Improvement in the thermal stability of Mucor prainii-derived FAD-dependent glucose dehydrogenase via protein chimerization

Enzyme and Microbial Technology 132 (2020) 109387

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Improvement in the thermal stability of Mucor prainii-derived FADdependent glucose dehydrogenase via protein chimerization

T

Yosuke Masakari , Chiaki Hara, Yasuko Araki, Keiko Gomi, Kotaro Ito ⁎

Research and Development Division, Kikkoman Corporation, 399 Noda, Noda City, Chiba, 278-0037, Japan

ARTICLE INFO

ABSTRACT

Keywords: FAD-dependent Glucose dehydrogenase Chimeric enzymes Thermal stability

FAD-dependent glucose dehydrogenase (FAD-GDH, EC 1.1.5.9) is an enzyme utilized industrially in glucose sensors. Previously, FAD-GDH isolated from Mucor prainii (MpGDH) was demonstrated to have high substrate specificity for glucose. However, MpGDH displays poor thermostability and is inactivated after incubation at 45 °C for only 15 min, which prevents its use in industrial applications, especially in continuous glucose monitoring (CGM) systems. Therefore, in this study, a chimeric MpGDH (Mr144–297) was engineered from the glucose-specific MpGDH and the highly thermostable FAD-GDH obtained from Mucor sp. RD056860 (MrdGDH). Mr144–297 demonstrated significantly higher heat resistance, with stability at even 55 °C. In addition, Mr144–297 maintained both high affinity and accurate substrate specificity for D-glucose. Furthermore, eight mutation sites that contributed to improved thermal stability and increased productivity in Escherichia coli were identified. Collectively, chimerization of FAD-GDHs can be an effective method for the construction of an FADGDH with greater stability, and the chimeric FAD-GDH described herein could be adapted for use in continuous glucose monitoring sensors.

1. Introduction Currently, more than 350 million people worldwide suffer from diabetes. To avoid serious complications, patients with diabetes must measure and control their blood glucose concentration. Self-monitoring of blood glucose (SMBG) is therefore recommended for patients with diabetes to facilitate glycemic control and to prevent hypoglycemia; frequent SMBG is required to optimize glucose control in patients with serious diabetes. Recently, the development of continuous glucose monitoring (CGM) sensors has revolutionized diabetes management. CGM systems can measure subcutaneous glucose concentrations almost continuously—for example, every 1–5 min, for several consecutive days—greatly increasing the available information on glucose dynamics compared to standard SMBG-based monitoring and thereby improving glycemic control, enhancing quality of life, and reducing diabetes-related complications [1]. Glucose oxidase (GOD, EC 1.1.3.4) is widely used in SMBG sensors and CGM sensors [1,2] because it has high thermostability and substrate specificity. However, GOD requires oxygen as an electron acceptor and dissolved oxygen in the blood influences the accuracy of glucose concentration measurements [3]. To avoid this problem, several glucose dehydrogenases (GDHs)—including NAD(P)-dependent glucose dehydrogenase (NAD(P)-GDH, EC 1.1.1.47), pyrroloquinoline ⁎

quinone-dependent GDH (PQQ-GDH, EC 1.1.5.2), and FAD-dependent GDH (FAD-GDH, EC 1.1.5.9)—are also often utilized in SMBG sensors [4–7]. In particular, FAD-GDH is preferentially used in SMBG sensors because it does not require addition of a coenzyme and has strict substrate specificity [8]. FAD-GDHs derived from various fungal and one bacterial species—Aspergillus terreus [7], Aspergillus oryzae [9–11], Aspergillus flavus [12,13], Aspergillus niger [14], Glomerella cingulata [15], Mucor prainii [16], Mucor sp. RD056860 [17], Mucor hiemalis [18], Pycnoporus cinnabarinus [19], Thermoascus aurantiacus [20], Talaromyces emersonii [21], and Burkholderia cepacia [22]—have been identified. Patients undergoing intravenous drip and xylose absorption tests have maltose and xylose in their blood, which can interfere with glucose readings [23,24]. Mucor-derived FAD-GDHs—which react minimally with maltose and xylose relative to other FAD-GDHs or PQQ-GDHs—are therefore particularly suited to glucose sensors [16]. Numerous studies have aimed to modify specific characteristics of FAD-GDH to facilitate its use in SMBG and CGM sensors. In particular, the lower thermal stability of FAD-GDH relative to that of GOD makes FAD-GDH currently unusable in a CGM sensor. Recent efforts to improve the thermal stability of FAD-GDH utilized a variant with an introduced disulfide bond [25] and variants created by random mutagenesis [26]; however, the engineered FAD-GDHs were insufficiently

Corresponding author. E-mail address: [email protected] (Y. Masakari).

https://doi.org/10.1016/j.enzmictec.2019.109387 Received 15 June 2019; Received in revised form 28 July 2019; Accepted 30 July 2019 Available online 03 August 2019 0141-0229/ © 2019 Elsevier Inc. All rights reserved.

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stable for use in a CGM sensor [25]. Moreover, FAD-GDH wired to an electrode by a redox hydrogel made with an osmium polymer demonstrated higher catalytic performance when deglycosylated by pretreatment with Endo H or sodium periodate than without treatment [27,28]. These reports suggest that it may be preferable to produce unglycosylated GDH in Escherichia coli. In this study, various chimeric enzymes were constructed using FAD-GDH from M. prainii (MpGDH), which has higher substrate specificity, and FAD-GDH from Mucor sp. RD056860 (MrdGDH), which has higher thermal stability. Then, the FAD-GDH construct most suitable for SMBG and CGM sensors was determined. In addition, mutation sites on MpGDH that contributed to the observed improved thermal stability were identified. The expression levels in E. coli of MpGDH and its thermostable variant were also compared.

transformed with each expression plasmid. Recombinant BL21(DE3) E. coli were cultured in test tubes containing 2.5 mL of ZYP-5052 medium (0.5% glycerol, 0.05% glucose, 0.2% lactose, 50 mM (NH4)2SO4, 50 mM KH2PO4, 50 mM Na2HPO4, and 1 mM MgSO4) at 30 °C for 27 h [12,29]. Cells were harvested by centrifuging at 8,000 × g and 4 °C for 10 min, disrupted by ultrasonication in 20 mM potassium phosphate buffer (pH 6.5), then centrifuged at 20,000 × g for 10 min at 4 °C. The supernatants and precipitants were analyzed by SDS-PAGE. SuperSep™ Ace polyacrylamide precast gels (5–20%; FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) were used for electrophoresis and CLEARLY Protein Ladder (Takara Bio) was loaded into the gels as a standard size marker. Proteins were stained with Coomassie Brilliant Blue (CBB) R-250 (Quick-CBB; FUJIFILM Wako Pure Chemical Corporation).

2. Materials and methods

2.5. Enzyme assay

2.1. Construction of Saccharomyces cerevisiae transformants and chimeric enzyme expression vectors The gene coding for FAD-GDH from Mucor prainii (MpGDH) was isolated previously [16], and that from Mucor RD056860 (MrdGDH) was commercially synthesized according to the codon bias of Aspergillus sojae. These genes were cloned into the pYES2/CT vector. Saccharomyces cerevisiae INVSc1 and the vector pYES2/CT are components of the S.c. EasyComp™ Transformation Kit (Thermo Fisher Scientific, Waltham, MA, USA). Chimeric genes from plasmids pYES-MpGDH and pYES-MrdGDH were amplified with primers (Table S1) using PrimeSTAR® Max DNA Polymerase (Takara Bio, Shiga, Japan). DNA fragments were purified using a QIAquick® Gel Extraction kit (QIAGEN, Venlo, Netherlands). The purified DNA fragments were then fused using an In-Fusion® HD Cloning Kit (Takara Bio).

GDH activity was determined using a previously described method [16]. In brief, glucose dehydrogenase activity was assayed in a reaction mixture (1.5 mL) containing 90 mM phosphate buffer (pH 7.0), 0.1 mM 2,6-dichlorophenolindophenol (DCIP) dihydrate, 0.2 mM phenazine methosulfate (PMS), 200 mM glucose, and 50 μL of the enzyme solution at 37 °C. For substrate specificity analysis, enzyme activities were assayed in the presence of 50 mM glucose, 50 mM maltose (Kanto Chemical Co., Inc.; Tokyo, Japan), or 50 mM xylose (Sigma-Aldrich, St. Louis, MO, USA). The activity was calculated by monitoring the decrease in absorbance of DCIP at 600 nm using a U-3900 spectrophotometer (Hitachi High-Tech Fielding Corporation, Tokyo, Japan). One unit of enzymatic activity was defined as the amount of enzyme that caused the reduction of 1 μmol of DCIP per minute under the assay conditions. The Km values were determined by the above method with 0–100 mM concentrations of glucose and fitting the results to the Michaelis-Menten equation.

2.2. Site-directed mutagenesis

2.6. Measurement of thermal stability

Site-directed mutagenesis was performed using a KOD -PlusMutagenesis Kit (Toyobo, Osaka, Japan) with pYES-MpGDH as the template. Mutant PCR products were digested by DpnI (New England Biolabs, Ipswich, MA, USA) at 37 °C for 1 h and introduced into E. coli JM109 (Nippon Gene, Tokyo, Japan).

Enzyme solutions containing the various mutants of FAD-GDHs were each diluted to 1 U/mL using 100 mM potassium phosphate buffer (pH 7.0). Thermal stability of the MpGDH, MrdGDH, and chimeric FADGDHs was determined after incubation for 15 min at a range of temperatures from 35 °C to 60 °C. Glucose oxidase (Type VII from Aspergillus niger, Sigma-Aldrich) was also incubated for 15 min at a range of temperatures from 50 °C to 60 °C. The residual activity was calculated as activity relative to the initial activity. All reactions were performed in triplicate.

2.3. Enzyme production in S. cerevisiaeand purification INVSc1 cells containing recombinant plasmids were grown at 30 °C for 24 h using Sc-U medium (6.7 g/L yeast nitrogen base without amino acids and 1.92 g/L Yeast Synthetic Drop-out Medium Supplements without uracil) containing raffinose (20 g/L). Subsequently, 1 mL of the pre-culture liquid was added to 4 mL of Sc-U medium containing raffinose (7.5 g/L) and D-galactose (25 g/L), then cultured at 30 °C for 16 h. Cells were pelleted by centrifugation for 10 min at 2,500 × g, then supernatants were harvested and buffer-exchanged into 100 mM potassium phosphate (pH 6). These supernatants were used for GDH enzyme assays. FAD-GDHs were purified by size-exclusion chromatography using a HiLoad Superdex200 26/60 column (GE Healthcare, Chicago, IL, USA) previously equilibrated with 20 mM potassium phosphate buffer and 150 mM NaCl, pH 6.

2.7. 3D model construction A 3D structural model of MpGDH was generated by homology modeling using the SWISS-MODEL [[30], https://swissmodel.expasy. org/interactive]. The crystal structure used as template was FAD-dependent glucose dehydrogenase from A. flavus (PDB ID: 4YNU). PyMol 0.99rc6 was used for molecular visualization. 3. Results 3.1. Construction of chimeric GDHs and evaluation of their thermal stability

2.4. Construction of E. colitransformants and enzyme production

Fig. 1 shows the multiple sequence alignment of MpGDH and other known FAD-dependent glucose dehydrogenases, including MrdGDH from Mucor RD056860, AfGDH from A. flavus [12,13], TaGDH from Thermoascus aurantiacus NBRC 6766 [20], and glucose oxidase from A. niger (AnGOD) (PDB ID: 1CF3). MpGDH exhibited low sequence identity (about 30%) with AfGDH, AtGDH, and AnGOD, but MpGDH comprises highly conserved regions near the FAD binding motif and the key active-site residues, His570 and His613 [16,31,32]. It is difficult to

The expression vector pET-22b(+) (EMD MilliporeSigma, Burlington, MA, USA) and BL21(DE3) competent E. coli (Nippon Gene) were used to produce unglycosylated recombinant enzymes. The codonoptimized fad-gdh for MpGDH, MpGDH-8 T, and MrdGDH were obtained by artificial gene synthesis, then were inserted into the pET-22b (+) vector using an In-Fusion® HD Cloning Kit. BL21(DE3) E. coli were 2

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Fig. 1. Comparison of amino acid sequences between MpGDH, other known FAD-dependent glucose dehydrogenases, and glucose oxidase. Multiple sequence alignment was performed using ClustalW. MrdGDH, AfGDH, and TaGDH are FAD-dependent glucose dehydrogenases derived from Mucor RD056860, Aspergillus flavus, and Thermoascus aurantiacus, respectively. AnGOD is a glucose oxidase derived from Aspergillus niger. Conserved amino acid residues are framed. Arrows indicate the points of recombination. Diamonds represent the site-specific mutagenized residues used in this study. Arrowheads show the active sites in AfGDH and AnGOD, closed arrowheads indicate the essential residues for AfGDH and AnGOD activity, and open arrowheads indicate the other residues.

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Fig. 3. Thermal stabilities of MpGDH, MrdGDH, and chimeric FAD-GDHs. Each FAD-GDH sample (1 U/mL) was incubated at the indicated temperatures for 15 min in 100 mM potassium phosphate buffer (pH 7.0). The residual activities are indicated as relative to the untreated samples. n = 3. *P < 0.05, **P < 0.01 versus MrdGDH, Student’s t-test.

Fig. 2. Construction of chimeric FAD-GDHs used in this study. White bars and gray bars indicate the regions derived from Mucor prainii FAD-GDH and Mucor RD056860 FAD-GDH, respectively. Upper numbers refer to amino acid positions in MpGDH. Lower numbers refer to amino acid positions in MrdGDH. MpGDH, MrdGDH, and all of the chimeric variants were expressed in S. cerevisiae INVSc1.

Table 1 Substrate specificity, specific activity, and Km values of D-glucose for MpGDH, MrdGDH, and Mr144–297.

construct a chimera using, for example, MpGDH and TaGDH, because Mucor-derived GDH contains characteristic sequences that other GDHs and GODs do not possess. While MpGDH and MrdGDH have 73% sequence identity, MpGDH has lower thermal stability and higher substrate specificity for glucose than does MrdGDH [16,17]. However, since no information regarding which regions of MpGDH contribute to stability exists, several chimeric enzymes were constructed to find regions responsible for stability (Figs. 1 and 2). First, MpGDH was replaced stepwise from its N-terminus with the corresponding sequence of MrdGDH—for example, Mr1–67 consisted of MrdGDH residues 1–67 and MpGDH residues 72-641. Amino acid positions showing high homology between MpGDH and MrdGDH were maintained to avoid constructing a chimeric GDH whose structure was markedly collapsed (Fig. 1). The native MpGDH, MrdGDH, and all chimeric GDHs constructed in this study were functionally expressed in S. cerevisiae. Under neutral pH conditions, MpGDH was completely inactivated after heat treatment at 45 °C, whereas MrdGDH was stable up to 50 °C (Fig. 3). The chimeric GDHs displayed a range of stabilities (Fig. 3). Since Mr1–67 and Mr1143 were approximately as stable as MpGDH, the mutation sites contributing to high thermal stability were likely not in the N-terminus of MrdGDH. In contrast, Mr1-297 had higher thermostability than MpGDH. Subsequently, Mr144–297—in which positions 1 to 143 of Mr1-297 were replaced with positions 1 to 147 of MpGDH—was constructed. After incubation at 60 °C for 15 min, the residual activities of MrdGDH, Mr1-297, and Mr144–297 were 0.1%, 19.8%, and 47.6%, respectively. Mr144–297 showed the highest stability, strongly suggesting that the significant region in MrdGDH for stabilization was from Phe144 to Leu297.

Enzyme variant

Relative activity (%)a

MpGDH MrdGDH Mr144-297

0.47 ± 0.04 0.99 ± 0.20 892 ± 38 2.32 ± 0.05 1.53 ± 0.03 537 ± 7 0.41 ± 0.01 1.11 ± 0.11 546 ± 7

maltose

Specific activity (U/mg)b

Km (mM)

xylose 39.9 ± 0.8 12.4 ± 3.3 36.6 ± 3.4

Values are presented as mean ± standard deviation. Activity of each FAD-GDH for D-glucose was set as 100%. a Relative activity was measured after incubation in 90 mM phosphate buffer (pH 7.0) with 50 mM substrate at 37 °C. b Specific activity was measured after incubation in 90 mM phosphate buffer (pH 7.0) with 200 mM glucose at 37 °C.

2.32 ± 0.05%, respectively. As with MpGDH, the chimeric GDH Mr144–297 displayed minimal activity with maltose (Table 1). The relative activities of MpGDH, MrdGDH, and Mr144–297 were all under 2% with xylose; Mr144–297 was slightly less reactive toward xylose than was MrdGDH. The Km values of Mr144–297 and MpGDH for Dglucose were almost equal, demonstrating that Mr144–297 maintained the high substrate specificity of MpGDH. The specific activity of Mr144–297 was the same as that of MrdGDH, suggesting that amino acid residues involved in the catalytic efficiency were present in the region from Phe144 to Leu297. 3.3. Identification of amino acid residues that contribute to improved stability To precisely investigate the thermostability-enhancing mutations, the amino acids that differed between MpGDH and Mr144–297 were compared. Stability tests for three chimeric GDHs—Mr144-186, Mr187236, and Mr237-297—were performed. Mr144-186 and Mr237-297 were slightly more stable than MpGDH, but all three GDHs were almost completely inactivated after heat treatment at 45 °C (Fig. 4A). In contrast, the stability of Mr187-236 was much higher than that of either Mr144-186 or Mr237-297. Subsequently, to identify the amino acid residues that contributed to the improved thermal stability, residues 191–240 of MpGDH and Mr187-236 were compared and found to have 20 differing amino acids (Fig. 2). Each differing residue was replaced

3.2. Substrate specificity, specific activity, and Km It is crucial that methods of blood glucose measurement are specific to glucose, and therefore, do not respond to other saccharides, especially maltose (23). The relative activities of MpGDH and MrdGDH with maltose relative to those with D-glucose were 0.47 ± 0.04% and 4

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Fig. 4. (A) Thermal stabilities of MpGDH, octuple mutant (MpGDH-8 T), and chimeric FAD-GDHs. Each FAD-GDH sample (1 U/mL) was incubated at the indicated temperatures for 15 min in 100 mM potassium phosphate buffer (pH 7.0). n = 3. *P < 0.05, **P < 0.01 versus MrdGDH, Student’s ttest (B) Thermal stabilities of single-site MpGDH mutants. Residual activities of FAD-GDHs were assayed after heat treatment at 40 °C for 15 min. n = 3. *P < 0.05, **P < 0.01 versus MpGDH, Student’s t-test.

with the corresponding amino acid in MrdGDH using site-directed mutagenesis. Each of the mutants S192 P, S196 N, I212 L, A218H, I226 T, D228 N, V232A, and Q233R was more thermostable than was wild-type MpGDH—I226 T was particularly thermostable (Fig. 4B). To investigate the effects of combinatorial mutations, an octuple mutant (MpGDH-8 T) was obtained by site-directed mutagenesis. MpGDH-8 T showed thermostability as high as that of the chimeric GDH Mr187-236 (Fig. 4A).

then the soluble and insoluble fractions were analyzed by SDS-PAGE (Fig. 5). Insoluble fraction samples of both GDHs resulted in a strongly stained band of about 60,000 Da. MrdGDH was also detectable in the insoluble fraction (Fig. 5). In contrast, a 60,000 Da band was solely detectable in the soluble fraction of MpGDH-8 T. The productivity of MpGDH and MpGDH-8 T expressed in E. coli cells was 0.0066 ± 0.0053 U/mL and 5.0 ± 0.2 U/mL, respectively, indicating the productivity of MpGDH-8 T was approximately 750-fold higher than that of MpGDH. Interestingly, the productivity of MrdGDH expressed in E. coli cells was 0.93 ± 0.90 U/mL, which was approximately 5-fold lower than that of MpGDH-8 T.

3.4. Expression of MpGDH-8T in E. coli The signal peptides of MpGDH and MrdGDH were each predicted to be 20 amino acids from their respective N-terminus by the SignalP server [[33], http://www.cbs.dtu.dk/services/SignalP/] and were therefore removed from the sequences of MpGDH, MpGDH-8 T, and MrdGDH prior to E. coli expression. E. coli cells overexpressing wildtype MpGDH or the thermostable variant MpGDH-8 T were prepared,

4. Discussion Using a simple chimerization method, variants of MpGDH were engineered to greatly improve its stability. Mr144–297—which was engineered to replace residues 144–297 with the corresponding Fig. 5. SDS-PAGE of recombinant MpGDH, MpGDH-8 T, and MrdGDH. All FAD-GDHs were expressed in E. coli, then disrupted by ultrasonication and separated into supernatant and precipitant fractions. The volume of supernatant loaded into a gel is 5% relative to that of precipitant. The arrow indicates the expected molecular weight of MpGDH and MrdGDH. M: molecular mass markers; sup: supernatant fraction; ppt: precipitant fraction.

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Fig. 6. (A) Overall 3D structure of the MpGDH model, determined by SWISS-MODEL using the structure of FAD-GDH derived from A. flavus (AfGDH, PDB ID: 4YNU) as a template. Pink: Phe148 to Phe191, Arg244 to Ile300; cyan: Ser192 to Gln233; orange: side chains of S192, S196, I212, A218, I226, D228, V232, and Q233; yellow (sticks): FAD; red (sticks): D-glucono-1,5-lactone. (B) Crystal structure surrounding AfGDH Thr202. Water molecules (red spheres) interact with Asn93 and Tyr554. The dashed lines indicate hydrogen bonds. (C) 3D structural model around Ile226 of MpGDH.

residues of MrdGDH—was the most thermostable chimeric GDH. Mr144–297 was more thermostable than AnGOD, which is used in current CGM sensors [[34], Fig. S1]. Based on the investigation of thermal stability of AnGOD, the parameters for its thermal inactivation were elucidated, and the half-life of AnGOD was estimated to be 9 days at 37 °C using the Arrhenius plot [25,35]. Therefore, Mr144–297 is expected to be stable for more than 9 days at 37 °C. Previous attempts have utilized random mutagenesis screening and DNA shuffling as methods to improve thermal stability, but these methods may also affect other functions [36]. Designing chimeric enzymes by crossover in homologous regions of enzymes with high sequence similarity and known properties is a powerful alternative approach for enzyme stabilization [37,38]. Mr144–297 had good substrate specificity due to its minimal reactivity to both maltose and xylose (Table 1). The 3D model of MpGDH—predicted based on the tertiary structure of FAD-GDH derived from A. flavus (AfGDH) [31]—suggests that the region from Phe148 to

Ile300 of MpGDH is located on the molecular surface and is far from the active site (Fig. 6A, pink and cyan). The predicted location of these residues is consistent with the observation that their substitution with Phe144 to Ile297 of MrdGDH did not affect substrate specificity but did increase stability. Since it is crucial for a therapeutically relevant GDH to not react to maltose and xylose in blood glucose sensors, Mr144–297 could prove very useful. Future work will focus on testing the stability of a sensor utilizing Mr144–297 and confirming its efficacy in accurately measuring blood glucose concentration in interstitial fluid. Subsequently, the region corresponding to MrdGDH in Mr144–297 was divided into three, and site-specific mutations were introduced to identify the amino acid residues responsible for improving thermostability (Fig. 4). This detailed investigation of Mr144–297 revealed that the mutations that contributed most to improved thermostability were concentrated in the region from Ser192 to Gln233 of MpGDH (Fig. 4B). However, Mr187-236 and MpGDH-8 T were still less stable than Mr144–297, indicating that thermal stability could be further 6

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improved by changes to other regions (Figs. 3 and 4A). The positions of these eight identified residues on the three-dimensional structure of MpGDH were inferred from the model structure (Fig. 6A, cyan). The side chains of Ser192, Ser196, Ala218, Asp228, and Gln233 were predicted to be present on the molecular surface. Substitution of threonine for isoleucine at residue 226 contributed the most to improvement of thermal stability. Thr226 is completely conserved in the other GDHs, while AnGOD and Pro192 are conserved in the thermostable GDH, TaGDH (Fig. 1). In addition, although the overall amino acid sequence homology between MpGDH and AfGDH is approximately 31%, the amino acid residues around Ile226 of MpGDH were predicted to be similar to those of AfGDH (Fig. 6B and C). According to the structure of AfGDH, Thr202—which is predicted to correspond to Ile226 in MpGDH—forms water-mediated hydrogen bonds with Asn93 and Tyr554 (Fig. 6B). Intramolecular hydrogen bonds contribute to protein stability [39,40]. The residues corresponding to Asn93 and Tyr554 in AfGDH are Asn119 and Tyr619 in MpGDH; therefore, if the I226 T substitution enabled formation of new hydrogen bonds via Asn119 and a water molecule, this could explain the improved observed thermal stability. An I226S mutant also displayed stability that was improved to the same degree as in the I226 T mutant (Fig. S2). However, a T222I substitution in MrdGDH worsened the thermal stability (Fig. S3). The additional hydroxyl group introduced by I226 T or I226S is important for enhancing thermal stability, likely because it supports the formation of new hydrogen bonds. AfGDH can be overexpressed in E. coli and detected in the soluble fraction, although it is comparatively difficult to express several other FAD-GDHs (12)—including MpGDH (Fig. 5). Unglycosylated enzymes, such as those expressed in E. coli, have higher reactivity when used in electrochemical sensors [27,28,41,42]. In this study, the expression level of MpGDH-8 T in E. coli cells was much higher than that of wildtype MpGDH. Protein deactivation induced during culturing can be suppressed by improving the thermal stability, and thereby result in high expression [43]. However, two T. aurantiacus-derived GDHs have minimal expression as soluble proteins in E. coli despite high heat resistance [20]. Additionally, it has been reported that the relationship between thermal stability and expression level is low and that solubility is important for increasing expression level in other proteins [44–46]. The thermal stability of MrdGDH was slightly lower than that of MpGDH-8 T and the expression level in E. coli was 5-fold lower (Fig. 4A). In the predicted structure of MpGDH-8 T, His218 and Arg233 are located on the molecular surface, suggesting they might affect solubility. In fact, when positions 218 and 233 in MpGDH-8 T were substituted with hydrophobic amino acids (H218 F/R233 V), the expression level in E. coli decreased by approximately 60% (data not shown). These results suggest that both stability and surface hydrophilicity of an enzyme are important factors for its robust expression in E. coli. Although MrdGDH had the equivalent corresponding residues—His214 and Arg229—the expression level of MrdGDH was lower than that of MpGDH-8 T. Therefore, it would likely be necessary to compare the crystal structures of both MpGDH and MrdGDH to accurately elucidate the exact mechanism behind the enhanced expression in E. coli. In conclusion, chimerization of MpGDH and MrdGDH made it possible to construct Mr144–297, which was the most thermostable Mucorderived FAD-GDH studied and had excellent substrate specificity. The main amino acid residues responsible for thermal stability were also successfully identified. In the future, substitution with other amino acids at these residues may further improve stability. Moreover, the secondary structure surrounding these mutations is presumed to be highly similar to that of other FAD-GDHs and GOD from Penicillium amagasakiense [16]. Therefore, similar mutations might be utilized successfully to improve the thermal stability of GOD and FAD-GDHs other than MpGDH. In addition, the expression level of MpGDH-8 T in E. coli was improved drastically relative to that of wild-type MpGDH. The expression level of MpGDH was improved by mutation, suggesting the possibility of applying the mutation protocol described herein to

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