can maintain its substrate decomposition activity through life activities It is because. In recent years, a method has been developed in which a wastewater treatment apparatus capable of continuously maintaining a substrate for a microorganism and the biofuel cell are simultaneously operated to perform wastewater treatment and electricity production. For example, Korean Patent Laid-Open Publication No. 2013-0029530 discloses an energy-standing type advanced wastewater treatment apparatus in which a microbial fuel cell and a microbial electrolysis cell are fused and a wastewater treatment method through the apparatus. However, even if the biofuel cell using such a microorganism is maintained at a constant level, the resistance value is increased to increase the temperature of the biofuel cell itself, and the increase in temperature decreases the activity of the microorganism. As a result, Biofuel cells using microorganisms also suffered from a short life span. In order to solve such a problem, it is necessary to use a microorganism having heat resistance. Since such a heat-resistant microorganism is not easily supplied with a substrate and culture conditions are limited, development of a biofuel cell using a heat- It is true. Therefore, researches for solving these problems have been actively carried out, but there are no results yet.

Under these circumstances, the present inventors have made extensive efforts to develop a biofuel cell that can be driven even under high temperature conditions, and as a result, it has been found that a biofuel cell comprising an aldehyde dehydrogenase, which is a heat-resistant enzyme as an oxidizing enzyme immobilized on an anode electrode, It has been confirmed that not only can it be driven in a wide temperature range but also the electric productivity is further improved under high temperature conditions, and the present invention has been completed.

It is an object of the present invention to provide a biofuel cell comprising an aldose saccharide dehydrogenase.

Another object of the present invention is to provide a method for producing electricity using the biofuel cell.

The inventors of the present invention have focused on a biofuel cell using an enzyme that is not a microorganism while carrying out various studies to develop a biofuel cell that can be driven even under high temperature conditions. Generally, a biofuel cell using an enzyme has a disadvantage that its driving period is shorter than that of a biofuel cell using microorganisms due to degradation of enzymes. On the other hand, compared to a biofuel cell using microorganisms, the substrate is simple, It is advantageous in that the biofuel cell can be simplified since components such as various carbon sources and nitrogen sources necessary for culturing are not required. In addition, when an enzyme is immobilized on an anode electrode or a cathode electrode of a biofuel cell using a fixed medium such as agarose, the fixed medium can maintain the enzyme reaction environment and maintain the activity of the enzyme for a long time, In the case of microorganisms, essential components necessary for the survival of microorganisms are required, but in the case of heat-resistant enzymes, such essential components are not necessary.

Thus, the present inventors searched for a heat-resistant enzyme suitable for fixing to an anode electrode of a biofuel cell using an enzyme, and as a result, selected the aldose dehydrogenase. In particular, the Thermus Thermophilus HJ6 Thermophilus HJ6 strain exhibits an enzyme activity in a relatively wide range of pH 5.0 to 9.5, exhibits enzyme activity in a wide temperature range of 30 to 90 ° C, and is most suitable for pH 7.0 to 7.5 and 70 ° C In addition, pyrroloquinoline quinone (PQQ), which is a cofactor, was found not to decrease enzyme activity even after 100 minutes of exposure at 85 ° C. As a result of fixing the aldehyde dehydrogenase to the anode electrode and testing the current production by the oxidation - reduction reaction, it was confirmed that the current productivity increased as the temperature condition increased.

Therefore, it has been found that the use of a biofuel cell containing aldose dehydrogenase can solve the problem due to the temperature increasing in proportion to the period of use of the biofuel cell.

As one embodiment for achieving the above-mentioned object, the present invention provides a biofuel cell comprising an aldehyde dehydrogenase.

Specifically, the biofuel cell comprising the aldose saccharide dehydrogenase of the present invention comprises (a) an anode electrode to which an aldose saccharide dehydrogenase derived from a strain of Thermus thermophilus HJ6 is immobilized; (b) a first medium containing a substrate of the dehydrogenase enzyme of aldose and providing an enzyme reaction environment of aldose sugar dehydrogenase; (c) an anode reaction vessel containing the anode electrode and the first medium; (d) a cathode electrode to which a reducing enzyme is immobilized; (e) a second medium containing a substrate of the reductase and providing an enzyme reaction environment of the reductase; (f) a cathode reaction tank containing the cathode and the second medium; And (g) a wire connecting the anode electrode and the cathode electrode.

The term "biofuel cell " of the present invention refers to a cell that causes an oxidation-reduction reaction by utilizing the functions of an organism, such as an enzyme and a microorganism, and specifically includes an enzyme or microorganism A means for converting the chemical energy generated in the process of conducting the catalytic reaction of the substrate by the metabolism into electrical energy and a means for recovering the converted electrical energy and consequently an enzyme or a microorganism is used to conduct electricity from the substrate Means a device that produces. Generally, a biofuel cell includes an anode electrode to which an oxidizing enzyme or a producing microorganism thereof is fixed, a cathode electrode to which a reducing enzyme or a producing microorganism thereof is fixed, a substrate capable of reacting with the enzyme or a microorganism, And a reaction chamber in which the respective electrodes and the culture medium are contained. When each of the reaction vessels is filled with a medium containing the substrate of each enzyme, the substrate is reacted with an oxidizing enzyme or a microorganism immobilized on the anode electrode to generate hydrogen ions and electrons, And the reducing enzyme immobilized on the cathode electrode receiving the electrons converts the dissolved oxygen into water by using electrons as a side reaction while oxidizing the substrate to consume the transferred electrons, The current flows to the anode electrode.

Since the biofuel cell can produce a current when hydrogen ions and electrons are generated in the substrate by an enzyme or a microorganism, the current can be continuously generated when the substrate is continuously supplied to the reaction tank. The biofuel cell includes an enzyme fuel cell that uses an electrode reaction of an organic compound using an enzyme as a catalyst, a microbial fuel cell that converts a fuel into a compound that is likely to cause an electrode reaction by a microbial reaction, Solar cells, and the like.

In the present invention, the biofuel cell includes an anode electrode to which an aldose sugar dehydrogenase is immobilized, and thus can be interpreted as an enzyme cell containing an aldehyde dehydrogenase.

The term "aldose dehydrogenase" of the present invention means a kind of quinoprotein glucose dehydrogenase (GDH: EC 1.1.5.2) dependent on pyrroloquinoline quinone (PQQ). The aldose sugar dehydrogenase is known to exhibit an activity of oxidizing glucose and other aldoses while reducing PQQ to produce aldino-delta-lactones corresponding to them. The enzyme has a high turnover number , And the electrochemical regeneration of the PQQ cofactor is easily achieved without the negative effects of oxygen partial pressure and product inhibition. The specific amino acid sequence of the dehydrogenase of aldose or the nucleotide sequence information of the gene encoding the same can be obtained from a known database such as GenBank of NCBI (GenBank Accession No. AFN86158 etc.). For example, the aldose dehydrogenase A protein expressed from the amino acid sequence of SEQ ID NO: 1 or the DNA sequence of SEQ ID NO: 2.

In the present invention, the aldose dehydrogenase can be an aldose saccharide dehydrogenase derived from a Somers' thermophilus HJ6 strain, as long as it can be used in a biofuel cell. Preferably at a pH of 5.0 to 9.5 and at a temperature of 30 to 90 DEG C and exhibiting activity exhibiting heat resistance that does not decrease enzyme activity even when exposed to 85 DEG C for 100 minutes when PQQ is present, It may be an aldose dehydrogenase derived from S. thermophilus HJ6 strain, and most preferably an aldose dehydrogenase comprising the amino acid sequence of SEQ ID NO: 1.

In addition, as long as heat resistance and current productivity can be improved as compared with the aldose sugar dehydrogenase, a mutant protein in which some amino acids of the amino acid sequence of SEQ ID NO: 1 are mutated by addition, substitution, deletion, And may be included in the category of dehydrogenase enzymes.

In particular, the aldose saccharide dehydrogenase provided in the present invention may include a polypeptide having a sequence that differs from the amino acid sequence of SEQ ID NO: 1 by one or more amino acid residues. Amino acid exchange in proteins and polypeptides that do not globally alter the activity of the molecule is known in the art. The most commonly occurring exchanges involve amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thy / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu and Asp / Gly. In addition, the protein may include a protein having increased structural stability or increased protein activity due to mutation or modification of the amino acid sequence, such as heat, pH and the like.

Finally, the aldose dehydrogenase can be produced by a chemical peptide synthesis method known in the art, or by amplifying the gene encoding the aldose dehydrogenase by PCR (polymerase chain reaction) Followed by expression and cloning into an expression vector.

The anode electrode to which the aldehyde dehydrogenase is immobilized is not particularly limited as long as the aldehyde dehydrogenase can be immobilized and electrons generated from the aldehyde dehydrogenase can be transported by the electric wire. For example, platinum, stainless steel mesh, carbon felt, carbon paper or the like can be used.

The substrate of the aldose sugar dehydrogenase is not particularly limited as long as it can be reduced by aldose sugar dehydrogenase to generate electrons, but it may preferably be various sugar compounds, more preferably monosaccharide, alcohol Glucose, D-galactose, D-fructose, L-arabinose, D-mannose, D-xylose, D-ribose, methanol, ethanol , 2-deoxy-glucose, glucoseamine, glucose 6-phosphate, maltose, a-lactose, D-sucrose, D-cellobiose and maltotriose can be used singly or in combination.

Meanwhile, the first medium may further include PQQ as a cofactor of the dehydrogenase enzyme of aldose. The PQQ not only improves the heat resistance of the aldose saccharide dehydrogenase but also increases the amount of current generated by the enzyme reaction.

Meanwhile, the reducing enzyme immobilized on the cathode electrode constituting the biofuel cell of the present invention is not particularly limited as long as it can consume electrons. Preferably, the reducing enzyme is selected from the group consisting of lacase, bilirubin oxidase (BOD) Various dehydrogenases such as ascorbate oxidase and ceruloplasmine can be used alone or in combination.

The cathode electrode to which the reducing enzyme is immobilized is not particularly limited as long as the reducing enzyme can be immobilized and electrons can be transferred from the electric wire to the reducing enzyme. Preferably, a carbon felt, carbon paper, carbon cloth, Or an air-cathode may be used.

The substrate of the reducing enzyme is not particularly limited as long as the reducing enzyme can consume electrons by the enzyme reaction. For example, when a lactase is used as a reducing enzyme, catechol, 2,6-dimethoxy-phenol, 4-methylcatechol, ABTS (2 , 2'-azino bis (3-ethylbenzthiazoline-6-sulfonate), guaiacol, 4-hydroxyindole and Syringaldazine.

The first medium and the second medium containing the anode and the cathode provide a reaction environment in which the aldose sugar dehydrogenase and the reducing enzyme immobilized on each electrode can exhibit normal enzymatic activity, The buffer solution may preferably be a buffer solution which maintains a pH of from 5.0 to 9.5. For example, a Tris buffer solution of pH 7.5 may be used.

According to another aspect of the present invention, there is provided a method of producing electricity using a biofuel cell including an aldehyde dehydrogenase.

Specifically, a method for producing electricity using a biofuel cell including aldose dehydrogenase of the present invention comprises the steps of: (a) supplying a first medium to an anode reaction tank of the biofuel cell; Supplying a medium; And (b) an aldose dehydrogenase enzyme immobilized on the anode electrode generates electrons using the substrate contained in each of the supplied media, and the produced electrons move to the cathode electrode through the electric wire, And an electron is consumed by the enzyme to generate an electric current. At this time, the conditions of the used substrate, medium and the like are the same as those described above.

The biofuel cell of the present invention is capable of producing a current in a wide temperature range of 25 to 90 ° C due to the characteristics of the aldehyde dehydrogenase contained therein, As the temperature increases, the amount of generation of the current increases further.

According to an embodiment of the present invention, a glassy carbon electrode to which the dehydrogenase is immobilized is placed in an enzyme reaction solution (50 mM Tris-HCl buffer (pH 7.5)), and 100 mM D-glucose And 1 μM PQQ, the current generation level was increased when glucose was added, and the current was increased to the highest level when glucose and a subsidiary PQQ were added (Fig. 3). Further, as a result of performing the same current potential curve analysis at 25 and 75 ° C, it was confirmed that as the temperature increases, a high reduction peak is generated and the current generation amount is increased (FIG. 4).

The biofuel cell comprising the anode electrode fixed with aldose dehydrogenase provided by the present invention can use an alcohol such as methanol or ethanol as a substrate as compared with the conventional biofuel cell and exhibits improved heat resistance In the environment where the temperature is increased, the electric productivity is increased, so that it can be widely used in the development of various biofuel cells.

Fig. 1 is an electrophoresis image showing the result of SDS-PAGE analysis of the expressed and purified aldose sugar dehydrogenase. In Fig. 1, lane 1 represents a marker, lane 2 represents a supernatant obtained after heat treatment, lane 3 represents HisTrap HP column. < / RTI >
FIG. 2A is a graph showing changes in the activity of dehydrogenase per aldose per pH change. FIG.
FIG. 2B is a graph showing changes in the activity of dehydrogenase on aldolase according to the reaction temperature. FIG.
FIG. 2C is a graph showing changes in enzyme activity of aldose sugar dehydrogenase with heat treatment time, in which (●) represents aldose sugar dehydrogenase without treatment with PQQ, and (▲) represents aldehyde sugar dehydrogenase Lt; / RTI >
FIG. 3 is a graph showing a result of analyzing the effect of glucose, a substrate, and PQQ, which is a substrate, on current production using a cyclic voltamogram using a glycocalone electrode to which an aldose dehydrogenase is immobilized, (-) represents the case where the electrode is immersed in a buffer solution containing glucose, (-) represents the case where the electrode coated with aldose dehydrogenase is immersed in a buffer solution, And the electrode is immersed in a buffer solution containing glucose and PQQ.
FIG. 4 is a graph showing a result of current curve analysis using a glacier carbon electrode to which an aldehyde dehydrogenase is immobilized according to a change in temperature, and (...) represents a case where the temperature of the enzyme reaction liquid is 25 ° C. ━) indicates the case where the temperature of the enzyme reaction solution is 75 캜.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example  1: Recombination Aldose  Production of sugar dehydrogenase

Example  1-1: Aldose  Production of a sugar dehydrogenase gene and production of the recombinant vector for gene expression

Summers Thermophilus HJ6 ( Thermus thermophilus HJ6) was applied to a GeneAll GENEx genomic kit (GeneAll Biotechnology, Seoul, Korea) to extract genomic DNA.

A primer capable of amplifying GDH (glucose dehydrogenase) of another Somus thermophilus strain was synthesized and PCR was performed using the primer synthesized with the extracted genomic DNA to obtain an amplified DNA fragment. At this time, the PCR was carried out 30 times at 95 ° C for 30 seconds, 55 ° C for 60 seconds and 72 ° C for 60 seconds.

Primer-1: 5'-GTCGGGGAAGGGGGTGGCTG-3 '(SEQ ID NO: 3)

Primer-2: 5'-AGGTCCGCTTCCGCAAGAGG-3 '(SEQ ID NO: 4)

The DNA fragment thus obtained was cloned into a pGEM-T vector to prepare an aldolase dehydrogenase gene introduction vector pGEM-asd. The nucleotide sequence of the aldose saccharide dehydrogenase gene contained therein was analyzed with an ABI Prism 3700 genetic analyzer (Perkin-Elmer Applied Biosystems, USA). As a result, the aldolase dehydrogenase gene (SEQ ID NO: 2) was expected to encode a protein consisting of 352 amino acids (SEQ ID NO: 1) composed of an ORF (open reading frame) of 1059 bp, The expected molecular weight of the osteoside dehydrogenase protein was about 38.6 kDa and the isoelectric point was estimated to be about 10.17.

Using the above-described pGEM-asd as a template, PCR was performed using the following primers to amplify a DNA fragment of 1.07 kb.

Primer-3: 5'-GGGGTGGCATATGGACCGGAGGCGCTTTCT-3 '(SEQ ID NO: 5)

Primer-4: 5'-CCGAAGCTTAAGGAGGCGTAGCACCCGG-3 '(SEQ ID NO: 6)

The amplified DNA fragment of 1.07 kb was digested with NdeI / HindIII restriction enzyme and introduced into a pET-21a vector (Novagen) treated with the same restriction enzymes to prepare an aldose dehydrogenase expression vector pET-asd Respectively. The expression vector was designed to produce a fusion protein in which the His-tag is attached to the C-terminus of the dehydrogenase enzyme of aldose.

Example  1-2: Aldose  Production of sugar dehydrogenase

A transformant was prepared by introducing the aldose sugar dehydrogenase expression vector pET-asd prepared in Example 1-1 into E. coli BL21 Codon-Plus (DE3), and the transformant was inoculated into OD 600, and 0.5 mM IPTG (isopropyl- beta -D-thiogalactopyranoside) was added thereto, followed by further incubation at 25 ° C for 16 hours. After completion of the culturing, the culture was centrifuged to obtain cells, which were suspended in buffer A (50 mM Tris-HCl pH 7.5, 500 mM NaCl, 20 mM imidazole) and disrupted to obtain a cell lysate. The obtained cell lysate was centrifuged (27,000 × g, 30 minutes) to obtain a supernatant. The supernatant thus obtained was heat-treated at 75 ° C. for 20 minutes, and the heat-treated supernatant was centrifuged again to obtain a supernatant. The resulting supernatant was applied to a HisTrap HP column (GE Healthcare), washed with buffer A, and eluted with a gradient of 0-500 mM imidazole concentration gradient to elute the fusion protein at a rate of 1 ml / min to obtain an active fraction Respectively. The obtained active fractions were concentrated and dialyzed with 20 mM phosphate buffer to remove impurities, followed by SDS-PAGE analysis (FIG. 1).

Fig. 1 is an electrophoresis image showing the result of SDS-PAGE analysis of the expressed and purified aldose sugar dehydrogenase. In Fig. 1, lane 1 represents a marker, lane 2 represents a supernatant obtained after heat treatment, lane 3 represents HisTrap HP column. < / RTI > As shown in FIG. 1, it was confirmed that the dehydrogenase of aldose was approximately 37 kDa in size.

Meanwhile, 20 mM Tris-HCl buffer (pH 7.5) containing pyrroloquinoline quinone (PQQ) was added to the produced aldose sugar dehydrogenase (1.3 mg / ml) Min, and the unbound PQQ was removed using a Sephadex G-25 column (PD-10; GE Healthcare).

Example  1-3: Aldose  Enzyme activity analysis of glucose dehydrogenase

The pH effect and the reaction temperature effect on the enzyme activity of the dehydrogenase produced in Example 1-2. Heat resistance and substrate specificity were analyzed.

Example  1-3-1: pH  Effect analysis

The aldose sugar dehydrogenase produced in Example 1-3 was incubated under the conditions of pH 5.0 to 9.5 (50 mM sodium acetate (pH 5.0-6.0), 50 mM sodium phosphate (pH 6.0-7.5), 50 mM Tris-HCl ~ 9.5)), the optimal pH range was analyzed (Figure 2a).

FIG. 2A is a graph showing changes in the activity of dehydrogenase per aldose per pH change. FIG. As shown in FIG. 2A, it was confirmed that the enzyme activity was 50% or more at pH 6.5 to 9.0, and the optimum enzyme activity was shown at pH 7.0 to 7.5.

Example  1-3-2: Analysis of reaction temperature effect

The aldehyde dehydrogenase produced in Example 1-2 was reacted at 30 to 90 ° C and the optimal temperature range was analyzed (Fig. 2b).

FIG. 2B is a graph showing changes in the activity of dehydrogenase on aldolase according to the reaction temperature. FIG. As shown in FIG. 2B, it showed excellent enzyme activity at 60 to 80 ° C and optimum activity at 70 ° C.

Example  1-3-3: Heat resistance analysis

The aldose sugar dehydrogenase produced in Example 1-2 was added to 50 mM Tris-HCl buffer (pH 7.5), treated at 85 ° C for 100 minutes with or without 1 μM PQQ, And its enzyme activity was analyzed (Fig. 2C). At this time, enzyme activity measurement was carried out at 70 ° C and pH 7.5.

FIG. 2C is a graph showing changes in enzyme activity of aldose sugar dehydrogenase with heat treatment time, in which (●) represents aldose sugar dehydrogenase without treatment with PQQ, and (▲) represents aldehyde sugar dehydrogenase Lt; / RTI > As shown in FIG. 2C, when the aldose dehydrogenase without PQQ treatment was heat-treated at 85 ° C for 25 minutes, the enzyme activity was reduced to half. However, the PQQ-treated aldose dehydrogenase was subjected to heat treatment at 85 ° C for 100 minutes It was confirmed that the enzyme activity did not decrease.

Considering that the optimum growth temperature of the Somersemester thermophilus HJ6 strain expressing the aldose sugar dehydrogenase is 80 ° C, the aldose dehydrogenase has a certain influence on the heat resistance characteristics of the Somershermophilus strain HJ6 Respectively.

Example  1-3-4: Substrate specificity analysis

The substrate specificity of the aldose sugar dehydrogenase produced in Example 1-2 was analyzed using various kinds of monosaccharides, alcohols, substituted glucose and polysaccharides as substrates of aldose dehydrogenase (Table 1). At this time, the kinetic value indicating the relationship between the enzyme activity and the substrate concentration was calculated using the Michaelis-Menten equation of the EZ-Fit program.

Substrate specificity of aldose dehydrogenase temperament Reaction rate
(units / mg) For glucose
Relative speed kcat (/ S) Km (M) kcat / Km
(mM / S / M) Monosaccharide
D-glucose
D-galactose
D-fructose
L-arabinose
D-Mannos
D-xylose
D-ribose
340
600
110
760
660
750
200
One
1.8
0.3
2.3
2
2.2
0.6
1054
823
404
1755
1627
1681
-
0.21
0.11
0.34
0.16
0.13
0.14
ND
5021
7488
1190
10972
12518
12011
1059 Alcohol
Methanol
ethanol
51
157
0.2
0.5
76
998
0.15
0.52
506
1919 Substituted glucose
2-deoxy-glucose
Glucosamine
Glucose 6-phosphate
1644
270
143
4.9
0.8
0.4
1141
197
192
0.04
0.02
0.08
29483
12989
2543 saccharose
Maltose
? -lactose
D-cross
D-cellobiose
450
620
55
1200
1.3
1.9
0.2
3.9
1022
1953
65
1042
0.17
0.19
0.09
0.07
6012
10280
730
14893 Polysaccharide
Maltotrios
640
1.9
772
0.13
6160

As shown in Table 1, it was confirmed that the aldehyde dehydrogenase can use various sugars as well as alcohol as a substrate. These results were confirmed to be attributable to the characteristics of only the aldose sugar dehydrogenase provided by the present invention which can not be found in other homologous enzymes. In particular, it was confirmed that D-galactose, L-arabinose, D-mannose and D-xylose showed more excellent substrate specificity than D-glucose and disaccharides and trisaccharides except sucrose exhibited superior substrate specificity than glucose Respectively. From the above results, it was confirmed that the aldehyde dehydrogenase of the present invention exhibited dehydrogenase activity for various sugars including aldehydes.

Example  2: Aldose  Electrochemical activity analysis of glucose dehydrogenase

Example  2-1: Fixed Aldose  Current Potential Curve Using Hydrolytic Dehydrogenase cyclic  voltamogram analysis

To analyze the effect of aldose dehydrogenase on the analysis of cyclic voltamogram using an electrochemical analyzer (WPG100e, WonaTech, Korea).

First, an Ag / AgCl electrode (Bioanalytical Systems, BAS MF-2079) as a reference electrode; Platinum wire (Bioanalytical Systems, BAS MF-1052) as a counter electrode; And a glassy carbon electrode (diameter, 3.0 mm) (CH Instruments, CHI104) were firstly washed with 0.3 mu m alumina slurry and then secondly washed with a 0.05 mu m alumina slurry , Treated with distilled water under ultrasound, and then dried at room temperature.

Single-walled carbon nanotubes (0.7-1.3 nm in diameter, Sigma-Aldrich) were dissolved in DMF (N, N-dimethylformamide) at a concentration of 1 mg / ml and dispersed by ultrasonication. 4 [mu] l of the dispersed single-walled nanotube solution was coated on the glacier carbon electrode and dried at room temperature. Then, 1% BSA (bovine serum albumin) solution and aldose dehydrogenase solution (15 mg / ml) produced in Example 1-2 were mixed at a ratio of 1: 2 to obtain a mixed solution, 6 μl was coated on a single-walled nanotube-coated glacier carbon electrode, and 2 μl of a 40 mM glutaraldehyde solution was added thereto to cross-link the enzyme to single-walled nanotubes to form an electrode coated with aldose dehydrogenase Respectively.

The electrode thus prepared was immersed in 50 mM Tris-HCl buffer (pH 7.5), and 100 mM D-glucose and 1 μM PQQ were added to the buffer under the single buffer condition, 100 mM D-glucose was added to the buffer, The current potential curve (scanning speed: 10 mV / s) was analyzed under the added conditions (Fig. 3).

FIG. 3 is a graph showing a result of analyzing the effect of glucose, a substrate, and PQQ, which is a substrate, on current production using a cyclic voltamogram using a glycocalone electrode to which an aldose dehydrogenase is immobilized, (-) represents the case where the electrode is immersed in a buffer solution containing glucose, (-) represents the case where the electrode coated with aldose dehydrogenase is immersed in a buffer solution, And the electrode is immersed in a buffer solution containing glucose and PQQ. As shown in FIG. 3, when the substrate glucose was added, the current generation level was increased, and when the substrate glucose and the auxiliary agent PQQ were added, it was confirmed that the current was generated to the highest level.

Example  2-2: Current potential curve analysis with temperature change

As described above, since the activity of the dehydrogenase of aldose is greatly changed according to the reaction temperature, the effect of the change of the reaction temperature on the current potential curve analysis using the fixed aldose sugar dehydrogenase was examined.

Specifically, current-potential curve analysis was performed using the same method as in Example 2-1 except that the temperature of the enzyme reaction solution was changed to 25 and 75 ° C (FIG. 4).

FIG. 4 is a graph showing a result of current curve analysis using a glacier carbon electrode to which an aldehyde dehydrogenase is immobilized according to a change in temperature, and (...) represents a case where the temperature of the enzyme reaction liquid is 25 ° C. ━) indicates the case where the temperature of the enzyme reaction solution is 75 캜. As shown in FIG. 4, it was confirmed that the higher the temperature of the enzyme reaction solution, the higher the reduction peak.

Therefore, it was found that the aldehyde dehydrogenase provided by the present invention has an effect of increasing the current generation as the reaction temperature is increased.

<110> Dong-eui University Industry-Academic Cooperation Foundation <120> Biofuel cell comprising aldose sugar dehydrogenase <130> KPA140483-KR <160> 6 <170> Kopatentin 2.0 <210> 1 <211> 352 <212> PRT <213> Artificial Sequence <220> <223> recombinant thermophilic aldose sugar dehydrogenase <400> 1 Met Asp Arg Arg Phe Leu Val Gly Leu Leu Gly Leu Gly Leu Ala   1 5 10 15 Arg Gly Gln Gly Leu Arg Val Glu Glu Val Val Gly Gly Leu Glu Val              20 25 30 Pro Trp Ala Leu Ala Phe Leu Pro Asp Gly Gly Met Leu Ile Ser Glu          35 40 45 Arg Pro Gly Arg Ile Arg Leu Phe Arg Glu Gly Arg Leu Ser Thr Tyr      50 55 60 Ala Glu Leu Pro Val Tyr His Arg Gly Glu Ser Gly Leu Leu Gly Leu  65 70 75 80 Ala Leu His Pro Arg Phe Pro Arg Glu Pro Tyr Val Tyr Ala Tyr Arg                  85 90 95 Thr Val Ala Glu Gly Gly Leu Arg Asn Gln Val Val Arg Leu Arg His             100 105 110 Leu Gly Glu Arg Gly Val Leu Asp Arg Val Val Leu Asp Gly Ile Pro         115 120 125 Ala Arg Pro His Gly Leu His Ser Gly Gly Arg Ile Ala Phe Gly Pro     130 135 140 Asp Gly Met Leu Tyr Val Thr Thr Gly Glu Val Tyr Glu Arg Glu Leu 145 150 155 160 Ala Gln Asp Leu Ala Ser Leu Gly Gly Lys Ile Leu Arg Leu Thr Pro                 165 170 175 Glu Gly Glu Pro Ala Pro Gly Asn Pro Phe Leu Gly Arg Arg Gly Ala             180 185 190 Arg Pro Glu Val Tyr Ser Leu Gly His Arg Asn Pro Gln Gly Leu Ala         195 200 205 Trp His Pro Arg Thr Gly Glu Leu Phe Ser Ser Glu His Gly Pro Ser     210 215 220 Gly Glu Leu Gly Tyr Gly His Asp Glu Val Asn Leu Ile Val Pro Gly 225 230 235 240 Gly Asn Tyr Gly Trp Pro Arg Val Val Gly Arg Gly Asn Asp Pro Arg                 245 250 255 Tyr Arg Asp Pro Leu Tyr Val Trp Pro Gln Gly Phe Pro Pro Gly Asn             260 265 270 Leu Ala Phe Phe Arg Gly Asp Leu Tyr Val Ala Gly Leu Arg Gly Gln         275 280 285 Ala Leu Leu Arg Leu Val Leu Glu Gly Gly Arg Gly Arg Trp Arg Val     290 295 300 Leu Arg Val Glu Thr Ala Leu Ser Gly Phe Gly Arg Leu Arg Glu Val 305 310 315 320 Gln Phe Gly Pro Asp His Ala Leu Tyr Val Thr Thr Ser Asn Arg Asp                 325 330 335 Gly Arg Gly Gln Val Arg Pro Gly Asp Asp Gln Val Leu Arg Leu Leu             340 345 350 <210> 2 <211> 1059 <212> DNA <213> Artificial Sequence <220> <223> recombinant thermophilic aldose sugar dehydrogenase <400> 2 gtggaccgga ggcgctttct cgtggggctt ctcggcctgg gcctcgcccg ggggcagggg 60 ctacgggtgg aggaggtggt ggggggcctc gaggtcccct gggccctggc cttcctgccg 120 gacgggggga tgctcatcgc ggagaggccc gggaggatcc ggctctttag ggagggcagg 180 ctttccacct acgccgagct tcccgtctac caccgggggg agtctgggct tttgggcctc 240 gccctccacc ctaggtttcc cgaggcgccc tacgtctacg cctaccgcac cgtggcggaa 300 gggggcctga ggaaccaggt ggtgcgcctg aggcacctgg gggaaagggg ggtcttggac 360 cgggtggtcc tggacgggat ccccgcccgg ccccacggcc tccactcggg ggggcgcatc 420 gccttcggcc ccgacgggat gctctacgtg accacggggg aggtctacga gcgggagctc 480 gcccaggacc tcgcctcctt gggggggaag atcctccgcc tcaccccgga aggggagccc 540 gccccgggaa accccttcct ggggcgaagg ggggcgaggc ccgaggtgta tagcctgggc 600 caccgcaacc cccagggcct cgcctggcac ccaaagaccg gggagctttt ctccagcgag 660 cacgggccga gcggggagca gggctatggc cacgacgagg tgaacctgat cgtccccggg 720 gggaactacg gctggccgag ggtggtgggg cgggggaacg acccccggta ccgggacccc 780 ctctacttct ggccccaggg cttccccccg gggaacctcg ccttcttccg gggggacctc 840 tacgtggcgg gcctccgggg gcaggccctc ttgaggctcg tcctggaggg ggagaggggc 900 cgctggcggg ttttgcgggt ggagaccgcc ctttccggct tcggccgcct tagggaggtg 960 caggtgggcc ccgatggggc cctttacgtc accacctcca accgggacgg gaggggccag 1020 gtgcgccccg gggacgaccg ggtgctacgc ctcctttag 1059 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 gtcggggaag ggggtggctg 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 aggtccgctt ccgcaagagg 20 <210> 5 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 ggggtggcat atggaccgga ggcgctttct 30 <210> 6 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 ccgaagctta aggaggcgta gcacccgg 28

Claims (12)

(a) an anode electrode fixed with an aldose sugar dehydrogenase derived from a strain of Thermus thermophilus HJ6;
(b) a first medium containing a substrate of the dehydrogenase enzyme of aldose and providing an enzyme reaction environment of aldose sugar dehydrogenase;
(c) an anode reaction vessel containing the anode electrode and the first medium;
(d) a cathode electrode to which a reducing enzyme is immobilized;
(e) a second medium containing a substrate of the reductase and providing an enzyme reaction environment of the reductase;
(f) a cathode reaction tank containing the cathode and the second medium; And
(g) a wire connecting the anode electrode and the cathode electrode.
The method according to claim 1,
The aldose dehydrogenase exhibited activity under pH conditions of pH 5.0 to 9.5 and temperature conditions of 30 to 90 ° C. When exposed to 85 ° C. for 100 minutes with pyrroloquinoline quinone (PQQ) And exhibits heat resistance such that the enzyme activity is not reduced.
The method according to claim 1,
Wherein the aldose saccharide dehydrogenase comprises the amino acid sequence of SEQ ID NO: &lt; RTI ID = 0.0 &gt; 1. &lt; / RTI &gt;
The method according to claim 1,
Wherein the anode electrode is made of a material selected from the group consisting of platinum, stainless steel mesh, carbon felt, carbon paper, and combinations thereof.
The method according to claim 1,
The substrate of the aldose dehydrogenase is selected from the group consisting of D-glucose, D-galactose, D-fructose, L-arabinose, D-mannose, D-xylose, D-ribose, 2- Wherein the saccharide is a saccharide selected from the group consisting of glucose 6-phosphate, maltose,? -Lactose, D-sucrose, D-cellobiose, maltotriose and combinations thereof.
The method according to claim 1,
Wherein the substrate of the aldose sugar dehydrogenase is methanol, ethanol or a combination thereof.
The method according to claim 1,
Wherein the reducing enzyme is a lacase, a bilirubin oxidase (BOD), an ascorbate oxidase, ceruloplasmine, or a combination thereof.
The method according to claim 1,
Wherein the cathode electrode is made of a material selected from the group consisting of carbon felt, carbon paper, carbon cloth, and combinations thereof, or air-cathode.
The method according to claim 1,
Wherein each of said media is a buffer maintaining a pH of 5.0 to 9.5.
The method according to claim 1,
Wherein the first medium further comprises pyrroloquinoline quinone (PQQ) as a cofactor.
The method according to claim 1,
Wherein the biofuel cell operates at 25 to &lt; RTI ID = 0.0 &gt; 90 C. &lt; / RTI &gt;
(a) supplying a first medium to an anode reaction tank of the biofuel cell according to any one of claims 1 to 11, and supplying a second medium to the cathode reaction tank; And
(b) an aldehyde dehydrogenase fixed on the anode electrode produces electrons using the substrate contained in each of the supplied media, and the produced electrons move to the cathode electrode through the electric wire, and the reducing enzyme To produce an electric current. &Lt; Desc / Clms Page number 24 &gt;

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