Isolation and Characterization of a Novel Laccase for Lignin Degradation, LacZ1

ABSTRACT

Lignin is a complex natural organic polymer and is one of the primary components of lignocellulose. The efficient utilization of lignocellulose is limited because it is difficult to degrade lignin. In this study, we screened a lacz1 gene fragment encoding laccase from the macrotranscriptome data of a microbial consortium WSC-6, which can efficiently degrade lignocellulose. The reverse transcription-quantitative PCR (RT-qPCR) results demonstrated that the expression level of the lacz1 gene during the peak period of lignocellulose degradation by WSC-6 increased by 30.63 times compared to the initial degradation period. Phylogenetic tree analysis demonstrated that the complete lacz1 gene is derived from a Bacillus sp. and encoded laccase. The corresponding protein, LacZ1, was expressed and purified by Ni-chelating affinity chromatography. The optimum temperature was 75°C, the optimum pH was 4.5, and the highest enzyme activity reached 16.39 U/mg. We found that Cu²⁺ was an important cofactor needed for LacZ1 to have enzyme activity. The molecular weight distribution of lignin was determined by gel permeation chromatography (GPC), and changes in the lignin structure were determined by ¹H nuclear magnetic resonance (¹H NMR) spectra. The degradation products of lignin by LacZ1 were determined by gas chromatography and mass spectrometry (GC-MS), and three lignin degradation pathways (the gentian acid pathway, benzoic acid pathway, and protocatechuic acid pathway) were proposed. This study provides insight into the degradation of lignin and new insights into high-temperature bacterial laccase.

IMPORTANCE Lignin is a natural aromatic polymer that is not easily degraded, hindering the efficient use of lignocellulose-rich biomass resources, such as straw. Biodegradation is a method of decomposing lignin that has recently received increasing attention. In this study, we screened a gene encoding laccase from the lignocellulose-degrading microbial consortium WSC-6, purified the corresponding protein LacZ1, characterized the enzymatic properties of laccase LacZ1, and speculated that the degradation pathway of LacZ1 degrades lignin. This study identified a new, high-temperature bacterial laccase that can degrade lignin, providing insight into lignin degradation by this laccase.

INTRODUCTION

Lignocellulose is the largest renewable biomass resource in the world, with an annual output of about 1,500 million tons (1). It is a product associated with agricultural production, and straw is one of the primary sources of lignocellulose. However, large amounts of straw are burned or discarded in many countries, while biomass recycling has become an effective method of reducing this straw waste (2). Lignin is an aromatic polymer and one of the primary components of lignocellulose. Lignin is primarily composed of p-coumaryl (H), guaiacyl (G), and syringyl (S) monomers and is connected by a β-O-4 bond, β-5 bond, and β-β bond (3). Lignin accounts for approximately 20 to 32% of the natural lignocellulose structure and is the second most abundant natural organic polymer, after cellulose (4, 5). Lignin strengthens the plant structure to prevent cellulose and hemicellulose from being hydrolyzed. The complex lignin structure also makes it difficult for lignocellulose to be decomposed and transformed by an organism, limiting the efficient use of agricultural waste. Biological treatment is often needed when using lignin-rich agricultural waste, including for papermaking, composting, and biogas (6–8). Lignin degradation improves the flexibility of paper, the nutrient content of organic fertilizers, and methane output during biogas fermentation. The key to using biological methods to decompose lignin is to obtain microbial resources that efficiently decompose and transform lignin and mine the enzymes with lignin-degrading ability in microorganisms.

Few microorganisms can naturally degrade lignin and produce lignin-degrading enzymes, including fungi, actinomycetes, and bacteria (9–11). They can secrete extracellular lignin degradation oxidases, which are primarily divided into lignin peroxidase (EC 1.11.1.14), manganese peroxidase (EC 1.11.1.13), and laccase (EC 1.10.3.2) (12–14). Laccase contains copper ions, which makes it a kind of multicopper oxidase. The molecular structure of laccase contains four copper ions, which can be divided into three types according to their magnetic and spectroscopic properties. Type I Cu²⁺ and type II Cu²⁺ are single-electron, paramagnetic acceptors, while type III Cu²⁺ are a coupled-ion, two-electron acceptor, diamagnetic pair (15). The substrate catalyzed by laccase is oxidized through the coordinated transfer of electrons by four copper ions. Laccase is widely found in fungi, bacteria, and plants. Of them, fungal laccase is primarily derived from white-rot fungi, while research on bacterial laccase is primarily concentrated on Streptomyces, Pseudomonas, and Bacillus (16). Oxygen can be used as an oxidizing agent to oxidize phenolic substrates (17), while nonphenolic substrates can also be oxidized under certain conditions, including as oxidation mediators (18). Both lignin peroxidase and manganese peroxidase rely on H₂O₂ as a mediator. Oxidation reactions (19) can increase the scope of laccase’s application and industrial production. The oxidation reaction catalyzed by laccase includes the degradation of polymers and the oxidative coupling reaction of phenolic compounds. In addition to degrading lignin, it can also catalyze the oxidation of biphenols, polyphenols, aminophenols, polyamines, and aryl diamines (20, 21). Laccase is the only enzyme that can degrade lignin on its own. There are fewer examples of bacterial laccase than of fungal laccase, though many bacterial laccases that can degrade lignin have not yet been discovered.

This paper reports a new lignin-degrading bacterial laccase, LacZ1, derived from the high-temperature lignocellulose-degrading microbial consortium WSC-6, which was obtained during previous laboratory research (22). By analyzing the high-throughput sequencing data of how the microbial consortium WSC-6 degrades lignocellulose during different periods, a bacterial laccase lacz1 gene fragment from the AA1 family was found. Reverse transcription-quantitative PCR (RT-qPCR) was used to detect changes in gene expression during the degradation of lignin by WSC-6. The protein was purified by cloning and expressing the gene, and the physicochemical properties of laccase were determined and optimized. The gas chromatography and mass spectrometry (GC-MS) method was used to determine the products of laccase lignin degradation and was combined with gel permeation chromatography (GPC) and ¹H nuclear magnetic resonance (¹H NMR) spectrum data to identify the metabolic pathway of lignin. The results demonstrate that laccase LacZ1 significantly degrades lignin and has a higher optimum temperature than other reported laccases. This study provides evidence that lignin is degraded by laccase under high-temperature conditions.

RESULTS

Fold change in gene expression and functional verification.

The amplification efficiency of the primers for the lacz1 gene and 16S rRNA genes were 101.7% and 95.5%, respectively. All primers can be used in subsequent experiments. RT-qPCR analysis was used to obtain the cycle threshold (C_T) values of the lacz1 gene fragment and 16S rRNA, while the expression ratio of WSC-6 at the initial, peak, and late stages of rice straw degradation was calculated using the 2^−ΔΔCT method. Our results demonstrated that the expression level of the lacz1 gene fragment at the degradation peak was 30.63 times higher than at the initial degradation stage and that the expression level at the end of degradation was 6.07 times higher than at the initial degradation stage (Fig. 1A). The increased gene expression at the degradation peak indicates that the protein encoded by the gene played an important role in rice straw degradation by WSC-6. The lacz1 gene was annotated as laccase from the AA1 family in the macrotranscriptome data (Table 1), indicating that this gene could be related to lignin degradation in rice straw. The lacz1 gene was amplified by PCR, ligated into the plasmid pMD18-T to construct the recombinant plasmid pMD18-T-lacz1, and subsequently transformed into Escherichia coli DH5α. The successful construction of the recombinant plasmid pMD18-T-lacz1 was verified by the results of plasmid sequencing and agarose gel electrophoresis, while the size of the lacz1 gene derived from the enrichment WSC-6 was 1,548 bp. The lacz1 gene was ligated into the plasmid pET-28a(+) to construct the recombinant plasmid pET28a-lacz1 and subsequently transformed into E. coli BL21(DE3). The successful construction of the recombinant plasmid pET28a-lacz1 was verified by agarose gel electrophoresis. To determine that the protein encoded by the lacz1 gene can degrade lignin, we measured changes in rice straw lignin content by the resting cells at 30°C. Our results demonstrated that the lignin contents in rice straw were 10.4%, 4.2%, 1.8%, and 1.0% at 0 h, 8 h, 16 h, and 24 h, respectively, and that the degradation rate of lignin reached 90.1% at 24 h. The lignin contents of the two control groups, E. coli BL21(DE3) transformed with empty plasmid and the uninoculated control group, did not significantly decrease (Fig. 1B). This demonstrates that the protein LacZ1, encoded by the lacz1 gene, can effectively degrade lignin.

FIG 1.

Fold change in gene expression and functional verification. (A) The 2^−ΔΔCT method was used to calculate the fold change of the expression of the lacz1 gene fragment at different stages of rice straw degradation by the microbial consortium WSC-6. Twelve hours is the initial stage of degradation, 72 h is the peak of degradation, and 168 h is the end of degradation. (B) Resting cells verified the ability of the lacz1 gene to degrade lignin in rice straw. Black represents the treatment group of E. coli BL21(DE3) transformed with the recombinant plasmid pET28a-lacz1, red represents the treatment group of E. coli BL21(DE3) transformed with the empty plasmid pET-28a(+), and blue represents the treatment group not inoculated.

TABLE 1.

Macrotranscriptome data of the AA1 family

GenBank accession no.	Protein	Strain	Gene fragment size (bp)
KYH23774.1	Laccase	Halalkalicoccus paucihalophilus	536
WP_014102799.1	Copper oxidase	Micavibrio aeruginosavorus	240
WP_042353229.1	Copper oxidase	Bacillus massiliogorillae	621
WP_003386914.1	Multicopper oxidase	Brevibacillus borstelensis	3,085
WP_024028661.1	Bilirubin oxidase	Bacillus vireti	535
WP_054110202.1	Copper-binding protein	Brevundimonas sp.	315
XP_007867185.1	Laccase hybrid	Gloeophyllum trabeum	381
KLJ09648.1	Hypothetical protein	Emmonsia parva	616
WP_017436327.1	Copper oxidase	Geobacillus caldoxylosilyticus	901
EGQ24147.1	Spore coat protein A	Sporosarcina newyorkensis	2,405
WP_051651387.1	Copper-binding protein	Brevundimonas bacteroides	318
XP_008024136.1	Hypothetical protein	Setosphaeria turcica	305
WP_006472662.1	Multicopper oxidase	Ochrobactrum	1,097
KKF95926.1	Iron transport multicopper oxidase	Ceratocystis platani	413

Sequence analysis of LacZ1.

The annotated transcriptome data indicated that the lacz1 gene could be derived from the AA1 family, which encodes laccase. To identify the source of the protein LacZ1, encoded by the lacz1 gene, we compared LacZ1 with the reported laccase and constructed a phylogenetic tree. Our results demonstrated that the homology of LacZ1 with laccase derived from Bacillus was 57.1% to 50.8% and that the homology with laccase derived from fungus was 33.3% to 19.9%. This suggests that the protein LacZ1 was derived from a Bacillus sp. in the microbial consortium WSC-6 (Fig. 2A). Four copper-binding LacZ1 sites were identified by analyzing the reported laccase amino acid sequences. L1 was located at amino acids 100 to 107, L2 was at amino acids 149 to 154, L3 was at amino acids 419 to 424, and L4 was at amino acids 491 to 497 (Fig. 2B). In contrast, we found that the copper-binding sites of LacZ1 were identical with the laccase from the Bacillus sp. but that the copper-binding sites L3 and L4 differed from those of laccase derived from E. coli and fungi.

FIG 2.

Amino acid sequence analysis. (A) The red font indicates the laccase LacZ1 used in this study, while the laccase used for comparison comes from the NCBI database. (B) The conserved binding domain of the nucleotide sequence was compared by ESPript 3. L1, L2, L3, and L4 are the copper-binding sites of LacZ1.

Purification and characterization of LacZ1.

The lacz1 gene was ligated into the expression plasmid pET-28a(+) and transformed into E. coli BL21(DE3). The protein was purified after induction at 16°C for 12 h. SDS-PAGE demonstrated that the protein that eluted with 300 mM imidazole had the highest purity, with a protein size of 59.7 kDa (Fig. 3A). The enzyme activity of LacZ1 was measured using a 0.1 M sodium acetate buffer with 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as the substrate. When 10 mM copper sulfate was added to the sodium acetate buffer, enzyme activity increased from 0.10 U/mg to 16.39 U/mg, or 159.44 times (Fig. 3B). The results demonstrated that Cu²⁺ was an important cofactor for LacZ1 to have enzyme activity, and copper sulfate was added to the sodium acetate buffer for all subsequent enzyme activity determination tests. The enzyme activity was measured in a sodium acetate buffer of pH 3.0 to 7.0, and the results demonstrated that LacZ1 had the highest enzyme activity in a sodium acetate buffer with pH 4.5 (Fig. 3C). The enzyme activity of LacZ1 at different temperatures was measured in a sodium acetate buffer at pH 4.5. The enzyme activity of LacZ1 was highest at a temperature of 75°C (Fig. 3D). When the temperature did not exceed 75°C during the thermal stability measurement for LacZ1, the enzyme activity stabilized within 1 h; when the temperature exceeded 75°C, the enzyme activity gradually decreased within 1 h (Fig. 3E). We added 0.5 mM concentrations of different metal ions (Zn²⁺, Ba²⁺, Mn²⁺, K⁺, Ca²⁺, Pb²⁺, and Mg²⁺) into the sodium acetate buffer to assess how the metal ions affected the enzyme activity of LacZ1. The results demonstrate that metal ions inhibited enzyme activity to various degrees and that Pb²⁺ had the largest inhibitory effect on enzyme activity, reducing enzyme activity by up to 40.3% (Fig. 3F). The kinetic parameters of LacZ1 were analyzed using ABTS as a substrate. The K_m of LacZ1 for the substrate ABTS was 0.35 mM, and the V_max was 20.61 μM/min (Fig. 3G).

FIG 3.

Purification and characterization of the protein LacZ1. (A) SDS-PAGE analysis of LacZ1. Lane M, molecular size markers; lane 1, sonicated E. coli BL21(DE3)-pETD28a-lacz1; lane 2, 20 mM imidazole elution; lane 3, 40 mM imidazole elution; lane 4, 60 mM imidazole elution; lane 5, 80 mM imidazole elution; lane 6, 200 mM imidazole elution; lane 7, 300 mM imidazole elution. (B) Effect of Cu²⁺ on LacZ1 activity. (C) Effect of pH on LacZ1 activity. (D) Effect of temperature on LacZ1 activity. (E) Effect of metal ions on LacZ1 activity. (F) Thermal stability of LacZ1 activity. (G) Kinetic analysis of LacZ1.

Analysis of lignin degradation by LacZ1.

We used purified laccase LacZ1 to degrade lignin in rice straw and found that the lignin content in rice straw continuously decreased at pH 7.0. The initial lignin content in rice straw was 10.2%; the lignin content decreased to 6.4% when it was incubated with the enzyme for 1 h and decreased to 4.5% after 8 h. The degradation rate of lignin was 55.8% when incubated with the enzyme for 8 h. After 8 h of incubation, the lignin in the rice straw was functionally no longer degraded. The lignin content in rice straw was 4.4% at 24 h, and the degradation rate of lignin was 56.7%. The lignin content in rice straw also continuously decreased at pH 4.5. After incubation for 1 h, the lignin content decreased from 10.3% to 5.0%, and after incubation for 8 h, the lignin content decreased to 3.3%, for a lignin degradation rate of 67.5%. After 8 h of incubation, the lignin in the rice straw was no longer degraded. The lignin content in rice straw was 3.3% after 24 h, for a lignin degradation rate of 68.2%. Lignin was not degraded in the control group without enzyme (Fig. 4A).

FIG 4.

Lignin degradation by LacZ1. (A) Degradation of lignin in rice straw by LacZ1. (B) The mass change of LacZ1 degradation of lignin.

Using purified laccase LacZ1 to degrade lignin demonstrated that lignin mass continuously decreased at pH 7.0 and pH 4.5. After 8 h of incubation, the lignin mass decreased slightly. At pH 7.0, the weight loss rates of lignin were 25.5%, 42.2%, and 45.7% at 1 h, 8 h, and 24 h after incubation, respectively. At pH 4.5, the weight loss rates of lignin were 41.2%, 56.8%, and 59.3% at 1 h, 8 h, and 24 h after incubation, respectively. Lignin was not degraded in the control group without enzyme (Fig. 4B).

Molecular weight distribution analysis of lignin degradation by LacZ1.

The purified laccase LacZ1 was used to degrade lignin, and the molecular weight distribution was determined via GPC. The results demonstrated that at 0 min, 30 min, and 60 min of degradation, number average molecular weight (Mn) decreased from 68,166 Da to 54,727 Da to 40,255 Da, weight average molecular weight (Mw) decreased from 85,700 Da to 71,308 Da to 56,913 Da, and peak molecular weight (Mp) decreased from 52,621 Da to 41,302 Da to 29,671 Da, respectively. The polydispersity coefficient (PDI) was obtained by Mw/Mn. PDI increased from 1.26 to 1.30 to 1.41 during lignin degradation (Table 2 and Fig. 5). The decreases in Mn, Mw, and Mp indicate that the macromolecular substances in the sample were continuously degraded and that the molecular weight gradually decreased. The PDI increase indicates that the molecular weight distribution of the sample widened and that the number of small-molecule compounds gradually increased. The results demonstrated that laccase can depolymerize lignin into monomers.

TABLE 2.

Molecular weight distribution of lignin degradation by LacZ1

Time (min)	Mn (Da)	Mw (Da)	Mp (Da)	PDI
0	68,166	85,700	52,621	1.26
30	54,727	71,308	41,302	1.30
60	40,255	56,913	29,671	1.41

FIG 5.

GPC diagram of lignin degradation by LacZ1. (A) Lignin degraded for 0 min. (B) Lignin degraded for 30 min. (C) Lignin degraded for 60 min. MV, millivolt.

Structural-change analysis of lignin degradation by LacZ1.

After laccase treatment, ¹H NMR was used for analysis. There was no significant difference in the absorption peaks, but the signal intensity of some absorption peaks changed significantly (Fig. 6). Laccase weakened the hydrogen signal in the aromatic ring of lignin, which indicated that hydrogen on the aromatic lignin ring was replaced after treatment. Laccase also weakened the signal of the β-O-4 bond, β-5 bond, and β-β bond, indicating that the primary linking bond of lignin was ruptured and that the macromolecules were depolymerized. As lignin degraded, the hydrogen signal on the methoxyl group decreased from 12.27 to 7.29, indicating that demethoxylation occurred during lignin degradation. The hydrogen in aromatic acetates and aliphatic acetates were partially reduced and the hydrogen in other aliphatic compounds showed no obvious change (Table 3).

FIG 6.

¹H NMR diagram of lignin degradation by LacZ1. (A) Lignin degraded for 0 min. (B) Lignin degraded for 30 min. (C) Lignin degraded for 60 min.

TABLE 3.

¹H NMR analysis of lignin degradation by LacZ1

Signal (ppm)	Assignment	Intensity
		0 min	30 min	60 min
4.79	D2O
7.80–6.50	H in aromatic	2.49	1.32	1.74
4.70–4.25	Hα and Hβ in β-O-4	3.00	1.77	0.42
4.00–3.50	H in methoxyl	12.27	7.59	7.29
3.50–3.00	Hα and Hβ in β-5	2.87	1.48	1.21
3.00–2.55	Hα and Hβ in β-β	1.18	0.26	0.09
2.50–2.20	H in aromatic acetates	1.70	0.57	0.52
2.20–2.00	H in aliphatic acetates	1.16	0.44	0.45
1.50–0.80	H in other aliphatic compounds	3.59	3.45	3.25

Analysis of lignin degradation products degraded by LacZ1.

The products of the LacZ1 degradation of lignin were determined via GC-MS, including nine monophenolic compounds and lactic acid (Table 4 and Fig. 7). The content of 4-hydroxybenzoic acid was the highest in all degradation products. Three pathways of lignin degradation were identified based on the degradation products, including the gentisate acid pathway, benzoic acid pathway, and protocatechuic acid pathway (see Fig. S1 in the supplemental material). The benzoic acid pathway had two different metabolic pathways. In the gentisate acid pathway, the carboxymethyl on 2-(3-hydroxyphenyl)acetic acid was converted to carboxyl to generate 3-hydroxybenzoic acid, and the 6-position of 3-hydroxybenzoic acid was subjected to hydroxylation to generate gentisic acid. In the protocatechuic acid pathway and the benzoic acid pathway, the aldehyde on 4-hydroxy-3,5-dimethoxybenzaldehyde was oxidized to carboxy to generate 4-hydroxy-3,5-dimethoxybenzoic acid, after which 4-hydroxy-3,5-dimethoxybenzoic acid removed the methoxy at the 5 position to generate 4-hydroxy-3-methoxybenzoic acid. 4-Hydroxy-3-methoxybenzoic acid can be metabolized in two ways: first, the methoxy at the 3 position can undergo demethylation to produce protocatechuic acid; second, the 3 position can undergo demethoxylation to generate 4-hydroxybenzoic acid. Another benzoic acid pathway is the ester on ethyl 2-hydroxy-2-(4-hydroxyphenyl)acetate, which was hydrolyzed and subjected to decarboxylation and oxidation to generate 4-hydroxybenzoic acid. No benzoic acid was detected in the degradation products of LacZ1-degrading lignin, indicating that LacZ1 could not dehydroxylate 4-hydroxybenzoic acid to generate benzoic acid.

TABLE 4.

Products of lignin degradation by LacZ1

No.	Abundance scan (min)	Compound	Chemical formula
1	9.117	Ethyl 2-hydroxy-2-(4-hydroxyphenyl)acetate	C10H12O4
2	9.381	2,5-Dihydroxybenzoic acid	C7H6O4
3	10.379	3-Hydroxybenzoic acid	C7H6O3
4	10.469	4-Hydroxybenzoic acid	C7H6O3
5	10.586	2-(3-Hydroxyphenyl)acetic acid	C8H8O3
6	11.321	4-Hydroxy-3,5-dimethoxybenzaldehyde	C9H10O4
7	11.924	Lactic acid	C3H6O3
8	11.990	4-Hydroxy-3-methoxybenzoic acid	C8H8O4
9	12.606	3,4-Dihydroxybenzoic acid	C7H6O4
10	13.393	4-Hydroxy-3,5-dimethoxybenzoic acid	C9H10O5

FIG 7.

Mass spectrum of lignin degradation products by LacZ1. (1) Ethyl 2-hydroxy-2-(4-hydroxyphenyl)acetate; (2) 2,5-dihydroxybenzoic acid; (3) 3-hydroxybenzoic acid; (4) 4-hydroxybenzoic acid; (5) 2-(3-hydroxyphenyl)acetic acid; (6) 4-hydroxy-3,5-dimethoxybenzaldehyde; (7) lactic acid; (8) 4-hydroxy-3-methoxybenzoic acid; (9) 3,4-dihydroxybenzoic acid; (10) 4-hydroxy-3,5-dimethoxybenzoic acid.

DISCUSSION

Lignocellulose is one of the most abundant renewable resources in the world and is also the primary component of agricultural waste. The annual global output of lignocellulose is approximately 2 × 10¹¹ tons (23). China has a large demand for food, resulting in significant straw resources. The incineration of agricultural waste is one of the primary causes of haze in the autumn and winter, which reduces environmental quality and can pose a threat to people’s health. Therefore, it is important to efficiently use and/or decompose straw waste. Lignin is one of the primary components of lignocellulose, has a complex structure, and is difficult to degrade, limiting its conversion, utilization, and efficient lignocellulose utilization. Current methods of lignin removal include physical, chemical, and biological methods (24, 25). The physical method produces low levels of pollution and is simple to operate, but it is expensive and requires a lot of energy. The chemical method removes lignin more completely but produces liquid waste that harms the environment. The biological treatment of lignin does not produce pollution and requires less energy. In this study, a high-temperature bacterial laccase, LacZ1, that can degrade lignin was purified from the lignocellulose-degrading microbial consortium WSC-6. Analysis of amino acid sequences indicates that LacZ1 is derived from Bacillus in the microbial consortium WSC-6. This new laccase provides resources for the biodegradation of lignin under high-temperature conditions, helps identify the biodegradation pathway of lignin, and provides a reference for the future enzymatic degradation of straw.

Our results demonstrated that the lignin degradation rate of resting cells was 33.4% higher than that of the purified enzyme maintained at pH 7.0 for 24 h. However, the lignin degradation rate of resting cells was only 4.1% higher than that of the purified enzyme maintained at pH 7.0 for 8 h. The activity of the purified enzyme gradually decreased under high temperatures, while the purified enzyme gradually inactivated after 8 h. The lignin degradation rate of the purified enzyme at pH 4.5 was 6.7% and 10.8% higher than those of the resting cells and the purified enzyme at pH 7.0, respectively. This indicates that the ability of the purified enzyme to degrade lignin is stronger under the optimum conditions than under other conditions.

Previous research assessing the microbial consortium WSC-6 demonstrated that the abundance of Bacillus organisms gradually increased during lignocellulose degradation by WSC-6 (26), which was consistent with the assumption that LacZ1 was derived from Bacillus. Laccase is a kind of copper oxidase and has four copper-binding sites. Adding Cu²⁺ to the degradation process can promote laccase activity. Other studies of laccase also found this characteristic (27–29). The laccase pH range means that there is higher enzymatic activity only in acidic environments and that lignin degradation is reduced in alkaline environments. Compared with previously reported optimum temperatures (30–32), our optimum temperature of 75°C indicates that LacZ1 is one of the most heat-resistant kinds of laccase. LacZ1 could not maintain high enzyme activity at high temperatures for 24 h, but its lignin degradation ability was strong enough after 8 h, and enzyme activity did not decrease after 1 h. Therefore, LacZ1 also possesses thermal stability. During straw resource utilization, a variety of treatment methods are performed under high- or medium-temperature conditions; laccase can adapt to high-temperature treatment conditions, meaning that it is highly practical.

In this study, the laccase LacZ1 metabolites used to degrade lignin were primarily monophenolic compounds. During laccase action, lignin loses hydrogen ions and exists as a free radical, which greatly increases its reaction activity. Laccase then breaks the C_αC_β bonds of the alkyl and aryl groups in the activated lignin, depolymerizing and degrading the lignin and releasing its substructure (33–35). The activated lignin substructure is then oxidatively modified and demethoxylated. ¹H NMR confirmed that laccase LacZ1 breaks the β-O-4 bond, β-5 bond, and β-β bond in lignin, completing the depolymerization of macromolecules. No downstream monophenol products were found in the metabolites, indicating that the laccase cannot break the C-C σ bond of the benzene ring for the next degradation step. The highest content of 4-hydroxybenzoic acid was found in the metabolites, indicating that the laccase could not complete the dihydroxylation reaction and that 4-hydroxybenzoic acid was the final product of the benzoic acid pathway. Benzoic acid has been detected in the metabolites of many strains reported to degrade lignin (36, 37), but it has not been detected in the metabolites of laccase LacZ1 degrading lignin. This could be because the degradation of 4-hydroxybenzoic acid to benzoic acid requires action from other enzymes and that LacZ1 alone cannot complete further degradation. While LacZ1 cannot completely degrade lignin into small molecular compounds, the degradation of LacZ1 into single phenolic compounds was sufficient to improve the resource utilization efficiency of straw.

LacZ1 is a novel laccase and differs from the reported bacterial laccases in several ways. Lacz1 has high lignin degradation ability without ABTS as a mediator. Most of the reported bacterial laccases require ABTS as a mediator to have strong lignin degradation ability (38–40). LacZ1 can break the β-O-4 bond, β-5 bond, and β-β bond in the lignin structure at the same time, and lignin was degraded by a single enzyme through three different pathways. Most of the reported bacterial laccases can break only one or two kinds of lignin bonds (41, 42). As laccase LacZ1 binds with lignin, the lignin macromolecules are gradually depolymerized and oxidized. However, the lignin is not completely degraded into small molecular acids. The complete lignocellulose degradation pathway of the microbial consortium WSC-6 can be clarified by assessing other enzymes related to the degradation of lignin and cellulose in the consortium, thereby achieving complete degradation of lignocellulose. In conclusion, laccase Lacz1 is an efficient high-temperature biomaterial that can be used to degrade lignin for industrial production in the future.

MATERIALS AND METHODS

Enrichment.

The microbial consortium WSC-6 was obtained from the Heilongjiang Provincial Key Laboratory of Environmental Microbiology and Recycling of Argo-waste in the Cold Region. It is known that the composite microbial system can significantly degrade lignocellulose at 50°C. E. coli DH5α was used for plasmid construction, and E. coli BL21(DE3) was used for protein expression.

Chemicals and media.

Rice straws were obtained from the experimental field of Heilongjiang Bayi Agricultural University (Daqing, China). Lignin (99% purity) and ABTS (99% purity) were purchased from Macklin (Shanghai, China). The lignin was extracted from wood, and its structure was the same as that of lignin in rice straw. Both are angiosperm lignin and contain three lignin monomers: p-coumarin (H), guaiacol (G) and syringyl (S). All reagents used were analytical purity or chromatographic purity, and all solvents used were of the highest purity available. Peptone-cellulose (PCS) medium (1 g/liter yeast extract, 5 g/liter tryptone, 2 g/liter CaCO₃, 5 g/liter NaCl, and 5 g/liter rice straw) was used to culture the microbial consortium WSC-6. Luria-Bertani (LB) medium (5 g/liter yeast extract, 10 g/liter tryptone, 10 g/liter NaCl) was used for culture of E. coli. Solid agar plates were prepared by adding 1.5% (wt/vol) agar to the LB liquid medium.

RT-qPCR analysis.

The total RNA of lignocellulose degraded by WSC-6 at different stages was extracted by the TRIzol method (TianGen, China). The selection of lignin degradation period refers to the previous research results for lignocellulose degradation by WSC-6. The initial degradation time was 12 h, the peak degradation time was 72 h, and the final degradation time was 168 h (26). After DNA removal with DNase I (Thermo Scientific), reverse transcription was carried out using the Promega GoScript reverse transcription system (product no. A5001; Promega). The cDNA product was diluted to the same concentration for the RT-qPCR analysis. Primers for lignin degradation lacz1 gene fragment designed by Beacon Designer 7 are as follows: lacz1-F (5′-CGTAGAGCCTGATAGTGATG-3′) and lacz1-R (5′-GTTGAGCCTGGTGATTCC-3′). Primers for 16S rRNA gene are as follows: 515-F (5′-GTGCCAGCMGCCGCGGTAA-3′) and 909-R (5′-CCCCGYCAATTCMTTTRAGT-3′). RT-qPCR was performed using the CFX96 real-time PCR detection system (Bio-Rad, Hercules, CA) with real master mix (SYBR green) (TianGen, China). The standard curve was constructed with gradient-diluted DNA as the template to determine the amplification efficiency of primers. The amplification efficiency of the primers should be within 90% to 105%. The RT-qPCR system was as follows: 2× Talent qPCR premix (with SYBR green I), 10 μl; cDNA, 0.4 μl; primer-F, 0.4 μl; primer-R, 0.4 μl; and double-distilled water (ddH₂O), 8.8 μl. The RT-qPCR program was as follows: initial denaturation at 95°C for 3 min, followed by 39 cycles of denaturation at 95°C for 20 s, annealing at 56°C (lignin degradation gene lacz1 fragment) or 59°C (16S rRNA gene) for 20 s, and elongation at 68°C for 15 s. The melting-curve analysis was as follows: temperature maintained at 95°C for 30 s, change to 55°C for 60 s, increase by 0.5°C every 5 s, and finally holding at 95°C. The fold change in gene expression was calculated using the 2^−ΔΔCT method, using the 16S rRNA gene as the reference gene.

Cloning and expression of gene.

Comparison of the nucleotide sequence fragment of lignin degradation function in metagenomics data with the NCBI database for Nucleotide BLAST obtained the most similar complete gene. Primers for the lacz1 gene designed by Primer Premier 5 are as follows: pMD18T-F (5′-ATGGAATTGTCCAAATTTGTCGACA-3′) and pMD18T-R (5′-TTAATGTTTTTCGGGTTCCTTTACC-3′). The PCR system was as follows: 2× Phanta Max master mix, 10 μl; DNA, 0.5 μl; primer-F, 0.6 μl; primer-R, 0.6 μl; and ddH₂O, 8.3 μl. The PCR-amplified gene was ligated into pMD18-T to generate the recombinant plasmid pMD18-T-lacz1. The recombinant plasmid was transformed into E. coli DH5α. Plasmid was extracted by TIANprep rapid miniplasmid kit (TianGen, China) and sequenced, obtaining the lacz1 gene from composite microbial system WSC-6. The lacz1 gene was amplified from the genomic DNA of WSC-6 using Pfu DNA polymerase (Vazyme, China). The primers, which are the NdeI (underlined) and XhoI (underlined) recognition sites, were as follows: pET28a-F (5′-TGCCGCGCGGCAGCCATATGGAATTGTCCAAATTTGT-3′) and pET28a-R (5′-TGGTGGTGGTGGTGCTCGAGTTAATGTTTTTCGGGTTCCT-3′). The PCR-amplified gene was ligated into pET-28a(+) to generate the recombinant plasmid pET28a-lacz1. The recombinant plasmid was transformed into E. coli BL21(DE3) for protein expression. The cells were cultured in LB medium at 200 rpm and 37°C for 4 h. Then, a final concentration of 0.4 mM isopropyl-β-d-thiogalactopyranoside (IPTG) was added into the medium, and the cells were further cultivated at 200 rpm and 16°C for 12 h. Centrifugation was performed to collect cells and the cells were resuspended in buffer (25 mM Tris, 300 mM NaCl, 20 mM imidazole [pH 8.0]), and then cells were disrupted using the Ultrasonic cell disruptor. LacZ1 protein with an N-terminal His tag was purified by Ni-chelating affinity chromatography. The protein was dialyzed in 1 mM EDTA for 12 h to remove salt ions. The residual imidazole in the eluted fractions was removed by ultrafiltration with 50 mM phosphate buffer (pH 8.0). SDS-PAGE was performed with electrophoresis buffer (3.02 g liter⁻¹ Tris, 18.8 g liter⁻¹ glycine, and 1.0 g liter⁻¹ SDS).

Lignin degradation by resting cells.

Resting cells were made by transforming E. coli BL21(DE3) with pET-28a-lacz1. Resting-cell assays were done using an optical density at 600 nm (OD₆₀₀) of 5.0 in 50 mM phosphate buffer (pH 7.0) with 5% rice straw at 200 rpm and 30°C. For contrast, empty E. coli BL21(DE3) and sterile phosphate buffer were used. Lignin content in rice straw was determined with a lignin content kit (QiYi, China).

Amino acid sequence data analysis.

The nucleotide sequence of LacZ1 was compared by NCBI with a protein BLAST search. The amino acid sequences of other laccases from bacteria and fungi were obtained from GenBank. Phylogenetic analysis was performed using MEGA 7. The conserved binding domain of nucleotide sequence was compared by ESPript 3.

Enzyme characterization.

LacZ1 activity was assayed by UV spectrophotometry. The total reaction system was 800 μl, including 740 μl 0.1 M sodium acetate buffer added with 10 mM CuSO₄, 40 μl 20 mM ABTS aqueous solution, and 20 μl enzyme solution. Sodium acetate buffers with different pH values from 3 to 7 were used to determine the optimal pH of this enzyme. Experiments to test the effects of temperature (30°C to 85°C) on this enzyme were carried out in sodium acetate buffer (pH 4.5). Different metal ions were added to determine the promotion and inhibition of this enzyme. The thermostability of this enzyme was determined after incubation at different temperatures (65°C to 85°C) for 1 h. Illustrations of the above-described data were made by Origin software. The kinetic parameters of this enzyme were analyzed by changing the concentration of ABTS aqueous solution. K_m and V_max were calculated by GraphPad Prism 5 software.

Lignin degradation by enzyme.

One gram rice straw and 200 μl laccase solution were added into 20 ml sodium acetate buffer at pH 4.5 and 7.0 and then incubated in a 75°C water bath. Samples were taken at 0 min, 15 min, 30 min, 45 min, 1 h, 8 h, 16 h, and 24 h. The degraded rice straw was washed with ethyl acetate, and the lignin content in the rice straw was determined with a lignin content kit (QiYi, China). The determination principle of the lignin content kit is that the phenolic hydroxyl group in lignin has a characteristic absorption peak at 280 nm after acetylation, and the absorbance value is positively correlated with the lignin content. The standard curve of the kit is y = 0.02776x + 0.0068 (R² = 0.9889). A total of 0.2 g lignin and 200 μl laccase solution were added into 20 ml sodium acetate buffer at pH 4.5 and 7.0 and then incubated in a 75°C water bath. The same sampling times as mentioned above were used. The degraded small-molecule metabolites were extracted with ethyl acetate, and then the lignin solution was dried to constant weight. The degradation rate of lignin was determined by weight loss method. The treatment group without enzyme served as the control.

Analysis of lignin degradation by GPC.

A total of 0.2 g lignin and 200 μl laccase solution were added into 20 ml sodium acetate buffer (pH 4.5) and then incubated for 1 h in a 75°C water bath. The degraded sample was lyophilized by a freeze dryer. Samples taken at 0 min, 30 min, and 60 min were used for GPC (Waters; 1515 refractive index detector) determination. A 0.1-g sample was dissolved in 1 ml ddH₂O for detection. An Ultrahydrogel linear column (7.8 by 300 mm) was used at a flow rate of 1.0 ml/min. The standard was kit poly (ethylene glycol). The detector temperature was 40°C, and the column temperature was 40°C.

Analysis of lignin degradation by ¹H NMR.

The reaction conditions for lignin degradation by laccase were the same as for the GPC test. The small molecular metabolites were extracted with ethyl acetate, and the lignin in water phase was freeze-dried with a freeze dryer. A 10-mg sample was dissolved in 0.5 ml D₂O and put into a 5-mm nuclear magnetic tube for ¹H NMR analysis. Tetramethylsilane (TMS) was used as the external standard. The experimental temperature was 298 K, and the number of scans was 16. TopSpin 3.6.3 software was used to analyze the data.

Analysis of lignin degradation products by GC-MS.

The reaction conditions for lignin degradation by laccase were the same as for the GPC test. After 20 ml ethyl acetate was added to stop the reaction, the supernatant was taken out and concentrated by nitrogen blowing. The sample was dissolved in 200 μl ethyl acetate and then N,O-Bis(trimethylsilyl)trifluoro-acetamide was added for derivatization at 60°C for 30 min. Metabolite analysis was done by GC-MS (Agilent 7890 gas chromatograph equipped with a 5977 mass spectrometer). An HP-5 silica capillary column (30 m by 0.25 mm by 0.25 μm) was used with helium as the carrier gas at a flow rate of 1.1 ml/min. The oven temperature program was set at 100°C (holding time 2 min) initially, increased to 250°C at 10°C/min, and then increased to 280°C at 30°C/min (43). Degradation product data were analyzed by GC-MSD software.