Trichoderma harzianum Produces a New Thermally Stable Acid Phosphatase, with Potential for Biotechnological Application

Acid phosphatases (ACPases) are produced by a variety of fungi and have gained attention due their biotechnological potential in industrial, diagnosis and bioremediation processes. These enzymes play a specific role in scavenging, mobilization and acquisition of phosphate, enhancing soil fertility and plant growth. In this study, a new ACPase from Trichoderma harzianum, named ACPase II, was purified and characterized as a glycoprotein belonging to the acid phosphatase family. ACPase II presents an optimum pH and temperature of 3.8 and 65°C, respectively, and is stable at 55°C for 120 min, retaining 60% of its activity. The enzyme did not require metal divalent ions, but was inhibited by inorganic phosphate and tungstate. Affinity for several phosphate substrates was observed, including phytate, which is the major component of phosphorus in plant foods. The inhibition of ACPase II by tungstate and phosphate at different pH values is consistent with the inability of the substrate to occupy its active site due to electrostatic contacts that promote conformational changes, as indicated by fluorescence spectroscopy. A higher affinity for tungstate rather than phosphate at pH 4.0was observed, in accordance with its highest inhibitory effect. Results indicate considerable biotechnological potential of the ACPase II in soil environments.


Introduction
Phosphorus (P) is an essential nutrient for normal growth and metabolic processes in plants and microorganisms. It is also involved in the biosynthesis of cellular components, such as nucleic acids, phospholipids and proteins [1,2]. Over 80% of the phosphorus applied to soil is lost due to its broad adsorption, precipitation or conversion to organic form [3,4]. In alkaline soils, phosphorus is encountered fixed in the tricalcium phosphate salt and in acid soils as molecules involved in cellular process, such as adenosine triphosphate (ATP), adenosine diphosphate (ADP) and adenosine monophosphate (AMP).
Considering the relevance and the potential biotechnological application of these phosphatases, efforts have been made to comprehend the molecular mechanisms and the relationship between structure and function. To date, understanding of uptake and phosphate release mechanisms in these enzymes remains unclear. In the present paper, we report the purification and functional characterization of a new enzyme presenting acid phosphatase activity from T. harzianum, named ACPase II.

Materials and Methods Materials
Culture media reagents were purchased from Himedia (India). Reagents for protein assays were purchased from Bio-Rad 1 (Quick Start Bradford, USA). Reagents for deglycosilation and substrates for enzymatic assays were purchased from Sigma Aldrich™ (USA).

Microorganism and culture conditions
Conidiospores from a single spore-derived pure culture for strain T. harzianum ALL42 (HS574263.1, GenBank) were provided by the Department of Biochemistry and Molecular Biology, Enzymology Laboratory, Federal University of Goiás, Goiânia, Goiás, Brazil. A stock culture of the microorganism was recovered on Malt Yeast Glucose (MYG) medium containing 0.5% (w/v) malt extract, 0.25% (w/v) yeast extract, 1.0% (w/v) glucose and 2.0% (w/v) agar, with incubation at 28°C. A sexual conidia were isolated in sterile saline solution, following centrifugation at 3000 g.

Enzymatic assay
Acid phosphatase activity was measured according to Leitão et al [25], using p-Nitrophenyl phosphate disodium salt hexahydrate (p-NPP) (Sigma Aldrich™) as substrate. One unit (1U) of acid phosphatase activity was defined as 1 μM of p-Nitrophenol (p-NP) formed per minute [36]. Protein concentration was determined by the Bradford method, using bovine serum albumin as standard [37].
Purification of acid phosphatase and analysis of acid phosphatase purity Crude extract containing enzymes was 10-fold concentrated by ultrafiltration using a 10 kDa membrane (Millipore, 63.5 mm). A 1 mg sample was applied onto a molecular exclusion Superdex 200 HiLoad 16/60 column (GE Healthcare), then equilibrated with 50 mM sodium acetate buffer, pH 4.8, using an AKTA Purifier fast protein liquid chromatography system (FPLC, GE Healthcare). Fractions presenting acid phosphatase activity were pooled and dialyzed against 50 mM sodium acetate buffer, pH 4.8,containing 3 M (NH 4 ) 2 SO 4 (buffer A). The samples were applied on a Hitrap Phenyl FF (High sub) column (GE Healthcare) previously equilibrated with buffer A. The column was washed with the same buffer and the bound proteins eluted using a decreasing gradient of 3 M to zero of (NH 4 ) 2 SO 4 in the same sodium acetate buffer described above. Fractions containing acid phosphatase activity were pooled, dialyzed against water and stored at -20°C.
Polyacrylamide gel electrophoresis under denaturing conditions (12%SDS-PAGE) was carried out to estimate protein purity and the molecular mass of the purified enzyme [38]. Protein was silver stained as described by Blum [39] and molecular mass was estimated using molecular weight markers β-galactosidase (116 kDa), Bovine serum albumin (66.2 kDa), Ovalbumin (45 kDa), Lactate dehydrogenase (35 kDa) and REase Bsp98l (25 kDa) (Thermo Scientific). The error of the molecular mass determination was estimated based on analysis of data for five 12% SDS-PAGE runs, with linear regression calculated using Image Lab 5 (Bio-Rad).

Protein identification byMALDI-TOF/TOF spectrometry
The band corresponding to the enzyme identified as ACPase II was excised from polyacrylamide gels and digested with trypsin Gold-Mass V582A (Promega), according to Schevchenko et al [39]. The resulting peptides of each spot were submitted to mass spectrometric analyses using an UltraFlexIII MALDI-TOF/TOF (Matrix-Assisted Laser Desorption/Ionization-Time of Flight), controlled with Flex Control 3.0 software (Bruker Daltonik). The sample was mixed with α-cyano-4hydroxycinnamic acid matrix solution (3:1, v/v) directly applied onto an MTP AnchorChip 400/384 target plate (Bruker Daltonik) and dried at room temperature. Peptides presenting mono isotopic masses were obtained in reflector mode with external calibration using the Protein Calibration Standard (Bruker Daltonik). Peptide MS/MS spectra were obtained by means of LIFT fragmentation. The software Flex Analysis 3.0 (Bruker Daltonik) and PepSeq (Waters) were used for mass spectrometric data analysis. Peptide primary structures were inferred by means of manual interpretation of fragmentation. The obtained sequences were then searched against the NCBI nr protein database (www.ncbi.nlm.nih.gov) using the algorithm Blastp. The identified protein sequences were analyzed using the Peptide Mass tool from the ProtParam Server (www.expasy.org) in order to predict theoretical molecular weight [40].

Deglycosylation of the acid phosphatase
The purified enzyme was treated with the enzymatic protein Deglycosylation kit, according to the manufacturer's protocol (Sigma Aldrich™). An ACPase sample containing 100 μg of protein was added to the reaction buffer and denaturation solution and boiled for 5 minutes. The sample was then incubated for 3 hours at 37°C with PNGase F and O-glycosidase. The deglycosylated protein was analyzed by12% SDS-PAGE, as described above.

Biochemical characterization of the purified ACPase II
The effect of pH on ACPase II activity was determined by varying the pH from3.2 to 5.6 using 50 mM glycine-HCl (pH 3.2-3.4) and 50 mM sodium acetate (pH 3.6-5.6) buffer. The effect of temperature on the enzymatic activity was determined at pH 3.8over a temperature range from 35 to 80°C. Thermostability was determined by pre-incubation of the purified enzyme at pH 4.0, 50, 55 and 60°C for 120 min and returning the temperature to60°C (optimum temperature). Kinetics parameters were estimated using p-NPP at concentrations ranging from 0.033 to 1.0μM. Activities were determined by the standard procedure and kinetics parameters (K m and V max ) calculated from the fitted Michaelis-Menten curve by nonlinear regression/enzymatic kinetics/ Michaelis-Menten enzyme kinetics via the program GraphPad Prism (version 6.01, 2000). The ability of ACPase II to hydrolyze different substrates (Sigma Aldrich™), i.e. p-NPP, ATP, ADP, AMP, fructose 6-phosphate, glucose 1-phosphate, phenyl phosphate, β-glucose phosphate and phytic acid, was evaluated at substrate concentrations of 5 mM [41]. The effects of inorganic phosphate and sodium tungstate on ACPase II activity were also determined after pre-incubation of purified enzyme with the respective compound (5 mM) for 10 min at 60°C.

Fluorescence Spectroscopy Assays
Fluorescence measurements of 0.66 μM ACPase II were performed at pH 4.0-5.5 (10 mM sodium acetate buffer), pH 6.5-9.0 (10 mM Tris buffer), and 25°C using the Jasco FP-6500 Spectrofluorimeter (Jasco Analytical Instruments, Tokio, Japan) coupled to a Jasco ETC-273T Peltier system (Jasco Analytical Instruments) with water circulation. Both the excitation and emission slits were fitted at 10.0 nm, with excitation and emission wavelengths at 295 nm and 300-400 nm, respectively.
ACPase II spectral changes were investigated in the presence of sodium phosphate (0-660 μM) and sodium tungstate (0-660 μM) with fluorescence quenching as a function of pH 4.0, 7.0 and 8.5. The averages of four fluorescence spectra were recorded and processed using the program Spectra Manager (Jasco Analytical Instruments, Tokio, Japan). The fluorescence emissions were fitted according to the classic Stern-Volmer equation (Eq 1) as follow [42]: where F and F 0 representthe fluorescence intensities in the presence and absence of quencher, respectively, K SV is Stern-Volmer constant and [Q] is the concentration of the quencher. In order to calculate the dissociation constants for ACPase-related phosphate and tungstate complex, the equilibrium between free and bound molecules is assumed to be proportional to the fluorescence intensity as [43]. The dissociation constants (K D ) for the equilibrium between free and bound ions on ACPase II were calculated by nonlinear regression, again using GraphPad Prism (version 6.01, 2000).

Statistical analysis
All experiments were performed in triplicate. Statistical analysis in enzymatic assays was performed based on standard deviation. Differences were considered to be significant at a value of p <0.05.
In this work, the amount of 10 7 spores.ml -1 of T. harzianum was cultivated in medium supplemented with glucose (15 g.L -1 ) without the addition of inorganic phosphate (pH adjusted to 4.0) for 90 hours. The highest levels of ACPases (14.3 U.mg -1 ) were obtained from culture supernatants at 48 hours (Fig 1). Additionally, a decrease of ACPases secretion in the presence of inorganic phosphate was observed.

Purification and characterizationof ACPase II from T. harzianum
In most reports on fungal ACPase, two or more chromatography steps are generally required for protein purification [13-14, 21, 23, 24, 53]. Similarly, a new acid phosphatase from T. harzianum, named ACPase II, was purified in our study by using two steps. Firstly, two distinguishable acid phosphatase activity peaks (I and II) (Fig 2A) from crude extract of enzymes, corresponding to the fractions 9-11 and 14-15, were obtained by size exclusion chromatography using a Superdex 200 column on an AKTA Purifier (GE Healthcare).
The activity found in peak I (Fig 2A and inset) corresponded to the eluted ACPase II (fractions 9 to 11) and that from peak II corresponded to the eluted ACPase I (fractions 14 to 15), as purified by Leitão et al [25]. This was assumed based on the differences in molecular mass of ACPase I (58 kDa) and ACPase II (90 kDa), as previously estimated through 12% SDS-PAGE (S1A Fig), together with respective activities presented in a zymogram gel (S1B Fig). Secondly, the ACPase II was purified by hydrophobic chromatography (HC) in Hitrap Phenyl FF, as shown in Fig 2B. Both resins preserved the biological activity of ACPase II, resulting in a 21.7-fold purification, with overall yield of 43.3% and specific activity of 1030.0 U.mg -1 . A starting material of 200 ml of T. harzianum protein crude extract (0.175 mg/ml) was concentrated to 3.314 mg in 1ml and applied onto a superdex 200 column ( Table 1).
The molecular mass of ACPase II was estimated by 12% SDS-PAGE ( Fig 2B) due its selfassociation tendency in solution, as indicated by size exclusion chromatography and dynamic light scattering (data not shown). ACPase II was eluted in a void volume and presented hydrodynamic radius of 14.3 nm and molecular mass of 326 kDa, compatible with a tetramer.
In order to assess whether ACPase II is a glycoprotein, the purified enzyme was treated with PNGase and O-glucosidase. After SDS PAGE, a defined band was obtained corresponding to a protein of 58 ± 5 kDa, according to the almost of predicted values presented in the S1 Table. The enzyme was characterized as a glycoprotein, composed of approximately 30% carbohydrate (Fig 3). Analysis of the peptide sequence using NetNGlyc 1.0 and NetOGlyc 3.1 showed two potential N-glycosylation and four potential O-glycosylation sites (S2B Fig). ACPase II sequencing by Maldi-Tof/Tof spectrometry Identification and classification of the enzyme was carried out based on the amino acid sequence of peptides obtained from protein hydrolysis with trypsin, using MALDI-TOF/TOF spectrometry (S2A Fig). Seven peptides were sequenced and compared to sequences from  GenBank. The amino acid sequences and molecular mass matched phytases from T. harzianum, histidine acid phytase from Trichoderma pleuroticola and a hypothetical protein from Trichoderma virens available on the NCBI database (S1 Table and S2B Fig). It is important to note that all of histidine phytase are refereed as histidine acid phosphatase and, to date, no sequence of acid phosphatase from T. harzianum has been found in the NCBI database. The molecular mass observed through SDS-PAGE (Fig 2B) did not match any theoretical molecular weight from similar sequences, indicating post-translational modifications, such as the identified N-glycosylation and O-glycosylation sites (Fig 3 and S2B Fig), or partial sequences of this protein on the database. However, after deglycosilation with PNGase F and O-glycosidase, the ACPase II shows a molecular mass of 68 ± 5 kDa, according to the predicted value (S1 Table). The sequence of peptides derived from ACPase II displayed a conserved histidine composing

Biochemical characterization of ACPase II
The optimum pH of ACPase II was 3.8 (Fig 4A), in agreement with data observed for fungal acid phosphatases from M. anisopliae [24], A. niger [56], A. oryzae [57] and A. nidulans [58], but distinct to those with optimum pH of 2.5 from A. ficuum [59] and A. fumigatus [55]. The optimum temperature of 65°C (Fig 4B) was similar to that for ACPases from M. hiemalis [23] and A. niger [55], but lower than observed for ACPases from M. anisopliae [24] and A. caespitosus [13]. Thermostability of ACPase II was analyzed as a criterion for potential of the characterized enzymes from this fungus in industrial application. ACPase II was stable for at least 120 min at 50°C and 55°C respectively, retaining 60% of maximum activity at 50°C and 45% of maximum activity at 55°C (Fig 4C). ACPase II showed a greater affinity for p-NPP compared to ACPase I [25] and other ACPases from Penicillium chrysogenum (111 μM), M. hiemalis (434 μM), A. bisporus (370 μM) and A. caespitosus (29 μM) [13,21,23,60], based on K m and V max values of 0.027 μM and 83.93 U/mg, respectively ( Fig 4D and Table 2). As ACPase II was not significantly inhibited by metal chelators, such as EDTA, the enzyme does not appear to require metal divalent ions as cofactors for catalytic activity (Table 3). In addition, the enzyme was not inhibited by sodium tartrate, was partially inhibited by inorganic phosphate and strongly inhibited by sodium tungstate (Table 3). Inorganic phosphate is a common inhibitor of most ACPases from fungi and in some cases is associated with mechanisms involved in enzyme regulation [13, 21-23, 55, 60]. Tungstate is a specific inhibitor of tyrosine phosphatase [24]. In both cases, the inhibition of ACPase II seems to be related with conformational changes of the protein resulting in steric hindrance surrounding of the active site. Fluorescence quenching provides evidences for this protein conformational change, as discussed below. Affinity of ACPase II for different substrates was observed, which may reflect a broad range of physiological functions in fungi, mainly associated with survival and environment adaptation. Additionally, ACPase II was not phosphate-specific, since it was able to cleave a broad range of phosphate esters, including p-NPP, a general substrate for acid phosphatases, ATP, ADP and AMP (Table 3). ACPase II was more efficient in cleaving phenyl sodium phosphate, D-glucose-1-phosphate, ATP, ADP, and AMP.
In summary, in contrast to ACPase I, based on the biochemical features, ACPase II from T. harzianum is an enzyme belonging to the acid phosphatase families, with higher activity than known ACPases from different sources. Potential practical applications of this purified phosphatase from T. harzianum may include bioremediation, biocontrol, plant nutrients uptake, aggregate stability, and carbon cycling and sequestration, as also observed with other fungal phosphatases [30,31,51,52]. However, for these biotechnological applications, among those biochemical features, the structural characterization is important to be investigated.

Structural characterization of ACPase II
The structural proprieties of ACPase II were assessed by fluorescence in order to investigate the protein structure/function relationship. The fluorescence spectra of ACPase II as a function of pH were to a shorter wavelength (336 to 327 nm), from pHs 4.5-9.0 to pH 4.0. This data is compatible with changes on tryptophan residues from exposed to buried environments as a function of acidic pH. The intensity of emission bands were also very pH dependent, increasing or decreasing in conformity with changes in the solvent polarity surrounding the tryptophan residues. This is likely due to the ionization of charged side chains and their molecular reorientation, leading to groups of spectra between pH 5.0-5.5, 6.0-7.0, 7.0-9.0 (Fig 5). These results indicate that ACPase II is more stable at pH 4.0, when compared with others pHs. It is interesting to note that this structural feature is related to the differences in enzyme activities, with highest values observed at pHs ranging from 3.8 to 4.0 (Fig 4A).

Effects of tungstate and phosphate ions on ACPase II structure and activity
The strategy employed in this work relied initially on purification and biochemical characterization of ACPase II from T. harzianum and its structural properties at pH 4.0. Indeed, we investigated the inhibitory effect of reactive ions, as tungstate and phosphate, correlated to structural properties. Inhibition of ACPase II by tungstate and phosphate observed at different pHs, was likely due to an inability of the substrate to occupy its active site. Tungstate and phosphate are polyatomic ions containing, respectively, an oxoanion of tungsten and phosphorus surrounded by four oxygen atoms which form several non covalent interactions in proteins, especially electrostatic interactions. Due their chemical properties, these compounds inhibit ACPase II (Table 3) from making electrostatic contacts, thus leading to inaccessibility of substrate for active site.
A fluorescence quenching assay was used to investigate conformational changes of ACPase II as a function of pH and affinity of compounds to ACPase II. This technique is a well-established tool that allows detection of conformational changes in proteins and protein-ligand interactions by monitoring solvent accessibility and the microenvironment of tryptophan residues under different conditions [61,62]. Specifically, changes of tryptophan emission spectra occur in response to protein conformational changes, protein-protein association, ligand binding, denaturation, etc, in which affect the environment surrounding the side chain of this residue [42].
Here in, the addition of increasing concentrations of tungstate and phosphate to ACPase II resulted in progressive decrease of fluorescence intensity at 330 nm, pH 4.0, 7.0 and 8.5 (data shown for pH 8.5; Fig 6A and 6B, inset), that were considered to estimate Stern-Volmer and dissociation constants (Figs 6 and 7). The Stern-Volmer plot profile and Ksv constants were consistent with a static fluorescence quenching process, as consequence of pH and proteinoxoanion complex formation. Phosphate and tungstate inhibitors binding to ACPase II also induce spectral shifts to shorter wavelength (from 332 nm to 328 nm) as seen at pH 8.5 (Fig 6A  and 6B, inset), indicating the tryptophan changing from exposed to buried environment.
According to the Stern-Volmer adjustment, a linear correlation was observed for compounds at different pHs (Fig 6A and 6B), supporting the existence of a static quenching process involving ACPase II, phosphate and tungstate complex formation. Fluorescence quenching corresponded to a single population of tryptophan residues in the ACPase II [42]. These results may be due to electrostatic, as well as unknown interactions in the vicinity of the fluorophore in the presence of these compounds that were responsible for conformational changes of the protein [63].
Tungstate showed a greater access to ACPase II at pH 4.0 and 7.0 than at pH 8.5, as indicated by K sv values (Table 4). In contrast, phosphate showed greater K sv values at pH 8.5 than in acidic and neutral conditions. It is noteworthy that the residual charge of the protein as a function of pH resulted in conformational changes that do not conceal the tryptophan residues at pH 7.0 and 8.5, with an exception at pH 4.0 (Fig 5). These conformational changes at pH 7.0, as well as tryptophan microenvironment changes at pH 4.0, allowed for easy access of tungstate, although not at pH 8.5. On the other hand, they were responsible for the easy access of phosphate at pH 8.5. Comparatively, increase in pH leads to a greater protein conformational change that interferes in the access of tungstate and facilitates the access of phosphate.
In order to calculate the dissociation constant of the ACPase II in complex with phosphate and tungstate, the equilibrium between free (Bo) and bounded (B) protein was assumed to be proportional to the fluorescence intensity, as B/Bo / F/Fo [42,43]. Fo is the initial fluorescence intensity and F is the fluorescence intensity in the presence of ligand (Table 4 and Fig 7).
The exact mechanism by which tungstate and phosphate inhibit ACPase II is still not understood. However, the binding of these compounds to ACPase II likely promotes protein low conformational changes, as indicated partially by the fluorescence quenching assay and a blue shift of emission bands. These findings are supported by buried rather than exposed  Table 4.  The increasing concentrations of tungstate ranging from 0 to 660 μM led to a progressive reduction in the fluorescence intensities. These data were considered in estimation of the dissociation constant (K D ), as presented in Table 4. tryptophan residues in ACPase II (blue shift of the emission band at~328 nm) that were only accessed by occurrence of electrostatic barriers for tungstate at pH 8.5 and for phosphate at pH 4.0 and 7.0. However, K D values are only slightly different for phosphate, suggesting that the microenvironment around the buried tryptophan presents low perturbations upon ion binding ( Table 4).
As also observed, the values for K sv for tungstate at pH 8.5 are very different. It is an indicative of differences in enzyme structure mainly concerning the ionized charged amino acid residues, such as histidine, which affect the tryptophan environment. All these results are associated with the highest affinity of the enzyme for tungstate at pH 4.0 and 7.0 and for phosphate at pH 8.5 (Table 4).
Considering the overall results of this study, tryptophan residues may be involved in the active sites of the enzyme, participating both in the binding and/or hydrolysis of the substrate and in the maintenance of the integrity of the active site. Additionally, fluorescence quenching shows that affinity of tungstate is higher than phosphate at optimum pH 4.0, in accordance with its inhibitory effect on ACPase II.

Conclusion
T. harzianum is a filamentous fungus with considerable biotechnological potential in industrial processes and applications in sustainable agriculture. The data reported here reveal such biotechnological application, with ACPase II from T. harzianum appropriate for the removal of phosphates from several substrates including phytate, an important molecule present in soil environments and in many plant species that are used for animal feed. Potential also exists for the development of strategies for ACPase II that enables maximal enzymatic rates, efficient production through heterologous expression, and increased thermal stability.