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Enzymatic hydrolysis of lignin by ligninolytic enzymes and analysis of the hydrolyzed lignin products
Journal Pre-proofs Enzymatic hydrolysis of lignin by ligninolytic enzymes and analysis of the hydrolyzed lignin products Sitong Zhang, Jianlong Xiao, Gang Wang, Guang Chen PII: DOI: Reference:
S0960-8524(20)30244-3 https://doi.org/10.1016/j.biortech.2020.122975 BITE 122975
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Bioresource Technology
Received Date: Revised Date: Accepted Date:
21 December 2019 5 February 2020 5 February 2020
Please cite this article as: Zhang, S., Xiao, J., Wang, G., Chen, G., Enzymatic hydrolysis of lignin by ligninolytic enzymes and analysis of the hydrolyzed lignin products, Bioresource Technology (2020), doi: https://doi.org/ 10.1016/j.biortech.2020.122975
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Enzymatic hydrolysis of lignin by ligninolytic enzymes and analysis of the hydrolyzed lignin products
Sitong Zhanga,b,1, Jianlong Xiaoa,1, Gang Wanga,b, Guang Chena,b,* aCollege bKey
of Life Sciences, Jilin Agricultural University, Changchun, China
Laboratory of Straw Biology and Utilization, The Ministry of Education,
Changchun, China
*Corresponding author, e-mail: [email protected] (G. Chen) 1
These authors contributed equally to this work and should be considered co-first authors.
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ABSTRACT The degradation of alkali lignin was studied using three types of pure enzyme, Lac, LiP, and MnP, using alkali lignin as substrate. The alkali lignin removal rate was found to be 28.98% when Lac, LiP, and MnP were cultured together for alkali lignin degradation. Changes in the structure and composition before and after degradation were characterized by scanning electron microscopy, Fourier-transform infrared spectroscopy, nitrogen adsorption, and gas chromatography–mass spectrometry. The degradation product pathways were analyzed. The enzyme was proven to degrade alkali lignin, resulting in destruction of the alkali lignin structure, ring-opening of the macromolecular benzene ring structure and groups in alkali lignin, and chemical bond cleavage. This study explains the principle of alkali lignin enzymatic hydrolysis and provides a theoretical basis for the biodegradation of lignin.
Keywords: Lignin; Lignin-degrading enzyme; Biodegradation; Chemical bond disruption; Degradation mechanism
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1. Introduction China has a large agricultural industry that produces massive amounts of agricultural waste each year, especially crop straw. The annual production of crop straw by the Chinese agricultural industry is 1.04 billion tons, which amounts to one-third of the total crop straw production worldwide (Li et al., 2018), with this number continuing to increase annually (Yan et al., 2006). Lignocellulose resources produced in China are not used effectively, with most incinerated, leading not only to a waste of resources but also severe environmental pollution (Liu et al., 2018). Lignocellulose is composed primarily of three closely associated components, namely, lignin, cellulose, and hemicellulose (Li et al., 2019). Owing to this close association, lignin degradation is a prerequisite for the effective use of lignocellulose resources. Degrading lignin in raw materials and releasing cellulose and hemicellulose is crucial for the full utilization of lignocellulose resources (Bilal et al., 2017). Lignin is the second most abundant component of lignocellulose after cellulose. Lignin is an amorphous aromatic polymer with a high molecular weight that is formed with phenylpropane as the basic structural unit (Chen et al., 2018). The complex structure of lignin makes it extremely difficult to degrade, with efficient degradation of lignin required for efficient biomass resource utilization. Among available lignin degradation methods (Bilal et al., 2018), biodegradation has multiple advantages, such as low energy consumption, mild reaction conditions, and environmental friendliness compared with physical and chemical methods (Wang et al., 2017). Therefore, biodegradation of lignin has become a popular area of research in recent years (Asina et al., 2016; Brodeur et al., 2011). Lignin biodegradation requires the participation of various microorganisms, including fungi, bacteria, and 3
actinomycetes, among which fungi play a more prominent role (Asgher et al., 2016). During growth, the hyphae formed by fungi penetrate into the plant cell wall, and the enzymes produced by fungal hyphae catalyze single-electron oxidations of the structural unit of lignin to generate free radicals. These free radicals form micromolecular compounds through a series of nonenzymatically catalyzed free-radical chain reactions, eventually entering the tricarboxylic acid cycle to produce CO2 (Bugg et al., 2011). Lignin can be degraded by many enzymes, including laccase (Lac), lignin peroxidase (LiP) (Bilal and Iqbal, 2019), manganese peroxidase (MnP), cellobiose dehydrogenase, aryl alcohol oxidase, and aryl alcohol dehydrogenase (Perez et al., 2002). Lac, LiP, and MnP degrade lignin particularly effectively (Iandolo et al., 2011). To date, many studies have used microorganisms or their enzymes to degrade lignin and then explored the structural and compositional changes in lignin before and after degradation at the microscale. These studies have used scanning electron microscopy (SEM) (Zhang et al., 2019), the Brunauer–Emmett–Teller (BET) method (Chu et al., 2019), Fourier-transform infrared (FT-IR) spectroscopy (Halder et al., 2019), and gas chromatography–mass spectrometry (GC–MS) (El Fels et al., 2014) to analyze the products of lignin degradation. However, after identifying the lignin degradation products, these studies did not attempt to characterize the mechanisms underpinning the enzymatic hydrolysis of lignin by Lac, LiP, and MnP. Therefore, further research into lignin degradation products is required. In the present study, the degradation of alkali lignin as substrate by three enzymes (Lac, LiP, and MnP), both alone and in different combinations, was studied for the first time.
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Changes in the structure and composition before and after degradation were characterized by scanning electron microscopy (SEM), FT-IR spectroscopy, nitrogen adsorption, and GC–MS. The effect of Lac, LiP, and MnP on lignin degradation and its mechanism were preliminarily analyzed to provide a theoretical basis for lignin biodegradation.
2. Materials and methods 2.1. Materials and instruments Alkali lignin was purchased from Solarbio Science & Technology Co., Ltd. (Beijing, China). Lac (enzymatic activity, 312 U/L) was purchased from Yuanye Biotechnology Co., Ltd. (Shanghai, China). LiP (enzymatic activity, 289 U/L) and MnP (enzymatic activity, 307 U/L) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Enzyme activity assays were performed as described previously (Bourbonnais and Paice, 1990; MingTienT and KentKirk, 1988; Ruiz-Duenas et al., 1999). The following instruments were used in the study: JSM-6700F scanning electron microscope (JEOL, Tokyo, Japan); Autosorb iQ specific surface area and pore size analyzer (Quantachrome, Florida, FL, USA); IRTracer-100 Fourier-infrared spectrophotometer (Shimadzu, Kyoto, Japan); and GCMS-QP2010 ultra gas chromatograph–mass spectrometer (Shimadzu). 2.2. Experimental methods 2.2.1. Construction of alkali lignin standard curve 5
Alkali lignin solutions were prepared at concentrations of 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 mg/L. The absorbance at 280 nm (Chang et al., 2014) was measured and recorded, with three parallels per group. According to the measured values and the corresponding concentrations of alkali lignin, the standard curve equation of alkali lignin was obtained, as follows: y = 0.0045x + 0.0675 (R2 = 0.9994) 2.2.2. Enzymatic hydrolysis of alkali lignin An alkali lignin solution (2 g/L) was prepared and adjusted to pH 5. This solution was added to microcentrifuge tubes (1 mL per tube). Different enzyme solutions were then added to the tubes according to Table 1 (sterile water was added to make the volume of each sample the same, ensuring the repeatability of the experiment). The reaction was conducted at 30 °C for 8 h. Eight treatments were conducted in this experiment (with Test group 1 as the blank group), with each comprising three repeats (Table 1). 2.2.3. Determination of alkali lignin degradation amount After enzymatic hydrolysis, the absorbance of the alkali lignin solution was measured at 280 nm (Chang et al., 2014). The concentration of alkali lignin was calculated from the absorbance, and the average amount of alkali lignin degradation in each experimental group was calculated using Eq. (1): Amount of alkali lignin degraded = (C1 C2)/C1 100%
(1)
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where C1 is the concentration of alkali lignin in the control (test group 1) and C2 is the concentration of alkali lignin in the test sample. 2.2.4. Structural and compositional changes to alkali lignin before and after degradation The enzymatically hydrolyzed alkali lignin solution was dried to a constant weight, and SEM, FT-IR spectroscopy, and nitrogen adsorption analysis of the dried sample were performed. A sample (0.1 ± 0.0005 g) was accurately weighed into a 2-mL centrifuge tube, and chromatographically pure methanol (1 mL) was added. The sample was ultrasonically extracted for 30 min and centrifuged at 10,000 g, and the supernatant was subjected to GC– MS analysis. The analytical conditions were as follows: Column, DB-5MS (Agilent J&W Scientific, Folsom, CA, USA; 30 m 0.25 mm 0.25 m); inlet temperature, 250 °C; split ratio, 10:1; carrier gas, high-purity helium; and flow rate, 1 mL/min. The ramping program started at 40 °C for 2 min, which was then increased to 320 °C at a rate of 10 °C/min, followed by 10 min at 320 °C. The ion source temperature was set to 220 °C and the interface temperature was 280 °C. The solvent removal time was 3 min and scanning was performed in the range of m/z 45–500. The NIST standard mass spectral library was used to identify chromatographic peaks. All search results were checked and replenished with reference to the relevant standard atlas and related literature, and the relative percentage content of each component was calculated using the chromatographic peak area normalization method. 2.2.5 Data processing 7
Data variance analysis of the alkali lignin degradation rate was conducted using SPSS 22.0 software, and plotted using Origin 2017 software. 3. Results and discussion 3.1. Amount of alkali lignin degraded The three enzymes, Lac, LiP, and MnP, were used separately or in various combinations to degrade alkali lignin, and the amount of alkali lignin degraded was calculated (Fig. 1). When individual enzymes were used for enzymatic hydrolysis, the amount of alkali lignin degraded by Lac reached 22.15%, which was substantially higher than that for either LiP or MnP. When two enzymes were used to degrade alkali lignin, the amount of alkali lignin degraded by a combination including Lac was much higher than that without Lac, and higher than that using Lac alone. The amount of alkali lignin degraded was highest, at 28.98%, when the three enzymes (Lac, LiP, and MnP) were used in combination (Fig. 1). Studies have shown that lignin biodegradation is completed mainly through a series of ligninolytic enzymes produced by microorganisms, the most important of which are Lac, LiP, and MnP. Bilal and others have immobilized lignin-degrading enzymes for lignin degradation in various agricultural crop straws. The results showed that the lignin degradation effect was best in sorghum, with a highest lignin degradation rate of 57.3%. In this study, to ensure the repeatability of the experiment, the amount of enzyme was restricted such that the alkali lignin degradation rate was lower than that reported by Bilal (Bilal et al., 2017b). Microorganisms that produce multiple ligninolytic enzymes degrade lignin more efficiently compared with microorganisms that produce a single ligninolytic enzyme. Knezevic et al. 8
found that, when white rot fungi degrades lignin in plants, the coexistence of two or more enzymes contributes to effective lignin degradation, with degradation by single enzymes inferior compared with the synergy of multiple enzymes (Knezevic et al., 2013). The results of the present study further confirm this conclusion. Based on two-dimensional electrophoresis and LC–MS, Abbas et al. found that, when fungi degrades lignin in oak, at least 40 enzymes are involved in lignin degradation (Abbas et al., 2005). This finding further corroborated that synergy between enzymes exists during the microbial degradation of lignin. 3.2. Structure of alkali lignin before and after degradation 3.2.1. Nitrogen adsorption and SEM observations The SEM results showed that, compared with the original alkali lignin, the structure of the treated alkali lignin was destroyed. The surface became rough, uneven, and decomposed into many fine structures, in sharp contrast to the smooth surface of primitive alkali lignin, which produced many fine pores. The appearance of pores on the surface was caused by the action of enzymes degrading alkali lignin. Sun et al. found that the surface of corn straw treated by T. hirsute yj9 also became rough, with similar small holes appearing (Sun et al., 2011). To further quantify the surface pore size of alkali lignin before and after enzyme treatment, the specific surface areas of the original alkali lignin and alkali lignin degraded by a mixed culture of the three enzymes were measured by nitrogen adsorption (BET method), and determined to be 3.919 and 18.188 m2, respectively. The specific surface area of alkali lignin after treatment was nearly five times higher than that before treatment, which demonstrated that Lac, LiP, and MnP had a good degradation effect on alkali lignin.
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3.2.2. FT-IR spectroscopy of alkali lignin The FT-IR spectra of alkali lignin before and after enzymatic hydrolysis were assigned according to the literature. The absorption peak at 2930 cm1 represented antisymmetric stretching vibrations of the CH3– and –CH2– groups in lignin. The intensity of this peak decreased substantially after enzymatic hydrolysis, indicating that lignin was degraded (Ohra-aho and Linnekoski, 2015). The peak at 1650 cm1, representing conjugated carbonyl groups (C=O) in lignin, also showed decreased peak intensity after enzymatic hydrolysis, indicating the ring-opening or substitution of aromatic rings. Lignin degradation might also cause lignin residues to polymerize with other organic intermediates produced by degradation to form humus. The peaks at ~1240 cm1 represented lignin degradation products, such as phenol, ethers, and alcohols. The observed increase in peak intensity for these bands indicated an increased amount of lignin degradation products (Omoike and Chorover, 2006). Furthermore, the absorption peak at 1125 cm1 was characteristic of a syringyl unit, while no obvious peak was observed at 1168 cm1, indicating that no para-hydroxyphenyl units were present. This result indicated that the alkali lignin sample might be a syringyl–guaiacyl-type lignin. The peak at 1037 cm1 peak represented C–H vibrations of the guaiacyl unit. The decrease in intensity of this peak showed that the guaiacyl structure was disrupted, further indicating lignin degradation (Pandey and Pitman, 2003). Based on previous studies and combined with the infrared spectra of alkali lignin before and after enzymatic hydrolysis, representative chemical bonds in alkali lignin were disrupted, while chemical bonds present in lignin degradation products increased substantially following
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enzymatic treatment. These results indicated that the combination of ligninolytic enzymes facilitated alkali lignin degradation. 3.2.3. GC–MS analysis of alkali lignin GC–MS analysis was performed to identify the various degradation products. The NIST standard mass spectral library was used to assign chromatographic peaks to compounds in the MS spectra. The amount of each degradation product was determined from the corresponding peak area in the chromatogram. Lac oxidizes lignin in the presence of oxygen in air. One electron is extracted from lignin, leading to the generation of free radicals from the substrate molecules, and oxygen molecules are reduced to water. The generated free radicals are unstable and undergo polymerization or depolymerization reactions, causing lignin degradation. The complete catalytic reaction is an electron transfer oxidation, which involves four consecutive one-electron oxidations. The reduced substrate binds to type-I Cu2+, allowing it to extract an electron from the substrate. The extracted electron is transferred to type-II Cu2+ via the Cys– His pathway (Baldrian, 2006) and then passed to O2 bound to this site, reducing O2 to water. In the complete reaction, four consecutive one-electron oxidations are required to fully reduce Lac. Therefore, four substrate molecules are oxidized to produce four substrate free radicals (Su et al., 2018). The large amount of free radicals produced undergo nonenzymatic reactions, such as demethoxy, decarboxyl, and C–C chain breaks. Eventually, the lignin macromolecules are depolymerized into monomers. Therefore, a large amount of aromatics are produced, which can be used by Lac as substrates to continue enzymatic hydrolysis. The
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resulting free radicals replace hydrogen atoms in the side chains of the aromatics to form alcohols. Therefore, degradation products will include a large amount of alcohols and aromatics. Because Lac does not require strong oxidants to participate in the enzymatic hydrolysis reaction, it is generally thought that enzymatic hydrolysis by Lac is the initiation step of lignin degradation. Based on GC–MS analysis, single-enzyme hydrolysis of alkali lignin by Lac yielded 40 degradation products, which were classified into eight groups, namely, acids, alcohols, alkanes, ketones, esters, phenols, aldehydes, and aromatics. Most of the degradation products were small molecules, such as aromatic acids, aromatic esters, and aliphatic compounds. The alcohol and aromatic contents were highest, accounting for 26.05% and 26.14% of the total degradation products, respectively. This result indicated that Lac degraded alkali lignin and disrupted its structure, causing ring-opening reactions and chemical bond cleavage in benzene ring structures and groups of large molecules in alkali lignin. This finding was consistent with previous reports (Feng et al., 2019; Munk et al., 2018; Rich et al., 2016). The most abundant alcohol was 2,2-dimethyl-1,3-butanediol. Owing to the acidic environment of the enzymatic hydrolysis reaction, 2,2-dimethyl-1,3-butanediol can react with acid to undergo pinacol rearrangement, producing 3,3-dimethyl-2-butanone and water. However, no 3,3-dimethyl-2-butanone was detected in the degradation products, probably because it is not soluble in methanol, which was used as the solvent for dissolution and analysis during sample pretreatment. When LiP was used for single-enzyme hydrolysis of alkali lignin, 40 degradation products were also identified. These products belonged to eight groups, namely, acids, 12
alcohols, alkanes, ketones, esters, phenols, aldehydes, and aromatics. The aromatic and ketone contents were highest, accounting for 34.77% and 22.23% of the total degradation products, respectively. The main chemical bond between lignin molecules is the -O-4-type bond (Ma et al., 2018). LiP catalyzes the decomposition of both benzene rings A and B in synthetic compounds with the β-O-4 bond, mainly through C–C cleavage to form 3,4-dimethoxybenzyl alcohol (veratryl alcohol) and 2-methoxyphenol (Hammel et al., 1993). Although 3,4-dimethoxybenzyl alcohol was not detected in the degradation products, its derivatives 3,4-dimethoxymethylacetic acid and 3,4-dimethoxymethylbenzene were detected. This observation was due to 3,4-dimethoxybenzyl alcohol being a substrate for LiP-catalyzed reactions, resulting in its continuous decomposition via oxidation, which leads to aromatic ring-opening in lignin and methoxyphenol derivative formation. The 2-methoxyphenol content present after lignin degradation was not high, accounting for only 2.93% of the total degradation products. However, the aromatics among the degradation products comprised a several 2-methoxyphenol derivatives, including 2-methoxy-4-ethylphenol, 2-methoxy-4-vinylphenol, 1,2,3-trimethoxy-5-methyl-Benzene, 3,3,5-dimethoxyacetophenone, 3,4,5-trimethoxyphenol, 2,5-dimethoxy-1,4-benzenediol, and 2,6-dimethoxy-4-(2-propenyl)phenol. These 2-methoxyphenol derivatives were all produced by continuous reactions between 2-methoxyphenol and other moieties, such as alcohols, aldehydes, and alkanes, in the degradation products. After single-enzyme hydrolysis with MnP alone, 40 products from alkali lignin degradation were identified, which belonged to seven groups, namely, acids, alcohols, alkanes, ketones, phenols, aldehydes, and aromatics. The aromatic and ketone contents were 13
highest, accounting for 55.70% and 21.54% of the total degradation products, respectively. Acetosyringone was the most abundant monophenol degradation product identified. The mechanism of action of MnP in nature involves the enzymatic oxidation of Mn2+ (present in abundance in nature) to Mn3+. The resulting Mn3+ chelates with glycolate and oxalate present in the system, thus existing in a stable state. These chelated Mn3+ ions can be used as a small-molecular-weight medium, which penetrates into the lignin structure, non-specifically attacking and oxidizing the phenol structure in lignin molecules. Therefore, lignin containing phenol structures forms phenoxy radicals that are unstable and easy to decompose via nonenzymatic reactions. Furthermore, these radical groups undergo a series of reactions that cause some decomposition of the lignin polymer (Agarwal et al., 2018). However, the reaction conditions for enzymatic hydrolysis of alkali lignin lacked Mn2+, which is required by MnP for activity. Therefore, the activity of MnP was noticeably lower compared with those of Lac and LiP. When alkali lignin was enzymatically hydrolyzed with double-enzyme combinations, the production of alcohols, aldehydes, and acids, and the reduction of phenols and aromatics by the Lac–LiP and Lac–MnP groups relative to the blank group was considerably higher compared with those by the LiP–MnP group. The main reason for this difference was that after Lac uses O2 taken from the environment as an oxidant to enzymatically hydrolyze alkali lignin, the resulting intermediates, such as carboxyl radicals with strong oxidizing properties, can act as oxidants for LiP and MnP to initiate the enzymatic hydrolysis reaction. Therefore, the Lac–LiP and Lac–MnP combinations exhibited stronger enzymatic hydrolysis compared with the LiP–MnP group (Fig. 2). 14
Among alcohols in the degradation products, which were produced by the replacement of hydrogen atoms in the side chain of aromatics by strongly oxidative hydroxyl radicals, 2,2-dimethyl-1,3-butanediol was the most abundant compound. The 2,2-dimethyl-1,3-butanediol content was highest in the Lac–LiP group among the double-enzyme combinations, followed by that in the Lac–MnP group. The 2,2-dimethyl-1,3-butanediol content was lowest in the LiP–MnP group, and even lower than that from hydrolysis by Lac only. The amount of degradation product 2,2-dimethyl-1,3-butanediol represents the degree of alkali lignin degradation. In the Lac–LiP group, both Lac and LiP degraded lignin readily after initiation by the oxidant generated by Lac. In the groups containing MnP, the degradation effect of MnP was not evident owing to the absence of Mn2+ in the reaction solution. This finding was in agreement with the experimental results for the degradation rate of alkali lignin. The combined use of the three enzymes to degrade alkali lignin yielded roughly the same result when compared with the hydrolysis reactions using two enzymes. Nonetheless, alkali lignin degradation was more complete in the Lac–LiP–MnP group owing to the increase in enzyme dose and the strong oxidative intermediates produced by enzymatic hydrolysis with Lac initiating the enzymatic hydrolysis reactions of LiP and MnP. In this study, the enzymatic hydrolysis products of alkali lignin from each group were analyzed by GC–MS. Based on the analysis of the degradation products, not only were the experimental results for the alkali lignin degradation rate explained, but also the mechanisms underpinning lignin degradation by Lac, LiP, and MnP, and combinations of these three enzymes, were explored. 15
4. Conclusions In this study, the degradation effect of laccase on lignin was found to be the most obvious, while the effect of the three enzymes in combination on lignin degradation was the best, achieving 28.98% degradation. Lignin degradation enzymes mainly destroyed the apparent structure and key chemical bonds of lignin, with most of the degradation products being aromatic small molecular compounds. Lignin degradation enzymes were proven to lead to ring-opening reactions of macromolecular benzene ring structures and groups in alkali lignin as well as chemical bond cleavage. This result provides a theoretical basis for the biodegradation of lignin. E-supplementary data for this work can be found in the electronic version of this paper online Declaration of competing interests The authors declare that they have no known competing financial interests or personal relationships that could influence the work reported in this paper.
FUNDING
This study was funded by the National Key R&D Program of China ( 2017YFD0501005).
Acknowledgments
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We thank the National Key R&D Program of China, the Key Laboratory of Straw Biology and Utilization, The Ministry of Education, for its support. We also thank Simon Partridge, PhD, from Liwen Bianji, Edanz Editing China (www.liwenbianji.cn/ac), for editing the English text of a draft of this manuscript.
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[25]Ma, R., Guo, M., Zhang, X., 2018. Recent advances in oxidative valorization of lignin. Catal. Today, 302(50-60. [26]MingTienT, KentKirk, 1988. Lignin Peroxidase of Phanerochaete chrysosporium. Method. Enzymol.161), 238-249. [27]Munk, L., Andersen, M.L., Meyer, A.S., 2018. Influence of mediators on laccase catalyzed radical formation in lignin. Enzyme Microb. Tech., 116(48-56. [28]Ohra-aho, T., Linnekoski, J., 2015. Catalytic pyrolysis of lignin by using analytical pyrolysis-GC–MS. J. Anal. Appl. Pyrol., 113(186-192. [29]Omoike, A., Chorover, J., 2006. Adsorption to goethite of extracellular polymeric substances from Bacillus subtilis. Geochim. Cosmochim. Ac., 70(4), 827-838. [30]Pandey, K.K., Pitman, A.J., 2003. FTIR studies of the changes in wood chemistry following decay by brown-rot and white-rot fungi. Int. Biodeter. Biodegr., 52(3), 151-160. [31]Perez, J., Munoz-Dorado, J., de la Rubia, T., Martinez, J., 2002. Biodegradation and biological treatments of cellulose, hemicellulose and lignin: an overview. [32]Rich, J.O., Anderson, A.M., Berhow, M.A., 2016. Laccase-mediator catalyzed conversion of model lignin compounds. Biocatalysis and Agricultural Biotechnology, 5(111-115. [33]Ruiz-Duenas, F.J., Guillen, F., Camarero, S., Perez-Boada, M., Martinez, M.J., Martinez, A.T., 1999. Regulation of peroxidase transcript levels in liquid cultures of the ligninolytic fungus Pleurotus eryngii. Appl Environ Microbiol, 65(10), 4458-63. [34]Su, J., Fu, J., Wang, Q., Silva, C., Cavaco-Paulo, A., 2018. Laccase: a green catalyst for the biosynthesis of poly-phenols. Crit. Rev. Biotechnol., 38(2), 294-307. [35]Sun, F., Li, J., Yuan, Y., Yan, Z., Liu, X., 2011. Effect of biological pretreatment with Trametes hirsuta yj9 on enzymatic hydrolysis of corn stover. Int. Biodeter. Biodegr., 65(7), 931-938. [36]Wang, X., Wang, G., Yu, X., Chen, H., Sun, Y., Chen, G., 2017. Pretreatment of corn stover by solid acid for d -lactic acid fermentation. Bioresource Technol., 239(490-495. [37]Yan, X., Ohara, T., Akimoto, H., 2006. Bottom-up estimate of biomass burning in mainland China. Atmos. Environ., 40(27), 5262-5273. [38]Zhang, X., Li, Y., Hou, Y., 2019. Preparation of magnetic polyethylenimine lignin and its adsorption of Pb(II). Int. J. Biol. Macromol., 141(1102-1110.
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20
Table 1 Amount of enzymes added to each experimental group.
Test group
1
2
3
4
5
6
7
8
Laccase (L)
–
10
–
–
10
10
–
10
–
–
10
–
10
–
10
10
–
–
–
10
–
10
10
10
Lignin peroxidase (L) Manganese peroxidase (L) “–” indicates that the volume was made up with sterile water.
21
35
Degradation rate %
30 25 20 15 10 5 0 Lac
LiP
MnP
Lac、LiP
Lac、MnP
LiP、MnP
Lac、LiP、MnP
Different combinations of enzymes
Fig. 1. Degradation rate of alkali lignin by laccase (Lac), lignin peroxidase (LiP), or manganese peroxidase (MnP), and in the presence of different combinations of the three enzymes.
22
12000000
Lac、Lip Lac、Mnp
10000000 Peak area of product
Lip、Mnp 8000000
6000000
4000000
2000000
0 Alcohol
Aldehydes
Esters Main products
Phenols
Aromatic
Fig. 2. Main products of enzymatic hydrolysis of alkali lignin using combinations of two enzymes: Laccase (Lac), lignin peroxidase (LiP), and manganese peroxidase (MnP).
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Sitong Zhang：Experimental design, review & editing Jianlong Xiao：Validation, Data analysis，writing - original draft Gang Wang：Data analysis, resources Guang Chen：Resources, Writing - Review & Editing, Supervision,Data Curation.
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Highlights 1. Alkali lignin degradation by three enzymes was examined. 2. Structural and compositional changes to degraded alkali lignin were characterized. 3. Degradation products were identified by GC-MS, and production pathways analyzed. 4. Synergy among ligninolytic enzymes gave the highest degradation levels of lignin. 5. Enzymes enabled ring-opening of benzene structures and rupture of chemical bonds.
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S0960-8524(20)30244-3 https://doi.org/10.1016/j.biortech.2020.122975 BITE 122975
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
21 December 2019 5 February 2020 5 February 2020
Please cite this article as: Zhang, S., Xiao, J., Wang, G., Chen, G., Enzymatic hydrolysis of lignin by ligninolytic enzymes and analysis of the hydrolyzed lignin products, Bioresource Technology (2020), doi: https://doi.org/ 10.1016/j.biortech.2020.122975
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Enzymatic hydrolysis of lignin by ligninolytic enzymes and analysis of the hydrolyzed lignin products
Sitong Zhanga,b,1, Jianlong Xiaoa,1, Gang Wanga,b, Guang Chena,b,* aCollege bKey
of Life Sciences, Jilin Agricultural University, Changchun, China
Laboratory of Straw Biology and Utilization, The Ministry of Education,
Changchun, China
*Corresponding author, e-mail: [email protected] (G. Chen) 1
These authors contributed equally to this work and should be considered co-first authors.
1
ABSTRACT The degradation of alkali lignin was studied using three types of pure enzyme, Lac, LiP, and MnP, using alkali lignin as substrate. The alkali lignin removal rate was found to be 28.98% when Lac, LiP, and MnP were cultured together for alkali lignin degradation. Changes in the structure and composition before and after degradation were characterized by scanning electron microscopy, Fourier-transform infrared spectroscopy, nitrogen adsorption, and gas chromatography–mass spectrometry. The degradation product pathways were analyzed. The enzyme was proven to degrade alkali lignin, resulting in destruction of the alkali lignin structure, ring-opening of the macromolecular benzene ring structure and groups in alkali lignin, and chemical bond cleavage. This study explains the principle of alkali lignin enzymatic hydrolysis and provides a theoretical basis for the biodegradation of lignin.
Keywords: Lignin; Lignin-degrading enzyme; Biodegradation; Chemical bond disruption; Degradation mechanism
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1. Introduction China has a large agricultural industry that produces massive amounts of agricultural waste each year, especially crop straw. The annual production of crop straw by the Chinese agricultural industry is 1.04 billion tons, which amounts to one-third of the total crop straw production worldwide (Li et al., 2018), with this number continuing to increase annually (Yan et al., 2006). Lignocellulose resources produced in China are not used effectively, with most incinerated, leading not only to a waste of resources but also severe environmental pollution (Liu et al., 2018). Lignocellulose is composed primarily of three closely associated components, namely, lignin, cellulose, and hemicellulose (Li et al., 2019). Owing to this close association, lignin degradation is a prerequisite for the effective use of lignocellulose resources. Degrading lignin in raw materials and releasing cellulose and hemicellulose is crucial for the full utilization of lignocellulose resources (Bilal et al., 2017). Lignin is the second most abundant component of lignocellulose after cellulose. Lignin is an amorphous aromatic polymer with a high molecular weight that is formed with phenylpropane as the basic structural unit (Chen et al., 2018). The complex structure of lignin makes it extremely difficult to degrade, with efficient degradation of lignin required for efficient biomass resource utilization. Among available lignin degradation methods (Bilal et al., 2018), biodegradation has multiple advantages, such as low energy consumption, mild reaction conditions, and environmental friendliness compared with physical and chemical methods (Wang et al., 2017). Therefore, biodegradation of lignin has become a popular area of research in recent years (Asina et al., 2016; Brodeur et al., 2011). Lignin biodegradation requires the participation of various microorganisms, including fungi, bacteria, and 3
actinomycetes, among which fungi play a more prominent role (Asgher et al., 2016). During growth, the hyphae formed by fungi penetrate into the plant cell wall, and the enzymes produced by fungal hyphae catalyze single-electron oxidations of the structural unit of lignin to generate free radicals. These free radicals form micromolecular compounds through a series of nonenzymatically catalyzed free-radical chain reactions, eventually entering the tricarboxylic acid cycle to produce CO2 (Bugg et al., 2011). Lignin can be degraded by many enzymes, including laccase (Lac), lignin peroxidase (LiP) (Bilal and Iqbal, 2019), manganese peroxidase (MnP), cellobiose dehydrogenase, aryl alcohol oxidase, and aryl alcohol dehydrogenase (Perez et al., 2002). Lac, LiP, and MnP degrade lignin particularly effectively (Iandolo et al., 2011). To date, many studies have used microorganisms or their enzymes to degrade lignin and then explored the structural and compositional changes in lignin before and after degradation at the microscale. These studies have used scanning electron microscopy (SEM) (Zhang et al., 2019), the Brunauer–Emmett–Teller (BET) method (Chu et al., 2019), Fourier-transform infrared (FT-IR) spectroscopy (Halder et al., 2019), and gas chromatography–mass spectrometry (GC–MS) (El Fels et al., 2014) to analyze the products of lignin degradation. However, after identifying the lignin degradation products, these studies did not attempt to characterize the mechanisms underpinning the enzymatic hydrolysis of lignin by Lac, LiP, and MnP. Therefore, further research into lignin degradation products is required. In the present study, the degradation of alkali lignin as substrate by three enzymes (Lac, LiP, and MnP), both alone and in different combinations, was studied for the first time.
4
Changes in the structure and composition before and after degradation were characterized by scanning electron microscopy (SEM), FT-IR spectroscopy, nitrogen adsorption, and GC–MS. The effect of Lac, LiP, and MnP on lignin degradation and its mechanism were preliminarily analyzed to provide a theoretical basis for lignin biodegradation.
2. Materials and methods 2.1. Materials and instruments Alkali lignin was purchased from Solarbio Science & Technology Co., Ltd. (Beijing, China). Lac (enzymatic activity, 312 U/L) was purchased from Yuanye Biotechnology Co., Ltd. (Shanghai, China). LiP (enzymatic activity, 289 U/L) and MnP (enzymatic activity, 307 U/L) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Enzyme activity assays were performed as described previously (Bourbonnais and Paice, 1990; MingTienT and KentKirk, 1988; Ruiz-Duenas et al., 1999). The following instruments were used in the study: JSM-6700F scanning electron microscope (JEOL, Tokyo, Japan); Autosorb iQ specific surface area and pore size analyzer (Quantachrome, Florida, FL, USA); IRTracer-100 Fourier-infrared spectrophotometer (Shimadzu, Kyoto, Japan); and GCMS-QP2010 ultra gas chromatograph–mass spectrometer (Shimadzu). 2.2. Experimental methods 2.2.1. Construction of alkali lignin standard curve 5
Alkali lignin solutions were prepared at concentrations of 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 mg/L. The absorbance at 280 nm (Chang et al., 2014) was measured and recorded, with three parallels per group. According to the measured values and the corresponding concentrations of alkali lignin, the standard curve equation of alkali lignin was obtained, as follows: y = 0.0045x + 0.0675 (R2 = 0.9994) 2.2.2. Enzymatic hydrolysis of alkali lignin An alkali lignin solution (2 g/L) was prepared and adjusted to pH 5. This solution was added to microcentrifuge tubes (1 mL per tube). Different enzyme solutions were then added to the tubes according to Table 1 (sterile water was added to make the volume of each sample the same, ensuring the repeatability of the experiment). The reaction was conducted at 30 °C for 8 h. Eight treatments were conducted in this experiment (with Test group 1 as the blank group), with each comprising three repeats (Table 1). 2.2.3. Determination of alkali lignin degradation amount After enzymatic hydrolysis, the absorbance of the alkali lignin solution was measured at 280 nm (Chang et al., 2014). The concentration of alkali lignin was calculated from the absorbance, and the average amount of alkali lignin degradation in each experimental group was calculated using Eq. (1): Amount of alkali lignin degraded = (C1 C2)/C1 100%
(1)
6
where C1 is the concentration of alkali lignin in the control (test group 1) and C2 is the concentration of alkali lignin in the test sample. 2.2.4. Structural and compositional changes to alkali lignin before and after degradation The enzymatically hydrolyzed alkali lignin solution was dried to a constant weight, and SEM, FT-IR spectroscopy, and nitrogen adsorption analysis of the dried sample were performed. A sample (0.1 ± 0.0005 g) was accurately weighed into a 2-mL centrifuge tube, and chromatographically pure methanol (1 mL) was added. The sample was ultrasonically extracted for 30 min and centrifuged at 10,000 g, and the supernatant was subjected to GC– MS analysis. The analytical conditions were as follows: Column, DB-5MS (Agilent J&W Scientific, Folsom, CA, USA; 30 m 0.25 mm 0.25 m); inlet temperature, 250 °C; split ratio, 10:1; carrier gas, high-purity helium; and flow rate, 1 mL/min. The ramping program started at 40 °C for 2 min, which was then increased to 320 °C at a rate of 10 °C/min, followed by 10 min at 320 °C. The ion source temperature was set to 220 °C and the interface temperature was 280 °C. The solvent removal time was 3 min and scanning was performed in the range of m/z 45–500. The NIST standard mass spectral library was used to identify chromatographic peaks. All search results were checked and replenished with reference to the relevant standard atlas and related literature, and the relative percentage content of each component was calculated using the chromatographic peak area normalization method. 2.2.5 Data processing 7
Data variance analysis of the alkali lignin degradation rate was conducted using SPSS 22.0 software, and plotted using Origin 2017 software. 3. Results and discussion 3.1. Amount of alkali lignin degraded The three enzymes, Lac, LiP, and MnP, were used separately or in various combinations to degrade alkali lignin, and the amount of alkali lignin degraded was calculated (Fig. 1). When individual enzymes were used for enzymatic hydrolysis, the amount of alkali lignin degraded by Lac reached 22.15%, which was substantially higher than that for either LiP or MnP. When two enzymes were used to degrade alkali lignin, the amount of alkali lignin degraded by a combination including Lac was much higher than that without Lac, and higher than that using Lac alone. The amount of alkali lignin degraded was highest, at 28.98%, when the three enzymes (Lac, LiP, and MnP) were used in combination (Fig. 1). Studies have shown that lignin biodegradation is completed mainly through a series of ligninolytic enzymes produced by microorganisms, the most important of which are Lac, LiP, and MnP. Bilal and others have immobilized lignin-degrading enzymes for lignin degradation in various agricultural crop straws. The results showed that the lignin degradation effect was best in sorghum, with a highest lignin degradation rate of 57.3%. In this study, to ensure the repeatability of the experiment, the amount of enzyme was restricted such that the alkali lignin degradation rate was lower than that reported by Bilal (Bilal et al., 2017b). Microorganisms that produce multiple ligninolytic enzymes degrade lignin more efficiently compared with microorganisms that produce a single ligninolytic enzyme. Knezevic et al. 8
found that, when white rot fungi degrades lignin in plants, the coexistence of two or more enzymes contributes to effective lignin degradation, with degradation by single enzymes inferior compared with the synergy of multiple enzymes (Knezevic et al., 2013). The results of the present study further confirm this conclusion. Based on two-dimensional electrophoresis and LC–MS, Abbas et al. found that, when fungi degrades lignin in oak, at least 40 enzymes are involved in lignin degradation (Abbas et al., 2005). This finding further corroborated that synergy between enzymes exists during the microbial degradation of lignin. 3.2. Structure of alkali lignin before and after degradation 3.2.1. Nitrogen adsorption and SEM observations The SEM results showed that, compared with the original alkali lignin, the structure of the treated alkali lignin was destroyed. The surface became rough, uneven, and decomposed into many fine structures, in sharp contrast to the smooth surface of primitive alkali lignin, which produced many fine pores. The appearance of pores on the surface was caused by the action of enzymes degrading alkali lignin. Sun et al. found that the surface of corn straw treated by T. hirsute yj9 also became rough, with similar small holes appearing (Sun et al., 2011). To further quantify the surface pore size of alkali lignin before and after enzyme treatment, the specific surface areas of the original alkali lignin and alkali lignin degraded by a mixed culture of the three enzymes were measured by nitrogen adsorption (BET method), and determined to be 3.919 and 18.188 m2, respectively. The specific surface area of alkali lignin after treatment was nearly five times higher than that before treatment, which demonstrated that Lac, LiP, and MnP had a good degradation effect on alkali lignin.
9
3.2.2. FT-IR spectroscopy of alkali lignin The FT-IR spectra of alkali lignin before and after enzymatic hydrolysis were assigned according to the literature. The absorption peak at 2930 cm1 represented antisymmetric stretching vibrations of the CH3– and –CH2– groups in lignin. The intensity of this peak decreased substantially after enzymatic hydrolysis, indicating that lignin was degraded (Ohra-aho and Linnekoski, 2015). The peak at 1650 cm1, representing conjugated carbonyl groups (C=O) in lignin, also showed decreased peak intensity after enzymatic hydrolysis, indicating the ring-opening or substitution of aromatic rings. Lignin degradation might also cause lignin residues to polymerize with other organic intermediates produced by degradation to form humus. The peaks at ~1240 cm1 represented lignin degradation products, such as phenol, ethers, and alcohols. The observed increase in peak intensity for these bands indicated an increased amount of lignin degradation products (Omoike and Chorover, 2006). Furthermore, the absorption peak at 1125 cm1 was characteristic of a syringyl unit, while no obvious peak was observed at 1168 cm1, indicating that no para-hydroxyphenyl units were present. This result indicated that the alkali lignin sample might be a syringyl–guaiacyl-type lignin. The peak at 1037 cm1 peak represented C–H vibrations of the guaiacyl unit. The decrease in intensity of this peak showed that the guaiacyl structure was disrupted, further indicating lignin degradation (Pandey and Pitman, 2003). Based on previous studies and combined with the infrared spectra of alkali lignin before and after enzymatic hydrolysis, representative chemical bonds in alkali lignin were disrupted, while chemical bonds present in lignin degradation products increased substantially following
10
enzymatic treatment. These results indicated that the combination of ligninolytic enzymes facilitated alkali lignin degradation. 3.2.3. GC–MS analysis of alkali lignin GC–MS analysis was performed to identify the various degradation products. The NIST standard mass spectral library was used to assign chromatographic peaks to compounds in the MS spectra. The amount of each degradation product was determined from the corresponding peak area in the chromatogram. Lac oxidizes lignin in the presence of oxygen in air. One electron is extracted from lignin, leading to the generation of free radicals from the substrate molecules, and oxygen molecules are reduced to water. The generated free radicals are unstable and undergo polymerization or depolymerization reactions, causing lignin degradation. The complete catalytic reaction is an electron transfer oxidation, which involves four consecutive one-electron oxidations. The reduced substrate binds to type-I Cu2+, allowing it to extract an electron from the substrate. The extracted electron is transferred to type-II Cu2+ via the Cys– His pathway (Baldrian, 2006) and then passed to O2 bound to this site, reducing O2 to water. In the complete reaction, four consecutive one-electron oxidations are required to fully reduce Lac. Therefore, four substrate molecules are oxidized to produce four substrate free radicals (Su et al., 2018). The large amount of free radicals produced undergo nonenzymatic reactions, such as demethoxy, decarboxyl, and C–C chain breaks. Eventually, the lignin macromolecules are depolymerized into monomers. Therefore, a large amount of aromatics are produced, which can be used by Lac as substrates to continue enzymatic hydrolysis. The
11
resulting free radicals replace hydrogen atoms in the side chains of the aromatics to form alcohols. Therefore, degradation products will include a large amount of alcohols and aromatics. Because Lac does not require strong oxidants to participate in the enzymatic hydrolysis reaction, it is generally thought that enzymatic hydrolysis by Lac is the initiation step of lignin degradation. Based on GC–MS analysis, single-enzyme hydrolysis of alkali lignin by Lac yielded 40 degradation products, which were classified into eight groups, namely, acids, alcohols, alkanes, ketones, esters, phenols, aldehydes, and aromatics. Most of the degradation products were small molecules, such as aromatic acids, aromatic esters, and aliphatic compounds. The alcohol and aromatic contents were highest, accounting for 26.05% and 26.14% of the total degradation products, respectively. This result indicated that Lac degraded alkali lignin and disrupted its structure, causing ring-opening reactions and chemical bond cleavage in benzene ring structures and groups of large molecules in alkali lignin. This finding was consistent with previous reports (Feng et al., 2019; Munk et al., 2018; Rich et al., 2016). The most abundant alcohol was 2,2-dimethyl-1,3-butanediol. Owing to the acidic environment of the enzymatic hydrolysis reaction, 2,2-dimethyl-1,3-butanediol can react with acid to undergo pinacol rearrangement, producing 3,3-dimethyl-2-butanone and water. However, no 3,3-dimethyl-2-butanone was detected in the degradation products, probably because it is not soluble in methanol, which was used as the solvent for dissolution and analysis during sample pretreatment. When LiP was used for single-enzyme hydrolysis of alkali lignin, 40 degradation products were also identified. These products belonged to eight groups, namely, acids, 12
alcohols, alkanes, ketones, esters, phenols, aldehydes, and aromatics. The aromatic and ketone contents were highest, accounting for 34.77% and 22.23% of the total degradation products, respectively. The main chemical bond between lignin molecules is the -O-4-type bond (Ma et al., 2018). LiP catalyzes the decomposition of both benzene rings A and B in synthetic compounds with the β-O-4 bond, mainly through C–C cleavage to form 3,4-dimethoxybenzyl alcohol (veratryl alcohol) and 2-methoxyphenol (Hammel et al., 1993). Although 3,4-dimethoxybenzyl alcohol was not detected in the degradation products, its derivatives 3,4-dimethoxymethylacetic acid and 3,4-dimethoxymethylbenzene were detected. This observation was due to 3,4-dimethoxybenzyl alcohol being a substrate for LiP-catalyzed reactions, resulting in its continuous decomposition via oxidation, which leads to aromatic ring-opening in lignin and methoxyphenol derivative formation. The 2-methoxyphenol content present after lignin degradation was not high, accounting for only 2.93% of the total degradation products. However, the aromatics among the degradation products comprised a several 2-methoxyphenol derivatives, including 2-methoxy-4-ethylphenol, 2-methoxy-4-vinylphenol, 1,2,3-trimethoxy-5-methyl-Benzene, 3,3,5-dimethoxyacetophenone, 3,4,5-trimethoxyphenol, 2,5-dimethoxy-1,4-benzenediol, and 2,6-dimethoxy-4-(2-propenyl)phenol. These 2-methoxyphenol derivatives were all produced by continuous reactions between 2-methoxyphenol and other moieties, such as alcohols, aldehydes, and alkanes, in the degradation products. After single-enzyme hydrolysis with MnP alone, 40 products from alkali lignin degradation were identified, which belonged to seven groups, namely, acids, alcohols, alkanes, ketones, phenols, aldehydes, and aromatics. The aromatic and ketone contents were 13
highest, accounting for 55.70% and 21.54% of the total degradation products, respectively. Acetosyringone was the most abundant monophenol degradation product identified. The mechanism of action of MnP in nature involves the enzymatic oxidation of Mn2+ (present in abundance in nature) to Mn3+. The resulting Mn3+ chelates with glycolate and oxalate present in the system, thus existing in a stable state. These chelated Mn3+ ions can be used as a small-molecular-weight medium, which penetrates into the lignin structure, non-specifically attacking and oxidizing the phenol structure in lignin molecules. Therefore, lignin containing phenol structures forms phenoxy radicals that are unstable and easy to decompose via nonenzymatic reactions. Furthermore, these radical groups undergo a series of reactions that cause some decomposition of the lignin polymer (Agarwal et al., 2018). However, the reaction conditions for enzymatic hydrolysis of alkali lignin lacked Mn2+, which is required by MnP for activity. Therefore, the activity of MnP was noticeably lower compared with those of Lac and LiP. When alkali lignin was enzymatically hydrolyzed with double-enzyme combinations, the production of alcohols, aldehydes, and acids, and the reduction of phenols and aromatics by the Lac–LiP and Lac–MnP groups relative to the blank group was considerably higher compared with those by the LiP–MnP group. The main reason for this difference was that after Lac uses O2 taken from the environment as an oxidant to enzymatically hydrolyze alkali lignin, the resulting intermediates, such as carboxyl radicals with strong oxidizing properties, can act as oxidants for LiP and MnP to initiate the enzymatic hydrolysis reaction. Therefore, the Lac–LiP and Lac–MnP combinations exhibited stronger enzymatic hydrolysis compared with the LiP–MnP group (Fig. 2). 14
Among alcohols in the degradation products, which were produced by the replacement of hydrogen atoms in the side chain of aromatics by strongly oxidative hydroxyl radicals, 2,2-dimethyl-1,3-butanediol was the most abundant compound. The 2,2-dimethyl-1,3-butanediol content was highest in the Lac–LiP group among the double-enzyme combinations, followed by that in the Lac–MnP group. The 2,2-dimethyl-1,3-butanediol content was lowest in the LiP–MnP group, and even lower than that from hydrolysis by Lac only. The amount of degradation product 2,2-dimethyl-1,3-butanediol represents the degree of alkali lignin degradation. In the Lac–LiP group, both Lac and LiP degraded lignin readily after initiation by the oxidant generated by Lac. In the groups containing MnP, the degradation effect of MnP was not evident owing to the absence of Mn2+ in the reaction solution. This finding was in agreement with the experimental results for the degradation rate of alkali lignin. The combined use of the three enzymes to degrade alkali lignin yielded roughly the same result when compared with the hydrolysis reactions using two enzymes. Nonetheless, alkali lignin degradation was more complete in the Lac–LiP–MnP group owing to the increase in enzyme dose and the strong oxidative intermediates produced by enzymatic hydrolysis with Lac initiating the enzymatic hydrolysis reactions of LiP and MnP. In this study, the enzymatic hydrolysis products of alkali lignin from each group were analyzed by GC–MS. Based on the analysis of the degradation products, not only were the experimental results for the alkali lignin degradation rate explained, but also the mechanisms underpinning lignin degradation by Lac, LiP, and MnP, and combinations of these three enzymes, were explored. 15
4. Conclusions In this study, the degradation effect of laccase on lignin was found to be the most obvious, while the effect of the three enzymes in combination on lignin degradation was the best, achieving 28.98% degradation. Lignin degradation enzymes mainly destroyed the apparent structure and key chemical bonds of lignin, with most of the degradation products being aromatic small molecular compounds. Lignin degradation enzymes were proven to lead to ring-opening reactions of macromolecular benzene ring structures and groups in alkali lignin as well as chemical bond cleavage. This result provides a theoretical basis for the biodegradation of lignin. E-supplementary data for this work can be found in the electronic version of this paper online Declaration of competing interests The authors declare that they have no known competing financial interests or personal relationships that could influence the work reported in this paper.
FUNDING
This study was funded by the National Key R&D Program of China ( 2017YFD0501005).
Acknowledgments
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We thank the National Key R&D Program of China, the Key Laboratory of Straw Biology and Utilization, The Ministry of Education, for its support. We also thank Simon Partridge, PhD, from Liwen Bianji, Edanz Editing China (www.liwenbianji.cn/ac), for editing the English text of a draft of this manuscript.
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Table 1 Amount of enzymes added to each experimental group.
Test group
1
2
3
4
5
6
7
8
Laccase (L)
–
10
–
–
10
10
–
10
–
–
10
–
10
–
10
10
–
–
–
10
–
10
10
10
Lignin peroxidase (L) Manganese peroxidase (L) “–” indicates that the volume was made up with sterile water.
21
35
Degradation rate %
30 25 20 15 10 5 0 Lac
LiP
MnP
Lac、LiP
Lac、MnP
LiP、MnP
Lac、LiP、MnP
Different combinations of enzymes
Fig. 1. Degradation rate of alkali lignin by laccase (Lac), lignin peroxidase (LiP), or manganese peroxidase (MnP), and in the presence of different combinations of the three enzymes.
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12000000
Lac、Lip Lac、Mnp
10000000 Peak area of product
Lip、Mnp 8000000
6000000
4000000
2000000
0 Alcohol
Aldehydes
Esters Main products
Phenols
Aromatic
Fig. 2. Main products of enzymatic hydrolysis of alkali lignin using combinations of two enzymes: Laccase (Lac), lignin peroxidase (LiP), and manganese peroxidase (MnP).
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Sitong Zhang：Experimental design, review & editing Jianlong Xiao：Validation, Data analysis，writing - original draft Gang Wang：Data analysis, resources Guang Chen：Resources, Writing - Review & Editing, Supervision,Data Curation.
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Highlights 1. Alkali lignin degradation by three enzymes was examined. 2. Structural and compositional changes to degraded alkali lignin were characterized. 3. Degradation products were identified by GC-MS, and production pathways analyzed. 4. Synergy among ligninolytic enzymes gave the highest degradation levels of lignin. 5. Enzymes enabled ring-opening of benzene structures and rupture of chemical bonds.
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