Bacillus sp. GLN Laccase Characterization: Industry and Biotechnology Implications

Abstract

Laccases are multicopper oxidases that exhibit a broad substrate spectrum, making them excellent biocatalysts for clean technological processes. The study isolated novel laccase-producing bacteria from decomposed wood samples and characterized the enzyme for potential industrial and biotechnological applications. The results showed that three bacteria, SP-2, SP-1, and WP-2, out of the eight isolated bacteria, oxidized both guaiacol and α-naphthol in the plate assay and exhibited extracellular laccase activity of 7.0 ± 0.01, 6.67 ± 0.02, and 7.40 ± 0.04 (U/mL), respectively. WP-2 was selected for further study and was identified as Bacillus sp. GLN (accessioned number: MK290989). Strain GLN maximally secreted laccase 48 h post-fermentation, with an enzyme yield of 36.83 ± 2.47 U/mL in optimized conditions. The crude laccase was optimally active at pH 9.0 and 90 °C and showed excellent pH and thermal stability, retaining approximately 65% residual activity at 100 °C for 270 min. GLN laccase demonstrated remarkable stability after treatment with organic solvents and metal ions, retaining more than 50% of its original activity in the presence of 100 mM inhibitors. The data from this study highlight the relevance of Bacillus sp. GLN and its laccase in promoting clean technology.

1. Introduction

Microorganisms play crucial roles in the development of clean technologies across various sectors of the economy due to their versatility and metabolic diversity. They contribute to a sustainable environment by degrading pollutants into non-toxic or less toxic compounds [1,2]. Microbial technology’s flexibility makes it a pivotal element in sustainability efforts, addressing various challenges across different sectors [3]. Microorganisms mediate various bioprocesses through the synthesizing and secretion of enzymes and other metabolites. Microbial enzymes are preferred over plant and animal enzymes, because they are more stable, exhibit superior catalytic activity, and are easier to produce [4]. Among the diverse enzymes microbes produce, lignin-modifying enzymes, such as laccases, play significant roles in lignocellulosic biomass valorization and bio-remediation technology. Laccases are multicopper oxidases endowed with the catalytic potential to oxidize a broad spectrum of substrates, making them excellent biotools for detoxifying recalcitrant industrial pollutants. They act as either major players or ancillary agents in the biorefinery processing of lignocellulosic biomass into products, including fuels, materials, and chemicals, thereby fostering a circular economy [5,6].

Fungal laccases have been the subject of extensive study due to their high redox potential. Nevertheless, they face numerous limitations, including low enzyme production and ineffectiveness under extreme pH levels, temperatures, and high concentrations of salts, metals, or organic solvents, which impact their industrial prospects [7]. Fungi demonstrate a slow generation time and tend to accumulate high cell biomass, making their enzyme production and downstream processing tedious endeavors. On this ground, bacterial laccases are increasingly gaining significance due to their robust tendencies to address the shortcomings of fungal laccases. Bacterial laccase research is currently on an upward trajectory due to its excellent properties, which promote its candidacy in diverse bioprocess technologies, including textile dye degradation, biobleaching, pulp delignification, and biosensor development, among other applications [8,9]. Bacterial genetic manipulation is a straightforward technique that creates a robust bacterial system with high enzyme yield capability, offering great benefits over their fungal counterparts.

Laccase production is widespread among Gram-negative and Gram-positive bacterial strains. However, special attention has been given to the laccases from Bacillus spp. [10,11], Thermus spp. [12,13,14], and Geobacillus spp. [15,16,17,18] due to their robust biocompatibility and hyperstability profile under various process conditions. Thermal stable laccases are important in harsh industrial processing, as they maintain their catalytic conformation at high temperatures, while converting their substrates into products [8]. An enhanced enzymatic efficiency at elevated temperatures speeds up bioprocessing and minimizes the risk of microbial contamination, especially in the food and pharmaceutical industries [11]. Enhanced extracellular laccase productivity by wild bacterial strains is advantageous from an economic perspective compared to recombinant systems, which are capital-intensive to develop. Most thermophilic bacteria optimally secrete thermostable laccases in the production medium at high temperatures, partly due to the natural condition of the site of isolation. Therefore, exploring the ecological milieu of under-exploited temperate and subtropical zones could yield mesophilic bacterial strains with novel laccases, facilitating innovative and sustainable developments. Consequently, this study described the isolation of bacteria from decomposed wood biomass in a temperate climatic region of the Amathole Mountains in South Africa and assessed the bacteria for laccase production. The study further evaluated the best-performing bacterium identified as Bacillus sp. GLN for optimal enzyme production by constructing improved physicochemical conditions. The properties of the novel laccase, vis-à-vis pH and thermal activity and stability, organic solvents’ compatibility, inhibitors’ impact, and metal ions’ stability profile were determined. Bacillus sp. GLN produced hyperthermophilic and alkaline laccase under mesophilic conditions, and this result suggests that the bacterium and enzyme hold immense industrial relevance and commercial prospects.

2. Materials and Methods

2.1. Collection and Processing of Decaying Wood Samples

Wood samples were extracted from decaying wood trunks that littered the Forest Reserve in the Amathole Mountains. The samples were pulverized into smaller pieces using a laboratory ceramic mortar and pestle. The pulverized samples underwent pre-treatment utilizing an air-drying technique at ambient temperature for 48 h. The desiccated samples were transferred into sterile containers and kept at room temperature for subsequent analysis.

2.2. Bacterial Isolation and Purification

Ten grams (10 g) of the crushed wood samples were suspended in 90 mL of sterile distilled water in 250 mL Erlenmeyer flasks and mixed thoroughly. A volume of hundred microliters (100 µL) of 5-fold serially diluted pulverized sample suspension was spread plated on nutrient agar (Merck chemicals (Pty) Ltd., Modderfontein, Gauteng, South Africa) amended with nystatin (50 µg/L). The plates were subsequently transferred in an incubator (Scientific Engineering (Pty) Ltd., Avondale, Cape Town, South Africa) set at 37 °C. After 24 h of incubation, visible colonies were isolated based on their morphological characteristics and further purified by streaking on newly prepared plates until an axenic bacterial colony was obtained.

2.3. Initial Evaluation of the Bacterial Isolates for Laccase Activity

The initial screening of the isolates for potential laccase activity was carried out using the previously described method [11]. Axenic cultures of the respective isolates were grown in Luria Bertani (Neogen Culture Media, Lansing, MI, USA), adequately enriched with 1 mM copper sulphate (CuSO₄) and 3 g/L lignin (Sigma-Aldrich, St. Louis, MO, USA). The media flasks were incubated for 18 h at 30 °C under agitation (130 rpm). Following incubation, 5 µL of the broth was placed at the center of basal salt medium (BSM) plates. The plates contained 0.3 g/L K₂HPO₄, 0.4 g/L KH₂PO₄, 0.5 g/L MgSO₄·7H₂O, 0.2 g/L CaCl₂, 0.5 g/L (NH₄)₂SO₄, 4 g/L glucose, 0.1 g/L CuSO₄·5H₂O, and 15 g/L agar (Merck chemicals (Pty) Ltd., Modderfontein, Gauteng, South Africa). The media were either amended with guaiacol (0.02%; w/v) or α-naphthol (0.005%; w/v) (Sigma-Aldrich, St. Louis, MO, USA). The inoculated plates were incubated for 7 days at 30 °C. Bacterial strains that produced reddish-brown or deep purple coloration on the agar plates due to the oxidation of guaiacol or α-naphthol (Sigma-Aldrich, St. Louis, MO, USA) by the extracellularly produced laccase were chosen for subsequent investigations.

2.4. Quantitative Evaluation of the Bacterial Strains with Positive Laccase Activity

Strains that showed positive results for laccase activity in the initial evaluation study were further examined for their ability to produce extracellular laccase using the methodology described by Unuofin et al. [19]. The bacterial strains were cultivated for 18 h in LB broth at 30 °C. Subsequently, the cells were collected through centrifugation (HERMLE Labortechnik GmbH, Siemensstr, Wehingen, Germany), and the resulting pellets were cleansed further by sterile saline washing. The samples were then standardized to match the 0.5 McFarland standard by checking the optical density of the bacterial suspensions at 600 nm using a spectrophotometer (PerkinElmer, Singapore, Singapore). Under aseptic conditions, 2% (v/v) of the bacterial solution was transferred into 40 mL of BSM sterilized by autoclaving. The BSM comprised 3 g/L lignin, 0.3 g/L K₂HPO₄, 0.4 g/L KH₂PO₄, 0.5 g/L MgSO₄, 0.2 g/L CaCl₂, 4 g/L glucose, and 0.1 g/L CuSO₄·5H₂O. The flasks were subsequently incubated at 30 °C under agitation (130 rpm) for 72 h. The crude extract was obtained by centrifuging the fermentation culture for 10 min at 15,000 rpm, and laccase activity was determined under standard assay conditions.

2.5. Determination of Laccase Activity and Protein Content

The oxidation of 2,2′-amino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) (Sigma-Aldrich, St. Louis, MO, USA) catalyzed by laccase was observed by spectrophotometry, following an established protocol [20]. In summary, ABTS solution (2 mM) was prepared in potassium phosphate buffer with pH (6.0) and strength (100 mM). A total of 50 µL of ABTS was transferred into a reaction vial containing 50 µL of the extract. The ABTS–enzyme mixture was left to stand for 10 min at 30 °C. The enzymatic reaction was halted using trichloroacetic acid (Merck (Pty) Ltd., Modderfontein, Gauteng, South Africa) (30 µL) at a concentration of 20% (v/v). A Synergy MX microtiter plate reader (BioTek Instrument Inc., Winooski, VT, USA) was used to measure substrate oxidation at 420 nm, and laccase activity was computed by applying the molar extinction coefficient (ε = 36,000 M⁻¹cm⁻¹). A control assay was conducted under identical conditions, using a heat-treated extract instead. Laccase activity was defined as one unit (U) when 1 µmol of ABTS was oxidized per minute under specified assay protocols. The protein concentration was determined using the standard Bradford assay [21].

2.6. Identification of the Potent Laccase Producer

A light microscope (Motic^®, Kowloon, Hong Kong) was utilized to examine the isolate’s characteristics. The Fungal/Bacterial deoxyribonucleic acid (DNA) kit™ (Zymo Research, Irvine, CA, USA) was employed for genomic DNA extraction. The bacterial strain’s target region (16S) was amplified through a polymerase chain reaction using working conditions and a pair of primers reported elsewhere [11]. The amplicons underwent gel extraction using Zymo Research, Zymoclean™ Gel DNA Recovery Kit (Irvine, CA, USA) and purification with Zymo Research, ZR-96 DNA sequencing clean-up kit™ (Irvine, CA, USA) before bidirectional sequencing on ABI 3500xl Genetic Analyzer (Thermo Fisher Scientific, Waltham, MA, USA). Geneious v2020.1.1 was used to analyze the sequencing products, followed by a GenBank basic local alignment search tool to compare and extract related sequences. The molecular evolutionary genetics analysis program v12 [22] was utilized to conduct a phylogenetic analysis. The isolate was identified as Bacillus sp. GLN and accessioned MK290989.

2.7. Construction of Optimal Physicochemical Variables

To the cultivation media’s physical variables and chemical components, we used the classical method to study the effects of various factors. The impact of different initial pH conditions on the synthesis and secretion of laccase was examined over a range of 3.0 to 10.0, with a 1-unit increment. Various lignocellulosic agricultural byproducts, including sawdust, mandarin peels, corn stover, and pine bark, were tested at 0.5 g/L to assess their stimulatory influence on extracellular laccase production by the isolate. The agricultural waste yielding the best stimulatory effect was further varied (0 to 0.8 g/L) to ascertain the optimal concentration. Other inducers, including ferulic acid, benzoic acid, vanillic acid, and guaiacol (Sigma-Aldrich, St. Louis, MO, USA), were also assessed at 0.04% (v/v). The influence of nitrogen sources, including ammonium sulfate, sodium nitrate, yeast extract, and urea (Merck (Pty) Ltd., Modderfontein, Gauteng, South Africa), as well as carbon sources, such as sucrose, glucose, lactose, and fructose (Merck (Pty) Ltd., Modderfontein, Gauteng, South Africa), was investigated at a concentration of 0.5 g/L. The most effective nitrogen and carbon sources were further examined at concentrations ranging from 0 to 2 g/L in increments of 0.5 g/L. Under optimized conditions, the time course of laccase production was evaluated over 168 h, with samples collected every 12 h to determine the medium laccase titer and protein concentration.

2.8. Characterization of the Produced Laccase

2.8.1. Determination of pH Optimal and Stability Profile of the Laccase

Laccase activity was evaluated in various buffers ranging from pH 2.0 to 10.0, with a strength of 100 mM. Different buffer solutions employed were glycine–HCl (pH 2.0–3.0), sodium acetate (pH 4.0–5.0), potassium phosphate (6.0–7.0), Tris–HCl (8.0–9.0), and sodium carbonate–bicarbonate (pH 10.0), as reported elsewhere [23]. The salts and acid used to prepare the buffers were purchased from Merck chemicals (Pty) Ltd. (Modderfontein, Gauteng, South Africa). For the pH stability study carried out over 180 min, pH values were selected based on the relative enzyme activity observed under various conditions, and they included 2.0, 5.0, 8.0, 9.0, and 10.0. The bacterial laccase was mixed with the corresponding buffer and incubated at 30 °C. Samples were collected at regular 30 min intervals to assess the remaining laccase activity. The standard enzyme activity determined before incubation was taken as 100%.

2.8.2. Determination of Temperature Optimal and Stability Profile of the Laccase

The enzyme activity assay was conducted over a temperature range of 30 °C to 100 °C, with measurements taken at 10 °C intervals. The enzyme’s heat resistance was then evaluated by exposing the enzyme solution to temperatures of 70, 80, 90, and 100 °C for 270 min. Samples were collected every 30 min to determine the remaining laccase activity. The laccase activity determined before pre-heating was taken as 100%.

2.8.3. The Impact of Organic Solvents and Inhibitors on Laccase Stability

The study evaluated the laccase’s tolerance to organic solvents by exposing the bacterial laccase to 10% or 20% (v/v) concentrations of organic solvents, including ethanol, methanol, propanol, and acetone (Merck chemicals (Pty) Ltd., Modderfontein, Gauteng, South Africa) at various incubation times of 1 h, 8 h, and 16 h. Additionally, the research also investigated the impact of multiple inhibitors (sodium dodecyl sulphate (SDS), ethylenediaminetetraacetic acid (EDTA), sodium azide (NaN₃), urea) (Merck (Pty) Ltd., Modderfontein, Gauteng, South Africa) on the stability of the bacterial laccase for 30 min at a concentration of 100 mM. For both experiments, the remaining enzyme activity was measured following the assay protocols stated before, and the assay without organic solvent or inhibitor was taken as 100%.

2.8.4. Determination of Metal Ions’ Influence on the Laccase Stability

Laccase stability was assessed in the presence of various metal ions, including CaCl₂, MgCl₂, FeCl₂, ZnSO₄, CuSO₄, NiCl₂, MnSO₄, and KCl (Merck (Pty) Ltd., Modderfontein, Gauteng, South Africa) at 1 mM. The remaining laccase activity was measured after the enzyme–metal ion mixture was incubated for 30 min.

2.9. Data Analysis

The experimental data generated in triplicates were analyzed using Statistical Package for Social Sciences (SPSS) version 23, with a statistically significant difference established at p < 0.05.

3. Results

3.1. Bacteria Isolation, Laccase Production, and Identity Confirmation

Bacterial strains with distinct morphological characteristics were selected from the cultivation plates. The isolated strains coded as SP-2, WP-3, WP-1, WP-4, SP-1, WP-2, WP-5, and SP-3 were qualitatively examined for potential laccase production (

Table 1. The qualitative evaluation of laccase production by decaying wood-associated isolates.

S/N	Isolate	Guaiacol	α-Naphthol
1.	SP-2	+	+
2.	WP-3	+	−
3.	WP-1	+	−
4.	WP-4	+	−
5.	SP-1	+	+
6.	WP-2	+	+
7.	WP-5	−	−
8.	SP-3	−	−

and Figure S1). Six (SP-2, WP-3, WP-1, WP-4, SP-1, WP-2) out of the eight isolates exhibited reddish-brown color zones on guaiacol amended plates. Three isolates (SP-2, SP-1, WP-2) showed a deep purple color on α-naphthol supplemented media due to oxidation by the secreted extracellular laccase. Additionally, three (SP-2, SP-1, WP-2) bacterial isolates oxidized both substrates by showing reddish-brown and deep purple color on guaiacol and α-naphthol plates, respectively. However, WP-5 and SP-3 could neither oxidize guaiacol nor α-naphthol on the plate assay. The quantitative assessment of the three isolates SP-2, SP-1, and WP-2 that oxidized both substrates on the plate assay showed respective extracellular laccase activity of 7.0 ± 0.01, 6.67 ± 0.02, and 7.40 ± 0.04 (U/mL), as shown in Figure 1. Considering the potent laccase activity shown by WP-2, it was designated for further analysis.

The microscopic characterization indicated that WP-2 is a bacilli (Figure 2A). Analysis of the 16S rRNA gene nucleotide sequence showed that WP-2 displayed high sequence homology (99%) with the Bacillus cereus sensu lato group (Figure 2B). Since it did not show complete discrimination among the species of this group, it was reasonably identified as Bacillus sp. and further assigned a unique strain name GLN. The nucleotide sequence of Bacillus sp. GLN was deposited in the nucleotide collection database with the accession number MK290989.

3.2. Process Variables Optimization for Enhanced Laccase Activity

The study showed that strain GLN produced laccase at a broad spectrum of pH, covering both the acid, neutral, and alkaline conditions with a maximum laccase activity of 8.79 ± 0.08 U/mL at pH 5.0 (Figure 3A). The inducers showed a remarkable effect on the extracellular laccase production by strain GLN compared to the control (6.8 ± 0.04 U/mL), with ferulic acid and guaiacol displaying the best enzyme induction with laccase activity of 15.39 ± 0.5 U/mL and 15.04 ± 1.14 U/mL, respectively (Figure 3B). The nitrogen sources added in the cultivation medium enhanced the laccase production compared to the control. The urea-supplemented medium showed the best enzyme activity (11.34 ± 0.24 U/mL), as shown in Figure 3C. Varying the amount of urea added in the fermentation showed that the extracellular laccase secretion by strain GLN was concentration-dependent, showing 10.37 ± 0.03 U/mL, 12.1 ± 0.038 U/mL, 13.29 ± 0.42 U/mL, and 16.79 ± 0.42 U/mL at 0.5 g/L, 1.0 g/L, 1.5 g/L, and 2.0 g/L, respectively (Figure 3D).

The assessment of conventional carbon sources’ effect on laccase production by strain GLN showed that lactose yielded the best result (14.35 ± 0.6 U/mL), followed by sucrose (12.29 ± 0.36 U/mL), glucose (12.21 U/mL), and fructose (9.11 ± 0.08 U/mL), while the lowest enzyme production was observed with the control experiment (7.66 ± 0.3 U/mL), as shown in Figure 4A. The study of laccase production at varying lactose concentrations indicated that 0.5 g/L exerted the best enhancement effect with an enzyme activity of 19.84 ± 0.09 U/mL (Figure 4B). Agro-waste biomass was also employed for formulating fermentation medium. The results showed that sawdust optimally influenced extracellular laccase production by strain GLN with enzyme activity of 14.84 ± 0.24 U/mL (Figure 4C). Additionally, pine bark, corn stover, and mandarin peel promoted the laccase production more than the control (6.28 ± 0.05 U/mL), showing enzyme activity of 12.12 ± 0.3, 10.57 ± 0.21, and 7.0 ± 0.33 (U/mL). The assessment of the impact of different concentrations of sawdust on laccase secretion by strain GLN revealed that 0.2 g/L improved the enzyme production, recording laccase activity of 23.87 ± 0.28 U/mL (Figure 4D). However, further increase in the medium concentration of sawdust caused a subsequent decline in the laccase yield, with enzyme activity of 21.33 ± 1.0, 18.91 ± 0.15, and 16.0 ± 0.98 (U/mL) observed at 0.4 g/L, 0.6 g/L, and 0.8 g/L, respectively.

3.3. Time-Dependent Profiling of Laccase Production by Bacillus sp. GLN

Laccase production in an optimized medium was monitored for 168 h, and the results indicated that Bacillus sp. GLN had enzyme production (10.31 ± 0.02 U/mL) after 12 h of cultivation. Strain GLN maximally secreted laccase 48 h post-fermentation, with enzyme yield of 36.83 ± 2.47 U/mL (Figure 5), which was a 5-fold increment compared to the initial enzyme activity (7.40 ± 0.04 U/mL) of the isolate before the optimization study. After 48 h, extracellular laccase concentration drastically decreased with further incubation. The maximum protein concentration of 1.55 ± 0.02 mg/mL was obtained in the cell-free extract after 72 h of fermentation (Figure 5).

3.4. pH Optimal and Stability Profile of Strain GLN Laccase

The crude laccase from Bacillus sp. GLN showed excellent enzyme activity at a broad pH spectrum with an optimum of 9.0 (Figure 6A). Generally, the enzyme displayed more than 70% activity relative to the optimum at all the pHs tested. The stability profile demonstrated that strain GLN laccase retained remarkable activity both in acid and alkaline conditions (Figure 6B). After 180 min of preincubation, the enzyme maintained residual activity of 77.32 ± 3.4%, 78.57 ± 8.87%, 91.69 ± 6.6%, 71.85 ± 2.17%, and 88.92 ± 2.16% at pHs 2.0, 5.0, 8.0, 9.0, and 10.0.

3.5. Temperature Optimal and Stability Profile of Strain GLN Laccase

The temperature evaluation showed that the catalytic property of the GLN laccase increased with increasing assay temperature and became optimum at 90 °C (Figure 7A). The crude laccase displayed robust catalysis at 100 °C, with 90% enzyme activity relative to the optimum. The temperature stability profiling showed that the GLN laccase was thermostable (Figure 7B). The thermostability evaluation also demonstrated that this enzyme was heat-activated, as it displayed unique activity peaks of 164.12 ± 7.47% at 210 min for 70 °C, 162.34 ± 0.75% at 180 min for 80 °C, 166.71 ± 7.49% at 120 min for 90 °C, and 148.49 ± 2.71% at 90 min for 100 °C. After 270 min of pre-heating, the enzyme retained 122.58 ± 4.97%, 100.4 ± 2.85%, 95.12 ± 3.09%, and 65.23 ± 4.36% of the original activity at 70 °C, 80 °C, 90 °C, and 100 °C, respectively (Figure 7B).

3.6. Influence of Organic Solvents, Inhibitors, and Metal Ions on Laccase Stability

The organic solvent stability of GLN laccase was studied using acetone, ethanol, methanol, and propanol at two concentrations for 1 h, 8 h, and 16 h; the results showed that the GLN laccase maintained at least 80%, 78%, and 82%, respectively, of its original activity in the various solvents (

Table 2. Influence of organic solvents on GLN laccase stability.

Organic Solvent	Concentration (%)	Residual Activity (%) a
		1 h	8 h	16 h
Control	-	100 ± 1.01	100 ± 1.32	100 ± 1.08
Acetone	10	80.18 ± 0.73	84.41 ± 0.09	92.53 ± 6.06
	20	90.77 ± 4.14	81.42 ± 1.01	86.61 ± 0.83
Ethanol	10	84.99 ± 1.1	78.95 ± 1.19	82.0 ± 1.29
	20	91.94 ± 0.46	88.56 ± 2.11	82.26 ± 1.10
Methanol	10	86.35 ± 1.01	89.67 ± 4.59	84.47 ± 0
	20	81.81 ± 1.56	87.52 ± 3.77	93.11 ± 2.66
Propanol	10	89.21 ± 3.58	84.47 ± 0	82.33 ± 2.30
	20	86.55 ± 1.84	88.17 ± 3.03	86.29 ± 5.70

^a The results were presented as mean values of three experimental analyses.

). The results further demonstrated, in some cases, that the enzyme regained stability after prolonged preincubation with the solvents. For example, acetone treatment at 10% showed 80.18 ± 0.73% after 1 h, 84.41 ± 0.09% after 8 h, and 92.53 ± 6.06% after 16 h. At the same time, methanol at 20% displayed a similar pattern of stability with 81.81 ± 1.56% at 1 h, 87.52 ± 3.77% at 8 h, and 93.11 ± 2.66% at 16 h (Table 2).

The enzyme stability was adversely affected after treatment with potential inhibitors at a higher concentration of 100 mM (

Table 3. Influence of enzyme inhibitors on GLN laccase stability.

Chemical Agents	Concentration (mM)	Residual Activity (%) a
Control	-	100 ± 0.76
SDS	100	53.67 ± 0.13
EDTA	100	58.33 ± 0.51
NaN3	100	65.14 ± 1.26
Urea	100	63.08 ± 3.17
NaCl	100	63.98 ± 0

^a The results were presented as mean values of three experimental analyses.

). GLN laccase retained 53.67 ± 0.13%, 58.33 ± 0.51%, 65.14 ± 1.26%, 63.08 ± 3.17, and 63.98 ± 0% of the original activity against SDS, EDTA, NaN₃, urea, and NaCl, respectively.

The influence of metal ions on the laccase’s catalytic property was assessed, and the results indicated that the enzyme maintained high residual activity (>85%), with CuSO₄ slightly enhancing the enzyme activity (110.68 ± 2.17%) compared to the control after 30 min of incubation (

Table 4. Metal ions’ influence on the GLN laccase stability.

Metal Ions	Concentration (mM)	Residual Activity (%) a
Control	-	100 ± 2.65
CaCl2	1	86.37 ± 3.337
MgCl2	1	96.42 ± 6.34
FeCl2	1	104.32 ± 1.53
MnSO4	1	96.76 ± 0.32
ZnSO4	1	92.67 ± 0.56
CuSO4	1	110.68 ± 2.17
NiCl	1	93.87 ± 0.64
KCl	1	93.70 ± 6.02

^a The results were presented as mean values of three experimental analyses.

).

4. Discussion

Bacterial laccases have shown a wealth of potential in sustainable development owing to their versatility. These enzymes demonstrated enhanced biocatalytic tendencies towards a broad range of substrates, high thermal stability, salt tolerance, optimal functionality at various pH conditions, organic solvent, and surfactant compatibility [24,25]. Due to the possible influence of environmental factors on the metabolic diversity of the microbial community, researchers have explored various habitats, including but not limited to wastewater samples [19], paper mills [26], decomposed wood samples [27,28], sawdust, dairy effluent soil samples [29], and textile industry effluent samples [30] for the isolation of bacteria with high laccase activities. This study used decaying wood biomass to isolate bacteria capable of secreting laccase in the extracellular medium. The exo-production of enzymes is advantageous economically, as it subsidizes downstream processing, while enhancing copious enzyme recovery. The qualitative assay has been considered relevant in evaluating multiple bacterial strains, as it identifies potential isolates and minimizes time and resource wastage. Studies on the laccase production potential of bacterial isolates have employed one substrate [30] or multiple substrates [27] when evaluating extracellular laccase activity using plate assay. The ability of the bacterial strains to oxidize more than one substrate during the qualitative screening is evidence of their broad substrate specificity and multifaceted application potential. Different substrates have been employed for the assessment of extracellular laccase production on solid media, and they include guaiacol, 1-naphthol, 2, 6-dimethoxy phenol, ABTS, syringaldazine, lignin, ferbamine, and tannic acid, among others [19,31,32,33]. The substrate oxidation pattern on the plate assay by the bacterial strains under investigation suggests their metabolic diversity. The extracellular laccase production by the bacterial strains that oxidized both guaiacol and α-naphthol on solid media further demonstrated the isolates’ peculiarity and their distinct enzyme machinery. WP-2 showed a robust expression system by displaying copious laccase titer in a submerged state fermentation.

The isolate WP-2 was identified as Bacillus sp. GLN based on its high percentage of sequence similarity with the B. cereus sensu lato group. This group of bacteria has been reported to possess numerous mobile genetic elements, which enhance their adaptation and versatility [34]. Consequently, this group has been implicated in numerous ecological functions due to their diverse metabolic capacities, contributing to environmental sustainability [35]. The significance of this group cannot be over-emphasized, as the members produce diverse and robust enzymes with promising candidacy in various sectors of the economy. However, some members of the B. cereus group have also been reported to possess toxin-producing potential, posing both safety concerns and high risks in food processing and other production avenues [36]. Therefore, it is imperative to determine the generally recognized as safe (GRAS) status of the present isolate in the future study. This approach will determine if the wild isolate can either be improved through molecular optimization to produce the required enzyme or if recombinant DNA technology can harness the laccase production potential.

Bacterial growth and enzyme production are markedly controlled by environmental conditions, suggesting that pH influences metabolic activities. The laccase production at a broad pH range by Bacillus sp. GLN is attractive from an industrial perspective as most bioprocesses suffer intermittent pH fluctuations due to the production/generation of variable compounds that overwhelm the standard buffering systems. Similar to the present finding, pH optimum at weakly acidic conditions of 5.0 has been reported to positively influence laccase production by Bacillus sp. NU2 [37] and Bacillus subtilis [33]. However, laccases production by Bacillus sp. BAB-4152 was negatively impacted at pH 5.0 but performed optimally at pH 8.0 [38]. Other credible reports have also demonstrated that some Bacillus spp. optimally produced extracellular laccases within neutral conditions [39,40].

Many factors, including the inductive effect of aromatic or phenolic compounds, strain preference, and metabolic capacities of the bacterial cells, greatly influence laccase production. Bacillus sp. GLN displayed optimal laccase potential in the presence of ferulic acid and guaiacol. This finding indicated that the presence of these phenolic compounds upregulated the expression of laccase-encoding genes in strain GLN. Extracellular laccase production has been favorably promoted when the fermentation medium was adequately supplemented with ferulic acid [41,42] or guaiacol [39,43].

Supplementation of the production media with a nitrogen source is essential in bacterial growth and active laccase expression for lignolytic biomass modification [44]. The level of the influence of varying concentrations of nitrogen sources on bacterial laccase production has been attributed to the strain’s peculiarity. Consequently, laccase secretion by different Bacillus spp. was optimally supported by 3% peptone for B. subtilis MTCC [45], 1% peptone for Bacillus sp. AKRC01 [46], 0.1% tryptone for Bacillus sp. [39], 0.1% yeast extract for Bacillus amyloliquefaciens [30] (El-Bendary et al., 2020), and 0.5% peptone for Bacillus aquimaris AKRC02 [47]. Meanwhile, laccase synthesis by another Bacillus sp. strain was positively impacted by urea [48]. The amendment of the production medium with two nitrogen sources, yeast extract (0.15%) coupling tryptone (0.15%) and yeast extract (0.4%) coupling tryptone (0.2%), was reported to promote laccase production by B. subtilis WPI [31] and Bacillus marisflavi BB4 [49], respectively. However, the utilization of multiple nitrogen sources in the production system is unsustainable from an economic perspective.

Similarly, carbon sources are essential for energy provision to facilitate microbial growth and metabolism. For this reason, optimizing the choice and concentration of carbon source for laccase production by Bacillus sp. GLN was crucial to enhance its performance. The tested carbon sources favored laccase synthesis, with lactose at 0.5 g/L, causing the maximum enzyme yield from strain GLN. Likewise, another report indicated that lactose enhanced extracellular laccase synthesis from Bacillus sp. [48]. Furthermore, laccase production by Bacillus spp. has been promoted in the presence of other carbon source variants, including 3% sucrose [45], 1% dextrose [39], 10% glucose [18], and 0.2% galactose [49].

Agro-residue utilization in fermentation systems presents the dual benefits of providing required nutrients for enzyme expression by microorganisms, while ensuring proper repurposing of the increasingly generated lignocellulosic biomass, which minimizes ecological degradation. The various biomass tested enhanced the laccase production by Bacillus sp. GLN compared to the control. Enzyme production using non-food agro-waste represents sustainable development and is attractive from an industrial point of view. GLN showed maximum laccase production with sawdust. However, the excellent performance of the isolate in the presence of sawdust was concentration-dependent, and the subsequent decline in laccase production observed at increased sawdust concentrations could be attributed to the low dissolved oxygen content and high viscous nature of the production medium. Reports have shown that Bacillus spp. responded differently to various agro-residues supplemented with the laccase production medium. Consequently, B. subtilis MTCC 2414 [45], B. aquimaris AKRC02 [28], Bacillus sp. PS [40], Bacillus sp. PK4 [50], and Bacillus sp. NU2 [51] achieved optimal laccase yield in the presence of wheat bran, rice bran, millet husks, groundnut shells, and tangerine peel, respectively.

Time course profiling indicated that laccase production by strain GLN became optimum at 48 h. This short timeline for the production peak is advantageous from industrial and biotechnological viewpoints, suggesting GLN’s commercial relevance. Identically, laccase production by B. cereus TY10 peaked at 48 h, with an extracellular enzyme yield of 1.61 U/mL [52]. In contrast, Bacillus aestuarii KSK [53], Bacillus sp. BAB-4151 [38], and B. cereus AKRC03 [54] showed maximum laccase production 60 h, 72 h, and 120 h post-incubation, respectively.

Strain GLN laccase showed remarkable catalysis at a broad range of pH spectrum with optimum at 9.0, and this attribute underpins its bioprocess application potential. Likewise, laccase from B. subtilis MTCC 2414 [45] and Bacillus sp. FM-86 [55] displayed excellent catalytic activity at pH 9.0. The stability study further substantiates the relevance of GLN laccase in various sustainable technologies that operate at conditions with unavoidable pH fluctuations. Bacterial laccases with a broad pH stability spectrum have been deemed indispensable assets in the green remediation of various industrial effluents. Tannery and textile effluents are rich in many compounds, with pH at the alkaline end of the spectrum, which suggests the future candidacy of alkalophilic laccase under investigation for the dephenolization of the principal organic and synthetic pollutants in various effluent samples. Conformational changes and structural adaptations have been reported as key mechanisms by which laccase from Bacillus pumilus ZB1 effectively mediated dye decolorization in alkaline conditions [56].

Even though fungal laccases have been extensively studied [57,58], the robust tendencies of bacterial laccases underscore their growing exploration for application in diverse industrial processes. GLN laccase has shown to be a hyperthermophilic enzyme by displaying optimal catalysis at 90 °C and retaining remarkable activity at 100 °C. Its optimum temperature for a biocatalytic function is likened to that recently reported for the laccase from Geobacillus stearothermophilus MB600 [18]. Bacillus tequilensis SN4 laccase also displayed optimal activity at 85 °C and could retain 50% of the enzyme activity at 100 °C [10]. Despite the fact that the South African region, Amathole Mountain Forest, where the sample used to isolate strain GLN was collected, experiences varying climates of subtropical and temperate conditions, with annual temperatures from 15 to 36 °C in summer and −2 to 26 °C in the winter, the isolate produced hyperthermophilic laccase at mesophilic conditions. Researchers have reported the expression of thermophilic laccase by the corresponding thermophilic bacteria from hot springs and the Antarctic, most of which belong to Geobacillus spp. [16,17,18]. The comparison of the biochemical properties of GLN laccase and other bacterial laccases are presented in

Table 5. Comparison of the biochemical characteristics of GLN laccase with other reported bacterial laccases.

Bacterial Source	Optimal Prod. Time (h)	Assay Substrate	Optimal pH	Optimal Temp. (°C)	Half-Life of GLN Laccase	Activity Enhancing Metal	References
Bacillus sp. GLN	48	ABTS	9	90	>270 min at 100 °C	Cu2+	This study
Geobacillus stearothermophilus MB600	72	Guaiacol	5	90	-	Cu2+, Ca2+, Cd2+, Li+	[18]
Bacillus tequilensis SN4	96	ABTS	5.5	85	1 h at 55 °C	Cu2+, Co2+	[10]
Lysinibacillus fusiformis	-	DMP	10.4	70	15.9 h at 60 °C	Cu2+	[59]
Bacillus sp. PC-3	36	ABTS	7	60	3.75 h at 60 °C	-	[11]
Bacillus subtilis	-	ABTS	3	75	2 h at 80 °C	-	[60]
Geobacillus thermocatenulatus MS5	96	ABTS	4–5	55–60	-	-	[61]
Geobacillus yumthangensis	-	ABTS	5	60	-	Cu2+	[16]
Geobacillus sp. ID17		Syringaldazine	7	55	30 min at 60 °C	Cu2+	[17]
Bacillus sp. FNT	-	Syringaldazine	6	70	>2 h at 60 °C	-	[62]
Catenuloplanes japonicus	-	DMP	9.2	70	60 min at 90 °C	-	[63]
Anoxybacillus ayderensis SK3-4	-	Syringaldazine	7	75	155 min at 65 °C	Cu2+, Mg2+	[64]
Thermobifida fusca	36	DMP	8	60	>3 h at 50 °C	-	[65]

Prod. = production; Temp. = temperature; DMP = 2,6-Dimethoxyphenol.

. The thermostability profile of GLN laccase indicated that the enzyme activity was thermally enhanced, and it retained more than the half-life after 100 °C heat treatment for 270 min. The production of hyperthermal–active–stable laccase by mesophilic Bacillus sp. GLN suggests a cost-effective process and, therefore, highlights the potential industrial significance of this isolate.

The solvent exposure of the GLN laccase at different concentrations indicated that the enzyme was sufficiently stable, irrespective of the treatment period and solvent concentration. Bacillus sp. GZB laccase demonstrated similar enzyme stability against 10% ethanol, acetone, and methanol but was structurally destabilized at 20%, as it retained 52.26%, 37.22%, and 62.54%, respectively, of the original activity [66]. Contrariwise, laccase from Bacillus sp. FM-78 showed lower residual activity in the presence of acetone, dimethylsulfoxide, ethanol, and methanol, recording between 53.42% and 73.21% at 10% (v/v) and 10.72% and 40.36% at 50% (v/v) solvent [55]. Additionally, the laccase under investigation showed more stability in 20% ethanol and methanol than Proteus vulgaris ATCC 6896 laccase, which has been promoted for various application potential [67]. The data from our study indicate that GLN laccase could be employed to detoxify and decolorize various industrial wastewater with significant doses of chemical solvents.

The stability profile showed that the potential enzyme inhibitors caused a decrease in enzyme catalytic efficiency, retaining between 53% and 65% of the original activity, though at elevated concentrations (100 mM) of the various chemical reagents tested. Our finding is superior to the report by Sharma and colleagues, where laccase from B. marisflavi BB4 retained 48% of the original after treatment with a small SDS concentration (0.2 mM) [49]. Likewise, Bacillus sp. FM-78 laccase suffered a strong inhibition ranging from 46% to 59% after pre-incubation with 1 mmol/L of EDTA, NaN₃, and SDS [55]. In addition, B. cereus UV25 laccase was significantly inhibited when EDTA, sodium azide, and SDS were introduced in the reaction mixture, which resulted in residual activity of 11.77%, 24.76%, and 17.28%, respectively [68]. Furthermore, the loss of biocatalytic function by 96% was reported after Bacillus sp. laccase treatment with 0.5 mM sodium azide [69]. The ability of the study laccase to retain >50% of the original activity after the treatment with a high concentration of potential inhibitors is noteworthy and suggests its commercial relevance.

The GLN laccase showed an excellent stability profile in the presence of the various metal ions examined. Biocatalytic enhancement was demonstrated in the presence of Cu²⁺, suggesting the role of this ion in the maintenance of active catalytic orientation of the laccase during enzyme reaction. A study on the formulation of functionalized copper oxide-laccase nanoparticles demonstrated that the nanoreactor exhibited excellent catalysis with a high turnover number compared to the free laccase [70]. Further, a probe into the cause via mass spectrometry showed that copper ions were lost during the reaction cycle of the free laccase. At the same time, the CuO nanoparticles acted as a source of copper ions for the laccase nanoreactor, thereby sustaining the enzyme structure and catalytic performance [70]. This report further sheds light on the performance enhancement demonstrated by laccases in the presence of Cu²⁺, as documented elsewhere [7,10,71]. The GLN laccase stability pattern against the tested metal ions is superior to the report of Sharma and co-workers on laccase from B. marisflavi BB4 [49]. Conversely, laccase from B. cereus UV25 was strongly inhibited by Fe²⁺ (28.16%), K⁺ (30.60%), and Zn²⁺ (53.49%) compared to GLN laccase, while Ca²⁺, Cu²⁺, Mg²⁺, and Mn²⁺ greatly promoted the enzyme activity [68]. The stability of GLN laccase in the presence of tested metal ions underpinned its potential application in industrial effluent treatment.

5. Conclusions

A decaying wood-associated bacterial strain identified as Bacillus sp. GLN copiously secreted active laccase after 48 h of fermentation under optimized process conditions. The enzyme exhibited optimal activity at pH 9.0 and 90 °C, with excellent pH and thermal stability. The activity and stability of the enzyme across a broad range of pH values, as well as its hyperthermophilicity, are noteworthy and warrant further investigation. The examination of the GLN laccase stability against organic solvents, metal ions, and potential inhibitors showed that the enzyme possesses properties that endeared it to possible commercial application in clean and sustainable developments. This study characterized the crude laccase from the bacterial isolate without further purification, as the purification of the enzyme contributes to the high production cost. However, crude enzyme analysis limits the evaluation of some important parameters that are paramount for assessing the application potential of the biocatalyst. Therefore, future studies will include determining the GRAS status of the strain GLN-upscaled production of laccase. Also, the detailed biochemical characterization of the enzyme, including purification study, kinetic parameters determination, zymogram analysis, and comparative study, will be implemented. In addition, GLN laccase’s capacity to degrade various industrially generated pollutants should also be investigated for suitable future applications.