Enhancing thermal tolerance of Aspergillus niger PhyA phytase directed by structural comparison and computational simulation

Enhancing thermal tolerance of Aspergillus niger PhyA phytase directed by structural comparison... Background: Phytase supplied in feeds for monogastric animals is important for improving nutrient uptake and reducing phosphorous pollution. High-thermostability phytases are particularly desirable due to their ability to withstand transient high temperatures during feed pelleting procedures. A comparison of crystal structures of the widely used industrial Aspergillus niger PhyA phytase (AnP) with its close homolog, the thermostable Aspergillus fumigatus phytase (AfP), suggests 18 residues in three segments associated with thermostability. In this work, we aim to improve the thermostability of AnP through site-directed mutagenesis. We identified favorable mutations based on structural comparison of homologous phytases and molecular dynamics simulations. Results: A recombinant phytase (AnP-M1) was created by substituting 18 residues in AnP with their AfP analogs. AnP-M1 exhibited greater thermostability than AnP at 70 °C. Molecular dynamics simulations suggested newly formed hydrogen bonding interactions with nine substituted residues give rise to the improved themostability. Thus, another recombinant phytase (AnP-M2) with just these nine point substitutions was created. AnP-M2 demonstrated superior thermostability among all AnPs at ≥70 °C: AnP-M2 maintained 56% of the maximal activity after incubation at 80 °C for 1 h; AnP-M2 retained 30-percentage points greater residual activity than that of AnP and AnP-M1 after 1 h incubation at 90 °C. Conclusions: The resulting AnP-M2 is an attractive candidate in industrial applications, and the nine substitutions in AnP-M2 are advantageous for phytase thermostability. This work demonstrates that a strategy combining structural comparison of homologous enzymes and computational simulation to focus on important interactions is an effective method for obtaining a thermostable enzyme. Keywords: Phytase, Thermostability, Site-directed mutagenesis, Homologous structural comparison, Molecular dynamics simulation Background be utilized directly [2]. Supplemental inorganic phosphate Phytate (myo-inositol hexakisphosphate) is the primary can partially compensate for the deficiency, but excessive form of stored phosphorus in most plants and conse- phytate phosphorus in animal excretions induces severe quently, in animal feeds [1]. Due to inadequate levels of ecological problems [3, 4]. The digestive enzyme phytase digestive enzymes in the gastrointestinal tracts of mono- can hydrolyze phytic acid or phytate into lower inositol gastric animals (swine, poultry, fish, etc.), phytate cannot phosphates [5]. Thus, phytases supplied in animal feeds not only improve phytate utilization in monogastric ani- mals, but also alleviate phytate phosphorus pollution [6]. * Correspondence: huangzunxi@163.com The first commercial phytase products were intro- Nanyu Han and Huabiao Miao contributed equally to this work. Equal contributors duced 20 years ago [7]. Currently, over 60% of feed School of Life Sciences, Yunnan Normal University, Kunming 650500, China products include phytase [8]. To lower production costs, Key Laboratory of Yunnan for Biomass Energy and Biotechnology of novel thermostable phytases capable of withstanding the Environment, Yunnan Normal University, Kunming 650500, China Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Han et al. BMC Biotechnology (2018) 18:36 Page 2 of 8 transient high temperatures (80–85 °C) of the feed pro- Results duction pelleting process are in high demand [9]. AnP-M1 showed improved thermostability The Aspergillus niger PhyA phytase (AnP) exhibits Inspired by the work done by Tao Xiang and coworkers superior enzymatic activity compared to other fungal [12], mainchain deviations were re-calculated. After phytases [5]. Specifically, AnP exhibited considerably superimposing AnP and AfP crystal structures, Cα higher specific activity (102.5 U/mg) than the Aspergillus atoms between analogous residues in three segments fumigatus phytase (AfP) (26.5 U/mg) [10]. Moreover, (A35-P42, R163-Q168, T248-K254 in AnP) were shown AnP is an acidic phosphatase that optimally active in the to have distances greater than 3 Å (Fig. 1a, b). This re- pH range prevalent in the digestive tract [2, 10]. How- sult is consistent with the findings from Tao Xiang et al. ever, when exposed to pelleting process conditions, AnP The first recombinant phytase (AnP-M1) was cre- loses 70–80% of its enzymatic activity [11]. In contrast, ated by substituting residues from AnP in the above the phytase from AfP, which shares 66% sequence iden- three segments with the corresponding residues in tity and has similar overall crystal structure [12], is well AfP by site-directed mutagenesis (35-ANESVISP-42, known for its heat resistance: AfP retains 90% of its 163-RAQPGQ-168, and 248-TSTVDTK-254 in AnP initial enzymatic activity after heating to 100 °C for correspond to 35-EDELSVSS-42, 163-GATNRA-168, 20 min [13]. Thus, AfP may act as a guide for AnP and 248-RTSDASQ-254 in AfP). Thermostability enzymatic engineering for both high activity and high assays revealed that AnP-M1 retained greater activity thermostability [14]. after treatment at 70 °C for 1 h compared to AnP Based on the above hypothesis, Tao Xiang and (Fig. 1c). AnP-M1 retained 51% activity while AnP coworkers proposed three segments that could contrib- retained 31% activity. ute to the thermostability of AfP from comparing crystal structures of AnP and AfP [12]. We confirmed these Hydrogen bonds in AnP-M1 govern the improved sites and constructed a recombinant phytase (AnP-M1) thermostability by substituting residues in the three segments in AnP Generally, creating an enzyme with improved thermosta- with residues at corresponding sites in AfP. As expected, bility usually requires combining multiple amino acid the resultant recombinant demonstrated higher thermo- exchanges, each of which slightly increases the unfolding stability than the parent AnP. We explored potential temperature of the protein. Some example mechanisms AnP-M1 heat resistant mechanisms using molecular dy- are: new hydrogen bonds, new disulfide bridges, β-turns namics (MD) simulations, and nine of the 18 mutated or flexible termini stabilization, hydrophobic packing en- residues in AnP-M1 exhibited potential interactions that hancement, or α-helix or β-sheet stabilization [15]. One could contribute to this. Subsequently, we created our study showed that the hydrogen bonding appears to be second recombinant phytase (AnP-M2) by substituting the most important factor for thermostability based on just the nine proposed thermostability-enhancing resi- analysis of enzymes from 16 families [16]. Specifically, dues into AnP. This new variant (AnP-M2) demon- increased hydrogen bonding interactions are observed in strated even greater thermal tolerance. the three segments of AfP based on structural analysis This work combines structural comparison and com- [12]. To identify the contribution of particular residues putational simulation to guide the design of a thermo- towards improving thermostability, we performed MD stable phytase. The most promising new variant has simulations of AnP and AnP-M1 at two temperatures particular potential for the animal feed industry. (50 °C and 70 °C). Both AnP and AnP-M1 were stable ac b Fig. 1 Structural comparison of homologous phytases and thermostability assays of the first recombinant. Structural comparison (a) and mainchain deviation (b) between AnP (tv_blue) and AfP (tv_yellow). Residual activity of AnP (black) and AnP-M1 (red) incubated at 70 °C for 1 h (c) Han et al. BMC Biotechnology (2018) 18:36 Page 3 of 8 during the 50 ns simulations at two temperatures, with thermostability for AnP-M1, and the mutated serines at the overall root-mean-square deviations (RMSD) of the position 39 and 42 in segment-1 are critical for the heavy atoms with respect to the initial structure less interactions. than 0.35 nm (Additional file 1: Figure S1). After investi- Hydrogen bond interactions within segment-2 residues gating the hydrogen bond patterns, we identified nine in AnP and AnP-M1 were also analyzed (Fig. 2b). AnP mutated residues in AnP-M1 that form new hydrogen R163 and D161 formed 1.74 and 1.70 hydrogen bonds at bonding interactions that could explain the improved 50 °C and 70 °C, respectively. In contrast, AnP-M1 G163 thermostability. and D161 formed no more than 0.8 hydrogen bonds at Hydrogen bonds connecting segment-1 residues both temperatures. In AnP, the probability of a hydrogen were monitored throughout the entire simulation in bond connecting Q165 and R129 was 89% at 50 °C, each phytase. Most hydrogen bonds connecting decreasing to 24% at 70 °C, indicating a flexible inter- 35-ANESVISP-42 and nearby residues in AnP per- action between Q165 and R129 in AnP at elevated sisted at both temperatures (Fig. 2a). The hydrogen temperature. In contrast, the hydrogen bond between bond connecting AnP A35 and S33 was well pre- T165 and R129 in AnP-M1 was well maintained and served at both temperatures (36% at 50 °C and 33% increased to 40% at 70 °C, suggesting a stable and favor- at 70 °C). The probability of hydrogen bonding able contact at higher temperature. Likewise, the prob- between AnP N36 and E37 is 42% at 50 °C and 40% ability of hydrogen bonding between AnP-M1 N166 and at 70 °C. The probability of hydrogen bonding be- R167 was approximately 20% at both temperatures, tween S38 and I40 in AnP increased to 93% at 70 °C. while no hydrogen bond was observed between AnP In AnP-M1, the formation probabilities of three P166 and G167 at either temperature. In summary, hydrogen bond pairs, E35-S33, D36-E37, and AnP-M1 segment-2 hydrogen bond pairs R129-T165 L38-V40, were less than those in AnP. and N166-R167 may contribute to the enhanced Interestingly, the probability of hydrogen bonding be- thermostability. tween S39 and E37 in AnP-M1 increased to 25% at 70 ° Hydrogen bonds formed within segment-3 residues C. Additionally, another hydrogen bond connecting in AnP (248-TSTVDTK-254) and AnP-M1 (248-RTS AnP-M1 S42 and D405 was well preserved at both tem- DASQ-254) were also compared (Fig. 2c). It is evident peratures: probabilities of hydrogen bonding between that R248 and D244 in AnP-M1 formed almost 2 S42 and D405 were 59 and 74% at 50 °C and 70 °C, re- hydrogen bonds at both temperatures, and the prob- spectively. However, hydrogen bonding pairs V39-E37 ability of this interaction is 4-fold greater than that and P42-D405 in AnP were rarely observed. In sum- between the corresponding AnP T248 and D244. mary, two newly formed hydrogen bond pairs, S39-E37 Almost no contact was monitored between residues and S42-D405, may contribute to the enhanced 248 and 249 in either AnP or AnP-M1. Although ab c Fig. 2 Statistics of hydrogen bonding with mutated residues. Statistics of hydrogen bonds connecting residues in segment 1–3(a-c, respectively) and nearby residues for AnP and AnP-M1 from simulations at 50 °C and 70 °C Han et al. BMC Biotechnology (2018) 18:36 Page 4 of 8 AnP-M1 S250 and Q254 formed 1.01 and 0.78 hydro- R248T, D251V, A252D, and Q254K). This nonuple gen bonds at the two temperatures, AnP T250 and mutant is denoted AnP-M2. K254 formed more hydrogen bonds at 70 °C (0.88), To evaluate thermostability, we measured the residual indicating a more stable interaction between AnP activities of AnP, AnP-M1 and AnP-M2 after 1 h incuba- T250 and K254 at 70 °C. Similarly, the hydrogen tion at three temperatures (70, 80, and 90 °C). The two bond between AnP-M1 S253 and S250 is not as mutants showed promising improvements in residual strong as that between AnP T253 and T250. In con- activity, and the nonuple mutant AnP-M2 exhibited the trast, D251, A252, and Q254 in AnP-M1 were associ- highest thermal tolerance. Specifically, AnP-M2 retained ated with higher probability in forming hydrogen 66% activity after 1 h incubation at 70 °C, AnP-M1 bonds with nearby residues than those in AnP. In retained 51% at the same condition, and AnP retained summary, AnP-M1 segment-3’shydrogen bonds markedly less (31%) (Fig. 4a). After 1 h incubation at within R248, D251, A252, and Q254 may contribute 80 °C, AnP-M2, AnP-M1 and AnP retained 58, 41 and to the improved thermostability. 21% activity, respectively (Fig. 4b). After incubation at In light of the above, not all the mutagenesis sites 90 °C for 1 h, AnP-M2 retained 30-percentage points demonstrated stable or increased hydrogen bonding in- greater residual activity than that of the AnP and teractions with nearby residues in AnP-M1, suggesting AnP-M1 (Fig. 4c). These results highlight that the nine that only a subset of the 18 point mutations contributed substitutions in AnP-M2 are advantageous for phytase to the improved thermostability. Comparing number of thermostability. hydrogen bonds formed with the substituted residues in AnP and AnP-M1, we concluded that S39 and S42 in Kinetic analysis of AnPs segment-1 (Fig. 3a, d); T165, N166, and R167 in Kinetic analysis showed apparent K values for AnP, segment-2 (Fig. 3b, e); and R248, D251, A252, and Q254 AnP-M1 and AnP-M2 of 289.8, 116.5 and 135.2 μM, re- in segment-3 (Fig. 3c, f) make the greatest contribution spectively (Table 1). The smaller Michaelis constant to the enhanced AnP-M1 thermostability. (K ) of both mutants indicate an increase in kinetic effi- ciency compared to that of AnP. Comparing k /K cat m AnP-M2 showed superior thermal tolerance among all values of three AnPs, AnP-M1 has the highest catalytic AnPs efficiency, followed by AnP and the AnP-M2. Kinetic To further increase AnP thermostability, we performed analysis suggests that the substitutions in AnP-M1 and another round of site-directed mutagenesis based on les- AnP-M2, which we designed primarily to improve ther- sons learned from our analysis of AnP-M1. In total, nine mostability, enhanced substrate binding affinity (K )by AnP residues were substituted to AfP residues at the two-fold; however, the k values of both mutants were cat equivalent sites (S39 V, S42P, T165Q, N166P, R167G, decreased to the same extent and k /K which cat m a b c d e f Fig. 3 Comparison of hydrogen bonding networks formed by mutated residues in AnP and AnP-M1. Illustration of the newly formed hydrogen bonding network in AnP-M1 (a-c) and analogous locations in AnP (d-f). Representative structures are cluster centers from clustering analysis Han et al. BMC Biotechnology (2018) 18:36 Page 5 of 8 ab c Fig. 4 Thermostability assays of all three AnPs. Residual activities of AnP (black), AnP-M1 (red), and AnP-M2 (blue) incubated at 70 °C (a), 80 °C (b), and 90 °C (c) for 1 h represents catalytic efficiency was slightly decreased for [18]. Residues 35–42 in AnP segment-1 are located AnP-M2 versus AnP-M1. within the fungal HAP’s unique N-terminal region. Therefore, the newly formed hydrogen bond pairs Discussion S39-E37 and S42-D405 in segment-1 may play a pivotal In order to create AnP variants with substantially in- role in enhancing the thermal tolerance of phytase mu- creased thermostability, we performed two rounds of tants through stabilizing the local structure in the rationally designed site-directed mutagenesis. A crystal N-terminal region. structure comparison between regular AnP and a particu- Although AnP-M2 has the highest thermostability, it larly thermostable homolog, AfP, indicated three segments has slightly decreased catalytic efficiency compared to that that may influence thermostability. MD simulations on a of AnP. The consensus catalytic motif 58-RHGARYP-64 mutant AnP created based these comparisons, AnP-M1, and substrate-binding motif 338-HD-339 are located in explored heat resistance mechanisms and pointed out the deep substrate-binding cleft at the interface of the nine particular thermostability-enhancing residues. We large α/β domain and the small α domain (Fig. 5a). Struc- created a final recombinant, AnP-M2, with superior ther- tural analysis shows that mutated sites from segment-3, mostability, enhanced substrate binding affinity, and located at the small α domain, are close to the catalytic decreased catalytic efficiency compared to other AnPs. and substrate-binding motifs, with a ~ 8 Å distance A previous study on phytase thermostability focused between the closest segment-3 and substrate-binding resi- on three residues derived from structural modeling, indi- dues (Fig. 5b). One previous effort to improve enzyme cated that hydrogen bond network and ionic interaction thermostability only mutated residues > 10 Å away from formed with the three residues support the high thermo- the active site in order to avoid such possible interference stability of AfP [17]. These three residues are present in [19]. Mutations in the AnP-M2 segment-3 may affect local both our full 18-residue homology-based mutant and re- movement, thus influencing catalysis. This conjecture re- fined 9-residue MD-based mutant. Similarly, our study quires experimental studies which could consist of single discovered that hydrogen bonding interactions formed point mutations of AnP-M2 segment-3 residues to identify with the nine substituted residues account for the catalytic efficiency influencing residue (s). This provides a improved thermostability. next step for designing a new variant with both high ther- The fungal histidine acid phytases (HAPs) possess an mostability and high catalytic efficiency. extra N-terminal region before the first β-strand of the α/β domain. This region folds into short helices and Conclusions loops. Structural studies suggest that the N-terminal re- Our work demonstrates the improvement of an gion in tetrameric phytase extend outward and form part enzyme using a multipart rational design strategy: of the interface of the dimer and tetramer structures identifying residues from homologs with desirable Table 1 Kinetics of AnP, AnP-M1 and AnP-M2 Enzymes K (μM) V (μmol/min/mg) k (/s) k /K (/s/μM) m max cat cat m AnP 289.8 ± 13.8 5954.2 ± 63.8 4885.4 ± 52.1 16.9 ± 1.7 AnP-M1 116.5 ± 9.2 3117.1 ± 55.1 2557.6 ± 44.9 22.0 ± 1.8 AnP-M2 135.2 ± 8.8 2484.4 ± 59.1 2042.6 ± 48.2 15.1 ± 1.8 Han et al. BMC Biotechnology (2018) 18:36 Page 6 of 8 Fig. 5 Illustration of mutated and catalytic residues in AnP-M2. Mutated residues in AnP-M2 are shown in spheres, substrate binding motif (orange) and catalytic motif (purple) are shown in cartoon (a). Distances between R248 in segment-3 of AnP-M2 and Y63 of the catalytic motif were shown in yellow dash (b) properties followed by determining important mecha- TransGen (Beijing, China). All other chemicals were of nisms from MD simulations and then final design analytical grade and commercially available. refinement. The final designed A.niger PhyA variant also provides new insight into the design of thermo- Gene cloning and site-directed mutagenesis stable phytases. Additional enzyme properties could The AnP sequence came from the Aspergillus niger also be modified using this combined method. PhyA gene (GenBank: Z16414) in the pMD19-T vector [20]. Genes of mutants (AnP-M1 and AnP-M2) were constructed by introducing mutations to AnP through Methods site-directed mutagenesis using the Fast MultiSite Materials System according to the manufacturer’s instructions. High fidelity DNA polymerase, restriction endonuclease PCR cycling conditions consisted of an initial step of (EcoRI, NotI) and dNTP from TaKaRa (Otsu, Japan). 5 min at 94 °C, followed by 30 cycles of 30 s at 94 °C, Plasmid mini-prep kit and DNA gel extraction kit from 1 min at 55 °C, and 3 min 30 s at 72 °C. Forward and Omega (Taipei, USA). One-step cloning kit from reverse primers for AnP-M1 and AnP-M2 are listed in Vazyme biotech (Nanjing, China). Fast MultiSite Muta- Additional file 2: Table S1. The AnP-M1 and AnP-M2 genesis System, Bradford protein assay kit, Escherichia PCR products in the pPIC9K vector were confirmed by coli Trans I-T1 cells and E.coli DMT cells from DNA sequencing. Han et al. BMC Biotechnology (2018) 18:36 Page 7 of 8 Protein expression and purification in the NPT ensemble (323 K/343 K, 1 bar). All systems P. pastoris GS115 competent cells were transformed were solvated with TIP3P waters in an octahedral box with the plasmids using electroporesis with a voltage of [26]. Sodium and chloride ions were added (100 mM) to 1500 V at time constant of 5 ms [21]. 1 mL transfor- neutralize the systems. Protonation states for histidines mants were cultured on YPD plates containing 250 μg/ were determined by the UCSF Chimera program [27]. ml G-418 at 30 °C for 1 day and then sub-cultured with The GROMACS program suite version 4.5.7 and Amber 100-fold dilution. The overnight YPD subculture was ff99SB force field were used in all simulations [28, 29]. transferred to 5 ml BMGY medium with initial OD = Bond length constraints were applied to all bonds that 0.2 and cultured overnight before transfer to BMMY contained hydrogen atoms based on the LINCS protocol inducible medium. The supernatant was collected for [30]. An integration step of 0.002 ps was used in all sim- analysis after 2 days of induction at 30 °C [22]. ulations. Electrostatic integrations were treated with For purification, cultures of AnP and mutant transfor- Particle Mesh Ewald method with a cutoff of 0.9 nm mants were centrifuged at 12,000×g for 10 min to with grid spacing for the FFT grid< 0.12 nm [31]. remove cell debris, and the crude phytases were concen- trated using an Amicon centrifugal filter device (cutoff Hydrogen bond analysis 10.000). The concentrates were purified using a Hydrogen bonds between mutational residues and nearby DEAE-Sepharose column (3 × 15 cm) equilibrated with residues in all simulation systems were analyzed by using 10 mM sodium acetate buffer (pH 5.5). The proteins g_hbond in the GROMACS suite. Geometrical criterions were eluted with elution buffer with a linear gradient of which include donor-acceptor distance (≤0.35 nm) and NaCl from 0 to 0.5 M. The fraction profiles of OD hydrogen-donor-acceptor angle (≤30°) are used to calculate and phytase activity were confirmed to contain the de- hydrogen bond. For each time frame, if both the sired protein peaks. The peak fractions were collected donor-acceptor distance and the hydrogen-donor-acceptor and stored at − 20 °C for further characterization [22]. angle satisfy the criterions, the number of hydrogen bond Purified protein samples were subjected to SDS-PAGE, will be counted as 1, otherwise 0. The number of hydrogen and enzyme concentration was determined by Bradford bonds was calculated based on the whole 50 ns simulation protein assay kit. (50,000 frames in total) in each system, and the error bar represents one standard error which was calculated based Enzyme activity characterization on the averaged number of hydrogen bonds every 10 ns in One phytase activity unit is defined as the amount of en- each system. zyme required to release 1 μmol phosphate from phytate per minute [23]. All assays in this work were performed Clustering analysis in triplicate. Thermostability was assayed by measuring Pair-wise clustering analysis was performed using residual enzyme activity after incubation at 70, 80 and gcluster in the GROMACS suite based on the 90 °C for 1 h at a phytase-optimal pH 5.5. root-mean-square deviation (RMSD) of the heavy Kinetic parameters (K , V ,and k ) for each atoms of protein. The snapshots to do the clustering m max cat phytase from a Michaelis-Menten rate expression analysis were selected 10 ps intervals from the simu- were determined in sodium acetate buffer (200 mM, lation trajectories (5000 snapshots in each system). pH 5.5) at 37 °C. The reactions were monitored at 12 different concentrations of sodium phytate ranging Calculation of mainchain deviations between AnP and AfP from 0.05 to 2.5 mM [22]. Kinetic parameters were Crystal structures of AnP and AfP were firstly superim- calculated by fitting to the Michaelis-Menten function posed according to the result of pair-wise sequence using Origin 8.5.1. alignment. Mainchain deviations were calculated for all equivalent mainchain Cα atoms based on their orthog- MD simulation details onal coordinates. The Aspergillus niger phytase X-ray crystal structure (PDB: 3K4P), which shares 97% sequence identity with Additional files the AnP phytase in the above material section, was used as the starting geometry for the AnP protein [24]. The Additional file 1: Figure S1. Root-mean-square deviations (RMSD) of SWISS-MODEL server was used to build the 18 point AnP and AnP-M1. RMSD of heavy atoms of AnP (black) and AnP-M1 mutation AnP-M1 [25]. After a 1000-step energy (green) as a function of simulation time at 50 °C, and RMSD of heavy atoms of AnP (red) and AnP-M1 (blue) as a function of simulation minimization, all the systems were equilibrated for 5 ns time at 70 °C. (PDF 956 kb) in the NPT ensemble followed by another 5 ns equilibra- Additional file 2: Table S1. Oligonucleotide primers for AnP-M1 and tion in the NVT ensemble by restraining all heavy AnP-M2. (DOCX 13 kb) atoms. Afterwards, each system was simulated for 50 ns Han et al. BMC Biotechnology (2018) 18:36 Page 8 of 8 Abbreviations 11. Wyss M, Pasamontes L, Remy R, Kohler J, Kusznir E, Gadient M, Muller F, van AfP: Aspergillus fumigatus phytase; AnP: Aspergillus niger PhyA phytase; E. Loon AP. Comparison of the thermostability properties of three acid coli: Escherichia coli; HAPs: Histidine acid phytases; MD: Molecular dynamics phosphatases from molds: aspergillus fumigatus phytase, A. Niger phytase, and A. Niger PH 2.5 acid phosphatase. Appl Environ Microbiol. Acknowledgements 1998;64:4446–51. We would like to thank Kevin Shi from the Massachusetts Institute of 12. Xiang T, Liu Q, Deacon AM, Koshy M, Kriksunov IA, Lei XG, Hao Q, Thiel DJ. Technology for providing language help. Crystal structure of a heat-resilient phytase from aspergillus fumigatus, carrying a phosphorylated histidine. J Mol Biol. 2004;339:437–45. Funding 13. Pasamontes L, Haiker M, Wyss M, Tessier M, van Loon AP. Gene cloning, This study is supported by the National Natural Science Foundation of China purification, and characterization of a heat-stable phytase from the fungus (No. 31660240 and No. 31660304), the National Key Research and Development aspergillus fumigatus. Appl Environ Microbiol. 1997;63:1696–700. Program of China (No. 2017YFB0308401), Applied Basic Research Foundation of 14. Tomschy A, Tessier M, Wyss M, Brugger R, Broger C, Schnoebelen L, van Yunnan Province (No. 2016FD018). Loon AP, Pasamontes L. Optimization of the catalytic properties of aspergillus fumigatus phytase based on the three-dimensional structure. Availability of data and materials Protein Sci. 2000;9:1304–11. All data generated or analyzed during this study are included in this 15. Jaenicke R, Schurig H, Beaucamp N, Ostendorp R. Structure and stability of published article and its supplementary information files. hyperstable proteins: glycolytic enzymes from hyperthermophilic bacterium Thermotoga maritima. Adv Protein Chem. 1996;48:181–269. Authors’ contributions 16. Vogt G, Woell S, Argos P. Protein thermal stability, hydrogen bonds, and ion NH carried out the computational prediction and molecular dynamics simulations. pairs. J Mol Biol. 1997;269:631–43. HM and TY performed the major experiments containing site-directed mutagenesis 17. Zhang W, Mullaney EJ, Lei XG. Adopting selected hydrogen bonding and and enzyme production. BX, YY and QW helped to purify and characterize the ionic interactions from aspergillus fumigatus phytase structure improves the phytases. NH and ZH wrote the manuscript. RZ and ZH revised this paper. All thermostability of aspergillus Niger PhyA phytase. Appl Environ Microbiol. authors read and approved the final manuscript. 2007;73:3069–76. 18. Ragon M, Hoh F, Aumelas A, Chiche L, Moulin G, Boze H. Structure of Ethics approval and consent to participate Debaryomyces castellii CBS 2923 phytase. Acta Crystallogr Sect F Struct Biol Not applicable. Cryst Commun. 2009;65:321–6. 19. Wijma HJ, Floor RJ, Jekel PA, Baker D, Marrink SJ, Janssen DB. Competing interests Computationally designed libraries for rapid enzyme stabilization. Protein The authors declare that they have no competing interests. Eng Des Sel. 2014;27:49–58. 20. van Hartingsveldt W, van Zeijl CM, Harteveld GM, Gouka RJ, Suykerbuyk ME, Luiten RG, van Paridon PA, Selten GC, Veenstra AE, van Gorcom RF, et al. Publisher’sNote Cloning, characterization and overexpression of the phytase-encoding gene Springer Nature remains neutral with regard to jurisdictional claims in (phyA) of aspergillus Niger. Gene. 1993;127:87–94. published maps and institutional affiliations. 21. Mullaney EJ, Locovare H, Sethumadhavan K, Boone S, Lei XG, Ullah AH. Site-directed mutagenesis of disulfide bridges in aspergillus Niger NRRL Author details 3135 phytase (PhyA), their expression in Pichia pastoris and catalytic School of Life Sciences, Yunnan Normal University, Kunming 650500, China. characterization. Appl Microbiol Biotechnol. 2010;87:1367–72. Key Laboratory of Yunnan for Biomass Energy and Biotechnology of 22. Liao Y, Li CM, Chen H, Wu Q, Shan Z, Han XY. Site-directed mutagenesis Environment, Yunnan Normal University, Kunming 650500, China. improves the thermostability and catalytic efficiency of aspergillus Niger Engineering Research Center of Sustainable and Utilization of Biomass N25 phytase mutated by I44E and T252R. Appl Biochem Biotechnol. Energy, Ministry of Education, Kunming 650500, China. 2013;171:900–15. 23. Tomschy A, Wyss M, Kostrewa D, Vogel K, Tessier M, Hofer S, Burgin H, Received: 5 January 2018 Accepted: 11 May 2018 Kronenberger A, Remy R, van Loon AP, Pasamontes L. 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Biochemical characterization of fungal phytases (myo-inositol hexakisphosphate phosphohydrolases): catalytic properties. Appl Environ Microbiol. 1999;65:367–73. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png BMC Biotechnology Springer Journals

Enhancing thermal tolerance of Aspergillus niger PhyA phytase directed by structural comparison and computational simulation

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Chemistry; Applied Microbiology; Biotechnology; Biochemical Engineering; Genetic Engineering; Plant Breeding/Biotechnology; Biomedical Engineering/Biotechnology
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Abstract

Background: Phytase supplied in feeds for monogastric animals is important for improving nutrient uptake and reducing phosphorous pollution. High-thermostability phytases are particularly desirable due to their ability to withstand transient high temperatures during feed pelleting procedures. A comparison of crystal structures of the widely used industrial Aspergillus niger PhyA phytase (AnP) with its close homolog, the thermostable Aspergillus fumigatus phytase (AfP), suggests 18 residues in three segments associated with thermostability. In this work, we aim to improve the thermostability of AnP through site-directed mutagenesis. We identified favorable mutations based on structural comparison of homologous phytases and molecular dynamics simulations. Results: A recombinant phytase (AnP-M1) was created by substituting 18 residues in AnP with their AfP analogs. AnP-M1 exhibited greater thermostability than AnP at 70 °C. Molecular dynamics simulations suggested newly formed hydrogen bonding interactions with nine substituted residues give rise to the improved themostability. Thus, another recombinant phytase (AnP-M2) with just these nine point substitutions was created. AnP-M2 demonstrated superior thermostability among all AnPs at ≥70 °C: AnP-M2 maintained 56% of the maximal activity after incubation at 80 °C for 1 h; AnP-M2 retained 30-percentage points greater residual activity than that of AnP and AnP-M1 after 1 h incubation at 90 °C. Conclusions: The resulting AnP-M2 is an attractive candidate in industrial applications, and the nine substitutions in AnP-M2 are advantageous for phytase thermostability. This work demonstrates that a strategy combining structural comparison of homologous enzymes and computational simulation to focus on important interactions is an effective method for obtaining a thermostable enzyme. Keywords: Phytase, Thermostability, Site-directed mutagenesis, Homologous structural comparison, Molecular dynamics simulation Background be utilized directly [2]. Supplemental inorganic phosphate Phytate (myo-inositol hexakisphosphate) is the primary can partially compensate for the deficiency, but excessive form of stored phosphorus in most plants and conse- phytate phosphorus in animal excretions induces severe quently, in animal feeds [1]. Due to inadequate levels of ecological problems [3, 4]. The digestive enzyme phytase digestive enzymes in the gastrointestinal tracts of mono- can hydrolyze phytic acid or phytate into lower inositol gastric animals (swine, poultry, fish, etc.), phytate cannot phosphates [5]. Thus, phytases supplied in animal feeds not only improve phytate utilization in monogastric ani- mals, but also alleviate phytate phosphorus pollution [6]. * Correspondence: huangzunxi@163.com The first commercial phytase products were intro- Nanyu Han and Huabiao Miao contributed equally to this work. Equal contributors duced 20 years ago [7]. Currently, over 60% of feed School of Life Sciences, Yunnan Normal University, Kunming 650500, China products include phytase [8]. To lower production costs, Key Laboratory of Yunnan for Biomass Energy and Biotechnology of novel thermostable phytases capable of withstanding the Environment, Yunnan Normal University, Kunming 650500, China Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Han et al. BMC Biotechnology (2018) 18:36 Page 2 of 8 transient high temperatures (80–85 °C) of the feed pro- Results duction pelleting process are in high demand [9]. AnP-M1 showed improved thermostability The Aspergillus niger PhyA phytase (AnP) exhibits Inspired by the work done by Tao Xiang and coworkers superior enzymatic activity compared to other fungal [12], mainchain deviations were re-calculated. After phytases [5]. Specifically, AnP exhibited considerably superimposing AnP and AfP crystal structures, Cα higher specific activity (102.5 U/mg) than the Aspergillus atoms between analogous residues in three segments fumigatus phytase (AfP) (26.5 U/mg) [10]. Moreover, (A35-P42, R163-Q168, T248-K254 in AnP) were shown AnP is an acidic phosphatase that optimally active in the to have distances greater than 3 Å (Fig. 1a, b). This re- pH range prevalent in the digestive tract [2, 10]. How- sult is consistent with the findings from Tao Xiang et al. ever, when exposed to pelleting process conditions, AnP The first recombinant phytase (AnP-M1) was cre- loses 70–80% of its enzymatic activity [11]. In contrast, ated by substituting residues from AnP in the above the phytase from AfP, which shares 66% sequence iden- three segments with the corresponding residues in tity and has similar overall crystal structure [12], is well AfP by site-directed mutagenesis (35-ANESVISP-42, known for its heat resistance: AfP retains 90% of its 163-RAQPGQ-168, and 248-TSTVDTK-254 in AnP initial enzymatic activity after heating to 100 °C for correspond to 35-EDELSVSS-42, 163-GATNRA-168, 20 min [13]. Thus, AfP may act as a guide for AnP and 248-RTSDASQ-254 in AfP). Thermostability enzymatic engineering for both high activity and high assays revealed that AnP-M1 retained greater activity thermostability [14]. after treatment at 70 °C for 1 h compared to AnP Based on the above hypothesis, Tao Xiang and (Fig. 1c). AnP-M1 retained 51% activity while AnP coworkers proposed three segments that could contrib- retained 31% activity. ute to the thermostability of AfP from comparing crystal structures of AnP and AfP [12]. We confirmed these Hydrogen bonds in AnP-M1 govern the improved sites and constructed a recombinant phytase (AnP-M1) thermostability by substituting residues in the three segments in AnP Generally, creating an enzyme with improved thermosta- with residues at corresponding sites in AfP. As expected, bility usually requires combining multiple amino acid the resultant recombinant demonstrated higher thermo- exchanges, each of which slightly increases the unfolding stability than the parent AnP. We explored potential temperature of the protein. Some example mechanisms AnP-M1 heat resistant mechanisms using molecular dy- are: new hydrogen bonds, new disulfide bridges, β-turns namics (MD) simulations, and nine of the 18 mutated or flexible termini stabilization, hydrophobic packing en- residues in AnP-M1 exhibited potential interactions that hancement, or α-helix or β-sheet stabilization [15]. One could contribute to this. Subsequently, we created our study showed that the hydrogen bonding appears to be second recombinant phytase (AnP-M2) by substituting the most important factor for thermostability based on just the nine proposed thermostability-enhancing resi- analysis of enzymes from 16 families [16]. Specifically, dues into AnP. This new variant (AnP-M2) demon- increased hydrogen bonding interactions are observed in strated even greater thermal tolerance. the three segments of AfP based on structural analysis This work combines structural comparison and com- [12]. To identify the contribution of particular residues putational simulation to guide the design of a thermo- towards improving thermostability, we performed MD stable phytase. The most promising new variant has simulations of AnP and AnP-M1 at two temperatures particular potential for the animal feed industry. (50 °C and 70 °C). Both AnP and AnP-M1 were stable ac b Fig. 1 Structural comparison of homologous phytases and thermostability assays of the first recombinant. Structural comparison (a) and mainchain deviation (b) between AnP (tv_blue) and AfP (tv_yellow). Residual activity of AnP (black) and AnP-M1 (red) incubated at 70 °C for 1 h (c) Han et al. BMC Biotechnology (2018) 18:36 Page 3 of 8 during the 50 ns simulations at two temperatures, with thermostability for AnP-M1, and the mutated serines at the overall root-mean-square deviations (RMSD) of the position 39 and 42 in segment-1 are critical for the heavy atoms with respect to the initial structure less interactions. than 0.35 nm (Additional file 1: Figure S1). After investi- Hydrogen bond interactions within segment-2 residues gating the hydrogen bond patterns, we identified nine in AnP and AnP-M1 were also analyzed (Fig. 2b). AnP mutated residues in AnP-M1 that form new hydrogen R163 and D161 formed 1.74 and 1.70 hydrogen bonds at bonding interactions that could explain the improved 50 °C and 70 °C, respectively. In contrast, AnP-M1 G163 thermostability. and D161 formed no more than 0.8 hydrogen bonds at Hydrogen bonds connecting segment-1 residues both temperatures. In AnP, the probability of a hydrogen were monitored throughout the entire simulation in bond connecting Q165 and R129 was 89% at 50 °C, each phytase. Most hydrogen bonds connecting decreasing to 24% at 70 °C, indicating a flexible inter- 35-ANESVISP-42 and nearby residues in AnP per- action between Q165 and R129 in AnP at elevated sisted at both temperatures (Fig. 2a). The hydrogen temperature. In contrast, the hydrogen bond between bond connecting AnP A35 and S33 was well pre- T165 and R129 in AnP-M1 was well maintained and served at both temperatures (36% at 50 °C and 33% increased to 40% at 70 °C, suggesting a stable and favor- at 70 °C). The probability of hydrogen bonding able contact at higher temperature. Likewise, the prob- between AnP N36 and E37 is 42% at 50 °C and 40% ability of hydrogen bonding between AnP-M1 N166 and at 70 °C. The probability of hydrogen bonding be- R167 was approximately 20% at both temperatures, tween S38 and I40 in AnP increased to 93% at 70 °C. while no hydrogen bond was observed between AnP In AnP-M1, the formation probabilities of three P166 and G167 at either temperature. In summary, hydrogen bond pairs, E35-S33, D36-E37, and AnP-M1 segment-2 hydrogen bond pairs R129-T165 L38-V40, were less than those in AnP. and N166-R167 may contribute to the enhanced Interestingly, the probability of hydrogen bonding be- thermostability. tween S39 and E37 in AnP-M1 increased to 25% at 70 ° Hydrogen bonds formed within segment-3 residues C. Additionally, another hydrogen bond connecting in AnP (248-TSTVDTK-254) and AnP-M1 (248-RTS AnP-M1 S42 and D405 was well preserved at both tem- DASQ-254) were also compared (Fig. 2c). It is evident peratures: probabilities of hydrogen bonding between that R248 and D244 in AnP-M1 formed almost 2 S42 and D405 were 59 and 74% at 50 °C and 70 °C, re- hydrogen bonds at both temperatures, and the prob- spectively. However, hydrogen bonding pairs V39-E37 ability of this interaction is 4-fold greater than that and P42-D405 in AnP were rarely observed. In sum- between the corresponding AnP T248 and D244. mary, two newly formed hydrogen bond pairs, S39-E37 Almost no contact was monitored between residues and S42-D405, may contribute to the enhanced 248 and 249 in either AnP or AnP-M1. Although ab c Fig. 2 Statistics of hydrogen bonding with mutated residues. Statistics of hydrogen bonds connecting residues in segment 1–3(a-c, respectively) and nearby residues for AnP and AnP-M1 from simulations at 50 °C and 70 °C Han et al. BMC Biotechnology (2018) 18:36 Page 4 of 8 AnP-M1 S250 and Q254 formed 1.01 and 0.78 hydro- R248T, D251V, A252D, and Q254K). This nonuple gen bonds at the two temperatures, AnP T250 and mutant is denoted AnP-M2. K254 formed more hydrogen bonds at 70 °C (0.88), To evaluate thermostability, we measured the residual indicating a more stable interaction between AnP activities of AnP, AnP-M1 and AnP-M2 after 1 h incuba- T250 and K254 at 70 °C. Similarly, the hydrogen tion at three temperatures (70, 80, and 90 °C). The two bond between AnP-M1 S253 and S250 is not as mutants showed promising improvements in residual strong as that between AnP T253 and T250. In con- activity, and the nonuple mutant AnP-M2 exhibited the trast, D251, A252, and Q254 in AnP-M1 were associ- highest thermal tolerance. Specifically, AnP-M2 retained ated with higher probability in forming hydrogen 66% activity after 1 h incubation at 70 °C, AnP-M1 bonds with nearby residues than those in AnP. In retained 51% at the same condition, and AnP retained summary, AnP-M1 segment-3’shydrogen bonds markedly less (31%) (Fig. 4a). After 1 h incubation at within R248, D251, A252, and Q254 may contribute 80 °C, AnP-M2, AnP-M1 and AnP retained 58, 41 and to the improved thermostability. 21% activity, respectively (Fig. 4b). After incubation at In light of the above, not all the mutagenesis sites 90 °C for 1 h, AnP-M2 retained 30-percentage points demonstrated stable or increased hydrogen bonding in- greater residual activity than that of the AnP and teractions with nearby residues in AnP-M1, suggesting AnP-M1 (Fig. 4c). These results highlight that the nine that only a subset of the 18 point mutations contributed substitutions in AnP-M2 are advantageous for phytase to the improved thermostability. Comparing number of thermostability. hydrogen bonds formed with the substituted residues in AnP and AnP-M1, we concluded that S39 and S42 in Kinetic analysis of AnPs segment-1 (Fig. 3a, d); T165, N166, and R167 in Kinetic analysis showed apparent K values for AnP, segment-2 (Fig. 3b, e); and R248, D251, A252, and Q254 AnP-M1 and AnP-M2 of 289.8, 116.5 and 135.2 μM, re- in segment-3 (Fig. 3c, f) make the greatest contribution spectively (Table 1). The smaller Michaelis constant to the enhanced AnP-M1 thermostability. (K ) of both mutants indicate an increase in kinetic effi- ciency compared to that of AnP. Comparing k /K cat m AnP-M2 showed superior thermal tolerance among all values of three AnPs, AnP-M1 has the highest catalytic AnPs efficiency, followed by AnP and the AnP-M2. Kinetic To further increase AnP thermostability, we performed analysis suggests that the substitutions in AnP-M1 and another round of site-directed mutagenesis based on les- AnP-M2, which we designed primarily to improve ther- sons learned from our analysis of AnP-M1. In total, nine mostability, enhanced substrate binding affinity (K )by AnP residues were substituted to AfP residues at the two-fold; however, the k values of both mutants were cat equivalent sites (S39 V, S42P, T165Q, N166P, R167G, decreased to the same extent and k /K which cat m a b c d e f Fig. 3 Comparison of hydrogen bonding networks formed by mutated residues in AnP and AnP-M1. Illustration of the newly formed hydrogen bonding network in AnP-M1 (a-c) and analogous locations in AnP (d-f). Representative structures are cluster centers from clustering analysis Han et al. BMC Biotechnology (2018) 18:36 Page 5 of 8 ab c Fig. 4 Thermostability assays of all three AnPs. Residual activities of AnP (black), AnP-M1 (red), and AnP-M2 (blue) incubated at 70 °C (a), 80 °C (b), and 90 °C (c) for 1 h represents catalytic efficiency was slightly decreased for [18]. Residues 35–42 in AnP segment-1 are located AnP-M2 versus AnP-M1. within the fungal HAP’s unique N-terminal region. Therefore, the newly formed hydrogen bond pairs Discussion S39-E37 and S42-D405 in segment-1 may play a pivotal In order to create AnP variants with substantially in- role in enhancing the thermal tolerance of phytase mu- creased thermostability, we performed two rounds of tants through stabilizing the local structure in the rationally designed site-directed mutagenesis. A crystal N-terminal region. structure comparison between regular AnP and a particu- Although AnP-M2 has the highest thermostability, it larly thermostable homolog, AfP, indicated three segments has slightly decreased catalytic efficiency compared to that that may influence thermostability. MD simulations on a of AnP. The consensus catalytic motif 58-RHGARYP-64 mutant AnP created based these comparisons, AnP-M1, and substrate-binding motif 338-HD-339 are located in explored heat resistance mechanisms and pointed out the deep substrate-binding cleft at the interface of the nine particular thermostability-enhancing residues. We large α/β domain and the small α domain (Fig. 5a). Struc- created a final recombinant, AnP-M2, with superior ther- tural analysis shows that mutated sites from segment-3, mostability, enhanced substrate binding affinity, and located at the small α domain, are close to the catalytic decreased catalytic efficiency compared to other AnPs. and substrate-binding motifs, with a ~ 8 Å distance A previous study on phytase thermostability focused between the closest segment-3 and substrate-binding resi- on three residues derived from structural modeling, indi- dues (Fig. 5b). One previous effort to improve enzyme cated that hydrogen bond network and ionic interaction thermostability only mutated residues > 10 Å away from formed with the three residues support the high thermo- the active site in order to avoid such possible interference stability of AfP [17]. These three residues are present in [19]. Mutations in the AnP-M2 segment-3 may affect local both our full 18-residue homology-based mutant and re- movement, thus influencing catalysis. This conjecture re- fined 9-residue MD-based mutant. Similarly, our study quires experimental studies which could consist of single discovered that hydrogen bonding interactions formed point mutations of AnP-M2 segment-3 residues to identify with the nine substituted residues account for the catalytic efficiency influencing residue (s). This provides a improved thermostability. next step for designing a new variant with both high ther- The fungal histidine acid phytases (HAPs) possess an mostability and high catalytic efficiency. extra N-terminal region before the first β-strand of the α/β domain. This region folds into short helices and Conclusions loops. Structural studies suggest that the N-terminal re- Our work demonstrates the improvement of an gion in tetrameric phytase extend outward and form part enzyme using a multipart rational design strategy: of the interface of the dimer and tetramer structures identifying residues from homologs with desirable Table 1 Kinetics of AnP, AnP-M1 and AnP-M2 Enzymes K (μM) V (μmol/min/mg) k (/s) k /K (/s/μM) m max cat cat m AnP 289.8 ± 13.8 5954.2 ± 63.8 4885.4 ± 52.1 16.9 ± 1.7 AnP-M1 116.5 ± 9.2 3117.1 ± 55.1 2557.6 ± 44.9 22.0 ± 1.8 AnP-M2 135.2 ± 8.8 2484.4 ± 59.1 2042.6 ± 48.2 15.1 ± 1.8 Han et al. BMC Biotechnology (2018) 18:36 Page 6 of 8 Fig. 5 Illustration of mutated and catalytic residues in AnP-M2. Mutated residues in AnP-M2 are shown in spheres, substrate binding motif (orange) and catalytic motif (purple) are shown in cartoon (a). Distances between R248 in segment-3 of AnP-M2 and Y63 of the catalytic motif were shown in yellow dash (b) properties followed by determining important mecha- TransGen (Beijing, China). All other chemicals were of nisms from MD simulations and then final design analytical grade and commercially available. refinement. The final designed A.niger PhyA variant also provides new insight into the design of thermo- Gene cloning and site-directed mutagenesis stable phytases. Additional enzyme properties could The AnP sequence came from the Aspergillus niger also be modified using this combined method. PhyA gene (GenBank: Z16414) in the pMD19-T vector [20]. Genes of mutants (AnP-M1 and AnP-M2) were constructed by introducing mutations to AnP through Methods site-directed mutagenesis using the Fast MultiSite Materials System according to the manufacturer’s instructions. High fidelity DNA polymerase, restriction endonuclease PCR cycling conditions consisted of an initial step of (EcoRI, NotI) and dNTP from TaKaRa (Otsu, Japan). 5 min at 94 °C, followed by 30 cycles of 30 s at 94 °C, Plasmid mini-prep kit and DNA gel extraction kit from 1 min at 55 °C, and 3 min 30 s at 72 °C. Forward and Omega (Taipei, USA). One-step cloning kit from reverse primers for AnP-M1 and AnP-M2 are listed in Vazyme biotech (Nanjing, China). Fast MultiSite Muta- Additional file 2: Table S1. The AnP-M1 and AnP-M2 genesis System, Bradford protein assay kit, Escherichia PCR products in the pPIC9K vector were confirmed by coli Trans I-T1 cells and E.coli DMT cells from DNA sequencing. Han et al. BMC Biotechnology (2018) 18:36 Page 7 of 8 Protein expression and purification in the NPT ensemble (323 K/343 K, 1 bar). All systems P. pastoris GS115 competent cells were transformed were solvated with TIP3P waters in an octahedral box with the plasmids using electroporesis with a voltage of [26]. Sodium and chloride ions were added (100 mM) to 1500 V at time constant of 5 ms [21]. 1 mL transfor- neutralize the systems. Protonation states for histidines mants were cultured on YPD plates containing 250 μg/ were determined by the UCSF Chimera program [27]. ml G-418 at 30 °C for 1 day and then sub-cultured with The GROMACS program suite version 4.5.7 and Amber 100-fold dilution. The overnight YPD subculture was ff99SB force field were used in all simulations [28, 29]. transferred to 5 ml BMGY medium with initial OD = Bond length constraints were applied to all bonds that 0.2 and cultured overnight before transfer to BMMY contained hydrogen atoms based on the LINCS protocol inducible medium. The supernatant was collected for [30]. An integration step of 0.002 ps was used in all sim- analysis after 2 days of induction at 30 °C [22]. ulations. Electrostatic integrations were treated with For purification, cultures of AnP and mutant transfor- Particle Mesh Ewald method with a cutoff of 0.9 nm mants were centrifuged at 12,000×g for 10 min to with grid spacing for the FFT grid< 0.12 nm [31]. remove cell debris, and the crude phytases were concen- trated using an Amicon centrifugal filter device (cutoff Hydrogen bond analysis 10.000). The concentrates were purified using a Hydrogen bonds between mutational residues and nearby DEAE-Sepharose column (3 × 15 cm) equilibrated with residues in all simulation systems were analyzed by using 10 mM sodium acetate buffer (pH 5.5). The proteins g_hbond in the GROMACS suite. Geometrical criterions were eluted with elution buffer with a linear gradient of which include donor-acceptor distance (≤0.35 nm) and NaCl from 0 to 0.5 M. The fraction profiles of OD hydrogen-donor-acceptor angle (≤30°) are used to calculate and phytase activity were confirmed to contain the de- hydrogen bond. For each time frame, if both the sired protein peaks. The peak fractions were collected donor-acceptor distance and the hydrogen-donor-acceptor and stored at − 20 °C for further characterization [22]. angle satisfy the criterions, the number of hydrogen bond Purified protein samples were subjected to SDS-PAGE, will be counted as 1, otherwise 0. The number of hydrogen and enzyme concentration was determined by Bradford bonds was calculated based on the whole 50 ns simulation protein assay kit. (50,000 frames in total) in each system, and the error bar represents one standard error which was calculated based Enzyme activity characterization on the averaged number of hydrogen bonds every 10 ns in One phytase activity unit is defined as the amount of en- each system. zyme required to release 1 μmol phosphate from phytate per minute [23]. All assays in this work were performed Clustering analysis in triplicate. Thermostability was assayed by measuring Pair-wise clustering analysis was performed using residual enzyme activity after incubation at 70, 80 and gcluster in the GROMACS suite based on the 90 °C for 1 h at a phytase-optimal pH 5.5. root-mean-square deviation (RMSD) of the heavy Kinetic parameters (K , V ,and k ) for each atoms of protein. The snapshots to do the clustering m max cat phytase from a Michaelis-Menten rate expression analysis were selected 10 ps intervals from the simu- were determined in sodium acetate buffer (200 mM, lation trajectories (5000 snapshots in each system). pH 5.5) at 37 °C. The reactions were monitored at 12 different concentrations of sodium phytate ranging Calculation of mainchain deviations between AnP and AfP from 0.05 to 2.5 mM [22]. Kinetic parameters were Crystal structures of AnP and AfP were firstly superim- calculated by fitting to the Michaelis-Menten function posed according to the result of pair-wise sequence using Origin 8.5.1. alignment. Mainchain deviations were calculated for all equivalent mainchain Cα atoms based on their orthog- MD simulation details onal coordinates. The Aspergillus niger phytase X-ray crystal structure (PDB: 3K4P), which shares 97% sequence identity with Additional files the AnP phytase in the above material section, was used as the starting geometry for the AnP protein [24]. The Additional file 1: Figure S1. Root-mean-square deviations (RMSD) of SWISS-MODEL server was used to build the 18 point AnP and AnP-M1. RMSD of heavy atoms of AnP (black) and AnP-M1 mutation AnP-M1 [25]. After a 1000-step energy (green) as a function of simulation time at 50 °C, and RMSD of heavy atoms of AnP (red) and AnP-M1 (blue) as a function of simulation minimization, all the systems were equilibrated for 5 ns time at 70 °C. (PDF 956 kb) in the NPT ensemble followed by another 5 ns equilibra- Additional file 2: Table S1. Oligonucleotide primers for AnP-M1 and tion in the NVT ensemble by restraining all heavy AnP-M2. (DOCX 13 kb) atoms. Afterwards, each system was simulated for 50 ns Han et al. BMC Biotechnology (2018) 18:36 Page 8 of 8 Abbreviations 11. Wyss M, Pasamontes L, Remy R, Kohler J, Kusznir E, Gadient M, Muller F, van AfP: Aspergillus fumigatus phytase; AnP: Aspergillus niger PhyA phytase; E. Loon AP. Comparison of the thermostability properties of three acid coli: Escherichia coli; HAPs: Histidine acid phytases; MD: Molecular dynamics phosphatases from molds: aspergillus fumigatus phytase, A. Niger phytase, and A. Niger PH 2.5 acid phosphatase. Appl Environ Microbiol. Acknowledgements 1998;64:4446–51. We would like to thank Kevin Shi from the Massachusetts Institute of 12. Xiang T, Liu Q, Deacon AM, Koshy M, Kriksunov IA, Lei XG, Hao Q, Thiel DJ. Technology for providing language help. Crystal structure of a heat-resilient phytase from aspergillus fumigatus, carrying a phosphorylated histidine. J Mol Biol. 2004;339:437–45. Funding 13. Pasamontes L, Haiker M, Wyss M, Tessier M, van Loon AP. Gene cloning, This study is supported by the National Natural Science Foundation of China purification, and characterization of a heat-stable phytase from the fungus (No. 31660240 and No. 31660304), the National Key Research and Development aspergillus fumigatus. Appl Environ Microbiol. 1997;63:1696–700. Program of China (No. 2017YFB0308401), Applied Basic Research Foundation of 14. Tomschy A, Tessier M, Wyss M, Brugger R, Broger C, Schnoebelen L, van Yunnan Province (No. 2016FD018). Loon AP, Pasamontes L. Optimization of the catalytic properties of aspergillus fumigatus phytase based on the three-dimensional structure. Availability of data and materials Protein Sci. 2000;9:1304–11. All data generated or analyzed during this study are included in this 15. Jaenicke R, Schurig H, Beaucamp N, Ostendorp R. Structure and stability of published article and its supplementary information files. hyperstable proteins: glycolytic enzymes from hyperthermophilic bacterium Thermotoga maritima. 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