Laccases (benzenediol: oxygen oxidoreductases, EC 1.10.3.2) are copper-containing oxidoreductases that can oxidize a wide range of phenolic and nonphenolic substrates, including ortho, meta, and para diphenols, specifically lignin, a lignocellulose component, and is employed as an attractive tool for the pre-treatment of biomass and its valorization. They are versatile oxidative biocatalysts that contain copper atoms in their active site and oxidize diverse substrates using only molecular oxygen as the known co-substrate instead of hydrogen peroxide, as in peroxidases (Agrawal et al., 2018; Agrawal and Verma, 2020). As green biocatalysts, laccases have been exploited for a potential application in broad biotechnological areas, viz., environmental remediation processes, biosensor design, synthesis of fine chemicals, food, cosmetic, and pharmaceutical industries, and synthetic dye decolorization capacity to detoxify a range of noxious and recalcitrant environmental pollutants (Yang et al., 2017; Becker and Wittmann, 2019; Zerva et al., 2019). However, most currently known laccases are difficult to overproduce in heterologous hosts (Kim et al., 2010). Abundant and inexpensive laccases are needed if the microbiological treatments are to compete with chemical treatments in industries to which they appeal.

Identification and engineering for more efficient and tolerant laccases for industrial applications are an ongoing effort to date. Laccase genes dwell in numerous biologically important taxa including plants, insects, lichen, bacteria, and fungi, with the Basidiomycetes class of fungi being the most important source (Hoegger et al., 2006; Arregui et al., 2019). It has been reported that microbial laccases are primarily involved in wood decay, lignin decomposition, and detoxification and linked to resistance to different environmental stresses (Arregui et al., 2019; Janusz et al., 2020). Fungal laccases have been studied in a variety of biotechnological applications due to their high redox potential (Abdel-Hamid et al., 2013; Upadhyay et al., 2016). However, their application is usually hampered due to the long fermentation periods, acidic pH optima, intolerance to extreme conditions, and difficulty in overproducing in heterologous hosts (Baldrian, 2006; Kim et al., 2010; Du et al., 2015). Meanwhile, small laccases, both three- and two-domain laccases, from bacterial sources have gained attention recently, due to their exceptional attributes such as ability to withstand wide temperature and pH ranges, ease in genetic manipulation, and tremendous stability even when inhibitory agents are present (Mukhopadhyay et al., 2013; Chauhan et al., 2017; Arregui et al., 2019; Gianolini et al., 2020; Sharma and Leung, 2021). In addition, a short generation time of bacteria makes it easier to scale up laccase production processes on a commercial scale (Brugnari et al., 2021; Akram et al., 2022). The major bacterial genera that have been reported to produce laccase-like multicopper oxidases include Streptomyces, Bacillus, Meiothermus, Gramella, Geobacillus, Aquisalibacillus, Lysinibacillus, Azospirillum, Rhodococcus, Ochrobactrum, Amycolatopsis, Pseudomonas, Stenotrophomonas, Iodidimonas, Alteromonas, and Nitrosomonas (Chauhan et al., 2017; Granja-Travez et al., 2018; Arregui et al., 2019; Jeon and Park, 2020; Yang et al., 2021).

Bacterial species under the genus Serratia, belonging to the family Enterobacteriaceae, has been identified for its numerous applications in biodegradation (Majumdar et al., 2020; An et al., 2021; Dabrowska et al., 2021). Serratia proteamaculans, a gram-negative, non-pigmented strain under this genus, was first reported to cause a leaf spot disease and is the only identified phytopathogen under the genus Serratia (Paine and Standsfield, 1919; Mahlen, 2011). It is widely distributed in nature and is frequently isolated from the gut microbiota of insects, including spiders and bark beetles (Bersanetti et al., 2005; Mikhailova et al., 2014). Serratia proteamaculans has been recently recognized for its capability to produce bio-degradative enzymes (Mehmood et al., 2009; Madhuprakash et al., 2015; Cano-Ramírez et al., 2016) and possess remarkable antagonistic traits against plant pathogens (Wang et al., 2014).

Even though its ability to synthesize extracellular enzymes such as chitinase, endoglucanase, and protease has been reported (Mikhailova et al., 2014; Madhuprakash et al., 2015; Cano-Ramírez et al., 2016), no research regarding its growth and extracellular laccase production has been reported so far. During an investigation of the cultivable microbial community from decomposed wood on the Ottawa River bank, a large number of bacterial and fungal strains were isolated. Among them, a new strain of Serratia proteamaculans, AORB19, has been isolated and characterized that showed significant laccase activities during the screening process. This study characterized the identified strain AORB19 and investigated its laccase production potential and the important culture parameters for enhanced laccase production under submerged culture conditions.

As the quest for economic, novel, and robust enzymes becomes increasingly important for biotechnological applications, more attention has been paid to systematic exploitation of microorganisms through natural biodiversity screenings and optimization of cultural conditions to produce abundant enzymes that are affordable as well as stable and active under desired operational conditions. By screening microorganisms of decayed wood stumps in this study, we have isolated a bacterial strain that naturally secreted a significant amount of laccase enzyme activities (Figure 5).

The selected strain was characterized phenotypically based on its morphological, physiological, and biochemical characteristics. Unlike many other species in the genus Serratia (Grimont et al., 1977), the identified bacterial strain produced non-pigmented colonies with a characteristic mucoid texture on nutrient agar. The biochemical reactions such as l-arabinose, ornithine and lysine decarboxylase, dulcitol, adonitol, d-arabitol, and d-sorbitol fermentation can aid to diversify different biogroups or species within the genus Serratia (Rafii, 2014). The bacterial strain was identified with a high probability score of 99.9% from the MicroScan version 4.4 x databases as Serratia liquifaciens complex which included S. proteamaculans, S. grimesii, and S. liquifaciens. As species-specific identification of Serratia liquifaciens complex was not included in the database, the results were further validated using 16S rRNA gene sequencing. The results of 16S rRNA gene sequencing were comparable with the biochemical characterization, and the species showed 99.9% homology to Serratia proteamaculans.

Aromatic compounds formed during the lignin degradation process are lethal to many microorganisms and can negatively affect productivities in the fermentation of lignocellulosic hydrolysates (Henson et al., 2018). However, while investigating the lignin-degrading potential, it was found that the isolated strain AORB19 was able to grow well with lignin as a sole carbon source in the culture media and produced copious laccase. In the study by Wang et al. (2020), it was reported that the cell growth in the lignin-based culture medium is linked to lignin degradation by extracellular enzymes. Thus, Serratia proteamaculans AORB19 possesses an important phenotype of lignin-degrading microorganism in terms of bacterial cell proliferation and laccase production in lignin-based media. Beyond this indication, the bacterial strain also exhibited exceptionally stable laccase activity in the culture medium for a prolonged period, suggesting an inherent tolerance of the strain and its enzymes to end-product accumulation during lignin degradation and metabolism.

Microbes produce extracellular enzymes to break down complicated organic materials into usable compounds that can be transported through the cell membrane (Ramin and Allison, 2019). The time-course analysis of strain AORB19 revealed extracellular laccase production throughout the growth phase following a short lag phase. During the exponential phase (12–36h), there was a marked increase in laccase production in the growth medium and peaked at the early stationary phase, reaching the maximum by 48 hours of incubation. The time for optimum laccase production varies depending on the microorganism. For Pseudomonas extremorientalis BU118 and Bacillus subtilis MTCC 2414, 24 h and 96 h were reported, respectively (Muthukumarasamy et al., 2015; Neifar et al., 2016).

The temperature and pH of the culture medium have a substantial impact on the strain's cell growth and metabolite synthesis. Although the production of laccase was estimated between pH 5 and 12, the optimal range of pH was about 7–10, which corresponded to the optimal cell growth in the study. It was noticed that the laccase production remained steady throughout the incubation period except minor ups and downs during the stationary phase of incubation. Laccase production from other species of Serratia has been reported around a neutral to alkaline pH range and in mesophilic temperature (Chandra et al., 2012). For strain AORB19, the optimal laccase production and cell growth occurred between 27 and 33°C similar to Bacillus sp., 30°C (Sondhi and Saini, 2019).

As nitrogen is a fundamental component of proteins and nucleic acids, it is widely known that it is one of the most important nutrients for microbial metabolism (Hernández et al., 2015). For strain AORB19, yeast extract was the most effective nitrogenous source and was consistent with a previous discovery regarding Bacillus subtilis DS (Kumar et al., 2018). In this study, maximum laccase production was noticed while using sucrose as the carbon source consistent with a study by Muthukumarasamy et al. (2015) for Bacillus subtilis MTCC 2414 strain. Cellulose and fructose significantly inhibited laccase production. While fructose is readily assimilable, cellulose may increase medium viscosity and thus lower oxygen supply and interfere with cell division and metabolic rate (Othman et al., 2018).

Laccase production of strain AORB19 was enhanced by the organic solvents; acetone and chloroform with acetone exhibited a higher impact. Such inductions by organic solvents have also been reported with Serratia marcescens and fungus GBPI-CDF-03, and the results were in concurrence with prior studies (Dhakar and Pandey, 2013; Kaira et al., 2015; Wu et al., 2019). Our study showed that several metal cations improved strain AORB19 laccase production. Copper was found to induce laccase production in MTCC2414 (Muthukumarasamy et al., 2015). At 0.5 mM, Cu²⁺ increased laccase production, but at 1 mM, Cu²⁺ initially lowered laccase production at 24 h and then increased enzyme production on further incubation. This reversal can be explained by the considerable toxicity of copper at greater concentrations, as well as the restriction of normal metabolic pathways caused by redox cycling of ions. As a result, laccase production could be viewed as a regulating system used by bacteria to survive the toxicity of inorganic materials like metal ions by changing their oxidation status under aerobic conditions (Kaur et al., 2019).

Laccase synthesis by microorganisms can be influenced by many factors such as chemical and nutritional composition of the media; the optimization approach used may be strain specific. In this study, factors [temperature 30 °C, pH 9, yeast extract (2g/l), Li⁺, Cu²⁺, Ca²⁺, and Mn²⁺(0.5mM), and acetone (5%)] were used to enhance laccase synthesis using a one-factor-at-a-time strategy, resulting in a 6.0-fold overall increase (73.3 U/L) in laccase yield. However, by employing a statistical approach of culture media optimization using five factors by response surface methodology, a 5.5-fold increase in laccase yield (58 U/L) was reported for Pseudomonas putida LUA15.1 (Verma et al., 2015). Similarly, Streptomyces psammoticus MTCC 7334 produced 3-fold increase in laccase using response surface methods (Niladevi et al., 2009). Furthermore, when compared with the unoptimized media (2.05 U/ml), a 4-fold increase in laccase production in Bacillus cereus TSS1 (9.03 U/ ml) was attained after altering the media variables using response surface approach (Rajeswari et al., 2015).

Taken together, a new bacterial strain Serratia proteamaculans AORB19 has been characterized that stably secreted high-level laccase activity after 24-h culture over an extended culture time. The combination of selected culture conditions resulted in a 6.0-fold increase in laccase production without the addition of any toxic inducers. This is, to our knowledge, the first report on the natural production and parameter selection for the expression of extracellular laccase by a Serratia proteamaculans strain. The isolated bacterial strain preferred weak alkaline pH and mesophilic culture conditions that are desired traits for many industrial applications. This first phase of research focused on identification and characterization of a new bacterial strain and screening of important factors that influence laccase production using the OFAT strategy. Future experiments will be designed to explore the impact of other important factors such as the amount of oxygen in the culture medium on enzyme activity and the interactions among different nutritional and conditional parameters and to identify the optimized conditions by the statistical method—Design of Experiment and Response Surface Methodology. The final optimized process should allow secretion of further increased amount of laccase that confers strain AORB19 greater potential to be either directly used in fermentation process or for laccase enzyme production.

In addition, the high-level enzyme expression would expedite the biochemical purification and characterization of the specific AORB19 laccase. A greater understanding of the functionality of this bacterial enzyme will likely lead to its practical application in lignin valorization and other biotechnologies that focus on cost-effective biomass conversion and environmental remediation.

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding authors.

NA carried out the majority of experiments and drafted the manuscript. FH set up laccase assay conditions and assisted in experiments. TY and WQ conceived the concept, designed the experiments, reviewed the manuscript, and coordinated the entire study. The final version of the manuscript was read and approved by all authors.

This manuscript represents the National Research Council Communication # 58306. The research of this project was funded by the NRC New Beginning Fund of the National Program Office Ideation Programs and the NRC Industrial Biotechnology Program to TY. NA was supported by the graduate scholarship of Lakehead University.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.