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Identification and characterization of a novel Old Yellow Enzyme from Bacillus subtilis str.168
Accepted Manuscript Title: Identification and characterization of a novel Old Yellow Enzyme from Bacillus subtilis str.168 Author: Xiqian Sheng Ming Yan Lin Xu Miao Wei PII: DOI: Reference:
S1381-1177(16)30069-8 http://dx.doi.org/doi:10.1016/j.molcatb.2016.04.011 MOLCAB 3359
To appear in:
Journal of Molecular Catalysis B: Enzymatic
Received date: Revised date: Accepted date:
19-1-2016 25-4-2016 26-4-2016
Please cite this article as: Xiqian Sheng, Ming Yan, Lin Xu, Miao Wei, Identification and characterization of a novel Old Yellow Enzyme from Bacillus subtilis str.168, Journal of Molecular Catalysis B: Enzymatic http://dx.doi.org/10.1016/j.molcatb.2016.04.011 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Identification and characterization of a novel Old Yellow Enzyme from Bacillus subtilis str.168 Xiqian Sheng; Ming Yan; Lin Xu; Miao Wei College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing, 211816, People's Republic of China *Corresponding author: Ming Yan, e-mail: [email protected] *Corresponding author. TEL.: +86-138-140-91121
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Graphical abstract
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Highlights: 1. Identifing a new “thermophilic-like” OYEs from Bacillus subtilis str.168。 2. Exhibiting high tolerance towards temperature and pH. 3. Exhibiting high activities towards maleimide, dimethyl maleate and acyclic ketones. 4. The YqiG can catalyze citral to (S)-citronellal with high e.e % ( >99%).
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Abstract We identified and characterized YqiG, a novel Old Yellow Enzyme (OYE) from Bacillus subtilis str.168, as a member of the “thermophilic-like” subfamily. It is most related to XenA from Pseudomonas putida, with 37.9% identity, but it exhibits certain differences in sequence and enzyme properties. The YqiG can reduce various activated alkenes and exhibits high temperature (60℃ for 12 h) and pH stability (pH 4.0 and 9.0 for 12 h), which indicates that it has a great potential for biocatalysis. However, it shows low tolerance toward organic solvents. Also the YqiG shows high activities towards maleimide, dimethyl maleate and acyclic ketones (55.5U/mg, 6.22U/mg and 2.97U/mg, respectively). Besides, The YqiG can catalyze citral to (S)-citronellal with a low rate but high ee % (> 99%).
Keywords: Old Yellow Enzyme, Thermophilic-like family, YqiG; Enzyme properties, Ene-reductase
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1. Introduction Old Yellow Enzyme [EC 1.6.99.1] was the first enzyme shown to contain flavin as a prosthetic group and was originally isolated from brewer’s bottom yeast by Warburg and Christian in 1933. In recent years, the OYE family has grown rapidly, and numerous OYE homologues with different characteristics have been identified [1]. Over the past 80 years of research, the OYEs have exhibited undeniably great potential for asymmetrically reducing a broad range of activated alkenes because they can generate up to two chiral centers when catalyzing C=C bond reductions [2, 3]. The OYEs are widely distributed in nature[4], especially in plants[5], fungi and bacteria[6-9]. These enzymes feature a broad substrate spectrum, including α, β-unsaturated aldehydes, ketones, nitriles, imides, nitro aromatics and carboxylic acids [1, 8, 10]. The enoate reductase stereospecifically reduces the C=C bond through hydrogen atom anti-addition [2, 11] . In 2010, Toogood et al. classified the OYEs into two categories: the “classical” family and the “thermophilic-like” family[1]. The “thermophilic-like” family often exhibited higher enzyme properties than the “classical” family [12], such as higher stability of pH and temperature. One difference between the two subfamilies is that the “classic” family usually forms monomers or dimers such as OYE1 from Saccharomyces pastorianus[13], 12-oxophytodienoate reductase from plants and MR from bacterica. but the “thermophilic-like” family often forms oligomers[1, 14]. Until now, the vast majority of OYE enzymes discovered belong to the “classic” family[5]. Only a few OYE members are “thermophilic-like” enzymes, including YqjM (P54550) 5
from Bacillus subtilis[15], XenA (AAF02538) from Pseudomonas putida[6], Chr-OYE3 (KJ019329) from Chryseobacterium sp. CA49[16], TOYE (ZP_00777979) from Thermoanaerobacter pseudethanolicus E39[17] , GeoOYE (Q5KXG9) from Geobacillus
kaustophilus[18]
and CrS
(YP_004203660)[19]
from Thermus
scotoductus. Based on the above sequences, we found a novel “thermophilic-like” OYE referred to as YqiG. Here, we identify and characterize YqiG from Bacillus subtilis; this is a new “thermophilic-like” OYE identified from Bacillus subtilis after YqjM (the first characterized “thermophilic-like” family OYE).
2. Materials and methods 2.1 Chemicals and solvents The chemicals and solvents used as substrates are analytical grade and from Aladdin. All of the enzymes used for cloning (including DNA polymerase, T4 DNA ligase and restriction enzymes) were purchased from Takara Biotechnology. The pET-22b (+) was from our own lab. 2.2 Cloning the yqiG gene from bacillus subtilis subst. subtilis. str. 168 The bacillus subtilis strain used as a PCR template was purchased from ATCC, and the yqiG DNA sequence was obtained from the UNIPROT database. The PCR experiments were performed using PrimerStar® DNA polymerase (forward primer, pET-22b (+): 5’- GGAATTCCATATGAATCCTAAGTATAAGCCA-3’; reverse primer: 5’- CATGCCATGGTTAATCTTTATAAGGCACCCA-3’). The reaction began with an initial denaturing step for 300 s at 95℃ followed by 30 cycles of 10 s at 98℃, 6
15 s at 56℃, and 90 s at 72℃ with a final extension step at 72℃ for 300 s. The yqiG gene was integrated into pET-22b (+) using the restriction sites NdeI and NcoI. The constructed vectors were transformed into E. coli DH5α. The plasmids were also transformed into E. coli BL21 (DE3) for expression after the vectors were sequenced 2.3 YqiG expression and purification Five milliliters of Luria-Bertani (LB) medium (1% tryptone, 0.5% yeast extract, and 1% NaCl) supplemented with 100 mgL-1 ampicillin was inoculated with a single YqiG colony for 12 h at 37℃. The YqiG-containing cells were then subcultured using 100 ml LB medium supplemented with 100 mgL-1 ampicillin at 37℃. Induction was initiated with the addition of 0.4mM isopropyl-β-D-thiogalactopyranoside (IPTG) at an OD600 of 0.6-0.8, and the cells were incubated for another 12h at 30℃. Cells harvested by centrifugation were suspended in 100mM potassium phosphate (pH7.0) and then broken with high pressure homogenizer (APV, Holland). Cell debris was removed by centrifugation at 12,000 rpm for 30min at 4℃.And then the supernatant was purified on ÄKTAprime plus (USA) with HiTrap DEAE FF column. The column was equilibrated with 20mM Tris-HCl buffer (pH7.0) containing 1M NaCl. The purified YqiG was stored at 4℃. 2.4 Protein analysis Protein concentration was determined with the BCA Protein Assay Kit (CWBIO, China). The molecular mass and the purity of the purified protein were estimated by SDS-PAGE. Free flavin was also determined by the Spectra Max M2. Free flavin 7
would be released after incubating in 100℃ boiling water bath for 10min. Molecular mass of native YqiG was determined using an ÄKTA purifier system with a Superdex 200pg 16/600 column. The flow rate for protein elution is 0.8ml/min. Thyroglobulin (670kDa), globulin (158kDa), ovalbumin (44kDa), myoglobin (17kDa), VB12 (1.35kDa) were used as molecular mass standards. 2.5 Enzyme assay The dimethyl maleate (DMM) was used as substrate for the determination of the enzymatic properties, and all measurements were used with purified YqiG on a Multimode Plate Reader (Spectra Max M2, Molecular Devices Corporation). And these experiments were implemented at least in triplicate. The enzyme activity was detected by monitoring NAD(P)H oxidation at 340 nm. These reactions were performed in 200µl system containing 10 mM substrate, 1 mM NADPH, 5 µl YqiG and phosphate buffer (100 mM, pH 7.0) at 30℃. To avoid a false positive NADPH consumption, we added two controls: the same reaction mixture without substrates and the same reaction mixture without YqiG. One unit of enzyme activity was defined as 1 μmol of NADPH oxidized per minute. To determine the influence of pH, we used sodium acetate (pH 4-6), potassium phosphate (pH 6-8), Tris-HCl (pH 8 -9) and sodium carbonate (pH 10) to determine the pH optima and the pH stability. And a temperature range of 20-60℃ was used for the determination of the optimum temperature. The effect of organic solvents was studied using ethanol, butyl acetate, isopropanol, n-hexanol, n-amyl alcohol, n-butanol, ethyl acetate, methanol, dimethyl sulfoxide (DMSO), iso-pentol alcohol 8
and n-hexane. The kinetic parameters were determined using the Michaelis-Menten equation, and 1 mM NADPH remained a constant with the substrate added at between 0.25-2 mM. We used maleimide as substrate when determined the kinetic parameters of NADPH and NADH. 2.6 Analytical procedures The product and enantiomeric excess (e.e%) were analyzed using the Aglient 7820 gas chromatograph (GC) equipped with a flame ionization detector (FID) and the FS-column HYDRODEX β-TBDAC (25 m,0.25 mm). 3. Results 3.1 Sequence and phylogenetic analyses The YqiG amino acid sequence was identified in the National Center for Biotechnology information (NCBI) databank using the basic local alignment search tool (BLAST). A phylogenetic analysis was performed for the YqiG with sixteen known OYE enzymes (Fig. 1). The results show that the YqiG did not cluster with other OYE enzymes but shared 37.9% identity with XenA from Pseudomonas putida. A phylogenetic tree shows that the YqiG belongs to the “thermophilic-like” subclass. A sequence analysis was performed for YqiG with twelve known OYE enzymes (Fig. 2). The YqiG consists of 373 amino acids with the predicted molecular weight 40 kDa. The sequence results showed certain shared invariant residues only within this subfamily, such as M37, Y40 and L98 []. But the YqiG still showed different positions with the “thermophilic-like” subclass, such as C38, R353 and R391. The data indicate that YqiG may be more similar to the “thermophilic-like” subclass than the “classic” 9
class. 3.2 Protein purification The protein was successfully expressed in E. coli BL21 (DE3) and purified using the HiTrap DEAE FF column. The purified enzyme presented the typical OYE yellow color. An SDS-PAGE analysis of the purified YqiG (Fig. 3) showed that its molecular mass is approximately 40 kDa. The native YqiG exhibited only one molecular mass around 40kDa. The results indicates that YqiG maybe existed as monomers. 3.3 Flavin content determination We obtained bright yellow supernatant after boiling for 10min and centrifugation for 5min. The UV-visible absorbance spectra of purified YqiG and free flavin released are shown in Fig.4. The spectrum of YqiG indicates a typical flavin-containing protein, exhibiting the maximum absorbance at 375 and 470nm with shoulders at 430 and 490nm [20]. The released flavin after protein denaturation showed absorbance maxima at 449nm, which was identical to the maximum of FMN. The results suggested that YqiG was FMN-containing protein. 3.3 Enzyme properties 3.3.1 Substrate spectrum To investigate the YqiG substrate spectra, 19 substrates with different electron withdrawing groups, including imides, α, β-unsaturated aldehydes, ketones, nitroalkenes, carboxylic acids and their derivatives, were tested in potassium phosphate buffer (pH 7.0). The activities toward the substrates are summarized in 10
Table 1. The YqiG was active toward imides, α,β-unsaturated aldehydes and ketones but not nitroalkenes and show little activity toward carboxylic acids. Most OYEs feature high activities toward cyclic ketones [7, 17, 21-23], but YqiG only exhibited little activity toward cyclic ketones, such as ketoisophorone (only 0.55 U/mg). However, YqiG showed high activities toward maleimide, dimethyl maleate and acyclic ketones, and the activity towards maleimide reached 55U/mg, which is higher than most other OYEs[24]. 3.3.2 Bioreduction with coenzyme regeneration To investigate YqiG enantioselectivity, asymmetric bioreduction with certain substrates was performed using a coenzyme regeneration system and glucose dehydrogenase at 30℃, and the ee values and conversions were detected and summarized in Table. 2. The YqiG can catalyze citral to (S)-citronellal with a low rate but high e.e % (> 99%). Also the YqiG showed high e.e.% (96%) but only 22% conversion toward ketoisophorone. 3.3.3 Determining kinetic parameters The kinetic parameters were detected through NADPH oxidation at 340 nm using a 200-µl system with 30 µg of enzyme, 1 mM NADPH, 0.2-2 mM substrate and phosphate buffer (100 mM, pH 7.0). The substrates were representatives for the substance classes: imides, α, β-unsaturated aldehydes, ketones and carboxylic acids. The kinetic parameters of substrates and NADPH are shown in Table 3. The results clearly showed that YqiG could only accept NADPH as cofactor. The kcat of NADPH reached 1788 whereas the kcat of NADH could not be detected. Moreover, the YqiG 11
clearly exhibited the greatest affinity for maleimide and Dimethyl maleate and the lowest affinity for mesaconic acid and citral. Also, the kcat/Km of 2-Cylohexen-1one was 21.8 s-1mM-1, which was higher than YqjM (15 s-1mM-1), MR (0.19 s-1mM-1) and TOYE (0.5 s-1mM-1) [14, 15, 17]. 3.3.4 The optimum temperature and temperature stability The effects of temperature were tested between 20℃ and 70℃ (Fig. 5A). The YqiG exhibited high activity between 30℃ and 45℃. After a water bath for 12 h, residual activity was used to determine the temperature stability of YqiG (Fig. 5B). The loss of activity was less than 10% between 30℃ and 40℃, whereas the residual activity rapidly decreased with an increase in temperature. 3.3.5 The optimum pH and pH stability The effects of pH were tested over a broad range, pH4-9 (Fig.6A). YqiG showed a broad pH range at pH 5.5 to pH 9.0 and the most activity between pH 6.5 and pH 7.5. After incubating in buffers with different pH values for 12 h, the residual activity was used to determine the YqiG pH stability (Fig. 6B). Over 75% of the activity remained between pH 6.0 and pH 9.0, and the residual activity rapidly decreased with a decrease in pH from pH 6.0 to pH 4.0. 3.3.6 Effect of different ions. The concentration of all ions were 5 mM in the 200-µl system (Fig. 7). No activity was detected when Fe2+, Cu2+ and Mn2+ were added to the reaction system. The reason might be that Fe2+, Cu2+ and Mn2+ were all strong oxidizing ions, which could consume much more oxygen than FMN. The effects of the remained eight salts 12
are shown as a fig. Clearly, Mg2+ increase the enzyme activity, but EDTA, Li+, Na+, K+ and Ba2+ exhibited no active effect on YqiG. 3.3.7 Effect of organic solvents Organic solvents are often used in biocatalysis to increase substrate solubility and product separation. We investigated the solvent stability of YqiG in several solvents with various logP values (from -1.3 to 3.5) at the concentrations 5%, 10% and 20% in the 200-µl system. Activity was not detected when ethanol, isopropanol, n-amyl alcohol, methanol and iso-pentol alcohol were added. The remaining six organic solvents exerted different effects on YqiG activity, as shown in Fig. 8. The data show that YqiG exhibits better activity in DMSO, which is a water-miscible solvent with a -1.3 logP. However, these organic solvents are not suitable for YqiG because it could not retain 60% of its activity at a solvent concentration of 20%, which suggests low tolerance toward organic solvents.
4. Discussion In this paper, a novel OYE from bacillus subtilis subst. subtilis. str. 168 referred to as YqiG was successfully cloned into BL21 (DE3) and purified using an ion exchange column. The YqiG featured a broad substrate spectrum, including α, β-unsaturated aldehydes, ketones, imides, carboxylic acids, and nitro aromatics. The YqiG showed high activity with imides but no obvious activity with nitro aromatics. A detailed study on “thermophilic-like” enzymes revealed that “thermophilic-like” enzymes exhibited greater stability and resistance in various aspects compared with 13
“classic” enzymes [17]. The optimum temperature for YqiG is 40℃,and it can tolerate 60℃ for 12 h with a loss of less than 50% enzyme activity. Further, YqiG exhibits a broad pH range. It can tolerate the pH values 6-9 for 12 h and retain more than 50% of its enzyme activity, which indicates that YqiG can maintain better catalytic activity for a considerably long time. Further, the above are characteristics of the “thermophilic-like” family, such as with Chr-OYE3, which can tolerate 55℃ for approximately 100 h until no activity remained[16]. Thus, we preliminarily classified the YqiG into the “thermophilic-like” family based on the phylogenetic tree and enzyme properties. YqiG exhibited low resistance toward various types of organic solvent, whereas other “thermophilic-like” enzymes feature a great resistance range [16, 19]; perhaps this discrepancy is due to the sequence difference of YqiG compared with the two families. It was often thought that members in “classic” family often exist as monomers or dimers whereas members in “thermophilic-like” family typically exist as higher oligomeric states[1]. But the molecular mass of native YqiG showed that YqiG only exist as monomers. The reason maybe that some residues about the subunit numbers are different from the “thermophilic-like” family but similar to the “classic” family. Further, YqiG features some other protein structure differences compared with “thermophilic-like” enzymes, in which protein thermostability plays an important role [25]. The “theromophlic-like” enzymes feature certain structural characteristics, such as shorter chain length (337-350 amino acids), high oligomeric dimer and tetramer 14
states and lower thermoliable residue levels. YqiG is 373 amino acids longer than other “thermophilic-like” enzymes, such as TOYE (337 amino acids), GeoOYE (340 amino acids), Chr-OYE3 (350 amino acids) and YqjM (338 amino acids). But the results of sequence analysis and the phylogenetic tree suggest that YqiG differs from the classic families in the key position, but closes to the “thermophilic-like” family. After the comparison between YqiG and the two subfamilies, it is obviously that there are more similarities between the YqiG and the “theromophlic-like” family than the “classic” family, such as temperature, pH, and some key positions, which indicates that YqiG should be classified into the “thermophilic-like” family.
5. Conclusions We have identified a novel OYE of the “thermophilic-like” family from Bacillus subtilis str.168 named YqiG, and we characterized this OYE as an ER that can reduce C=C bonds of various activated alkenes. Further, its high temperature and pH stability indicates a great potential for biocatalysis. Although the physiological role of YqiG is unclear, given the substrate spectrum and certain enzyme properties, it may be useful for further research and applications of this subfamily of enzymes.
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Acknowledgments This work was supported by the National Basic Research Program of China under Grant No. 2011CBA00804.
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Reference [1] H.S. Toogood, J.M. Gardiner, N.S. Scrutton, ChemCatChem, 2 (2010) 892-914. [2] M. Hall, C. Stueckler, H. Ehammer, E. Pointner, G. Oberdorfer, K. Gruber, B. Hauer, R. Stuermer, W. Kroutil, P. Macheroux, K. Faber, Advanced Synthesis & Catalysis, 350 (2008) 411-418. [3] E. Brenna, F.G. Gatti, D. Monti, F. Parmeggiani, S. Serra, Advanced Synthesis & Catalysis, 354 (2012) 105-112. [4] A. Fryszkowska, H. Toogood, M. Sakuma, J.M. Gardiner, G.M. Stephens, N.S. Scrutton, Adv Synth Catal, 351 (2009) 2976-2990. [5] K. Shimoda, N. Kubota, H. Hamada, Tetrahedron: Asymmetry, 15 (2004) 2443-2446. [6] Y. Yanto, H.H. Yu, M. Hall, A.S. Bommarius, Chemical communications, 46 (2010) 8809-8811. [7] N. Richter, H. Groger, W. Hummel, Applied microbiology and biotechnology, 89 (2011) 79-89. [8] J.F. Chaparro-Riggers, T.A. Rogers, E. Vazquez-Figueroa, K.M. Polizzi, A.S. Bommarius, Advanced Synthesis & Catalysis, 349 (2007) 1521-1531. [9] M. Kataoka, A. Kotaka, R. Thiwthong, M. Wada, S. Nakamori, S. Shimizu, Journal of biotechnology, 114 (2004) 1-9. [10] C.K. Winkler, G. Tasnadi, D. Clay, M. Hall, K. Faber, Journal of biotechnology, 162 (2012) 381-389. [11] A.D. Vaz, S. Chakraborty, V. Massey, Biochemistry, 34 (1995) 4246-4256. [12] H. Zhang, X. Gao, J. Ren, J. Feng, T. Zhang, Q. Wu, D. Zhu, Journal of Molecular Catalysis B: Enzymatic, 105 (2014) 118-125. [13] K. Stott, K. Saito, D.J. Thiele, V. Massey, The Journal of biological chemistry, 268 (1993) 6097-6106. [14] D.J. Opperman, B.T. Sewell, D. Litthauer, M.N. Isupov, J.A. Littlechild, E. van Heerden, Biochemical and biophysical research communications, 393 (2010) 426-431. [15] T.B. Fitzpatrick, N. Amrhein, P. Macheroux, The Journal of biological chemistry, 278 (2003) 19891-19897. [16] M.-Y. Xu, X.-Q. Pei, Z.-L. Wu, Journal of Molecular Catalysis B: Enzymatic, 108 (2014) 64-71. [17] B.V. Adalbjornsson, H.S. Toogood, A. Fryszkowska, C.R. Pudney, T.A. Jowitt, D. Leys, N.S. Scrutton, Chembiochem : a European journal of chemical biology, 11 (2010) 197-207. [18] M. Schittmayer, A. Glieder, M.K. Uhl, A. Winkler, S. Zach, J.H. Schrittwieser, W. Kroutil, P. Macheroux, K. Gruber, S. Kambourakis, J.D. Rozzell, M. Winkler, Advanced Synthesis & Catalysis, 353 (2011) 268-274. [19] D.J. Opperman, L.A. Piater, E. van Heerden, J Bacteriol, 190 (2008) 3076-3082. [20] F. Muller, S.G. Mayhew, V. Massey, Biochemistry, 12 (1973) 4654-4662. [21] N.K. Ahmed, R.L. Felsted, N.R. Bachur, The Journal of pharmacology and experimental therapeutics, 209 (1979) 12-19. [22] D. Clay, C.K. Winkler, G. Tasnadi, K. Faber, Biotechnology letters, 36 (2014) 1329-1333. [23] X. Gao, J. Ren, Q. Wu, D. Zhu, Enzyme and microbial technology, 51 (2012) 26-34. [24] A. Brige, D. Van den Hemel, W. Carpentier, L. De Smet, J.J. Van Beeumen, The Biochemical journal, 394 (2006) 335-344. [25] S. Ehira, H. Teramoto, M. Inui, H. Yukawa, Microbiology, 156 (2010) 1335-1341. 17
Figures: Captions to Figures: Fig. 1. Phylogenetic relationships between YqiG homologies and other OYEs with known functions. Amino acid sequences were used to generate the distance tree using ClustalX (Version 1.83). A distance neighbor-joining tree was then created using MEGA (Version 4.0). The OYE names are indicated to the right of the phylogenetic tree with NCBI accession numbers in parentheses. Fig. 2. Sequence analysis of YqiG homologues and other known OYEs. A sequence alignment was performed using ClustalX (Version 1.83) and graphically displayed using the following ESprit. Accession numbers: OYE1 (Q02899), KYE1 (P40952), OPR1 (Q9XG54), OPR3 (Q9FEW9), MR (AAC43569), PETNR (AAB38683), NemA (B7L5K3), XenA (AAF02538), Chr-OYE3 (KJ019329), TOYE (ZP_00777979), GeoOYE (Q5KXG9), and YqjM (P54550). The boxes shaded in red show the conserved residues, and the unshaded boxes indicate similar residues. The green arrows show the active residues for “classic” OYEs and “theromophilic-like” OYEs. The alignment numbering refers to the OYE1 sequence. Fig. 3. SDS-PAGE analysis of purified YqiG. Lane M: molecular weight marker, lane B: BL21(DE3)-pET22b(+)-YqiG, and lane YqiG: purified YqiG. Fig.4. UV visible absorption spectra of purified YqiG (solid line) and released flavin (dotted line). Fig. 5. YqiG temperature optima and stability. (A) Effect of temperature on YqiG enzyme activity. (B) YqiG temperature stability detected after a water bath for 12 h. 18
Fig. 6. YqiG pH optima and stability. (A) Effect of pH on YqiG enzyme activity. (B) YqiG pH stability detected after 12 h of incubation in buffers with different pH values. Sodium acetate (pH 4-6, black circles), potassium phosphate (pH 6-8, white circles), Tris-HCl (pH 8-9, black triangles) and sodium carbonate (pH 10, white triangle) were used to determine the pH optima and the pH stability. Fig. 7. The effect of salts was studied using dimethyl maleate (DMM) as a substrate. Eleven ions were used in this study. Fig. 8. The effect of organic solvents was studied using dimethyl maleate (DMM) as a substrate; eleven organic solvents were used in this study.
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Fig. 1
Fig. 1. Phylogenetic relationships between YqiG homologies and other OYEs with known functions.
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Fig. 2
21
Fig. 2. Sequence analysis of YqiG homologues and other known OYEs.
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Fig. 3 B
YqiG
M 116.0KDa 66.2KDa 45.0KDa 35.0KDa
25.0KDa 18.4KDa 14.4KDa Fig. 3. SDS-PAGE analysis of purified YqiG
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Fig. 4 0.5 Absorbance
0.4 0.3 0.2 0.1 0 310 360 410 460 510 560 610 Wavelength (nm)
Fig.4. UV visible absorption spectra of purified YqiG (solid line) and released flavin (dotted line).
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100%
3.50
Relative activity (%)
Enzyme activity （U/mg）
Fig. 5
3.00 2.50 2.00 1.50 1.00
60% 40% 20% 0%
0.50 -20%
0.00 20
40
60
B
80%
20 25 30 35 40 45 50 55 60 65 70 75
Temperature(℃)
Tenperature （℃）
Fig. 5. YqiG temperature optima and stability. (A) Effect of temperature on YqiG enzyme activity. (B) YqiG temperature stability detected after a water bath for 12 h.
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Fig. 6 5.00
100%
Enzyme activity (U/mg)
A Relative activity (%)
4.00
B
3.00
2.00
1.00
80% 60% 40% 20% 0%
3.5 4.5 5.5 6.5 7.5 8.5 9.5 10.5
3.5
pH (-)
4.5
5.5
6.5
7.5
8.5
9.5 10.5
pH (-)
Fig. 6. YqiG pH optima and stability. (A) Effect of pH on YqiG enzyme activity. (B) YqiG pH stability detected after 12 h of incubation in buffers with different pH values.
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Fig. 7 120%
Relative activty (%)
100% 80% 60% 40% 20% 0% Mg
Zn
Ca
EDTA
Li
Na
K
Ba
Ion
Fig. 7. The effect of salts was studied using dimethyl maleate (DMM) as a substrate. Eleven ions were used in this study.
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Relative enzyme activity (%)
Fig. 8
100% 80% butyl acetate 60%
n-hexanol n-butanol
40%
ethyl acetate DMSO
20% 0% 5%
10%
20%
Organic solvent (v/v) Fig. 8. The effect of organic solvents was studied using dimethyl maleate (DMM) as a substrate; eleven organic solvents were used in this study.
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Tables Table 1 YqiG substrate spectrum. nd: not detected. Substrate
Structure
Specific activity (Umg -1)
55.5 2.12
Maleimide
citraconic anhydride
nd 0.29 0.02
2-cyclopenten-1-one 3-Methyl-2-cyclopentenone
nd
3-Methyl-2-cyclohexen-1-one
nd
1-Acetyl-1-cyclohexene
nd
0.55 0.02
Ketoisophorone
Carvone
1.2 0.03
Pulegone
nd 2.97 0.09
3-Phenyl-2-methylpropenal
6.22 0.23
Dimethyl maleate
29
2.71 0.12
2-Methyl-2-pentenal
0.28 0.02
Citral Trans-2-hexen-1-al
nd
Itaconic acid
nd
0.17 0.02
Mesaconic acid
Nitrocyclohexene
nd
1-Phenyl-2-nitropropene
nd
2.5 0.075
2-Cylohexen-1one
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Table 2 Asymmetric bioreduction of YqiG in 2h. GC was used to determine the conversions and enantiomeric excesses (ee, %). Substrate
structure
conversion (%)
Maleimide
e.e(%)
>99(1h)
Carvone
21
Ketoisophorone
22
96(R)
3-phenyl-2-methylpropenal
72
43.3(R)
Citral
12
>99 (S)
31
86.3(2R,5R)
Table 3 YqiG kinetic parameters. ND: Not detected. Substrate
NADPH NADH
Km (mmol*L-1)
kcat (S-1)
kcat/Km(S-1mM-1)
0.15 0.01
711 7
1788 35
Nd
Nd
Nd
Maleimide
0.67 0.01
536 5
800 42
Dimethyl maleate
2.71 0.04
20.4 1.03
7.53 0.12
Carvone
1.59 0.20
19.9 0.97
12.5 0.22
0.18 0.02
3.43 0.15
19.05 0.31
1.41 0.08
20.2 0.88
14.3 0.54
2-Methyl-2-pentenal
1.01 0.03
17.5 0.64
17.3 0.76
2-cyclopenten-1-one
0.23 0.14
5.66 0.12
24.6 0.03
mesaconic acid
1.55 0.69
3.45 0.09
2.23 0.01
citral
2.08 0.54
13.9 0.54
6.68 0.04
2-Cyclohexen-1one
0.85 0.03
18.6 0.88
21.8 0.97
Ketoisophorone 3-phenyl-2-methylpropenal
32
S1381-1177(16)30069-8 http://dx.doi.org/doi:10.1016/j.molcatb.2016.04.011 MOLCAB 3359
To appear in:
Journal of Molecular Catalysis B: Enzymatic
Received date: Revised date: Accepted date:
19-1-2016 25-4-2016 26-4-2016
Please cite this article as: Xiqian Sheng, Ming Yan, Lin Xu, Miao Wei, Identification and characterization of a novel Old Yellow Enzyme from Bacillus subtilis str.168, Journal of Molecular Catalysis B: Enzymatic http://dx.doi.org/10.1016/j.molcatb.2016.04.011 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Identification and characterization of a novel Old Yellow Enzyme from Bacillus subtilis str.168 Xiqian Sheng; Ming Yan; Lin Xu; Miao Wei College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing, 211816, People's Republic of China *Corresponding author: Ming Yan, e-mail: [email protected] *Corresponding author. TEL.: +86-138-140-91121
1
Graphical abstract
2
Highlights: 1. Identifing a new “thermophilic-like” OYEs from Bacillus subtilis str.168。 2. Exhibiting high tolerance towards temperature and pH. 3. Exhibiting high activities towards maleimide, dimethyl maleate and acyclic ketones. 4. The YqiG can catalyze citral to (S)-citronellal with high e.e % ( >99%).
3
Abstract We identified and characterized YqiG, a novel Old Yellow Enzyme (OYE) from Bacillus subtilis str.168, as a member of the “thermophilic-like” subfamily. It is most related to XenA from Pseudomonas putida, with 37.9% identity, but it exhibits certain differences in sequence and enzyme properties. The YqiG can reduce various activated alkenes and exhibits high temperature (60℃ for 12 h) and pH stability (pH 4.0 and 9.0 for 12 h), which indicates that it has a great potential for biocatalysis. However, it shows low tolerance toward organic solvents. Also the YqiG shows high activities towards maleimide, dimethyl maleate and acyclic ketones (55.5U/mg, 6.22U/mg and 2.97U/mg, respectively). Besides, The YqiG can catalyze citral to (S)-citronellal with a low rate but high ee % (> 99%).
Keywords: Old Yellow Enzyme, Thermophilic-like family, YqiG; Enzyme properties, Ene-reductase
4
1. Introduction Old Yellow Enzyme [EC 1.6.99.1] was the first enzyme shown to contain flavin as a prosthetic group and was originally isolated from brewer’s bottom yeast by Warburg and Christian in 1933. In recent years, the OYE family has grown rapidly, and numerous OYE homologues with different characteristics have been identified [1]. Over the past 80 years of research, the OYEs have exhibited undeniably great potential for asymmetrically reducing a broad range of activated alkenes because they can generate up to two chiral centers when catalyzing C=C bond reductions [2, 3]. The OYEs are widely distributed in nature[4], especially in plants[5], fungi and bacteria[6-9]. These enzymes feature a broad substrate spectrum, including α, β-unsaturated aldehydes, ketones, nitriles, imides, nitro aromatics and carboxylic acids [1, 8, 10]. The enoate reductase stereospecifically reduces the C=C bond through hydrogen atom anti-addition [2, 11] . In 2010, Toogood et al. classified the OYEs into two categories: the “classical” family and the “thermophilic-like” family[1]. The “thermophilic-like” family often exhibited higher enzyme properties than the “classical” family [12], such as higher stability of pH and temperature. One difference between the two subfamilies is that the “classic” family usually forms monomers or dimers such as OYE1 from Saccharomyces pastorianus[13], 12-oxophytodienoate reductase from plants and MR from bacterica. but the “thermophilic-like” family often forms oligomers[1, 14]. Until now, the vast majority of OYE enzymes discovered belong to the “classic” family[5]. Only a few OYE members are “thermophilic-like” enzymes, including YqjM (P54550) 5
from Bacillus subtilis[15], XenA (AAF02538) from Pseudomonas putida[6], Chr-OYE3 (KJ019329) from Chryseobacterium sp. CA49[16], TOYE (ZP_00777979) from Thermoanaerobacter pseudethanolicus E39[17] , GeoOYE (Q5KXG9) from Geobacillus
kaustophilus[18]
and CrS
(YP_004203660)[19]
from Thermus
scotoductus. Based on the above sequences, we found a novel “thermophilic-like” OYE referred to as YqiG. Here, we identify and characterize YqiG from Bacillus subtilis; this is a new “thermophilic-like” OYE identified from Bacillus subtilis after YqjM (the first characterized “thermophilic-like” family OYE).
2. Materials and methods 2.1 Chemicals and solvents The chemicals and solvents used as substrates are analytical grade and from Aladdin. All of the enzymes used for cloning (including DNA polymerase, T4 DNA ligase and restriction enzymes) were purchased from Takara Biotechnology. The pET-22b (+) was from our own lab. 2.2 Cloning the yqiG gene from bacillus subtilis subst. subtilis. str. 168 The bacillus subtilis strain used as a PCR template was purchased from ATCC, and the yqiG DNA sequence was obtained from the UNIPROT database. The PCR experiments were performed using PrimerStar® DNA polymerase (forward primer, pET-22b (+): 5’- GGAATTCCATATGAATCCTAAGTATAAGCCA-3’; reverse primer: 5’- CATGCCATGGTTAATCTTTATAAGGCACCCA-3’). The reaction began with an initial denaturing step for 300 s at 95℃ followed by 30 cycles of 10 s at 98℃, 6
15 s at 56℃, and 90 s at 72℃ with a final extension step at 72℃ for 300 s. The yqiG gene was integrated into pET-22b (+) using the restriction sites NdeI and NcoI. The constructed vectors were transformed into E. coli DH5α. The plasmids were also transformed into E. coli BL21 (DE3) for expression after the vectors were sequenced 2.3 YqiG expression and purification Five milliliters of Luria-Bertani (LB) medium (1% tryptone, 0.5% yeast extract, and 1% NaCl) supplemented with 100 mgL-1 ampicillin was inoculated with a single YqiG colony for 12 h at 37℃. The YqiG-containing cells were then subcultured using 100 ml LB medium supplemented with 100 mgL-1 ampicillin at 37℃. Induction was initiated with the addition of 0.4mM isopropyl-β-D-thiogalactopyranoside (IPTG) at an OD600 of 0.6-0.8, and the cells were incubated for another 12h at 30℃. Cells harvested by centrifugation were suspended in 100mM potassium phosphate (pH7.0) and then broken with high pressure homogenizer (APV, Holland). Cell debris was removed by centrifugation at 12,000 rpm for 30min at 4℃.And then the supernatant was purified on ÄKTAprime plus (USA) with HiTrap DEAE FF column. The column was equilibrated with 20mM Tris-HCl buffer (pH7.0) containing 1M NaCl. The purified YqiG was stored at 4℃. 2.4 Protein analysis Protein concentration was determined with the BCA Protein Assay Kit (CWBIO, China). The molecular mass and the purity of the purified protein were estimated by SDS-PAGE. Free flavin was also determined by the Spectra Max M2. Free flavin 7
would be released after incubating in 100℃ boiling water bath for 10min. Molecular mass of native YqiG was determined using an ÄKTA purifier system with a Superdex 200pg 16/600 column. The flow rate for protein elution is 0.8ml/min. Thyroglobulin (670kDa), globulin (158kDa), ovalbumin (44kDa), myoglobin (17kDa), VB12 (1.35kDa) were used as molecular mass standards. 2.5 Enzyme assay The dimethyl maleate (DMM) was used as substrate for the determination of the enzymatic properties, and all measurements were used with purified YqiG on a Multimode Plate Reader (Spectra Max M2, Molecular Devices Corporation). And these experiments were implemented at least in triplicate. The enzyme activity was detected by monitoring NAD(P)H oxidation at 340 nm. These reactions were performed in 200µl system containing 10 mM substrate, 1 mM NADPH, 5 µl YqiG and phosphate buffer (100 mM, pH 7.0) at 30℃. To avoid a false positive NADPH consumption, we added two controls: the same reaction mixture without substrates and the same reaction mixture without YqiG. One unit of enzyme activity was defined as 1 μmol of NADPH oxidized per minute. To determine the influence of pH, we used sodium acetate (pH 4-6), potassium phosphate (pH 6-8), Tris-HCl (pH 8 -9) and sodium carbonate (pH 10) to determine the pH optima and the pH stability. And a temperature range of 20-60℃ was used for the determination of the optimum temperature. The effect of organic solvents was studied using ethanol, butyl acetate, isopropanol, n-hexanol, n-amyl alcohol, n-butanol, ethyl acetate, methanol, dimethyl sulfoxide (DMSO), iso-pentol alcohol 8
and n-hexane. The kinetic parameters were determined using the Michaelis-Menten equation, and 1 mM NADPH remained a constant with the substrate added at between 0.25-2 mM. We used maleimide as substrate when determined the kinetic parameters of NADPH and NADH. 2.6 Analytical procedures The product and enantiomeric excess (e.e%) were analyzed using the Aglient 7820 gas chromatograph (GC) equipped with a flame ionization detector (FID) and the FS-column HYDRODEX β-TBDAC (25 m,0.25 mm). 3. Results 3.1 Sequence and phylogenetic analyses The YqiG amino acid sequence was identified in the National Center for Biotechnology information (NCBI) databank using the basic local alignment search tool (BLAST). A phylogenetic analysis was performed for the YqiG with sixteen known OYE enzymes (Fig. 1). The results show that the YqiG did not cluster with other OYE enzymes but shared 37.9% identity with XenA from Pseudomonas putida. A phylogenetic tree shows that the YqiG belongs to the “thermophilic-like” subclass. A sequence analysis was performed for YqiG with twelve known OYE enzymes (Fig. 2). The YqiG consists of 373 amino acids with the predicted molecular weight 40 kDa. The sequence results showed certain shared invariant residues only within this subfamily, such as M37, Y40 and L98 []. But the YqiG still showed different positions with the “thermophilic-like” subclass, such as C38, R353 and R391. The data indicate that YqiG may be more similar to the “thermophilic-like” subclass than the “classic” 9
class. 3.2 Protein purification The protein was successfully expressed in E. coli BL21 (DE3) and purified using the HiTrap DEAE FF column. The purified enzyme presented the typical OYE yellow color. An SDS-PAGE analysis of the purified YqiG (Fig. 3) showed that its molecular mass is approximately 40 kDa. The native YqiG exhibited only one molecular mass around 40kDa. The results indicates that YqiG maybe existed as monomers. 3.3 Flavin content determination We obtained bright yellow supernatant after boiling for 10min and centrifugation for 5min. The UV-visible absorbance spectra of purified YqiG and free flavin released are shown in Fig.4. The spectrum of YqiG indicates a typical flavin-containing protein, exhibiting the maximum absorbance at 375 and 470nm with shoulders at 430 and 490nm [20]. The released flavin after protein denaturation showed absorbance maxima at 449nm, which was identical to the maximum of FMN. The results suggested that YqiG was FMN-containing protein. 3.3 Enzyme properties 3.3.1 Substrate spectrum To investigate the YqiG substrate spectra, 19 substrates with different electron withdrawing groups, including imides, α, β-unsaturated aldehydes, ketones, nitroalkenes, carboxylic acids and their derivatives, were tested in potassium phosphate buffer (pH 7.0). The activities toward the substrates are summarized in 10
Table 1. The YqiG was active toward imides, α,β-unsaturated aldehydes and ketones but not nitroalkenes and show little activity toward carboxylic acids. Most OYEs feature high activities toward cyclic ketones [7, 17, 21-23], but YqiG only exhibited little activity toward cyclic ketones, such as ketoisophorone (only 0.55 U/mg). However, YqiG showed high activities toward maleimide, dimethyl maleate and acyclic ketones, and the activity towards maleimide reached 55U/mg, which is higher than most other OYEs[24]. 3.3.2 Bioreduction with coenzyme regeneration To investigate YqiG enantioselectivity, asymmetric bioreduction with certain substrates was performed using a coenzyme regeneration system and glucose dehydrogenase at 30℃, and the ee values and conversions were detected and summarized in Table. 2. The YqiG can catalyze citral to (S)-citronellal with a low rate but high e.e % (> 99%). Also the YqiG showed high e.e.% (96%) but only 22% conversion toward ketoisophorone. 3.3.3 Determining kinetic parameters The kinetic parameters were detected through NADPH oxidation at 340 nm using a 200-µl system with 30 µg of enzyme, 1 mM NADPH, 0.2-2 mM substrate and phosphate buffer (100 mM, pH 7.0). The substrates were representatives for the substance classes: imides, α, β-unsaturated aldehydes, ketones and carboxylic acids. The kinetic parameters of substrates and NADPH are shown in Table 3. The results clearly showed that YqiG could only accept NADPH as cofactor. The kcat of NADPH reached 1788 whereas the kcat of NADH could not be detected. Moreover, the YqiG 11
clearly exhibited the greatest affinity for maleimide and Dimethyl maleate and the lowest affinity for mesaconic acid and citral. Also, the kcat/Km of 2-Cylohexen-1one was 21.8 s-1mM-1, which was higher than YqjM (15 s-1mM-1), MR (0.19 s-1mM-1) and TOYE (0.5 s-1mM-1) [14, 15, 17]. 3.3.4 The optimum temperature and temperature stability The effects of temperature were tested between 20℃ and 70℃ (Fig. 5A). The YqiG exhibited high activity between 30℃ and 45℃. After a water bath for 12 h, residual activity was used to determine the temperature stability of YqiG (Fig. 5B). The loss of activity was less than 10% between 30℃ and 40℃, whereas the residual activity rapidly decreased with an increase in temperature. 3.3.5 The optimum pH and pH stability The effects of pH were tested over a broad range, pH4-9 (Fig.6A). YqiG showed a broad pH range at pH 5.5 to pH 9.0 and the most activity between pH 6.5 and pH 7.5. After incubating in buffers with different pH values for 12 h, the residual activity was used to determine the YqiG pH stability (Fig. 6B). Over 75% of the activity remained between pH 6.0 and pH 9.0, and the residual activity rapidly decreased with a decrease in pH from pH 6.0 to pH 4.0. 3.3.6 Effect of different ions. The concentration of all ions were 5 mM in the 200-µl system (Fig. 7). No activity was detected when Fe2+, Cu2+ and Mn2+ were added to the reaction system. The reason might be that Fe2+, Cu2+ and Mn2+ were all strong oxidizing ions, which could consume much more oxygen than FMN. The effects of the remained eight salts 12
are shown as a fig. Clearly, Mg2+ increase the enzyme activity, but EDTA, Li+, Na+, K+ and Ba2+ exhibited no active effect on YqiG. 3.3.7 Effect of organic solvents Organic solvents are often used in biocatalysis to increase substrate solubility and product separation. We investigated the solvent stability of YqiG in several solvents with various logP values (from -1.3 to 3.5) at the concentrations 5%, 10% and 20% in the 200-µl system. Activity was not detected when ethanol, isopropanol, n-amyl alcohol, methanol and iso-pentol alcohol were added. The remaining six organic solvents exerted different effects on YqiG activity, as shown in Fig. 8. The data show that YqiG exhibits better activity in DMSO, which is a water-miscible solvent with a -1.3 logP. However, these organic solvents are not suitable for YqiG because it could not retain 60% of its activity at a solvent concentration of 20%, which suggests low tolerance toward organic solvents.
4. Discussion In this paper, a novel OYE from bacillus subtilis subst. subtilis. str. 168 referred to as YqiG was successfully cloned into BL21 (DE3) and purified using an ion exchange column. The YqiG featured a broad substrate spectrum, including α, β-unsaturated aldehydes, ketones, imides, carboxylic acids, and nitro aromatics. The YqiG showed high activity with imides but no obvious activity with nitro aromatics. A detailed study on “thermophilic-like” enzymes revealed that “thermophilic-like” enzymes exhibited greater stability and resistance in various aspects compared with 13
“classic” enzymes [17]. The optimum temperature for YqiG is 40℃,and it can tolerate 60℃ for 12 h with a loss of less than 50% enzyme activity. Further, YqiG exhibits a broad pH range. It can tolerate the pH values 6-9 for 12 h and retain more than 50% of its enzyme activity, which indicates that YqiG can maintain better catalytic activity for a considerably long time. Further, the above are characteristics of the “thermophilic-like” family, such as with Chr-OYE3, which can tolerate 55℃ for approximately 100 h until no activity remained[16]. Thus, we preliminarily classified the YqiG into the “thermophilic-like” family based on the phylogenetic tree and enzyme properties. YqiG exhibited low resistance toward various types of organic solvent, whereas other “thermophilic-like” enzymes feature a great resistance range [16, 19]; perhaps this discrepancy is due to the sequence difference of YqiG compared with the two families. It was often thought that members in “classic” family often exist as monomers or dimers whereas members in “thermophilic-like” family typically exist as higher oligomeric states[1]. But the molecular mass of native YqiG showed that YqiG only exist as monomers. The reason maybe that some residues about the subunit numbers are different from the “thermophilic-like” family but similar to the “classic” family. Further, YqiG features some other protein structure differences compared with “thermophilic-like” enzymes, in which protein thermostability plays an important role [25]. The “theromophlic-like” enzymes feature certain structural characteristics, such as shorter chain length (337-350 amino acids), high oligomeric dimer and tetramer 14
states and lower thermoliable residue levels. YqiG is 373 amino acids longer than other “thermophilic-like” enzymes, such as TOYE (337 amino acids), GeoOYE (340 amino acids), Chr-OYE3 (350 amino acids) and YqjM (338 amino acids). But the results of sequence analysis and the phylogenetic tree suggest that YqiG differs from the classic families in the key position, but closes to the “thermophilic-like” family. After the comparison between YqiG and the two subfamilies, it is obviously that there are more similarities between the YqiG and the “theromophlic-like” family than the “classic” family, such as temperature, pH, and some key positions, which indicates that YqiG should be classified into the “thermophilic-like” family.
5. Conclusions We have identified a novel OYE of the “thermophilic-like” family from Bacillus subtilis str.168 named YqiG, and we characterized this OYE as an ER that can reduce C=C bonds of various activated alkenes. Further, its high temperature and pH stability indicates a great potential for biocatalysis. Although the physiological role of YqiG is unclear, given the substrate spectrum and certain enzyme properties, it may be useful for further research and applications of this subfamily of enzymes.
15
Acknowledgments This work was supported by the National Basic Research Program of China under Grant No. 2011CBA00804.
16
Reference [1] H.S. Toogood, J.M. Gardiner, N.S. Scrutton, ChemCatChem, 2 (2010) 892-914. [2] M. Hall, C. Stueckler, H. Ehammer, E. Pointner, G. Oberdorfer, K. Gruber, B. Hauer, R. Stuermer, W. Kroutil, P. Macheroux, K. Faber, Advanced Synthesis & Catalysis, 350 (2008) 411-418. [3] E. Brenna, F.G. Gatti, D. Monti, F. Parmeggiani, S. Serra, Advanced Synthesis & Catalysis, 354 (2012) 105-112. [4] A. Fryszkowska, H. Toogood, M. Sakuma, J.M. Gardiner, G.M. Stephens, N.S. Scrutton, Adv Synth Catal, 351 (2009) 2976-2990. [5] K. Shimoda, N. Kubota, H. Hamada, Tetrahedron: Asymmetry, 15 (2004) 2443-2446. [6] Y. Yanto, H.H. Yu, M. Hall, A.S. Bommarius, Chemical communications, 46 (2010) 8809-8811. [7] N. Richter, H. Groger, W. Hummel, Applied microbiology and biotechnology, 89 (2011) 79-89. [8] J.F. Chaparro-Riggers, T.A. Rogers, E. Vazquez-Figueroa, K.M. Polizzi, A.S. Bommarius, Advanced Synthesis & Catalysis, 349 (2007) 1521-1531. [9] M. Kataoka, A. Kotaka, R. Thiwthong, M. Wada, S. Nakamori, S. Shimizu, Journal of biotechnology, 114 (2004) 1-9. [10] C.K. Winkler, G. Tasnadi, D. Clay, M. Hall, K. Faber, Journal of biotechnology, 162 (2012) 381-389. [11] A.D. Vaz, S. Chakraborty, V. Massey, Biochemistry, 34 (1995) 4246-4256. [12] H. Zhang, X. Gao, J. Ren, J. Feng, T. Zhang, Q. Wu, D. Zhu, Journal of Molecular Catalysis B: Enzymatic, 105 (2014) 118-125. [13] K. Stott, K. Saito, D.J. Thiele, V. Massey, The Journal of biological chemistry, 268 (1993) 6097-6106. [14] D.J. Opperman, B.T. Sewell, D. Litthauer, M.N. Isupov, J.A. Littlechild, E. van Heerden, Biochemical and biophysical research communications, 393 (2010) 426-431. [15] T.B. Fitzpatrick, N. Amrhein, P. Macheroux, The Journal of biological chemistry, 278 (2003) 19891-19897. [16] M.-Y. Xu, X.-Q. Pei, Z.-L. Wu, Journal of Molecular Catalysis B: Enzymatic, 108 (2014) 64-71. [17] B.V. Adalbjornsson, H.S. Toogood, A. Fryszkowska, C.R. Pudney, T.A. Jowitt, D. Leys, N.S. Scrutton, Chembiochem : a European journal of chemical biology, 11 (2010) 197-207. [18] M. Schittmayer, A. Glieder, M.K. Uhl, A. Winkler, S. Zach, J.H. Schrittwieser, W. Kroutil, P. Macheroux, K. Gruber, S. Kambourakis, J.D. Rozzell, M. Winkler, Advanced Synthesis & Catalysis, 353 (2011) 268-274. [19] D.J. Opperman, L.A. Piater, E. van Heerden, J Bacteriol, 190 (2008) 3076-3082. [20] F. Muller, S.G. Mayhew, V. Massey, Biochemistry, 12 (1973) 4654-4662. [21] N.K. Ahmed, R.L. Felsted, N.R. Bachur, The Journal of pharmacology and experimental therapeutics, 209 (1979) 12-19. [22] D. Clay, C.K. Winkler, G. Tasnadi, K. Faber, Biotechnology letters, 36 (2014) 1329-1333. [23] X. Gao, J. Ren, Q. Wu, D. Zhu, Enzyme and microbial technology, 51 (2012) 26-34. [24] A. Brige, D. Van den Hemel, W. Carpentier, L. De Smet, J.J. Van Beeumen, The Biochemical journal, 394 (2006) 335-344. [25] S. Ehira, H. Teramoto, M. Inui, H. Yukawa, Microbiology, 156 (2010) 1335-1341. 17
Figures: Captions to Figures: Fig. 1. Phylogenetic relationships between YqiG homologies and other OYEs with known functions. Amino acid sequences were used to generate the distance tree using ClustalX (Version 1.83). A distance neighbor-joining tree was then created using MEGA (Version 4.0). The OYE names are indicated to the right of the phylogenetic tree with NCBI accession numbers in parentheses. Fig. 2. Sequence analysis of YqiG homologues and other known OYEs. A sequence alignment was performed using ClustalX (Version 1.83) and graphically displayed using the following ESprit. Accession numbers: OYE1 (Q02899), KYE1 (P40952), OPR1 (Q9XG54), OPR3 (Q9FEW9), MR (AAC43569), PETNR (AAB38683), NemA (B7L5K3), XenA (AAF02538), Chr-OYE3 (KJ019329), TOYE (ZP_00777979), GeoOYE (Q5KXG9), and YqjM (P54550). The boxes shaded in red show the conserved residues, and the unshaded boxes indicate similar residues. The green arrows show the active residues for “classic” OYEs and “theromophilic-like” OYEs. The alignment numbering refers to the OYE1 sequence. Fig. 3. SDS-PAGE analysis of purified YqiG. Lane M: molecular weight marker, lane B: BL21(DE3)-pET22b(+)-YqiG, and lane YqiG: purified YqiG. Fig.4. UV visible absorption spectra of purified YqiG (solid line) and released flavin (dotted line). Fig. 5. YqiG temperature optima and stability. (A) Effect of temperature on YqiG enzyme activity. (B) YqiG temperature stability detected after a water bath for 12 h. 18
Fig. 6. YqiG pH optima and stability. (A) Effect of pH on YqiG enzyme activity. (B) YqiG pH stability detected after 12 h of incubation in buffers with different pH values. Sodium acetate (pH 4-6, black circles), potassium phosphate (pH 6-8, white circles), Tris-HCl (pH 8-9, black triangles) and sodium carbonate (pH 10, white triangle) were used to determine the pH optima and the pH stability. Fig. 7. The effect of salts was studied using dimethyl maleate (DMM) as a substrate. Eleven ions were used in this study. Fig. 8. The effect of organic solvents was studied using dimethyl maleate (DMM) as a substrate; eleven organic solvents were used in this study.
19
Fig. 1
Fig. 1. Phylogenetic relationships between YqiG homologies and other OYEs with known functions.
20
Fig. 2
21
Fig. 2. Sequence analysis of YqiG homologues and other known OYEs.
22
Fig. 3 B
YqiG
M 116.0KDa 66.2KDa 45.0KDa 35.0KDa
25.0KDa 18.4KDa 14.4KDa Fig. 3. SDS-PAGE analysis of purified YqiG
23
Fig. 4 0.5 Absorbance
0.4 0.3 0.2 0.1 0 310 360 410 460 510 560 610 Wavelength (nm)
Fig.4. UV visible absorption spectra of purified YqiG (solid line) and released flavin (dotted line).
24
100%
3.50
Relative activity (%)
Enzyme activity （U/mg）
Fig. 5
3.00 2.50 2.00 1.50 1.00
60% 40% 20% 0%
0.50 -20%
0.00 20
40
60
B
80%
20 25 30 35 40 45 50 55 60 65 70 75
Temperature(℃)
Tenperature （℃）
Fig. 5. YqiG temperature optima and stability. (A) Effect of temperature on YqiG enzyme activity. (B) YqiG temperature stability detected after a water bath for 12 h.
25
Fig. 6 5.00
100%
Enzyme activity (U/mg)
A Relative activity (%)
4.00
B
3.00
2.00
1.00
80% 60% 40% 20% 0%
3.5 4.5 5.5 6.5 7.5 8.5 9.5 10.5
3.5
pH (-)
4.5
5.5
6.5
7.5
8.5
9.5 10.5
pH (-)
Fig. 6. YqiG pH optima and stability. (A) Effect of pH on YqiG enzyme activity. (B) YqiG pH stability detected after 12 h of incubation in buffers with different pH values.
26
Fig. 7 120%
Relative activty (%)
100% 80% 60% 40% 20% 0% Mg
Zn
Ca
EDTA
Li
Na
K
Ba
Ion
Fig. 7. The effect of salts was studied using dimethyl maleate (DMM) as a substrate. Eleven ions were used in this study.
27
Relative enzyme activity (%)
Fig. 8
100% 80% butyl acetate 60%
n-hexanol n-butanol
40%
ethyl acetate DMSO
20% 0% 5%
10%
20%
Organic solvent (v/v) Fig. 8. The effect of organic solvents was studied using dimethyl maleate (DMM) as a substrate; eleven organic solvents were used in this study.
28
Tables Table 1 YqiG substrate spectrum. nd: not detected. Substrate
Structure
Specific activity (Umg -1)
55.5 2.12
Maleimide
citraconic anhydride
nd 0.29 0.02
2-cyclopenten-1-one 3-Methyl-2-cyclopentenone
nd
3-Methyl-2-cyclohexen-1-one
nd
1-Acetyl-1-cyclohexene
nd
0.55 0.02
Ketoisophorone
Carvone
1.2 0.03
Pulegone
nd 2.97 0.09
3-Phenyl-2-methylpropenal
6.22 0.23
Dimethyl maleate
29
2.71 0.12
2-Methyl-2-pentenal
0.28 0.02
Citral Trans-2-hexen-1-al
nd
Itaconic acid
nd
0.17 0.02
Mesaconic acid
Nitrocyclohexene
nd
1-Phenyl-2-nitropropene
nd
2.5 0.075
2-Cylohexen-1one
30
Table 2 Asymmetric bioreduction of YqiG in 2h. GC was used to determine the conversions and enantiomeric excesses (ee, %). Substrate
structure
conversion (%)
Maleimide
e.e(%)
>99(1h)
Carvone
21
Ketoisophorone
22
96(R)
3-phenyl-2-methylpropenal
72
43.3(R)
Citral
12
>99 (S)
31
86.3(2R,5R)
Table 3 YqiG kinetic parameters. ND: Not detected. Substrate
NADPH NADH
Km (mmol*L-1)
kcat (S-1)
kcat/Km(S-1mM-1)
0.15 0.01
711 7
1788 35
Nd
Nd
Nd
Maleimide
0.67 0.01
536 5
800 42
Dimethyl maleate
2.71 0.04
20.4 1.03
7.53 0.12
Carvone
1.59 0.20
19.9 0.97
12.5 0.22
0.18 0.02
3.43 0.15
19.05 0.31
1.41 0.08
20.2 0.88
14.3 0.54
2-Methyl-2-pentenal
1.01 0.03
17.5 0.64
17.3 0.76
2-cyclopenten-1-one
0.23 0.14
5.66 0.12
24.6 0.03
mesaconic acid
1.55 0.69
3.45 0.09
2.23 0.01
citral
2.08 0.54
13.9 0.54
6.68 0.04
2-Cyclohexen-1one
0.85 0.03
18.6 0.88
21.8 0.97
Ketoisophorone 3-phenyl-2-methylpropenal
32