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Wang, S., Lv, M., Yang, J., Zhou, Y., and Xu, B. (2018). "Effects and mechanism of metal ions on enzymatic hydrolysis of wheat straw after pretreatment," BioRes. 13(2), 2617-2631.

Abstract

The effects and mechanism on the enzymatic hydrolysis of lignocellulose by metal ions were investigated by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), dynamic light scattering (DLS), Fluorescence Spectra, and enzymatic kinetics and activity analyses of the enzymatic hydrolysis system. Fe3+ exerted the best enzymatic hydrolysis effect on pretreated wheat straw, and the highest reducing sugar conversion was 44.5%. SEM and FTIR indicated that alkali and ultrasonic pretreatments partially removed the lignin and hemicellulose from lignocellulose. DLS and fluorescence spectra results revealed that the electrostatic interaction between Fe3+ and the carboxyl group in the enzyme enlarged the enzyme’s steric structure. The data on enzymatic kinetics displayed that Fe3+ increased the maximum rate of reaction by 33.9% and the Michaelis-Menten by 5.72 g/L. The enzyme activity increase rate rose initially and then diminished, and the maximum increase rate was 36.1%.


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Effects and Mechanism of Metal Ions on Enzymatic Hydrolysis of Wheat Straw after Pretreatment

Shan Wang, Mengdie Lv, Jian Yang, Yawen Zhou,* and Baocai Xu *

The effects and mechanism on the enzymatic hydrolysis of lignocellulose by metal ions were investigated by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), dynamic light scattering (DLS), Fluorescence Spectraand enzymatic kinetics and activity analyses of the enzymatic hydrolysis system. Fe3+ exerted the best enzymatic hydrolysis effect on pretreated wheat straw, and the highest reducing sugar conversion was 44.5%. SEM and FTIR indicated that alkali and ultrasonic pretreatments partially removed the lignin and hemicellulose from lignocellulose. DLS and fluorescence spectra results revealed that the electrostatic interaction between Fe3+ and the carboxyl group in the enzyme enlarged the enzyme’s steric structure. The data on enzymatic kinetics displayed that Fe3+ increased the maximum rate of reaction by 33.9% and the Michaelis-Menten by 5.72 g/L. The enzyme activity increase rate rose initially and then diminished, and the maximum increase rate was 36.1%.

Keywords: Metal ions; Cellulose; Enzymatic hydrolysis

Contact information: School of Food Science and Chemical Engineering, Beijing Key Laboratory of Flavor Chemistry, Beijing Higher Institution Engineering Research Center of Food Additives and Ingredients, Beijing Technology and Business University, Beijing Technology and Business University, Beijing, 100048, China; *Corresponding authors: zhouyw@th.btbu.edu.cn; xubac@163.com

INTRODUCTION

Lignocellulose accounts for nearly half of the total bioresources in the world. The utilization and transformation of lignocellulose are highly important in solving many global problems, such as the energy crisis, resource shortage, environmental issues, and food safety. Cellulose degradation and conversion have become hot research topics in the field of science and technology (Farrell et al. 2006; Balat 2011).

The hydrolysis of lignocellulose to fermentable sugar is usually conducted by acid or enzyme. Acid hydrolysis is constantly associated with high pressure, low glucose yield, and undesirable furfural byproducts; it is expensive and not environment friendly (Suganuma et al. 2008; Rinaldi and Schüth 2009). Therefore, hydrolytic efficiency is improved and costs are reduced by optimizing the method through various additives. Adding metal ions has achieved valuable progress. Adding FeCl3 and FeCl2 to acid solutions improves sugar yield (Yan and Ren 1999; Nguyen and Tucker 2002).

Enzymatic hydrolysis has increasingly attracted much attention because of its mild reaction conditions, high selectivity, environmental friendliness, and low byproduct amount (Paljevac et al. 2007; Kristensen et al. 2007; Fang et al. 2010; Liu et al. 2011). However, the main problems of enzymatic hydrolysis are the high enzyme-level requirement, high cost, low enzyme activity, lack of economical and feasible fermentation technology, and low conversion rate.

Metal ions are employed to promote enzymatic hydrolysis at two stages, namely, pretreatment and enzymatic hydrolysis. There are substantial reports on the addition of metal ions during pretreatment (Liu and Wyman 2006; Li et al. 2009; Liu et al. 2009; Chen et al. 2010; Jing et al. 2011; Kamireddy et al. 2013; Kang et al. 2013). Adding NH4Cl and MgCl2 during pretreatment increases enzymatic digestibility and facilitates xylan removal. FeSO4 and FeCl3 aid hemicellulose removal from corn stalk and increase glucose yield from enzymatic hydrolysis by altering the composition and structure of corn stalk. Under the same conditions, the enzymatic digestibility yields of samples in the presence of FeCl3 and CuCl2 are higher than those of samples pretreated with sulfuric acid.

However, literature is scarce on metal ion addition during mashing. MgSO4, FeSO4, and other chemicals can increase the reducing sugar concentration for the enzymatic hydrolysis of rice straw (Ren et al. 2012). Fe2+ and Cu2+ promote the degradation of steam-exploded rice straw, whereas Mg2+ and Fe3+ inhibit the enzymatic hydrolysis of the same substrate (Li et al. 2015). Mg2+ and Ca2+ increase the sugar yield of corn stalk, and the corresponding yields are increased by 5.9 % and 4.8 %, respectively (Tong 2009). Mg2+ inhibits the ineffective adsorption of cellulase by lignin, and the substrate is wheat straw pretreated with dilute acid (Akimkulova et al. 2016). Strong ion chelators and polyethylene glycols mitigate the inhibition (Tejirian and Xu 2010).

Thus it can be seen that the effects of different metal ions on different cellulose substrates significantly differ, and the effects of metal ions on enzymatic catalysis are affected by many factors. Therefore, in order to get more regular achievement in this respect, investigating the effects of metal ions on enzymatic hydrolysis is very important. In addition, although the effects of inorganic salts on the cellulase hydrolysis have been studied, few studies have investigated the mechanism of the cellulase hydrolysis by metal salts, especially about Fe3+ metal ion.

In this work, the effects and mechanism of Fe3+ metal ion on enzymatic hydrolysis of wheat straw after pretreatment were investigated by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), dynamic light scattering (DLS), Fluorescence spectra, and enzymatic kinetics and enzymatic activity analyses of the enzymatic hydrolysis system. This work aims to clarify the effects of metal ions on enzymatic hydrolysis and the mechanisms of such effects.

EXPERIMENTAL

Materials

Wheat Straw was obtained from South China University of Technology. The materials were milled until passage through a 2 mm sieve and then screened to obtain the 40 to 60 mesh fraction. The straw was composed of 40.4% cellulose, 24.5% hemicellulose, 13.0% lignin and 7.2% ash. The cellulase was purchased from Beijing Solarbio Technology Corporation (Beijing, China), and the filter paper enzyme activity was 46 FPU/g.

Pretreatment of Wheat Straw

The alkali pretreatment operation was as follows. Wheat straw fractions at ranging from 40- to 60-mesh were placed in 3% sodium hydroxide aqueous solution for 48 h at room temperature. The solid liquid ratio was 10:1 (10 mL of sodium hydroxide solution and 1 g of wheat straw). The samples were washed continuously with water until they reached a pH of 7.

Ultrasonic pretreatment was performed on an ultrasonic cleaner (Elmasonic P60H, Shenzhen Feite Li Technology Co. Ltd, Shenzhen, China) 30 min after alkali pretreatment. The frequency is 37 KHz, and the power density is 1W/cm2. The other operations were the same as the alkaline pretreatment.

The wet wheat straws were dried to constant weight. Substrate after alkali pretreatment was composed of 65.8% cellulose, 12.0% hemicellulose, 9.2% lignin and 5.6% ash. Substrate after ultrasonic pretreatment was composed of 69.4% cellulose, 11.8% hemicellulose, 8.2% lignin, and 4.4% ash, and the experimental method based on literature (Soest 1963; Mo 2008).

Enzymatic Hydrolysis

All enzymatic hydrolysis experiments were carried out in an oscillator at 150 rpm equipped with a constant temperature thermostat (IS-REDA, Suzhou Jiemei Electronic Co Ltd, Suzhou, China). All experiments were performed in duplicate under the same conditions, and their average values and standard deviations were calculated.

Analytical Method for the Reducing Sugar

The reducing sugar concentrations were determined by the DNS method (Zhang and Yu 2017), i.e., the reduction of 3,5-dinitrosalicylic acid by the reducing sugar in the presence of bases and heat. The process produces a red-brown product. The amount of reducing sugar is directly related to the concentration of this red-brown product and is determined through a UV-visible light spectrophotometer (UV-3600, Shimadzu, National Tsing Hua University (Beijing) Technology Co., Ltd, Beijing, China) (Shi 2004). The reducing sugar conversion rate was calculated by Eq. 1,

(1)

SEM

The substrates (which included the non-pretreated substrate, alkali-pretreated substrate, ultrasonically pretreated substrate, substrate after enzymatic hydrolysis without added Fe3+, and substrate after enzymatic hydrolysis with added Fe3+) were observed using a scanning electron microscope (TESCAN VEGA II, S.R.O. Corporation, Brno, Czech Republic) at an acceleration voltage of 20 kV. Prior to analysis, the samples were mounted on an aluminum stub by using double-sided tape and coated with Au (JEOL JFC-1600 Auto Fine Coater, Tokyo, Japan) under vacuum.

FTIR

The FTIR spectra of the substrates were acquired using a Nicolet Is10 spectrophotometer (Thermo Scientific, Waltham, USA). The substrates were mixed with KBr and measured within the wavenumber range of 400 to 4000 cm−1 with an accumulation of 32 scans.

DLS

DLS measurements were performed with a Malvern Instrument Zetasizer Nano (Malvern, UK), equipped with a 22 mW He–Ne laser operating at a wavelength of 632.8 nm. The solutions were filtered through 450 nm filters. The scattering angle was 173°.

Kinetics of Enzyme-Catalyzed Reactions

The Michaelis-Menten equation was used to obtain the kinetics of enzyme-catalyzed reactions (Eq. 2),

V = Vmax s / (Ks) (2)

where s is the mass concentration of the substrate (g/L), V is the starting rate (g/(L·min)), Vmax is the maximum reaction rate (g/(L·min)), and Km is the Michaelisian constant (g/L). The enzymatic hydrolysis conditions were as follows: buffer pH of 4.8, cellulase mass concentration of 3 g/L, Fe3+ mass concentration of 0.3 g/L, and enzymatic hydrolysis time and temperature of 24 h and 50 °C, respectively.

Enzymatic Activity of Enzymatic Hydrolysis

The enzymatic activity of enzymatic hydrolysis was obtained by the relative increase in enzyme activity, as calculated using Eq. 3,

(3)

The enzymatic hydrolysis conditions were as follows: cellulose mass concentration of 3 g/L, Fe3+ mass concentration of 0.3 g/L, and substrate liquid mass fraction of 1.25 %.

Fluorescence Spectra

An F-4600 spectrofluorometer was used to measure enzymatic hydrolysis solutions without and with Fe3+ at 24 h and 36 h. The enzymatic hydrolysis conditions were as follows: mass concentration of cellulose was 3 g/L, mass concentration of Fe3+ was 0.3 g/L, and substrate liquid mass fraction was 1.25 %. The emission spectra of 300 to 650 nm were recorded for the excitation wavelength of 280 nm.

RESULTS AND DISCUSSION

Effect of Different Metal Ions

The effects of different metal ions on enzymatic hydrolysis at 50 °C are shown in Fig. 1. These metal ions demonstrated positive influences on enzymatic hydrolysis that depended on the metal ion concentrations. For example, at the concentration of 0.4 g/L, Fe3+ and Co2+ improved the conversions. In particular, Fe3+ increased the reducing sugar conversion by 10.6%. However, beyond 0.4 g/L, the reducing sugar conversion decreased with rising ion concentration. Although Co2+, Fe2+, and Mg2+ are all divalent cations, Co2+ and Fe2+ improved the conversions, whereas Mg2+ only slightly influenced the conversion. For the same cation Mg2+, the effect of MgSO4 on the conversion was slightly greater than that of MgCl2. Thus, the effects of metal ions not only depended on their charge but also on their types.

Fig. 1. Effects of different metal ions on the reducing sugar conversion

Surface Morphology

The morphological changes of substrates were examined by SEM to obtain insights into the structural modification of the surface. Figure 2 shows the SEM micrographs of non-pretreated, pretreated, and after enzymatic hydrolysis substrates. The images were amplified by factors of 800 and 1200. The non-pretreated substrates (micrographs a and b) showed compact and rigid structure. However, the substrates became a rough and rich fraction after alkali pretreatment (micrographs c and d), and the substrates became looser after ultrasonic pretreatment (micrographs e and f) (Li et al. 2010). Compared with the non-pretreated substrates, pretreated substrates appeared looser and exhibited more internal surfaces. It can be inferred that the partial removal of hemicelluloses and lignin destroyed the network formed by cellulose – hemicelluloses – lignin, leading to the disruption of the hydrogen bond between cellulose. The looser structure and increased surface area of pretreated substrates increase the accessibility of enzyme and allow for cellulase to penetrate, absorb, and hydrolyze the lignocellulose more easily, thus promoting the enzymatic hydrolysis efficiency (Zhang and Lynd2004; Hendriks and Zeeman 2009).

A comparison of the micrographs taken before and after enzymatic hydrolysis showed many holes in the substrate after enzymatic hydrolysis, and micrographs i and j reveal more holes than micrographs g and h. This illustrates that Fe3+ has an important effect on enzymatic hydrolysis.

Fig. 2. SEM micrographs of (a,b) non-pretreated substrate ×800 and ×1200, (c,d) alkali-pretreated substrate ×800 and ×1200, (e,f) ultrasonically pretreated substrate ×800 and ×1200, (g,h) substrate after enzymatic hydrolysis without added Fe3+ ×800 and ×1200, and (i,j) substrate after enzymatic hydrolysis with added Fe3+ ×800 and ×1200

FTIR Spectra

The FTIR spectra of different substrates are shown in Fig. 3. The relative intensities of absorption peaks of infrared spectra are shown in Table 1. The absorption peaks in the region near 3500 to 3300 cm−1 corresponded to the characteristic absorption peak of the intramolecular hydrogen bonding, and the relative intensity of the absorption peak decreased with substrate change. This result indicates that the hydrogen bonding is increasingly destroyed. Alkali pretreatment (Fig. 3b) and ultrasonic pretreatment (Fig. 3c) resulted in the breakdown of hydrogen bonds in the cellulose molecule. The relative intensities of the absorption peaks under Fe3+ addition (Fig. 3e) decreased relative to that in the absence of Fe3+ (Fig. 3d). This finding suggests that the added Fe3+ strongly degraded the intramolecular hydrogen bonding.

The absorption peak at 1735 cm−1 corresponded to the uronic ester groups and acetyl groups of hemicelluloses or the ester linkage of the carboxylic group of ferulic and p-coumaric acid present in hemicellulose and lignin (Li et al. 2010). This peak disappeared in the cellulose-rich fractions. Such alterations imply that the ester bonds were cleaved after alkali pretreatment (Fig. 3b), ultrasonic pretreatment (Fig. 3c), and enzymatic hydrolysis (Figs. 3d and 3e).

The absorption peak at 1637 cm−1 corresponded to the absorbed water in the substrates (Lu et al. 2004; Li et al. 2010). The absorption peak was split into two peaks after pretreatment and enzymatic hydrolysis (Fig. 3). The relative intensities of the absorption peaks decreased, as shown in Table 1, and this reduction indicated a decrease in water absorption.

The characteristic absorption peak at 1432 cm−1 corresponded to the structure of cellulose, and the relative intensity of absorption peak did not change considerably after pretreatment and enzymatic hydrolysis. This result revealed that the macromolecular structure of cellulose did not change drastically.

The absorption peak of the C–O–C groups from lignocellulose appeared at 1164 cm−1, and the relative intensity of the absorption peak decreased after pretreatment and enzymolysis (Table 1). This result was achieved because the lignin content decreased.

Peaks from 1058 to 1060 cm−1 corresponded to the stretching vibrations of the C=O groups from hemicellulose and lignin, and the relative intensity of the absorption peak decreased after pretreatment and enzymolysis (Table 1). This result indicated that the hemicellulose and lignin contents decreased after pretreatment and enzymatic hydrolysis.

Fig. 3. FTIR spectra of the (a) non-pretreated substrate, (b) alkali-pretreated substrate, (c) ultrasonically pretreated substrate, (d) substrate after enzymatic hydrolysis without added Fe3+, and (e) substrate after enzymatic hydrolysis with added Fe3+

Table 1. Relative Absorption Intensity in the Infrared Spectra

Note: The values correspond to the ratio of the absorbance of the corresponding wavenumber and the absorbance at 1372 cm−1 (Lu et al. 2005; Zhang et al. 2010b; Song et al. 2011).

Hydrated Particle Size

The DLS measurements of the hydrated particle size are presented in Fig. 4. The hydrated particle sizes of the different concentrations of cellulases were similar, and the average hydrated particle sizes were about 825 nm (Fig. 4a). The particle size increased with increasing Fe3+ concentration, but when the concentration of Fe3+ was 0.6 g/L and 1 g/L, the particle size was constant at 825 nm (Fig. 4b). The hydrated particle sizes in the mixture of different concentrations of Fe3+ and 3 g/L cellulase showed similar trends with different Fe3+concentrations (Fig. 4c). However, the particle size remained at 955 nm when Fe3+ concentration exceeded 0.4 g/L. The particle sizes were between 1100 and 1300 nm after enzymatic hydrolysis in Fig. 4d and were larger than that before enzymatic hydrolysis.

An enzyme molecule is a specific 3D structure consisting of helical folded sheets and unfolded amino acids. The electrostatic interaction between Fe3+ and the carboxyl group in the enzyme reduced the electrostatic repulsion between the carboxyl groups and thus enlarged the enzyme’s steric structure. Therefore, the particle sizes enlarged when Fe3+ was added.

Fig. 4. Particle sizes in the (a) enzyme solution, (b) different concentrations of Fe3+ solution, (c) different concentrations of Fe3+and 3 g/L enzyme mixture before enzymatic hydrolysis, and (d) different concentrations of Fe3+ and 3 g/L enzyme mixture after enzymatic hydrolysis

Effect of Fe3+ on Enzyme Kinetics

Figure 5 and Table 2 show the effect of Fe3+ on enzyme kinetics. Fe3+ increased the maximum reaction rate and the Michaelis-Menten constant of the enzymatic reaction. Adding Fe3+ also raised the maximum reaction rate by 33.9% and the Michaelis-Menten constant by 5.72 g/L.

The Michaelis–Menten constant Km represents the affinity between substrate and enzyme, and the Km value is inversely proportional to the affinities of the enzyme and substrate. Adding Fe3+ reduced the affinity of cellulase and cellulose because Fe3+ adsorbed onto the lignins and occupied the adsorption position of cellulose (Akimkulova et al. 2016 and Liu et al. 2010). As a result, the ineffective adsorption of cellulase decreased; hence, the enzyme activity and enzymatic intermediates of combination and dissociation were increased. The apparent Km value increased, but with decreased concentration of the intermediate product. This effect was beneficial to the enzymatic reaction, which was then promoted to the positive reaction direction. Ultimately, the dissociation of the intermediate product was enhanced (Zhang et al. 2010a).

Fig. 5. Effect of Fe3+ on enzyme kinetics

Table 2. Effect of Fe3+ on Enzyme Kinetic Parameters

Effect of Fe3+ on Enzymatic Activity

The effect of Fe3+ on the enzymatic activities of the enzymatic hydrolysis system is revealed in Fig. 6. Within the experimental time, Fe3+ promoted the enzymatic activity, and the relative increase rate of enzyme activity rose initially and then diminished. The highest relative increase rate of enzyme activity appeared at 24 h, and the maximum increase rate was 36.1%.

Fe3+ can significantly increase the activity of cellulase. Probably Fe3+is the activator of cellulase, and changes the conformation of cellulase protein, so that the number of active enzyme increases and reduces in non-productive enzyme adsorption. As a consequence, the cellulase enzyme activity increases (Li et al. 2009; Wang et al. 2012; Vasconcellos et al. 2016). At the same time, Fe3+ played a bridging role between the active site of cellulase and substrate, and promoted the combination of active site of cellulase and substrate, thus accelerating the reaction and improving the enzymatic hydrolysis efficiency.

Fig. 6. Effect of Fe3+ on enzymatic activity

Fluorescence Spectra

Fluorescence spectra of enzymatic hydrolysis solutions with and without Fe3+ are shown in Fig. 7. Because 280 nm was the maximum absorption wavelength of cellulase, so 280 nm is used as the excitation wavelength. The emission spectrum of 300 to 650 nm reflected the change of the molecular conformation of enzyme combined with others (Wu et al. 2008). In Fig. 7 it is apparent that the wavelengths of the maximum fluorescence intensity of the enzyme were 346.4 nm (24 h) and 344.80 nm (36 h) without Fe3+, which were the emission spectrum of tryptophan and tyrosine in cellulase (Podestá and Plaxton 2003; Sułkowska et al. 2004). The wavelengths were shifted to 435.90 nm (24 h) and 439.40 nm (36 h) with the addition of Fe3+; this shift was due to the combination of cellulase molecules and iron ions. In addition, due to the fluorescence quenching effect of Fe3+, the fluorescence intensities were obviously lower than those without Fe3+.

Fig. 7. Fluorescence spectrum of enzymatic hydrolysis solution

CONCLUSIONS

  1. Different metal ions had different effects on enzymatic hydrolysis of pretreated wheat straw. The hemicellulose and lignin can be partially removed by alkali and ultrasonic pretreatment. DLS and fluorescence spectra results revealed that there was interaction between Fe3+ and enzyme.
  2. Fe3+ increased the maximum reaction rate and the Michaelis-Menten constant. The relative increase rate of enzyme activity rose initially and then diminished.

ACKNOWLEDGMENTS

This work was funded by the National Key R&D Program of China (No 2017YFB0308701), the National Natural Science Foundation of China (Nos. 21203005, 21676003), the Science and Technology Program Key Project of Beijing Municipal Commission of Education (KZ201510011010), and the transformation of scientific and technological achievements-promotion plan project (TJSHG201510011020).

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Article submitted: October 29, 2017; Peer revision completed: December 30, 2017; Revisions accepted: February 13, 2018; Published: February 16, 2018

DOI: 10.15376/biores.13.2.2617-2631