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. 2020 May 7;9(3):182–190. doi: 10.1093/toxres/tfaa013

Exploring the influence of silver and lead on structure and function of xylanase: spectroscopic and calorimetric methods

Mingyang Jing 1,2, Rui Tang 1, Guangye Han 3, Shansheng Zhang 2, Rutao Liu 1,✉
PMCID: PMC7438702  PMID: 32850115

Abstract

Soil contamination with heavy metal could induce the alteration of soil ecological environments, and soil enzyme activities are sensitive indicators for the soil toxicology. Xylanase is one of predominant soil enzymes related to carbon nitrogen cycle. In this work, we explored the underlying mechanisms for conformational and enzymatic activity alterations of xylanase after silver and lead exposure at molecular level with systematical measurements including multiple spectroscopic methods, isothermal titration calorimetry, and enzymatic activity. Both silver and lead could loosen and unfold the skeleton of xylanase with the quenching of endogenous fluorescence. Silver interacted with xylanase forming larger-size aggregations through Van der Waals forces and hydrogen bonding, while lead interacted with xylanase forming larger-size aggregations through hydrophobic force. Silver and lead induced an obvious loss (67.1 and 56.31%) of the xylanase enzymatic activity, but silver has a greater impact on xylanase than that of lead. The xylanase enzymatic activity significantly decreased due to the conformational alterations. The negative effect of silver exposure on xylanase structure and function was more prominent than that of lead.

Keywords: silver, lead, xylanase, spectroscopic methods, calorimetry

Graphical Abstract

Graphical Abstract.

Graphical Abstract

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Introduction

Xylanase is an extracellular enzyme produced and released mainly by fungi in the aerobic environment. Xylanase is a class of enzymes that can catalyze the hydrolysis of ß-1,4 bonds of xylan molecules to monosaccharides [1]. Xylanase, amylase, cellulase, sucrase, protease, chitinase, and urease are predominant soil enzymes related to the carbon nitrogen cycle [2]. The biochemical reactions in soil are mostly mediated by various enzymes [3]. Thus, soil enzyme activities are sensitive indicators to the changes of soil properties and pollutants.

Heavy metals have been regarded as great endangerment to soil environment because of their indissolubility and bioaccumulation, which may adversely affect the soil ecosystem resulting in significant decrease in soil quality [4, 5]. Remarkable accumulation of heavy metals has been observed in some delta areas, such as the Mekong River Delta [6], the Pearl River Delta [7], and the Yellow River Delta [8]. Previous surveys showed that the mean content of silver and lead in soil in China reached 0.35 mg/kg [9] and 26 mg/kg [10], much higher than that of the whole world. In this study, silver and lead were selected as the representatives of heavy metals for different electric charges.

The toxic effects of heavy metal to enzymes have been explored by toxicologists since the 1990s. Researchers found that lead pollution could significantly enhance the catalase activity and inhibit the invertase and activities [11]. Soil enzyme activity was greatly depressed by conditions in the heavy metal-contaminated soil [12–14]. Previous studies usually measured the enzyme activities in the whole soils and rarely considered the direct interaction mechanism of heavy metal with single enzyme in soil at molecular level. In this work, multiple spectroscopic methods, three-dimensional fluorescence analysis, enzymatic activity, and calorimetric measurements were utilized to investigate the structure and function changes of xylanase under silver and lead exposure. Thermodynamic parameter analysis and binding modes of xylanase–silver or lead system were also performed to recognize the binding details. This study intended to characterize silver and lead effects on the structure and function of xylanase and understand the possible mechanism at molecular level.

Materials and Methods

Materials

Xylanase (1,4-β-xylanxylanohydrolase, EC 3.2.1.8, 6000 U/mg) from penicillium was purchased from Shanghai Yuanye Biotechnology Co., Ltd., which was dissolved in ultrapure water to form a 0.3 mg/l solution and then filtered with 0.45 micron filter membrane. Silver nitrate and lead acetate were provided by Tianjin Kermel Chemical Reagent Co., Ltd., China. A solution of acetate–acetate buffer (0.2 M) was used to stabilize the pH (5.4) containing a mixture of CH3COONa·3H2O and CH3COOH. 3,5-dinitrosalicylic acid (DNS), phenyl hydroxide, sodium potassium tartrate tetrahydrate, and sodium sulfite was obtained from J&K Scientific co., Ltd. Xylan was obtained from Sigma Chemical Company (St. Louis, MO). Ultrapure water (18.25 Ω) was used throughout the experiments. Chemicals used were all of analytical grade.

In all the experiments, the pH of the experimental system was set as 5.4 because the optimum pH for the action of xylanase was at 5.0–5.5 [15]. Moreover, heavy metal (lead) ions do not precipitate at pH = 5.4.2.2 UV–visible absorption spectra.

UV–visible spectra have been employed recently to reflect the structural changes of enzymes. The absorption spectra of xylanase exposed to different concentrations of heavy metals at 298 K were recorded by UV-2450 spectrophotometer (Shimadzu, Japan). Solutions were prepared as follows: 1.0 ml HAc–NaAc buffer (pH = 5.4), 4.0 ml xylanase solution, and different concentrations of heavy metal were made up to 10 ml with distilled water and stirred thoroughly. Corresponding concentration of heavy metal in buffer solution was used as reference. The optical path length was 1 cm and the scanning range was 190–350 nm.

Fluorescence measurements

To investigate the amino acid residue microenvironments, all measurements of fluorescence spectra were performed by an F-4600 fluorescence spectrophotometer (Hitachi, Japan) equipped with a xenon lamp and a 1.0 cm quartz cell. The corresponding excitation and emission slit widths of all fluorescence spectra were set at 5.0 nm, and the excitation wavelength was set at 280 nm. The scanning speed was 1200 nm/min and the scanning range was 290–450 nm. In this experiment, the composition of the reaction system included a series of concentrations of heavy metals, 0.3 mg/l xylanase, and 0.02 M HAc–NaAc buffer (pH = 5.4). PMT voltage was set at 700 V.

The synchronous fluorescence spectra of xylanase were measured by scanning the excitation and emission wavelengths simultaneously under fixed wavelength differences (Δλ = 60 nm, λex = 250–350 nm and Δλ = 15 nm, λex = 250–350 nm). To correct the inner filter effect (IEF) caused by reaction system, the following formula was introduced:

graphic file with name M1.gif

where Fcor and Fobs are the correct and observed fluorescence intensity, respectively. Aex and Aem represent the absorbances at excitation and emission wavelengths, respectively.

Resonance light scattering measurements were measured at λem = λex (from 200 to 450 nm).

The three-dimensional fluorescence spectra were measured with the following parameters: λem = 200–600 nm, λex = 200–450 nm, slit width of 5.0 nm, and PMT voltage of 475 V.

Xylanase enzymatic activity measurements

To determine the correlation between xylanase activity and heavy metal content, the 3,5-dinitrosalicylic acid (DNS) method that indicates the decomposition rate of xylooligosaccharides was selected in this study [1, 16]. Due to the fact that DNS gives a higher color response with reducing sugar, the changes in xylanase activity can be monitored by UV–visible absorption at 540 nm. Then, the inhibition or promotion rate of enzymatic activity was calculated using the following equation:

graphic file with name M2.gif

where ΔA is the reduction of the absorption value at 540 nm in absence of silver or lead and ΔA0 is the reduction of the absorption value at 540 nm in presence of silver or lead.

Isothermal titration calorimetry (ITC)

In a single reaction of xylanase binding to silver or lead, the enthalpy, entropy, and Gibbs free energy changes were measured simultaneously on a Microcal ITC200 calorimetry (GE, Fairfield, CT). HAc–NaAc buffer (0.02 M) was used as solvent for both xylanase and silver or lead solution. Then, the solutions were filtered by 0.22 μm filter membrane. The filtered heavy metal solution was loaded in the syringe (about 40 μl), and xylanase was added into the sample cell (about 280 μl). The heavy metal solution was injected 14 times, and the stirring speed was 1000 rpm. A blank experiment was performed by titrating heavy metal solution into 0.02 M HAc–NaAc buffer.

The concentration of xylanase was determined by the classic Coomassie method with bovine serum albumin (BSA) standard solution as the control.

Results and Discussion

Investigation of the conformational changes of xylanase

The backbone changes of xylanase induced by silver and lead exposure

UV–visible spectra has been employed to reflect the structural changes of enzymes in the presence of toxins. The UV–visible absorption spectra of xylanase in the absence and presence of silver and lead are shown in Fig 1A and B. Xylanase has two absorption peaks with the strong peak (206 nm) representing the framework conformation of the protein and the weak peak (280 nm) representing the aromatic amino acids (tryptophan, tyrosine, and phenylalanine) [17].

Figure 1.

Figure 1

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The UV–visible absorption spectra of the xylanase—silver(lead) system. Figure 1A: silver, Fig. 1B: lead; conditions: silver(lead):(μM): (a) 0, (b) 20, (c) 60, (d) 100, (e) 200, (f) 400, (g)600; pH = 5.4; T = 298 K.

As shown in Fig. 1A, the strong peak (206 nm) decreased accompanied by a redshift from 206 to 210 nm, with gradual increase of silver dose. The skeleton of xylanase was unfolded and loosened. Then, the internal hydrophobic region of xylanase began to spread and become more hydrophilic. The energy of the Π–Π* transition reduced, resulting in the redshift [18]. The rather weak peak at about 270 nm was increasing with the addition of silver, which illustrated that the microenvironment of the aromatic acid residues was changed.

As shown in Fig. 1B, the strong peak (206 nm) also decreased accompanied by a redshift from 206 to 213 nm with gradual increase of lead dose. Thus, the lead exposure also induced unfolding and loosening the skeleton of xylanase. The rather weak peak at about 270 nm was not influenced by the addition of lead, which illustrated that the microenvironment of the aromatic acid residues was unchanged.

Influence of silver and lead on the fluorescence intensity of xylanase

The aromatic amino acid residues (tyrosine, tryptophan, and phenylalanine) are the main source of endogenous fluorescence in proteins. Fluorescence spectroscopy can be utilized to investigate the change of microenvironment around the fluorophores [19]. The inner filter effect (IFE) has been considered in preliminary experiments, in which the sum of the absorption at 278 nm (excitation wavelength) and 335 (peak wavelength) caused no more than 5% error [20, 21]. Thus, the IFE was negligible in this study. The fluorescence spectra of xylanase at various concentrations of silver or lead are shown in Fig. 2. The fluorescence intensity of xylanase decreased regularly with the addition of silver from 20 to 400 μM, which depicted that the addition of silver results in conformational change, denaturation of xylanase through biomolecule binding [22]. Upon addition of lead (in Fig. 2B), the fluorescence intensity of xylanase was also quenched continuously indicating the conformational change of xylanase.

Figure 2.

Figure 2

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Fluorescence intensity of xylanase with different concentrations of silver and lead. Figure 2A: silver, Fig. 2B: lead; conditions: silver(lead):(μM): (a) 0, (b) 20, (c) 60, (d) 100, (e) 200, (f) 400; pH = 5.4; T = 298 K.

Effect of silver and lead on the synchronous fluorescence spectroscopy of xylanase

Synchronous fluorescence spectra is characteristic in the prominent advantages of reduced light scattering, simplified spectra, and improved selectivity, in which the basic information about the microenvironment in the vicinity of the chromospheres can be presented [23]. When excitation (λex) and emission (λem) wavelengths intervals (Δλ) is at stabilized 15 and 60 nm, the synchronous fluorescence presents the characteristic information about the polarity and hydrophobicity of tyrosine residues and tryptophan residues [24], respectively. The synchronous fluorescence spectra of xylanase are shown in Fig. 3.

Figure 3.

Figure 3

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Synchronous fluorescence spectra of xylanase with different concentrations of silver and lead. (1)Δλ = 60 nm, Fig. 3A: Silver, Fig. 3B: Lead; (2)Δλ = 15 nm, Fig. 3C: silver, Fig. 3D: lead; conditions: silver(lead):(μM): (a) 0, (b) 20, (c) 60, (d) 100, (e) 200, (f) 400; pH = 5.4; T = 298 K.

When Δλ was fixed at 60 nm, synchronous fluorescence spectra of Trp residues (Fig. 3A and B) decreased gradually with the addition of silver and lead but had negligible shift. The results express that silver and lead could bind to xylanase, but the microenvironment of Trp residues were not perturbed. The characteristic spectra of the Tyr residues (Δλ = 15 nm) for silver and lead system are shown in Fig. 3C and D. Moreover, the fluorescence intensity of Tyr residues decreased regularly with the addition of silver and lead in both systems. Thus, synchronous fluorescence spectra further confirmed the occurrence of fluorescence quenching in the xylanase–silver and xylanase–lead binding process.

Protein size changes characterized by resonance light scattering

Resonance light scattering (RLS) is a satisfactory technique to efficiently characterize chromophore aggregation in biochemical assays [25, 26]. Figure 4A and B shows the RLS spectra of silver–xylanase and lead–xylanase systems, respectively. The fluorescence intensity is proportional to dispersed particles [26]. The peak fluorescence intensity of RLS in silver–xylanase system increased obviously (from 158 to 4916) with gradual increase of silver dose, which was a clear indication about the formation of complex with larger particle size. The possible reason for the obvious increase particle size in silver–xylanase system is that silver could bind with xylanase forming larger copolymers [27, 28]. Previous studies have verified that silver could denature proteins [29, 30] that can explain the increase in particle size. As shown in Fig. 4B, the peak fluorescence intensity of RLS (from 130 to 456) increased at 0–100 μM, while the intensity began decrease with continuous increase of lead dose. The result revealed that aggregations were formed at low dose of lead, but the aggregations were destroyed effectively with continuous increase of lead dose. The reduction of agglomerate particle size is attributed to the damage of solvent shell on the surface of xylanase or disintegration of agglomerates that leads to dispersion [31].

Figure 4.

Figure 4

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RLS spectra of silver or lead–xylanase system; Fig. 4A: silver, Fig. 4B: lead; conditions: silver(lead):(μM): (a) 0, (b) 20, (c) 60, (d) 100, (e)200, (f)400; pH = 5.4; T = 298 K.

Although the intensity of RLS in lead–xylanase system was also enhanced as a whole, the enhancements were not as obvious as that of silver. Silver seems to be more inclined to coordinate with the amino acid residues of the polypeptide chain [32]. The size of the xylanase molecule changes confirmed silver and lead result in conformational alterations as the above-mentioned investigations.

The three-dimensional fluorescence analysis on structural changes of xylanase

Three-dimensional fluorescence measurement was performed to further understand the structural changes of xylanase intuitively and scientifically. As shown in Fig. 5, peak a, the resonance Rayleigh scattering peak (λem = λex) enhanced with the addition of silver and lead, while more significant enhancement occurred by silver exposure. Peak b, the second-order scattering peak (λem = 2 λex), enhanced slightly by silver and lead. Both the two peaks enhanced after the addition of silver and lead, indicating that the hydration radius increased [33] under the exposure of silver and lead. The results were corresponding to the resonance light scattering spectra above.

Figure 5.

Figure 5

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Three-dimensional fluorescence spectra of silver–xylanase and lead–xylanase system. Conditions: Xylanase: 0.3 mg/l; silver/(μM): A1, 0; A2, 20; A3, 100; lead/(μM): B1, 0; B2, 20; B3, 100; pH = 5.4; T = 298 K.

Figure 6.

Figure 6

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Xylanase activity alterations in silver or lead–xylanase system; Fig. 6A: silver, Fig. 6B: lead; conditions: pH = 5.4; T = 298 K.

Peak 1 (λex = 280 nm, λem = 335 nm) and peak 2 (λex = 230 nm, λem = 335 nm) reveals the intrinsic fluorescence of amino acid residues (Trp and Tyr) and polypeptide backbone structures [34], respectively. The change of fluorescence intensities of Peak 1 and Peak 2 in silver-xylanase and lead-xylanase systems were shown in Table 1. Peak 1 decreased after the addition of silver and lead, indicating that both of silver and lead quenched the endogenous fluorescence of xylanase and changed conformation of xylanase. The quench of peak 2 indicated that the polypeptide chain of xylanase was unfolded and the backbone structure was loosened after silver and lead exposure. The results were consistent with the fluorescence measurements and UV–visible experiments.

Table 1.

Three-dimensional characteristics of silver–xylanase and lead–xylanase systems

Toxicant μM Peak 1 Peak 2
Position Intensity Position Intensity
Silver 0 λex = 280 nm λem = 335 nm 351.9 λex = 230 nm λem = 335 nm 322.4
20 λex = 280 nm λem = 335 nm 351.0 λex = 230 nm λem = 335 nm 286.4
100 λex = 280 nm λem = 335 nm 309.4 λex = 230 nm λem = 335 nm 235.1
Lead 0 λex = 280 nm λem = 335 nm 425.9 λex = 230 nm λem = 335 nm 398
20 λex = 280 nm λem = 335 nm 401.3 λex = 230 nm λem = 335 nm 333.6
100 λex = 280 nm λem = 335 nm 373.3 λex = 230 nm λem = 335 nm 158.3
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Alterations of xylanase enzymatic activity induced by silver and lead exposure

Generally, the specific physiological function of proteins has a close relationship with the certain backbone structures of proteins [35]. And enzyme activity is one of the most important indicators of protein function. The impacts of silver and lead on the enzymatic activity of xylanase were shown in Fig. 5. The activity change of xylanase was calculated by setting the relative activity of xylanase as 100% in the absence of silver or lead. With the addition of silver or lead, the enzymatic activity of xylanase obviously decreased, but alterations were unconspicuous in the enzymatic activity at low concentrations of silver (lower than 20 μM) or lead (lower than 100 μM). The xylanase enzymatic activity significantly decreased at 400 μM silver or lead exposure to 31.10 or 56.31% of the initial level. The effect of silver on xylanase activity was more obvious than that of lead that were consistent with the changes of xylanase particle size. The decrease in xylanase activity in heavy metal polluted soils was detected in previous study [36] that affected the carbon nitrogen cycle [37]. According to the results above, the xylanase enzymatic activity significantly decreased due to the conformational alterations. The active site of xylanase is located in a cleft between β barrel and ribbon strand. The active site contains the vital catalytic residues Glu132 and Glu238 [38]. On the other hand, silver and lead might bind with the amino acid residues within the active center resulting in alterations of enzymatic activity. 3D structure of the xylanase (Fig. S1) has been presented in supplementary materials. Results in the fluorescence measurements indicated that silver and lead caused the intrinsic fluorescence of fluorophores(Trp, Tyr, and Phe residues). Coincidentally, residues Trp88, Trp268, andTrp276 are near the catalytic center of xylanase that might related to enzymatic activity.

Binding affinity and thermodynamic parameter determination

In this study, the thermodynamic properties in the reaction of silver or lead with xylanase were further studied by isothermal calorimetric titration (ITC). After deducting the dilution heat of the blank group, the experimental results of silver-titrated xylanase and lead-titrated xylanase are shown in Fig. 7A and B, respectively. Negative peak was observed when silver was titrated into xylanase solution, indicating the interaction was exothermic. In contrast, the interaction between lead and xylanase was endothermic. The ΔG of both reaction systems(shown in the attached Fig. 7) was less than zero, which meant that each process was spontaneous.

Figure 7.

Figure 7

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ITC profiles of the interaction between silver (A) and lead (B) with xylanase. Experimental conditions: C[xylanase] = 8 μM, C[silver] = C[lead] = 0.2 mM.

The titration curve conforms to the single binding site model. The binding mode between small molecule and protein is mainly determined by enthalpy change (ΔH) and entropy change (ΔS) of the reaction. The negative values of ΔH and ΔS observed in silver–xylanase system revealed binding process was dominated by Van der Waals forces and hydrogen bonding [39], while hydrophobic force was the dominant driving force in lead–xylanase system with the positive values of ΔH and ΔS [40]. Van der Waals forces/hydrogen bonding/hydrophobic force was also confirmed as dominant forces in the interactions between the metal ion and enzymes [41–43]. Similar studies have found the hydrophobic was the dominant driving forces in lead–gonadotropin and in cadmium–lysome systems [41, 44]. Previous study also stated that Cu2+ might spontaneously bind to xylanase by hydrophobic force, hydrogen bond, or Van der Waals forces [45]. Additionally, the binding constant KSilver-Xylanase is 1.08 × 103 and KLead-Xylanase is 2.5 × 103, which indicated that the binding force of silver and lead interacting with xylanase was relatively moderate. The diffusion rate of silver or lead to target sites in xylanase is faster based on the perspective of metabolic kinetics.

Conclusions

In the present study, the interaction mechanism of xylanase with heavy metals (silver and lead) was compared by multiple spectroscopic methods, isothermal titration calorimetry, and enzyme activity measurement. The results demonstrated the existence of binding interactions between xylanase and silver or lead. Van der Waals forces and hydrogen bonding were the dominant forces in the reaction between silver and xylanase, while hydrophobic force was the dominant force in the reaction between lead and xylanase. Both of silver and lead obviously quenched the endogenous fluorescence of xylanase, loosened, and unfold the skeleton of xylanase. The particle size of xylanase was increased after silver and lead exposure indicated the formation of aggregations. Silver and lead induced an obvious loss of the xylanase enzymatic activity. Silver has a greater impact on xylanase than lead. Our work confirms the direct interaction modes of silver and lead with xylanase at the molecular level and provides methodological support for soil toxicology.

Funding

This work is supported by National Natural Science Foundation of China (21477067, 51608304, 21777088, and U1806216), the Cultivation Fund of the Key Scientific and Technical Innovation Project, Research Fund for the Doctoral Program of Higher Education and Ministry of Education of China (708058, 20130131110016), independent innovation program of Jinan (201202083), and Science and Technology Development Plan of Shandong Province (2014GSF117027).

Conflict of interest

There are no conflicts to declare.

Supplementary Material

Supporting_information_tfaa013
Click here for additional data file. (879KB, doc)

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