Journal of Bioscience and Bioengineering VOL. xx No. xx, 1e6, 2015 www.elsevier.com/locate/jbiosc
Novel glucose dehydrogenase from Mucor prainii: Purification, characterization, molecular cloning and gene expression in Aspergillus sojae Ryoko Satake,* Atsushi Ichiyanagi, Keiichi Ichikawa, Kozo Hirokawa, Yasuko Araki, Taro Yoshimura, and Keiko Gomi Research and Development Division, Kikkoman Corporation, 399 Noda, Chiba 278-0037, Japan Received 19 December 2014; accepted 16 March 2015 Available online xxx
Glucose dehydrogenase (GDH) is of interest for its potential applications in the field of glucose sensors. To improve the performance of glucose sensors, GDH is required to have strict substrate specificity. A novel flavin adenine dinucleotide (FAD)-dependent GDH was isolated from Mucor prainii NISL0103 and its enzymatic properties were characterized. This FAD-dependent GDH (MpGDH) exhibited high specificity toward glucose. High specificity for glucose was also observed even in the presence of saccharides such as maltose, galactose and xylose. The molecular masses of the glycoforms of GDH ranged from 90 to 130 kDa. After deglycosylation, a single 80 kDa band was observed. The gene encoding MpGDH was cloned and expressed in Aspergillus sojae. The apparent kcat and Km values of recombinant enzyme for glucose were found to be 749.7 sL1 and 28.3 mM, respectively. The results indicated that the characteristics of MpGDH were suitable for assaying blood glucose levels. Ó 2015, The Society for Biotechnology, Japan. All rights reserved. [Key words: Diabetes; Glucose dehydrogenase; Glucose sensor; Flavin adenine dinucleotide; Substrate specificity]
The self-monitoring of blood glucose at home is important for management of diabetes. There has been a striking evolution in glucose monitoring technology since the first blood glucose tests for self-monitoring were introduced around 1980 (1). The currently available glucose monitoring sensors are mainly based on electronmediator-dependent glucose oxidoreductases. Glucose oxidase (GOD) (EC 1.1.3.4) has been widely used as a mediated amperometric glucose sensor based on its high thermostability and high glucose selectivity (2). However, errors in glucose measurement often occur because of variations in the concentration of dioxygen (O2) in the blood samples. To avoid this problem, glucose dehydrogenases have been used in glucose sensors: NAD-dependent glucose dehydrogenase (GDH), NADPdependent GDH [NAD(P)-dependent GDH, EC 1.1.1.47] and pyrroloquinoline quinine (PQQ)-dependent GDH (EC 1.1.99.17) (3e11). NAD(P)-dependent GDH has strict substrate specificity, though NAD(P) cofactor must be supplied exogenously together with specific artificial electron mediators to carry out the electrochemical measurement because NAD(P) cofactor is not bound to the enzyme. PQQ-GDH has high catalytic activities for glucose and can use a variety of electron acceptors as redox mediators except for O2 (12). However, PQQ-GDH has broad substrate specificity and thus, when using PQQ-GDH, blood glucose levels higher than the actual value are obtained when the specimen contains high maltose, icodextrin, galactose, or xylose (13). In a patient whose blood glucose level was measured using a
simplified self-monitoring blood glucose sensor employing PQQGDH during administration of a maltose-containing infusion, hypoglycemia accompanied by a disruption in consciousness was experienced when the insulin dose was adjusted based on the measured value. The strict accuracy guidelines for blood glucose meters were published by the International Organization for Standardization (ISO) (14). Recently, Fraeyman et al. (15) have reported the successful production of a mutant strain of PQQ-GDH which shows no cross-reactivity with maltose. Flavin adenine dinucleotide (FAD)-GDH was first discovered in 1951 in Aspergillus oryzae (16); however, a detailed characterization of GDH has not been completed. Since the use of FAD-GDH as an electrode catalyst in glucose sensors was published, GDHs from Burkholderia cepacia (17), Aspergillus terreus (18,19), A. oryzae (20) and Glomerella cingulata (21) have been studied. The advantage of employing FAD-GDH in glucose sensors is its strict substrate specificity toward glucose. Therefore, we tried screening for fungi to get novel FAD-GDH which had substrate specificity suited to glucose sensors. In this study, we discovered a novel FAD-GDH from Mucor prainii NISL0103 (MpGDH). MpGDH showed high substrate specificity toward glucose. Notably, its reactivity for xylose was much lower than any other FAD-GDH. We also describe the cloning of the gene encoding the MpGDH and its recombinant expression in Aspergillus sojae. MATERIALS AND METHODS
* Corresponding author. Tel.: þ81 4 7123 5989; fax: þ81 4 7123 5948. E-mail address: [email protected] (R. Satake).
Strains and culture media Escherichia coli JM109 was used for the recombinant plasmid construction. The pUTE300K0 plasmid, which was derived from pBR322 DNA (Takara, Shiga, Japan), was used for the gene cloning (22). A. sojae BM-7
1389-1723/$ e see front matter Ó 2015, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2015.03.012
Please cite this article in press as: Satake, R., et al., Novel glucose dehydrogenase from Mucor prainii: Purification, characterization, molecular cloning and gene expression in Aspergillus sojae, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.03.012
2
SATAKE ET AL.
J. BIOSCI. BIOENG.,
(pyrG-, niaD-, Dku70::niaD) (23) was used as the host strain for the recombinant expression of MpGDH. Malt extract medium consisting of 2.0% malt extract, 2.0% D-glucose and 0.1% polypeptone (pH 6.0) was used for screening. Yeast glucose (YD) medium containing 2.0% yeast extract and 4.0% D-glucose (pH 6.0) was used for the cultivation of M. prainii. The YDS medium for the cultivation of recombinant A. sojae was comprised 0.2% yeast extract, 2% D-glucose, 0.5% soypeptone, 0.1% KH2PO4 and 0.050% MgSO4 7H2O (pH 6.0). Enzyme activity and protein assays Glucose dehydrogenase activity was assayed in 90 mM phosphate buffer (pH 7.0), containing 0.1 mM 2,6-dichlorophenol indophenol dehydrate (DCIP) and 0.2 mM phenazine methosulfate (PMS) at 37 C in the presence of 50 mM glucose or various concentrations of substrates. The activity was calculated by monitoring the decrease in absorbance of DCIP at 600 nm using a Hitachi U-3010 spectrophotometer (Hitachi High-Tech Fielding, Tokyo, Japan). One unit of enzymatic activity was defined as the amount of enzyme that caused the reduction of 1 mmol of DCIP per minute under the assay conditions. The extinction coefficient of 20.4 cm2/mmol and 18.3 cm2/mmol were used for the calculation in phosphate buffer (pH 7.0) and phosphate buffer (pH 6.5), respectively. The glucose oxidase activity assay in 80 mM phosphate buffer (pH 7.0), 135 mM glucose, 0.2 mM 4-aminoantipyrine (4-AA), 0.3 mM N-ethyl-N-(2-hydroxy-3-sulfopropyl)3-methylaniline (TOOS) and 4 U/ml peroxidase (Kikkoman Biochemifa, Chiba, Japan) at 37 C. The activity was calculated by monitoring the increase in absorbance at 555 nm using a Hitachi U-3010 spectrophotometer (Hitachi HighTech Fielding). One unit of enzymatic activity was defined as the amount of enzyme that caused a reduction of 1 mmol of H2O2 per minute under the above assay conditions. An extinction coefficient of 39.2 cm2/mmol was used for the calculation. The protein concentration was determined using the absorbance at 280 nm. The absorbance of a protein at 280 nm depends on the content of tryptophan, tyrosine and cystine (24). After cloning of MpGDH, it was revealed MpGDH contained 9 of tryptophan and 31 of tyrosine. The molar absorption coefficient, ε, of MpGDH was calculated and the protein concentration was recalculated using the absorbance at 280 nm and ε. Screening for fungi producing glucose dehydrogenase Fungi isolated from soils or stock cultures collections were grown at 30 C in 3 ml of malt extract medium with shaking for several days. The mycelia were harvested and washed with 10 mM acetate buffer (pH 5.0). Subsequently, the washed mycelia were resuspended in 1 ml of 10 mM acetate buffer (pH 5.0) and disrupted using glass beads. Then insoluble extracts were removed by centrifugation (2000 g). The cell extracts were assayed for glucose dehydrogenase activity. Preparation of purified MpGDH M. prainii NISL0103 was cultured in 40 L of YD medium with shaking at 30 C for 4 days. The mycelia were harvested and washed with 10 mM acetate buffer (pH 5.0), and disrupted using glass beads (diameter 0.75 mm) with a Dyno-Mill (Shinmaru Enterprises, Osaka, Japan). Then the insoluble portions were removed by centrifugation (6000 g, 30 min). The resulting extract was concentrated and filtered in a hollow fiber crossflow module (AIP2013, 6 kDa cut-off, Asahi Kasei Chemicals) to remove low-molecular-mass components. Solid ammonium sulfate was added to the enzyme solution to a final concentration of 70%. The solution was stored at 4 C overnight and the precipitate was removed by centrifugation at 20,000 g. The resulting supernatant was applied to a hydrophobic interaction column, TOYOPEARL-Butyl 650C (Tosoh, Tokyo, Japan), which was equilibrated with 10 mM acetate buffer (pH 5.0) containing 2 M ammonium sulfate. Proteins were eluted within a liner gradient from 2 M to 0 M ammonium sulfate in 10 column volumes. Fractions containing GDH activity were pooled and concentrated using a Centricon Plus-70 (Merck Millipore, Billerica, MA, USA). The concentrated enzyme solution was loaded onto a cation exchange column, SP-Sepharose FF (GE Healthcare Bio-Sciences, Piscataway, NJ, USA), previously equilibrated with 10 mM acetate buffer (pH 4.5). Proteins were eluted within a linear salt gradient from 0 mM to 200 mM of KCl in 20 column volumes. The elution was dialyzed against 10 mM acetate buffer (pH 5.0) using a Centricon Plus-70. The purified native MpGDH was subjected to SDS-PAGE (SuperSep Ace 10e20%, Wako Pure Chemical Industries, Osaka, Japan). A broad band of MpGDH ranging between 90 and 130 kDa was excised and then internal sequencing was performed by APRO Life Science Institute Inc. MpGDH was deglycosylated using the Enzymatic Deglycosylation Kit (Prozyme, Hayward, CA, USA) according to the manufacturer’s instructions.
FIG. 1. SDS-PAGE of native MpGDH. Lane 1, MpGDH; lane 2, MpGDH treated by the Enzymatic Deglycosylation Kit; lane M, molecular mass markers (Molecular Weight Standards Low Range, Bio-Rad).
of maltose were between 120 mg/dl and 600 mg/dl. The final concentrations of galactose were between 60 mg/dl and 300 mg/dl. The final concentrations of xylose were between 40 mg/dl and 200 mg/dl. Cloning and sequencing of the GDH gene from M. prainii NISL0103 Total RNA was extracted from disrupted mycelia using the ISOGEN (NIPPON GENE, Tokyo, Japan) according to the manufacturer’s instructions. Degenerate RT-PCR was performed using the Prime Script RT-PCR Kit (Takara) according to the manufacturer’s instructions. The Oligo dT primer from the kit was used for the synthesis of the 1st strand DNA, and the degenerate primers based on the partial amino acids sequences were used for PCR. The PCR products (approximately 800 bp) were sequenced with a CEQ200XL DNA Analysis System (Beckman Coulter, Brea, CA, USA). To obtain the complete open reading frame (ORF), 50 and 30 rapid amplification of the cDNA ends (RACE) was performed using the 30 -Full Race Core Set (Takara) and 50 -Full RACE Core Set (Takara), respectively, according to the manufacturer’s instructions. Primers for the 30 RACE and 50 -RACE were designed from the DNA sequences obtained from degenerate RT-PCR products. The full-length mpgdh was amplified using Prime Script RT-PCR Kit with the total RNA as a template and the MpGdh-full_F and MpGdh-full_R primers with a NdeI site. The PCR products were digested with NdeI and inserted into the pUTE300K0 vector to yield p300K0-Mpgdh.
Measurement of enzyme stability Purified MpGDH was diluted to 80 U/ml using the proper buffer, and enzyme stability was determined by measuring the residual activity after incubation under the conditions described below. For measurement of pH stability, MpGDH was diluted in various buffers having different pH values between 2.5 and 9.0, and incubated at 25 C for 16 h. For measurement of thermal stability, MpGDH was diluted with 100 mM potassium acetate buffer (pH 5.0) and incubated for 15 min between 25 C and 60 C. To investigate the effect of some compounds on MpGDH stability, MpGDH was diluted in 100 mM potassium phosphate buffer (pH 7.0) with each compound at a final concentration of 50 mM and incubated at 40 C for 15 min. Prior to this test, some compounds were previously adjusted to pH 7.0 with NaOH as appropriate. Cross-reactivity test To investigate the effect of other saccharides on MpGDH activity, GDH activity was assayed in the presence of 100 mg/dl glucose and other saccharides: maltose, galactose, xylose or all of them. The final concentrations
FIG. 2. Characterization of spectra MpGDH showing both the oxidized (black) and reduced (gray) forms of the enzyme. To reduce the enzyme, glucose was used.
Please cite this article in press as: Satake, R., et al., Novel glucose dehydrogenase from Mucor prainii: Purification, characterization, molecular cloning and gene expression in Aspergillus sojae, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.03.012
VOL. xx, 2015
NOVEL GLUCOSE DEHYDROGENASE FROM M. PRAINII
3
FIG. 3. The effects of temperature on MpGDH activity and stability. (a) The MpGDH activity was assayed from 25 C to 55 C. (b) MpGDH was incubated from 25e60 C for 15 min.
Expression and production of MpGDH in A. sojae The expression cassette of MpGDH in A. sojae was composed of the translation elongation factor 1 (TEF1) promoter, which has been shown to work constitutively in A. oryzae (25), the region used to integrate into the alkaline protease gene (alp) locus and the pyrG marker encoding orotidine-50 -phosphate decarboxylase. This expression cassette was constructed by a fusion PCR method (26). Briefly, each fragment was amplified from A. sojae NBRC4239 genome DNA or p300K0-Mpgdh using X_first PCR primers (X indicates DNA names). The PCR products were purified by gel extraction. Then, each product was mixed and used as a template for fusion PCR. The fusion PCR reaction was carried out containing the template described above and the AsAlp_Nest_F and AsAlp_Nest_R primers. The PCR product from this reaction was used as the MpGDH expression cassette. A. sojae BM-7 was transformed with the cassette as previously described (27) and the transformants were selected on CzapekeDox minimal medium. The integration of the targeting vector was confirmed by PCR using the primers AsAlp_Check_F and AsPyrG_Check_R, AsPyrG_Check_F and MpGdh_first_R, or MpGdh_first_F and AsAlp_Check_R. A sojae harboring MpGDH was cultured in 80 L of YDS medium with shaking at 30 C for 4 days in jar fermentors. The recombinant MpGDH (recMpGDH) was purified according to the procedures used for the native enzyme.
activities (Table S2). All strains which showed glucose dehydrogenase activity were Zygomycetes species. Second, the activities toward maltose, xylose and galactose were assayed revealing that strain M. prainii NISL0103 expressed GDH with strict specificity toward glucose. Purification of MpGDH As MpGDH was mainly located in a cell body, the mycelia were disrupted and the insoluble extracts were removed. MpGDH was purified as described and found to have a specific activity of 169 U/mg. The purified MpGDH showed a broad band with an estimated molecular mass of 90e130 kDa (Fig. 1). After deglycosylation, a single band with an estimated molecular mass of 80 kDa was obtained.
Nucleotide sequence accession number The nucleotide sequence of mpgdh has been deposited in the GenBank database under the accession number FW588501.1.
The catalytic properties of MpGDH The purified enzyme had absorption maxima at 394 and 460 nm. After reduction with glucose, the peaks disappeared and the enzyme became colorless. MpGDH and GOD from Aspergillus niger (Kikkoman Biochemifa, Chiba, Japan) were assayed for their glucose oxidase and glucose dehydrogenase activities which were defined in Materials and
RESULTS AND DISCUSSION
TABLE 2. Cross-reactivity test result. Test
Screening for glucose dehydrogenase To search for dehydrogenases that react specifically toward glucose, stock cultures of soil microorganisms (500 strains) were cultured and screened. The cell extracts of 12 fungi showed glucose dehydrogenase
TABLE 1. Substrate specificity of native MpGDH. Substrate
Glucose Maltose Galactose Xylose Mannose Sucrose Trehalose Maltotriose Maltotetraose
MpGDH relative activity (%) 100 1.09 0.44 1.53 0.66 0.00 0.22 0.88 0.66
GDH from G. cingulata relative activitya (%) 100 2.3 0.2 31 0.9 e e e e
Enzyme activities were assayed according to the method described in Materials and methods in the presence of 50 mM saccharide. The activity for glucose was taken as 100%. a Cited from Sygmund et al. (21).
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
Additive saccharide (mg/dl)
Relative activity (%)
Maltose
Galactose
Xylose
0 120 240 360 480 600 0 0 0 0 0 0 0 0 0 0 120 240 360 480 600
0 0 0 0 0 0 60 120 180 240 300 0 0 0 0 0 60 120 180 240 300
0 0 0 0 0 0 0 0 0 0 0 40 80 120 160 200 40 80 120 160 200
100 100 101 101 101 102 99 100 100 100 100 98 99 99 98 98 100 101 102 102 102
Enzyme activities were assayed according to the method described in Materials and methods in the presence of 100 mg/dl glucose and other saccharides. The activity for 100 mg/dl glucose was taken as 100%.
Please cite this article in press as: Satake, R., et al., Novel glucose dehydrogenase from Mucor prainii: Purification, characterization, molecular cloning and gene expression in Aspergillus sojae, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.03.012
4
SATAKE ET AL.
J. BIOSCI. BIOENG.,
FIG. 4. Multiple alignments of MpGDH, other known FAD-dependent glucose dehydrogenases and glucose oxidase. Multiple alignments were performed using ClustalW online software (http://www.genome.jp/tools/clustalw/). AoGDH, AtGDH and GcGDH are FAD-dependent glucose dehydrogenases derived from Aspergillus oryzae, A. terreus and Glomerella cingulate, respectively. PaGOD is a glucose oxidase derived from Penicillium amagasakiense. Arrowheads show the active sites in PaGOD, closed arrowheads indicate the essential residues for PaGOD activity and open arrowheads indicate the other residues. The secondary structure of MpGDH predicted by Jpred 3 is depicted above the sequence and that of PaGOD assigned by the author of a PDB entry is depicted below the sequence (29). The arrowed lines and the cylinders show b-strand structures and a-helix structures, respectively. The read frame shows MpGDH-specific inserted regions expected by comparison with the secondary structure.
Please cite this article in press as: Satake, R., et al., Novel glucose dehydrogenase from Mucor prainii: Purification, characterization, molecular cloning and gene expression in Aspergillus sojae, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.03.012
VOL. xx, 2015 methods. While GOD from A. niger had 29.7 U/mg GDH activity and 100 U/mg GOD activity, MpGDH had 169 U/mg GDH activity and GOD activity was not detected. It was shown that MpGDH was unable to use oxygen as the electron acceptor and thus, it was not affected by the dissolved oxygen. The optimal temperature and pH for the enzyme activity were 30 Ce40 C and pH 6.0 to 7.5, respectively (Fig. 3a and Fig. S1a). Approximately 80% of the enzymatic activity was retained after incubation at 40 C for 15 min at pH 5.0 (Fig. 3b). Treatment at 25 C for 16 h showed that the enzyme was stable from pH 3.5 to 7.0 (Fig. S1b). Table S4 shows that the addition of carboxylate and sulfate compounds increases the thermal stability of MpGDH. The substrate specificity of MpGDH was also investigated (Table 1). The relative activity for each saccharide compared with glucose was under 2%. The relative activity of GDH from G. cingulata (GcGDH) was 2.3% for maltose and 31% for xylose (22). Compared with GcGDH, the substrate specificity of MpGDH was very strict. In particular, MpGDH had very low reactivity toward xylose. The effect of additive saccharide in a glucose solution on the measurement of blood glucose levels was also investigated. The results are summarized in Table 2. The maximum change of absorbance was only 2% and the effect of additive saccharides was minimal. This suggested that MpGDH could be used to precisely measure the glucose level even in the presence of maltose, galactose and/or xylose.
Cloning, sequencing, and analysis of gene and protein sequences To obtain MpGDH cDNA, a partial amino acid sequencing of MpGDH was carried out. Two internal peptide sequences were obtained (KVENFTPPTPAQIE, IRNSTDEWANYY), and the primers for degenerate PCR were designed. Next, the partial MpGDH cDNA were obtained from degenerate PCR. The cDNA was cloned using 30 - and 50 -RACE, and the complete DNA sequence and deduced amino acid sequence of MpGDH were determined (Fig. 4). The 1926 bp of the MpGDH gene encoded a protein of 642 amino acids with a calculated molecular weight of 69,537 Da. Two obtained internal peptide sequences (KVENFTPPTPAQIE, IRNSTDEWANYY) were located at 153 to 166 and 415 to 426 in the amino acid sequence, respectively. The motif was found at 38 to43 in the amino acid sequence. Based on the existence of this motif and the absorption spectrum of MpGDH shown in Fig. 2, it is strongly indicated that the cofactor of MpGDH is a flavin adenine dinucleotide (FAD). Fig. 4 shows the multiple sequence alignment of MpGDH and other known FAD-dependent glucose dehydrogenases, AoGDH from A. oryzae (20), AtGDH from A. terreus (19), GcGDH from G. cingulata (21), and glucose oxidase (PaGOD) from Penicillium amagasakiense (PDB ID; 1gpe). MpGDH has low sequence identity (about 30%) with other GDHs and GOD, but MpGDH contains highly conserved regions near the FAD binding motif, and the C terminus. Previously, it was reported that the key active-site residues of PaGOD were Y73, S114, F418, W430, R516, N518, H520 and H563 (28). H520 and H563, which form hydrogen bonds to the 1-OH group of glucose, were believed to be involved in the transfer of protons and electrons derived from the substrate, and R516 which was essential for the efficient binding of glucose played an important role in enzyme function (29). The critical residues R516, H520 and H563 are completely conserved in all these enzymes, but the other residues involved in the interaction with glucose are generally not conserved in MpGDH. Thus, MpGDH is expected to catalyze glucose oxidation in a similar fashion as PaGOD, but the residues which interact with glucose are thought to be different. In addition, the secondary structure of MpGDH predicted by Jpred 3 (30) generally corresponds to that of PaGOD (Fig. 4), although their primary structures share low sequence identity with each other.
NOVEL GLUCOSE DEHYDROGENASE FROM M. PRAINII
5
TABLE 3. Substrate specificity of recMpGDH. Substrate
Glucose Maltose Galactose Xylose Mannose Sucrose Trehalose Maltotriose Maltotetraose
MpGDH relative activity (%) 100 0.00 0.00 0.472 0.00 0.00 0.00 0.00 0.00
Recombinant GDH from A. oryzae relative activitya (%) 100 0.0 0.6 e 2.1 0.0 1.0 0.2 0.2
Enzyme activities were assayed according to the method described in Materials and methods in the presence of 4 mM saccharide. The activity for glucose was taken as 100%. a Cited from Tsuji et al. (20).
MpGDH contains unique inserted sequences in the region from N360 to T387, and from N475 to L484. Comparison of the secondary structures of MpGDH and PaGOD near the region from T348 to T386 in MpGDH demonstrates that b-strand, a-helix and b-strand are positioned sequentially in both enzymes. Therefore, the former MpGDH-specific inserted region predicted by considering the second structure (shown by the read frame in Fig. 4) is thought to correspond to the region from A342 to G350 in PaGOD. According to the PaGOD structure (PDB ID; 1gpe), the region from A342 to G350 is located on the loop between two b-sheets near the entrance of the substrate-binding pocket. In addition, comparison of the secondary structures of MpGDH and PaGOD near the region from I456 to V480 in MpGDH shows that two b-strands are positioned sequentially in both. Thus, the latter MpGDH-specific inserted region predicted by considering the secondary structure (shown by the read frame in Fig. 4) is thought to correspond to the region from T421 to G423 in PaGOD located between these two bstrands. Structural analysis of PaGOD indicated that the region from T421 to G423 was also located near the entrance of the substratebinding pocket and formed the b-turn between two b-sheets. Consequently, these results indicate that two MpGDH-specific inserted regions form a unique conformation near the entrance of the substrate-binding pocket, which might be involved in substrate interactions.
Expression and production of MpGDH in A. sojae The expression cassette of MpGDH was constructed by cloning the nucleotide sequence including pyrG and the MpGDH sequence into the alp locus under the control of the TEF1 promoter. Transformed A. sojae cells were obtained by using pyrG selection, and integration of the cassette into the genome was confirmed by PCR. Three positive transformants were tested for expression in a small scale experiment and the best clone produced 0.024 mg/ml MpGDH, which was 27-fold higher than the native strain. The GDH activity of the host strain was not detected. The recMpGDH was purified and further investigated. SDS-PAGE showed a broad band from 80 to 100 kDa. The molecular mass of recMpGDH was found to be 200 kDa by gel filtration chromatography. Therefore, MpGDH was assumed to be a dimeric enzyme. The catalytic properties of the enzyme were examined to obtain detailed information about substrate specificity. The apparent kcat and Km values for D-glucose in 35 mM phosphate buffer (pH6.5) at 37 C were found to be 749.7 s1 and 28.3 mM, respectively. The substrate specificity was also investigated. The activity for each substrate was assayed in the presence of 4 mM saccharide to compare the specificity of recMpGDH with that of the known recombinant GDH from A. oryzae (21). The result is summarized in Table 3. The recMpGDH showed high substrate specificity toward
Please cite this article in press as: Satake, R., et al., Novel glucose dehydrogenase from Mucor prainii: Purification, characterization, molecular cloning and gene expression in Aspergillus sojae, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.03.012
6
SATAKE ET AL.
J. BIOSCI. BIOENG.,
glucose. Compared with the recombinant GDH from A. oryzae, the substrate specificity of recMpGDH was stricter. Conclusion It is possible that during blood glucose monitoring, overestimation of blood glucose levels can occur for patients with diabetes. This is particularly important for patients that undergo high-calorie infusions containing maltose, a galactose tolerance test, or a xylose absorption test. Therefore, strict specificity toward glucose is required. We identified a novel FADdependent glucose dehydrogenase (MpGDH) that showed highly specific to glucose. We investigated the effect of additive saccharide in the glucose solution toward the measurement of blood glucose. Although MpGDH shows limited activity toward xylose, the effect of additive saccharide on the detection of glucose was quite minimal. We also obtained A. sojae implanted with the mpgdh gene, which could produce MpGDH at a 27-fold higher concentration than the native strain. Although further electrochemical analyses of recMpGDH are necessary, we expect that recombinant MpGDH is suitable for self-monitoring glucose sensor application. Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.jbiosc.2015.03.012 ACKNOWLEDGMENT We thank Kimie Takayama for experimental assistance. References 1. Mark, D. H.: The business of self-monitoring of blood glucose: a market profile, J. Diabetes Sci. Technol., 3, 1219e1223 (2009). 2. Wilson, R. and Turner, A. P. F.: Glucose oxidase: an ideal enzyme, Biosens. Bioelectron., 7, 165e185 (1992). 3. Ye, L., Hammerle, M., Olshoorn, A. J. J., Schuhmann, W., Schmidt, H. L., Duine, J. A., and Heller, A.: High current density “wired” quinoprotein glucose dehydrogenase electrode, Anal. Chem., 65, 238e241 (1993). 4. Katz, E., Schlereth, D. D., and Schmidt, H. L.: Reconstitution of the quinoprotein glucose dehydrogenase from its apoenzyme on a gold electrode surface modified with a monolayer of pyrroloquinoline quinine, Electroanal. Chem., 368, 165e171 (1994). 5. Laurinavicius, V., Kurtinaitiene, B., Liauksminas, V., Jankauskaite, A., Simkus, R., Meskys, R., Boguslavsky, L., Skotheim, T., and Tanenbaum, S.: Reagentless biosensor based on PQQ-dependent glucose dehydrogenase and partially hydrolyzed polyarbutin, Talanta, 52, 485e493 (2000). 6. Yamazaki, T., Kojima, K., and Sode, K.: Extended-range glucose sensor employing engineered glucose dehydrogenases, Anal. Chem., 72, 4689e4693 (2000). 7. Razumiene, J., Mesky, R., Gureviciene, V., Reshetova, M. D., and Ryabov, A. D.: 4-ferrocenylphenol as an electron transfer mediator in PQQdependent alcohol and glucose dehydrogenase catalyzed reactions, Electrochem. Commun., 2, 307e311 (2000). 8. Takahashi, Y., Igarashi, S., Nakazawa, Y., Tsugawa, W., and Sode, K.: Construction and characterization of glucose enzyme sensor employing engineered water soluble PQQ glucose dehydrogenase with improved thermal stability, Electrochemistry, 68, 907e911 (2000). 9. Yoshida, H., Iguchi, T., and Sode, K.: Construction of multichimeric pyrroloquinoline quinone glucose dehydrogenase with improved enzymatic properties and application in glucose monitoring, Biotechnol. Lett., 22, 1505e1510 (2000).
10. Pauly, H. E. and Pfleiderer, G.: Conformational and functional aspects of the reversible dissociation and denaturation of glucose dehydrogenase, Biochemistry, 16, 4599e4604 (1977). 11. Groton, L. and Dominguez, E.: Electrocatalytic oxidation of NAD(P)H at mediator-modified electrodes, Rev. Mol. Biotechnol., 82, 371e392 (2002). 12. D’Costs, E. J., Higgins, I. J., and Turner, A. P. F.: Quinoprotein glucose dehydrogenase and its application in an amperometric glucose sensor, Biosensors, 2, 71e87 (1986). 13. Sano, S., Sugiura, N., Ohno, T., Higashi, M., Kizawa, S., Sakakibara, H., Yamanouchi, K., and Hirooka, Y.: Influence of maltose on self-monitoring blood glucose meter used in Japan, Practice, 21, 91e96 (2004) (in Japanese). 14. The International Organization for Standardization: In vitro diagnostic test systems. Requirements for blood-glucose monitoring systems for self-testing in managing diabetes mellitus, ISO15197:2013 (2013). 15. Fraeyman, A., Claeys, G., and Zaman, Z.: Effect of non-glucose sugars and haematocrit on glucose measurements with Roche Accu-Chek Performa glucose strips, Ann. Clin. Biochem., 47, 494e496 (2010). 16. Ogura, Y.: Studies on the glucose dehydrogenase of Aspergillus oryzae, J. Biochem., 38, 75e84 (1951). 17. Sode, K., Tsugawa, W., Yamazaki, T., Watanabe, M., Ogasawara, N., and Tanaka, M.: A novel thermostable glucose dehydrogenase varying temperature properties by altering its quaternary structures, Enzyme Microb. Technol., 19, 82e85 (1996). 18. Tsujimura, S., Kojima, S., Kano, K., Ikeda, T., Sato, M., Sanada, H., and Omura, H.: Novel FAD-dependent glucose dehydrogenase for a dioxygeninsensitive glucose biosensor, Biosci. Biotechnol. Biochem., 70, 654e659 (2006). 19. Omura, K., Sanada, H., Yada, T., Morita, T., Kuyama, M., Ikeda, T., Kano, K., and Tsujimura, S.: Coenzyme-binding glucose dehydrogenase, WO 2004/ 058958 A1, 2004-2007-15 (2004) (in Japanese). 20. Tsuji, Y., Kitabayashi, M., Kishimoto, T., and Nishiya, Y.: Production method of glucose dehydrogenase from Aspergillus oryzae, JP2007-289148A, 2007-1108 (2007) (in Japanese). 21. Sygmund, C., Klausberger, M., Felice, A., and Ludwig, R.: Reduction of quinines and phenoxy radicals by extracellular glucose dehydrogenase from Glomerella cingulata suggests a role in plant pathogenicity, Microbiology, 157, 3203e3212 (2011). 22. Koyama, Y. and Nakano, E.: Cloning of the glp K gene of Escherichia coli K-12 and its overexpression using a ‘sleeper’ bacteriophage vector, Agric. Biol. Chem., 54, 1315e1316 (1990). 23. Ito, K., Koyama, Y., and Hanya, Y.: Identification of the glutaminase genes of Aspergillus sojae involved in glutamate production during soy sauce fermentation, Biosci. Biotechnol. Biochem., 77, 1832e1840 (2013). 24. Pace, C. N., Vajdos, F., Fee, L., Grimsley, G., and Gray, T.: How to measure and predict the molar absorption coefficient of a protein, Protein Sci., 4, 2411e2423 (1995). 25. Kitamoto, N., Yoshino, S., Ohmiya, K., and Tsukagoshi, N.: Sequence analysis, overexpression, and antisense inhibition of a beta-xylosidase gene, xylA, from Aspergillus oryzae KBN616, Appl. Environ. Microbiol., 65, 20e24 (1999). 26. Ogawa, M., Tokuoka, M., Jin, F. J., Takahashi, T., and Koyama, Y.: Genetic analysis of conidiation regulatory pathways in koji-mold Aspergillus oryzae, Fungal Genet. Biol., 47, 10e18 (2010). 27. Takahashi, T., Hatamoto, O., Koyama, Y., and Abe, K.: Efficient gene disruption in the koji-mold Aspergillus sojae using a novel variation of the positivenegative method, Mol. Gen. Genomics, 272, 344e352 (2004). 28. Wohlfahrt, G., Witt, S., Hendle, J., Schomburg, D., Kalisz, H. M., and Hecht, H. J.: 1.8 and 1.9 Å resolution structures of the Penicillium amagasakiense and Aspergillus niger glucose oxidases as a basis for modelling substrate complexes, Acta Crystallogr. D Biol. Crystallogr., 55, 969e977 (1999). 29. Witt, S., Wohlfahrt, G., Schomburg, D., Hecht, H. J., and Kalisz, H. M.: Conserved arginine-516 of Penicillium amagasakiense glucose oxidase is essential for the efficient binding of beta-D-glucose, Biochem. J., 347, 553e559 (2000). 30. Cole, C., Barber, J. D., and Barton, G. J.: The Jpred 3 secondary structure prediction server, Nucleic Acids Res., 36, W197eW201 (2008).
Please cite this article in press as: Satake, R., et al., Novel glucose dehydrogenase from Mucor prainii: Purification, characterization, molecular cloning and gene expression in Aspergillus sojae, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.03.012
Novel glucose dehydrogenase from Mucor prainii: Purification, characterization, molecular cloning and gene expression in Aspergillus sojae Ryoko Satake,* Atsushi Ichiyanagi, Keiichi Ichikawa, Kozo Hirokawa, Yasuko Araki, Taro Yoshimura, and Keiko Gomi Research and Development Division, Kikkoman Corporation, 399 Noda, Chiba 278-0037, Japan Received 19 December 2014; accepted 16 March 2015 Available online xxx
Glucose dehydrogenase (GDH) is of interest for its potential applications in the field of glucose sensors. To improve the performance of glucose sensors, GDH is required to have strict substrate specificity. A novel flavin adenine dinucleotide (FAD)-dependent GDH was isolated from Mucor prainii NISL0103 and its enzymatic properties were characterized. This FAD-dependent GDH (MpGDH) exhibited high specificity toward glucose. High specificity for glucose was also observed even in the presence of saccharides such as maltose, galactose and xylose. The molecular masses of the glycoforms of GDH ranged from 90 to 130 kDa. After deglycosylation, a single 80 kDa band was observed. The gene encoding MpGDH was cloned and expressed in Aspergillus sojae. The apparent kcat and Km values of recombinant enzyme for glucose were found to be 749.7 sL1 and 28.3 mM, respectively. The results indicated that the characteristics of MpGDH were suitable for assaying blood glucose levels. Ó 2015, The Society for Biotechnology, Japan. All rights reserved. [Key words: Diabetes; Glucose dehydrogenase; Glucose sensor; Flavin adenine dinucleotide; Substrate specificity]
The self-monitoring of blood glucose at home is important for management of diabetes. There has been a striking evolution in glucose monitoring technology since the first blood glucose tests for self-monitoring were introduced around 1980 (1). The currently available glucose monitoring sensors are mainly based on electronmediator-dependent glucose oxidoreductases. Glucose oxidase (GOD) (EC 1.1.3.4) has been widely used as a mediated amperometric glucose sensor based on its high thermostability and high glucose selectivity (2). However, errors in glucose measurement often occur because of variations in the concentration of dioxygen (O2) in the blood samples. To avoid this problem, glucose dehydrogenases have been used in glucose sensors: NAD-dependent glucose dehydrogenase (GDH), NADPdependent GDH [NAD(P)-dependent GDH, EC 1.1.1.47] and pyrroloquinoline quinine (PQQ)-dependent GDH (EC 1.1.99.17) (3e11). NAD(P)-dependent GDH has strict substrate specificity, though NAD(P) cofactor must be supplied exogenously together with specific artificial electron mediators to carry out the electrochemical measurement because NAD(P) cofactor is not bound to the enzyme. PQQ-GDH has high catalytic activities for glucose and can use a variety of electron acceptors as redox mediators except for O2 (12). However, PQQ-GDH has broad substrate specificity and thus, when using PQQ-GDH, blood glucose levels higher than the actual value are obtained when the specimen contains high maltose, icodextrin, galactose, or xylose (13). In a patient whose blood glucose level was measured using a
simplified self-monitoring blood glucose sensor employing PQQGDH during administration of a maltose-containing infusion, hypoglycemia accompanied by a disruption in consciousness was experienced when the insulin dose was adjusted based on the measured value. The strict accuracy guidelines for blood glucose meters were published by the International Organization for Standardization (ISO) (14). Recently, Fraeyman et al. (15) have reported the successful production of a mutant strain of PQQ-GDH which shows no cross-reactivity with maltose. Flavin adenine dinucleotide (FAD)-GDH was first discovered in 1951 in Aspergillus oryzae (16); however, a detailed characterization of GDH has not been completed. Since the use of FAD-GDH as an electrode catalyst in glucose sensors was published, GDHs from Burkholderia cepacia (17), Aspergillus terreus (18,19), A. oryzae (20) and Glomerella cingulata (21) have been studied. The advantage of employing FAD-GDH in glucose sensors is its strict substrate specificity toward glucose. Therefore, we tried screening for fungi to get novel FAD-GDH which had substrate specificity suited to glucose sensors. In this study, we discovered a novel FAD-GDH from Mucor prainii NISL0103 (MpGDH). MpGDH showed high substrate specificity toward glucose. Notably, its reactivity for xylose was much lower than any other FAD-GDH. We also describe the cloning of the gene encoding the MpGDH and its recombinant expression in Aspergillus sojae. MATERIALS AND METHODS
* Corresponding author. Tel.: þ81 4 7123 5989; fax: þ81 4 7123 5948. E-mail address: [email protected] (R. Satake).
Strains and culture media Escherichia coli JM109 was used for the recombinant plasmid construction. The pUTE300K0 plasmid, which was derived from pBR322 DNA (Takara, Shiga, Japan), was used for the gene cloning (22). A. sojae BM-7
1389-1723/$ e see front matter Ó 2015, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2015.03.012
Please cite this article in press as: Satake, R., et al., Novel glucose dehydrogenase from Mucor prainii: Purification, characterization, molecular cloning and gene expression in Aspergillus sojae, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.03.012
2
SATAKE ET AL.
J. BIOSCI. BIOENG.,
(pyrG-, niaD-, Dku70::niaD) (23) was used as the host strain for the recombinant expression of MpGDH. Malt extract medium consisting of 2.0% malt extract, 2.0% D-glucose and 0.1% polypeptone (pH 6.0) was used for screening. Yeast glucose (YD) medium containing 2.0% yeast extract and 4.0% D-glucose (pH 6.0) was used for the cultivation of M. prainii. The YDS medium for the cultivation of recombinant A. sojae was comprised 0.2% yeast extract, 2% D-glucose, 0.5% soypeptone, 0.1% KH2PO4 and 0.050% MgSO4 7H2O (pH 6.0). Enzyme activity and protein assays Glucose dehydrogenase activity was assayed in 90 mM phosphate buffer (pH 7.0), containing 0.1 mM 2,6-dichlorophenol indophenol dehydrate (DCIP) and 0.2 mM phenazine methosulfate (PMS) at 37 C in the presence of 50 mM glucose or various concentrations of substrates. The activity was calculated by monitoring the decrease in absorbance of DCIP at 600 nm using a Hitachi U-3010 spectrophotometer (Hitachi High-Tech Fielding, Tokyo, Japan). One unit of enzymatic activity was defined as the amount of enzyme that caused the reduction of 1 mmol of DCIP per minute under the assay conditions. The extinction coefficient of 20.4 cm2/mmol and 18.3 cm2/mmol were used for the calculation in phosphate buffer (pH 7.0) and phosphate buffer (pH 6.5), respectively. The glucose oxidase activity assay in 80 mM phosphate buffer (pH 7.0), 135 mM glucose, 0.2 mM 4-aminoantipyrine (4-AA), 0.3 mM N-ethyl-N-(2-hydroxy-3-sulfopropyl)3-methylaniline (TOOS) and 4 U/ml peroxidase (Kikkoman Biochemifa, Chiba, Japan) at 37 C. The activity was calculated by monitoring the increase in absorbance at 555 nm using a Hitachi U-3010 spectrophotometer (Hitachi HighTech Fielding). One unit of enzymatic activity was defined as the amount of enzyme that caused a reduction of 1 mmol of H2O2 per minute under the above assay conditions. An extinction coefficient of 39.2 cm2/mmol was used for the calculation. The protein concentration was determined using the absorbance at 280 nm. The absorbance of a protein at 280 nm depends on the content of tryptophan, tyrosine and cystine (24). After cloning of MpGDH, it was revealed MpGDH contained 9 of tryptophan and 31 of tyrosine. The molar absorption coefficient, ε, of MpGDH was calculated and the protein concentration was recalculated using the absorbance at 280 nm and ε. Screening for fungi producing glucose dehydrogenase Fungi isolated from soils or stock cultures collections were grown at 30 C in 3 ml of malt extract medium with shaking for several days. The mycelia were harvested and washed with 10 mM acetate buffer (pH 5.0). Subsequently, the washed mycelia were resuspended in 1 ml of 10 mM acetate buffer (pH 5.0) and disrupted using glass beads. Then insoluble extracts were removed by centrifugation (2000 g). The cell extracts were assayed for glucose dehydrogenase activity. Preparation of purified MpGDH M. prainii NISL0103 was cultured in 40 L of YD medium with shaking at 30 C for 4 days. The mycelia were harvested and washed with 10 mM acetate buffer (pH 5.0), and disrupted using glass beads (diameter 0.75 mm) with a Dyno-Mill (Shinmaru Enterprises, Osaka, Japan). Then the insoluble portions were removed by centrifugation (6000 g, 30 min). The resulting extract was concentrated and filtered in a hollow fiber crossflow module (AIP2013, 6 kDa cut-off, Asahi Kasei Chemicals) to remove low-molecular-mass components. Solid ammonium sulfate was added to the enzyme solution to a final concentration of 70%. The solution was stored at 4 C overnight and the precipitate was removed by centrifugation at 20,000 g. The resulting supernatant was applied to a hydrophobic interaction column, TOYOPEARL-Butyl 650C (Tosoh, Tokyo, Japan), which was equilibrated with 10 mM acetate buffer (pH 5.0) containing 2 M ammonium sulfate. Proteins were eluted within a liner gradient from 2 M to 0 M ammonium sulfate in 10 column volumes. Fractions containing GDH activity were pooled and concentrated using a Centricon Plus-70 (Merck Millipore, Billerica, MA, USA). The concentrated enzyme solution was loaded onto a cation exchange column, SP-Sepharose FF (GE Healthcare Bio-Sciences, Piscataway, NJ, USA), previously equilibrated with 10 mM acetate buffer (pH 4.5). Proteins were eluted within a linear salt gradient from 0 mM to 200 mM of KCl in 20 column volumes. The elution was dialyzed against 10 mM acetate buffer (pH 5.0) using a Centricon Plus-70. The purified native MpGDH was subjected to SDS-PAGE (SuperSep Ace 10e20%, Wako Pure Chemical Industries, Osaka, Japan). A broad band of MpGDH ranging between 90 and 130 kDa was excised and then internal sequencing was performed by APRO Life Science Institute Inc. MpGDH was deglycosylated using the Enzymatic Deglycosylation Kit (Prozyme, Hayward, CA, USA) according to the manufacturer’s instructions.
FIG. 1. SDS-PAGE of native MpGDH. Lane 1, MpGDH; lane 2, MpGDH treated by the Enzymatic Deglycosylation Kit; lane M, molecular mass markers (Molecular Weight Standards Low Range, Bio-Rad).
of maltose were between 120 mg/dl and 600 mg/dl. The final concentrations of galactose were between 60 mg/dl and 300 mg/dl. The final concentrations of xylose were between 40 mg/dl and 200 mg/dl. Cloning and sequencing of the GDH gene from M. prainii NISL0103 Total RNA was extracted from disrupted mycelia using the ISOGEN (NIPPON GENE, Tokyo, Japan) according to the manufacturer’s instructions. Degenerate RT-PCR was performed using the Prime Script RT-PCR Kit (Takara) according to the manufacturer’s instructions. The Oligo dT primer from the kit was used for the synthesis of the 1st strand DNA, and the degenerate primers based on the partial amino acids sequences were used for PCR. The PCR products (approximately 800 bp) were sequenced with a CEQ200XL DNA Analysis System (Beckman Coulter, Brea, CA, USA). To obtain the complete open reading frame (ORF), 50 and 30 rapid amplification of the cDNA ends (RACE) was performed using the 30 -Full Race Core Set (Takara) and 50 -Full RACE Core Set (Takara), respectively, according to the manufacturer’s instructions. Primers for the 30 RACE and 50 -RACE were designed from the DNA sequences obtained from degenerate RT-PCR products. The full-length mpgdh was amplified using Prime Script RT-PCR Kit with the total RNA as a template and the MpGdh-full_F and MpGdh-full_R primers with a NdeI site. The PCR products were digested with NdeI and inserted into the pUTE300K0 vector to yield p300K0-Mpgdh.
Measurement of enzyme stability Purified MpGDH was diluted to 80 U/ml using the proper buffer, and enzyme stability was determined by measuring the residual activity after incubation under the conditions described below. For measurement of pH stability, MpGDH was diluted in various buffers having different pH values between 2.5 and 9.0, and incubated at 25 C for 16 h. For measurement of thermal stability, MpGDH was diluted with 100 mM potassium acetate buffer (pH 5.0) and incubated for 15 min between 25 C and 60 C. To investigate the effect of some compounds on MpGDH stability, MpGDH was diluted in 100 mM potassium phosphate buffer (pH 7.0) with each compound at a final concentration of 50 mM and incubated at 40 C for 15 min. Prior to this test, some compounds were previously adjusted to pH 7.0 with NaOH as appropriate. Cross-reactivity test To investigate the effect of other saccharides on MpGDH activity, GDH activity was assayed in the presence of 100 mg/dl glucose and other saccharides: maltose, galactose, xylose or all of them. The final concentrations
FIG. 2. Characterization of spectra MpGDH showing both the oxidized (black) and reduced (gray) forms of the enzyme. To reduce the enzyme, glucose was used.
Please cite this article in press as: Satake, R., et al., Novel glucose dehydrogenase from Mucor prainii: Purification, characterization, molecular cloning and gene expression in Aspergillus sojae, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.03.012
VOL. xx, 2015
NOVEL GLUCOSE DEHYDROGENASE FROM M. PRAINII
3
FIG. 3. The effects of temperature on MpGDH activity and stability. (a) The MpGDH activity was assayed from 25 C to 55 C. (b) MpGDH was incubated from 25e60 C for 15 min.
Expression and production of MpGDH in A. sojae The expression cassette of MpGDH in A. sojae was composed of the translation elongation factor 1 (TEF1) promoter, which has been shown to work constitutively in A. oryzae (25), the region used to integrate into the alkaline protease gene (alp) locus and the pyrG marker encoding orotidine-50 -phosphate decarboxylase. This expression cassette was constructed by a fusion PCR method (26). Briefly, each fragment was amplified from A. sojae NBRC4239 genome DNA or p300K0-Mpgdh using X_first PCR primers (X indicates DNA names). The PCR products were purified by gel extraction. Then, each product was mixed and used as a template for fusion PCR. The fusion PCR reaction was carried out containing the template described above and the AsAlp_Nest_F and AsAlp_Nest_R primers. The PCR product from this reaction was used as the MpGDH expression cassette. A. sojae BM-7 was transformed with the cassette as previously described (27) and the transformants were selected on CzapekeDox minimal medium. The integration of the targeting vector was confirmed by PCR using the primers AsAlp_Check_F and AsPyrG_Check_R, AsPyrG_Check_F and MpGdh_first_R, or MpGdh_first_F and AsAlp_Check_R. A sojae harboring MpGDH was cultured in 80 L of YDS medium with shaking at 30 C for 4 days in jar fermentors. The recombinant MpGDH (recMpGDH) was purified according to the procedures used for the native enzyme.
activities (Table S2). All strains which showed glucose dehydrogenase activity were Zygomycetes species. Second, the activities toward maltose, xylose and galactose were assayed revealing that strain M. prainii NISL0103 expressed GDH with strict specificity toward glucose. Purification of MpGDH As MpGDH was mainly located in a cell body, the mycelia were disrupted and the insoluble extracts were removed. MpGDH was purified as described and found to have a specific activity of 169 U/mg. The purified MpGDH showed a broad band with an estimated molecular mass of 90e130 kDa (Fig. 1). After deglycosylation, a single band with an estimated molecular mass of 80 kDa was obtained.
Nucleotide sequence accession number The nucleotide sequence of mpgdh has been deposited in the GenBank database under the accession number FW588501.1.
The catalytic properties of MpGDH The purified enzyme had absorption maxima at 394 and 460 nm. After reduction with glucose, the peaks disappeared and the enzyme became colorless. MpGDH and GOD from Aspergillus niger (Kikkoman Biochemifa, Chiba, Japan) were assayed for their glucose oxidase and glucose dehydrogenase activities which were defined in Materials and
RESULTS AND DISCUSSION
TABLE 2. Cross-reactivity test result. Test
Screening for glucose dehydrogenase To search for dehydrogenases that react specifically toward glucose, stock cultures of soil microorganisms (500 strains) were cultured and screened. The cell extracts of 12 fungi showed glucose dehydrogenase
TABLE 1. Substrate specificity of native MpGDH. Substrate
Glucose Maltose Galactose Xylose Mannose Sucrose Trehalose Maltotriose Maltotetraose
MpGDH relative activity (%) 100 1.09 0.44 1.53 0.66 0.00 0.22 0.88 0.66
GDH from G. cingulata relative activitya (%) 100 2.3 0.2 31 0.9 e e e e
Enzyme activities were assayed according to the method described in Materials and methods in the presence of 50 mM saccharide. The activity for glucose was taken as 100%. a Cited from Sygmund et al. (21).
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
Additive saccharide (mg/dl)
Relative activity (%)
Maltose
Galactose
Xylose
0 120 240 360 480 600 0 0 0 0 0 0 0 0 0 0 120 240 360 480 600
0 0 0 0 0 0 60 120 180 240 300 0 0 0 0 0 60 120 180 240 300
0 0 0 0 0 0 0 0 0 0 0 40 80 120 160 200 40 80 120 160 200
100 100 101 101 101 102 99 100 100 100 100 98 99 99 98 98 100 101 102 102 102
Enzyme activities were assayed according to the method described in Materials and methods in the presence of 100 mg/dl glucose and other saccharides. The activity for 100 mg/dl glucose was taken as 100%.
Please cite this article in press as: Satake, R., et al., Novel glucose dehydrogenase from Mucor prainii: Purification, characterization, molecular cloning and gene expression in Aspergillus sojae, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.03.012
4
SATAKE ET AL.
J. BIOSCI. BIOENG.,
FIG. 4. Multiple alignments of MpGDH, other known FAD-dependent glucose dehydrogenases and glucose oxidase. Multiple alignments were performed using ClustalW online software (http://www.genome.jp/tools/clustalw/). AoGDH, AtGDH and GcGDH are FAD-dependent glucose dehydrogenases derived from Aspergillus oryzae, A. terreus and Glomerella cingulate, respectively. PaGOD is a glucose oxidase derived from Penicillium amagasakiense. Arrowheads show the active sites in PaGOD, closed arrowheads indicate the essential residues for PaGOD activity and open arrowheads indicate the other residues. The secondary structure of MpGDH predicted by Jpred 3 is depicted above the sequence and that of PaGOD assigned by the author of a PDB entry is depicted below the sequence (29). The arrowed lines and the cylinders show b-strand structures and a-helix structures, respectively. The read frame shows MpGDH-specific inserted regions expected by comparison with the secondary structure.
Please cite this article in press as: Satake, R., et al., Novel glucose dehydrogenase from Mucor prainii: Purification, characterization, molecular cloning and gene expression in Aspergillus sojae, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.03.012
VOL. xx, 2015 methods. While GOD from A. niger had 29.7 U/mg GDH activity and 100 U/mg GOD activity, MpGDH had 169 U/mg GDH activity and GOD activity was not detected. It was shown that MpGDH was unable to use oxygen as the electron acceptor and thus, it was not affected by the dissolved oxygen. The optimal temperature and pH for the enzyme activity were 30 Ce40 C and pH 6.0 to 7.5, respectively (Fig. 3a and Fig. S1a). Approximately 80% of the enzymatic activity was retained after incubation at 40 C for 15 min at pH 5.0 (Fig. 3b). Treatment at 25 C for 16 h showed that the enzyme was stable from pH 3.5 to 7.0 (Fig. S1b). Table S4 shows that the addition of carboxylate and sulfate compounds increases the thermal stability of MpGDH. The substrate specificity of MpGDH was also investigated (Table 1). The relative activity for each saccharide compared with glucose was under 2%. The relative activity of GDH from G. cingulata (GcGDH) was 2.3% for maltose and 31% for xylose (22). Compared with GcGDH, the substrate specificity of MpGDH was very strict. In particular, MpGDH had very low reactivity toward xylose. The effect of additive saccharide in a glucose solution on the measurement of blood glucose levels was also investigated. The results are summarized in Table 2. The maximum change of absorbance was only 2% and the effect of additive saccharides was minimal. This suggested that MpGDH could be used to precisely measure the glucose level even in the presence of maltose, galactose and/or xylose.
Cloning, sequencing, and analysis of gene and protein sequences To obtain MpGDH cDNA, a partial amino acid sequencing of MpGDH was carried out. Two internal peptide sequences were obtained (KVENFTPPTPAQIE, IRNSTDEWANYY), and the primers for degenerate PCR were designed. Next, the partial MpGDH cDNA were obtained from degenerate PCR. The cDNA was cloned using 30 - and 50 -RACE, and the complete DNA sequence and deduced amino acid sequence of MpGDH were determined (Fig. 4). The 1926 bp of the MpGDH gene encoded a protein of 642 amino acids with a calculated molecular weight of 69,537 Da. Two obtained internal peptide sequences (KVENFTPPTPAQIE, IRNSTDEWANYY) were located at 153 to 166 and 415 to 426 in the amino acid sequence, respectively. The motif was found at 38 to43 in the amino acid sequence. Based on the existence of this motif and the absorption spectrum of MpGDH shown in Fig. 2, it is strongly indicated that the cofactor of MpGDH is a flavin adenine dinucleotide (FAD). Fig. 4 shows the multiple sequence alignment of MpGDH and other known FAD-dependent glucose dehydrogenases, AoGDH from A. oryzae (20), AtGDH from A. terreus (19), GcGDH from G. cingulata (21), and glucose oxidase (PaGOD) from Penicillium amagasakiense (PDB ID; 1gpe). MpGDH has low sequence identity (about 30%) with other GDHs and GOD, but MpGDH contains highly conserved regions near the FAD binding motif, and the C terminus. Previously, it was reported that the key active-site residues of PaGOD were Y73, S114, F418, W430, R516, N518, H520 and H563 (28). H520 and H563, which form hydrogen bonds to the 1-OH group of glucose, were believed to be involved in the transfer of protons and electrons derived from the substrate, and R516 which was essential for the efficient binding of glucose played an important role in enzyme function (29). The critical residues R516, H520 and H563 are completely conserved in all these enzymes, but the other residues involved in the interaction with glucose are generally not conserved in MpGDH. Thus, MpGDH is expected to catalyze glucose oxidation in a similar fashion as PaGOD, but the residues which interact with glucose are thought to be different. In addition, the secondary structure of MpGDH predicted by Jpred 3 (30) generally corresponds to that of PaGOD (Fig. 4), although their primary structures share low sequence identity with each other.
NOVEL GLUCOSE DEHYDROGENASE FROM M. PRAINII
5
TABLE 3. Substrate specificity of recMpGDH. Substrate
Glucose Maltose Galactose Xylose Mannose Sucrose Trehalose Maltotriose Maltotetraose
MpGDH relative activity (%) 100 0.00 0.00 0.472 0.00 0.00 0.00 0.00 0.00
Recombinant GDH from A. oryzae relative activitya (%) 100 0.0 0.6 e 2.1 0.0 1.0 0.2 0.2
Enzyme activities were assayed according to the method described in Materials and methods in the presence of 4 mM saccharide. The activity for glucose was taken as 100%. a Cited from Tsuji et al. (20).
MpGDH contains unique inserted sequences in the region from N360 to T387, and from N475 to L484. Comparison of the secondary structures of MpGDH and PaGOD near the region from T348 to T386 in MpGDH demonstrates that b-strand, a-helix and b-strand are positioned sequentially in both enzymes. Therefore, the former MpGDH-specific inserted region predicted by considering the second structure (shown by the read frame in Fig. 4) is thought to correspond to the region from A342 to G350 in PaGOD. According to the PaGOD structure (PDB ID; 1gpe), the region from A342 to G350 is located on the loop between two b-sheets near the entrance of the substrate-binding pocket. In addition, comparison of the secondary structures of MpGDH and PaGOD near the region from I456 to V480 in MpGDH shows that two b-strands are positioned sequentially in both. Thus, the latter MpGDH-specific inserted region predicted by considering the secondary structure (shown by the read frame in Fig. 4) is thought to correspond to the region from T421 to G423 in PaGOD located between these two bstrands. Structural analysis of PaGOD indicated that the region from T421 to G423 was also located near the entrance of the substratebinding pocket and formed the b-turn between two b-sheets. Consequently, these results indicate that two MpGDH-specific inserted regions form a unique conformation near the entrance of the substrate-binding pocket, which might be involved in substrate interactions.
Expression and production of MpGDH in A. sojae The expression cassette of MpGDH was constructed by cloning the nucleotide sequence including pyrG and the MpGDH sequence into the alp locus under the control of the TEF1 promoter. Transformed A. sojae cells were obtained by using pyrG selection, and integration of the cassette into the genome was confirmed by PCR. Three positive transformants were tested for expression in a small scale experiment and the best clone produced 0.024 mg/ml MpGDH, which was 27-fold higher than the native strain. The GDH activity of the host strain was not detected. The recMpGDH was purified and further investigated. SDS-PAGE showed a broad band from 80 to 100 kDa. The molecular mass of recMpGDH was found to be 200 kDa by gel filtration chromatography. Therefore, MpGDH was assumed to be a dimeric enzyme. The catalytic properties of the enzyme were examined to obtain detailed information about substrate specificity. The apparent kcat and Km values for D-glucose in 35 mM phosphate buffer (pH6.5) at 37 C were found to be 749.7 s1 and 28.3 mM, respectively. The substrate specificity was also investigated. The activity for each substrate was assayed in the presence of 4 mM saccharide to compare the specificity of recMpGDH with that of the known recombinant GDH from A. oryzae (21). The result is summarized in Table 3. The recMpGDH showed high substrate specificity toward
Please cite this article in press as: Satake, R., et al., Novel glucose dehydrogenase from Mucor prainii: Purification, characterization, molecular cloning and gene expression in Aspergillus sojae, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.03.012
6
SATAKE ET AL.
J. BIOSCI. BIOENG.,
glucose. Compared with the recombinant GDH from A. oryzae, the substrate specificity of recMpGDH was stricter. Conclusion It is possible that during blood glucose monitoring, overestimation of blood glucose levels can occur for patients with diabetes. This is particularly important for patients that undergo high-calorie infusions containing maltose, a galactose tolerance test, or a xylose absorption test. Therefore, strict specificity toward glucose is required. We identified a novel FADdependent glucose dehydrogenase (MpGDH) that showed highly specific to glucose. We investigated the effect of additive saccharide in the glucose solution toward the measurement of blood glucose. Although MpGDH shows limited activity toward xylose, the effect of additive saccharide on the detection of glucose was quite minimal. We also obtained A. sojae implanted with the mpgdh gene, which could produce MpGDH at a 27-fold higher concentration than the native strain. Although further electrochemical analyses of recMpGDH are necessary, we expect that recombinant MpGDH is suitable for self-monitoring glucose sensor application. Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.jbiosc.2015.03.012 ACKNOWLEDGMENT We thank Kimie Takayama for experimental assistance. References 1. Mark, D. H.: The business of self-monitoring of blood glucose: a market profile, J. Diabetes Sci. Technol., 3, 1219e1223 (2009). 2. Wilson, R. and Turner, A. P. F.: Glucose oxidase: an ideal enzyme, Biosens. Bioelectron., 7, 165e185 (1992). 3. Ye, L., Hammerle, M., Olshoorn, A. J. J., Schuhmann, W., Schmidt, H. L., Duine, J. A., and Heller, A.: High current density “wired” quinoprotein glucose dehydrogenase electrode, Anal. Chem., 65, 238e241 (1993). 4. Katz, E., Schlereth, D. D., and Schmidt, H. L.: Reconstitution of the quinoprotein glucose dehydrogenase from its apoenzyme on a gold electrode surface modified with a monolayer of pyrroloquinoline quinine, Electroanal. Chem., 368, 165e171 (1994). 5. Laurinavicius, V., Kurtinaitiene, B., Liauksminas, V., Jankauskaite, A., Simkus, R., Meskys, R., Boguslavsky, L., Skotheim, T., and Tanenbaum, S.: Reagentless biosensor based on PQQ-dependent glucose dehydrogenase and partially hydrolyzed polyarbutin, Talanta, 52, 485e493 (2000). 6. Yamazaki, T., Kojima, K., and Sode, K.: Extended-range glucose sensor employing engineered glucose dehydrogenases, Anal. Chem., 72, 4689e4693 (2000). 7. Razumiene, J., Mesky, R., Gureviciene, V., Reshetova, M. D., and Ryabov, A. D.: 4-ferrocenylphenol as an electron transfer mediator in PQQdependent alcohol and glucose dehydrogenase catalyzed reactions, Electrochem. Commun., 2, 307e311 (2000). 8. Takahashi, Y., Igarashi, S., Nakazawa, Y., Tsugawa, W., and Sode, K.: Construction and characterization of glucose enzyme sensor employing engineered water soluble PQQ glucose dehydrogenase with improved thermal stability, Electrochemistry, 68, 907e911 (2000). 9. Yoshida, H., Iguchi, T., and Sode, K.: Construction of multichimeric pyrroloquinoline quinone glucose dehydrogenase with improved enzymatic properties and application in glucose monitoring, Biotechnol. Lett., 22, 1505e1510 (2000).
10. Pauly, H. E. and Pfleiderer, G.: Conformational and functional aspects of the reversible dissociation and denaturation of glucose dehydrogenase, Biochemistry, 16, 4599e4604 (1977). 11. Groton, L. and Dominguez, E.: Electrocatalytic oxidation of NAD(P)H at mediator-modified electrodes, Rev. Mol. Biotechnol., 82, 371e392 (2002). 12. D’Costs, E. J., Higgins, I. J., and Turner, A. P. F.: Quinoprotein glucose dehydrogenase and its application in an amperometric glucose sensor, Biosensors, 2, 71e87 (1986). 13. Sano, S., Sugiura, N., Ohno, T., Higashi, M., Kizawa, S., Sakakibara, H., Yamanouchi, K., and Hirooka, Y.: Influence of maltose on self-monitoring blood glucose meter used in Japan, Practice, 21, 91e96 (2004) (in Japanese). 14. The International Organization for Standardization: In vitro diagnostic test systems. Requirements for blood-glucose monitoring systems for self-testing in managing diabetes mellitus, ISO15197:2013 (2013). 15. Fraeyman, A., Claeys, G., and Zaman, Z.: Effect of non-glucose sugars and haematocrit on glucose measurements with Roche Accu-Chek Performa glucose strips, Ann. Clin. Biochem., 47, 494e496 (2010). 16. Ogura, Y.: Studies on the glucose dehydrogenase of Aspergillus oryzae, J. Biochem., 38, 75e84 (1951). 17. Sode, K., Tsugawa, W., Yamazaki, T., Watanabe, M., Ogasawara, N., and Tanaka, M.: A novel thermostable glucose dehydrogenase varying temperature properties by altering its quaternary structures, Enzyme Microb. Technol., 19, 82e85 (1996). 18. Tsujimura, S., Kojima, S., Kano, K., Ikeda, T., Sato, M., Sanada, H., and Omura, H.: Novel FAD-dependent glucose dehydrogenase for a dioxygeninsensitive glucose biosensor, Biosci. Biotechnol. Biochem., 70, 654e659 (2006). 19. Omura, K., Sanada, H., Yada, T., Morita, T., Kuyama, M., Ikeda, T., Kano, K., and Tsujimura, S.: Coenzyme-binding glucose dehydrogenase, WO 2004/ 058958 A1, 2004-2007-15 (2004) (in Japanese). 20. Tsuji, Y., Kitabayashi, M., Kishimoto, T., and Nishiya, Y.: Production method of glucose dehydrogenase from Aspergillus oryzae, JP2007-289148A, 2007-1108 (2007) (in Japanese). 21. Sygmund, C., Klausberger, M., Felice, A., and Ludwig, R.: Reduction of quinines and phenoxy radicals by extracellular glucose dehydrogenase from Glomerella cingulata suggests a role in plant pathogenicity, Microbiology, 157, 3203e3212 (2011). 22. Koyama, Y. and Nakano, E.: Cloning of the glp K gene of Escherichia coli K-12 and its overexpression using a ‘sleeper’ bacteriophage vector, Agric. Biol. Chem., 54, 1315e1316 (1990). 23. Ito, K., Koyama, Y., and Hanya, Y.: Identification of the glutaminase genes of Aspergillus sojae involved in glutamate production during soy sauce fermentation, Biosci. Biotechnol. Biochem., 77, 1832e1840 (2013). 24. Pace, C. N., Vajdos, F., Fee, L., Grimsley, G., and Gray, T.: How to measure and predict the molar absorption coefficient of a protein, Protein Sci., 4, 2411e2423 (1995). 25. Kitamoto, N., Yoshino, S., Ohmiya, K., and Tsukagoshi, N.: Sequence analysis, overexpression, and antisense inhibition of a beta-xylosidase gene, xylA, from Aspergillus oryzae KBN616, Appl. Environ. Microbiol., 65, 20e24 (1999). 26. Ogawa, M., Tokuoka, M., Jin, F. J., Takahashi, T., and Koyama, Y.: Genetic analysis of conidiation regulatory pathways in koji-mold Aspergillus oryzae, Fungal Genet. Biol., 47, 10e18 (2010). 27. Takahashi, T., Hatamoto, O., Koyama, Y., and Abe, K.: Efficient gene disruption in the koji-mold Aspergillus sojae using a novel variation of the positivenegative method, Mol. Gen. Genomics, 272, 344e352 (2004). 28. Wohlfahrt, G., Witt, S., Hendle, J., Schomburg, D., Kalisz, H. M., and Hecht, H. J.: 1.8 and 1.9 Å resolution structures of the Penicillium amagasakiense and Aspergillus niger glucose oxidases as a basis for modelling substrate complexes, Acta Crystallogr. D Biol. Crystallogr., 55, 969e977 (1999). 29. Witt, S., Wohlfahrt, G., Schomburg, D., Hecht, H. J., and Kalisz, H. M.: Conserved arginine-516 of Penicillium amagasakiense glucose oxidase is essential for the efficient binding of beta-D-glucose, Biochem. J., 347, 553e559 (2000). 30. Cole, C., Barber, J. D., and Barton, G. J.: The Jpred 3 secondary structure prediction server, Nucleic Acids Res., 36, W197eW201 (2008).
Please cite this article in press as: Satake, R., et al., Novel glucose dehydrogenase from Mucor prainii: Purification, characterization, molecular cloning and gene expression in Aspergillus sojae, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.03.012
