Antibiotics constitute one of the most widely prescribed and used drug categories globally. Due to their extensive usage and limited metabolism within the human body (with up to 70% excretion in an unchanged form), their release into the environment is steadily increasing, leading to elevated concentrations in water, soil, and sediments [63]. In raw wastewater (including hospital effluents) and treated wastewater, antibiotic concentrations exhibit a wide range, from a few ng/L to several tens of mg/L [64]. The rise of antibiotic resistance in clinically significant bacterial species poses a global public health challenge, necessitating the essential monitoring of antibiotic contamination in the environment.

Laccase efficacy has been extensively examined for various antibiotic classes, including sulfonamides, penicillins, fluoroquinolones, tetracyclines and others like trimethoprim. Antibiotics have been the focus of intensive studies concerning laccase, resulting in several reviews on this topic [30,65]. These studies encompass evaluations with crude laccase extracts or the cultivation of laccase-producing organisms in media containing antibiotics. In a study conducted by Becker et al. [66], the degradation of 38 antibiotics by laccase from T. versicolor (Sigma-Aldrich) was investigated, and their concentrations were assessed after the enzymatic reaction using HPLC. The efficiency of biotransformation varied based on the structural characteristics of the compounds. Notably, penicillins, such as amoxicillin (96.6%) and ampicillin (88.6%), fluoroquinolones (e.g., ofloxacin, ciprofloxacin, enrofloxacin, danofloxacin, and marbofloxacin) with removal rates ranging from 50.1 to 59.4%, and tetracyclines (oxytetracycline, chlortetracycline, doxycycline, and tetracycline) with removal rates ranging from 26.0 to 48.4%, were efficiently removed. The presence of the laccase mediator system (LMS) using syringaldehyde (SYR) increased the percentage of removal of most antibiotics to over 90%. However, the application of this mediator heightened the ecotoxicity of degradation products after the enzyme-mediated reaction, as indicated by a growth inhibition test with antibiotic-sensitive Bacillus subtilis and the Microtox test with Vibrio fischeri. It is noteworthy that laccase alone in a solution with antibiotics did not induce a similar increase in ecotoxicity. SYR likely forms derivatives in the presence of laccase, negatively impacting test organisms [66].

Sulfonamide antibiotics are widely used to treat livestock diseases and account for a high proportion of total antibiotic consumption worldwide. Laccase itself was not effective in the removal of sulfonamide antibiotics [49,66,67]. Sulfadimethoxine and sulfamonomethoxine were efficiently removed by the LMS using laccase from the white-rot fungus Perenniporia strain TFRI 707 and 2,2′-azino-di (3-ethyl-benzothiazoline sulfonic acid) (ABTS) or violuric acid (VA) as a redox mediator. Laccase from T. versicolor (Sigma-Aldrich) efficiently degraded 12 sulfonamide antibiotics with SYR (1 mM) [66]. In a study conducted by Alharbi et al. [68], the ability of T. versicolor laccase (Sigma-Aldrich) to degrade sulfamethoxazole at a concentration of 5 mg/L was tested, observing only 48% removal within 48 h. Shi et al. [49] observed 100% removal of the sulfonamide antibiotics sulfadiazine, sulfamethazine, and sulfamethoxazole by the LMS with SYR (1 mM) but no removal with laccase from Echinodontium taxodii itself. Weng et al. [67] found that deaniline and oxidative coupling are two detectable pathways for the conversion of sulfonamide antibiotics by the LMS. The coupling reaction of sulfonamide antibiotics with redox mediators was also determined via LC-MS in a study conducted by Shi et al. [49]. They observed the formation of 2,6-dimethoxybenzoquinone (2,6-DMBQ) with the tested sulfonamide antibiotics, where 2,6-DMBQ was formed after the oxidation of the natural mediators acetosyringone, SYR or syringic acid (SA) by E. taxodii laccase. A toxicity evaluation of the degradation products after treatment with T. versicolor laccase using the bioluminescent Photobacterium leiognathi (BLT-Screen) showed no toxic effect [68]. Furthermore, the degradation products of sulfonamide antibiotics did not have a negative effect on the growth of Staphylococcus aureus and E. coli [49], confirming the biotransformation of the antibiotic to a form without the original antibiotic effect.

Penicillins represent one of the most extensively used classes of antibiotics, primarily owing to their broad spectrum of clinical applications. Depending on their structure, penicillins can serve as substrates for laccase produced by both bacteria [69] and fungi [66]. The laccase from T. versicolor (Sigma-Aldrich) demonstrated efficient removal of amoxicillin and ampicillin (96.6 and 88.6%, respectively). Additionally, penicillin G, penicillin V, cloxacillin, and oxacillin were degraded using the LMS with SYR as a mediator at a concentration of 1 mM, resulting in removal rates ranging from 53.5 to 93.9% [66]. Moreover, laccase from B. subtilis, expressed in E. coli, exhibited the complete removal of ampicillin within two hours, as confirmed via HPLC analysis [69]. The authors subsequently proposed a degradation mechanism featuring two putative pathways, wherein the cleavage of the β-lactate ring occurs during ampicillin degradation by laccase, leading to the subsequent loss of antibiotic activity. The degradation mechanism was corroborated by ecotoxicity assays utilizing B. subtilis and E. coli.

Fluoroquinolones represent a substantial group of broad-spectrum bactericidal agents, characterized by a common bicyclic nuclear structure related to 4-quinolone and are employed in the treatment of bacterial infections. In a study conducted by Zou et al. [70], the efficacy of laccase from T. versicolor (Sigma-Aldrich) in degrading fluoroquinolones, specifically norfloxacin, enrofloxacin, and moxifloxacin, was investigated. The laccase was immobilized in a magnetically modified biochore, primarily facilitating antibiotic removal through sorption—a common phenomenon observed with immobilized laccases [71,72]. While the presence of laccase slightly increased the removal percentage (by 0.7–2.6%), the addition of ABTS as a redox mediator further enhanced removal by 11.0–16.8%. Additionally, the removal of ciprofloxacin by laccase from T. versicolor (Sigma-Aldrich) was found to be effective in the presence of a redox mediator, either p-coumaric acid (p-CA) (57%) or HBT (81%). It is noteworthy that a higher percentage of removal was observed in the phosphate buffer compared to the wastewater sample (approximately 1.6 times less) [71]. The proposed putative biotransformation mechanism for fluoroquinolones by laccase was elucidated through LC-MS analysis.

The typical biotransformation mechanism of fluoroquinolones involves processes such as the loss of ethyl/ethylene, defluorination, and demethylation or decarboxylation. In a study conducted by Zou et al. [71], the laccase-mediated pretreatment of ciprofloxacin resulted in only a negligible inhibition of B. subtilis, S. aureus, and Pseudomonas aeruginosa. This observation affirms the positive impact of laccase in the biotransformation of antibiotics.

Tetracycline antibiotics as broad-spectrum antibiotics are widely used to treat human and animal diseases, making them the second most widely used antibiotics in the world [73]. Laccase produced by bacteria and expressed in E. coli, as well as laccase from Myceliophthora thermophila, expressed in Aspergillus sp., have been tested [72,74]. Laccase from Bacillus amyloliquefaciens, expressed in E. coli, was able to efficiently remove tetracycline antibiotics such as tetracycline, doxycycline, and tigecycline with the efficiency of 86.1, 96.5 and 81.0%, respectively [72]. Tetracycline was biotransformed to a level below the detection limit of the HPLC analysis after a 2-h catalyzed reaction with laccase of B. subtilis, expressed in E. coli [69]. Laccase from M. thermophila exhibited the complete removal of tetracycline using SYR as a redox mediator even in salt water [74]. The authors proposed a putative mechanism of tetracycline biotransformation with two possible pathways, both leading to the opening of the tetracycline ring to form small acid molecules, resulting in a loss of antibiotic activity. In the case of tetracycline in the presence of laccase from B. amyloliquefaciens, the opening of the aromatic ring was observed in two of the three putative pathways, preceded by epimerization, demethylation, deamination, dehydrogenation, and hydroxylation in the second putative pathway or demethylation and dehydrogenation, dehydroxylation in the third pathway [72].

The same reactions, leading to the formation of degradation products with a smaller molecular size, have also been observed for tetracycline degraded by fungal laccase [74] and other tetracycline antibiotics such as doxycycline and tigecycline [72]. The loss of the antibiotic effect of tetracycline was also confirmed by ecotoxicity assays determined via the incubation of degradation products with B. subtilis and E. coli bacteria [69].

Trimethoprim (diaminopyrimidine) is an antibiotic used for treating and preventing urinary tract infections. In a study conducted by Alharbi et al. [68], the efficacy of laccase from T. versicolor (Sigma-Aldrich) in degrading trimethoprim at a concentration of 5 mg/L was investigated. The results demonstrated a 95% removal of this drug within 48 h. LC-MS was used in the study to identify degradation products, but none were detected in either the positive or negative scanning mode. Furthermore, testing against the bioluminescent P. leiognathi (BLT-Screen) after the enzyme-catalyzed reaction revealed no toxic effect, indicating that the laccase-catalyzed reaction did not lead to the formation of toxic products [68].

In summary, it can be concluded that bacterial or fungal laccase, without a redox mediator, exhibited efficient removal of penicillins, fluoroquinolones, and tetracyclines, with degradation involving the opening of the antibiotic’s aromatic rings. The degradation percentage varied based on the antibiotic’s structure and the presence of electron-donating or electron-withdrawing functional groups (Table 3). An enhanced degradation efficiency was observed with the use of the LMS employing SYR, HBT, or ABTS [66,67,71,74]. Sulfonamide antibiotics proved to be the most resistant to laccase-catalyzed degradation, requiring the presence of a redox mediator for a decrease in concentration. Putative degradation pathways indicated biotransformation through crosslinking with a redox mediator [49,67]. Importantly, the antibiotic effect was eliminated in all studied cases, but the choice of a mediator in the LMS is crucial to avoid the production of toxic products [69,70,72,74].

Drug	Concentration [mg/L]	Laccase Producer	Laccase Activity [U/L]	Concentration of Redox Mediator [mM]	Enzyme Reaction	Degradation/Transformation Efficiency	Ref.
Sulfamonomethoxine	50	Perenniporia TFRI 707	600	ABTS [1.0]	30 °C, pH 4.0, 0.5 h	100% (BT)	[67]
				VA [1.0]		100% (BT)
	0.01	Trametes versicolor	n.s.	-	25 °C, pH 7.0, 24 h	5.4% (n.s.)	[66]
				SYR [1.0]		96.1% (n.s.)
Sulfadimethoxine	50	Perenniporia TFRI 707	600	ABTS [1.0]	30 °C, pH 4.0, 0.5 h	100% (BT)	[67]
				VA [1.0]		100% (BT)
Sulfadiazine	0.01	Trametes versicolor	n.s.	-	25 °C, pH 7.0, 24 h	11.2% (n.s.)	[66]
				SYR [1.0]		99.7% (n.s.)
	50	Echinodontium taxodii	200	SA [1.0]	30 °C, pH 5.0, 0.5 h	100% (BT)	[49]
Sulfamethoxazole	50	Echinodontium taxodii	200	SA [1.0]	30 °C, pH 5.0, 0.5 h	100% (BT)	[49]
	5.0	Trametes versicolor	430–460	-	25 °C, pH 6.8–6.9, 8 h	56% (n.s.)	[68]
	0.01	Trametes versicolor	n.s.	-	25 °C, pH 7.0, 24 h	14.2% (n.s.)	[66]
				SYR [1.0]		97.2% (n.s.)
Tetracycline	0.01	Trametes versicolor	n.s.	-	25 °C, pH 7.0, 24 h	26.0% (n.s.)	[66]
				SYR [0.01]		85.2% (n.s.)
	100	Bacillus subtilis	34.3	-	25 °C, pH 5.5, 2 h	100% (D)	[69]
	100	Bacillus amyloliquefaciens	2000	-	30 °C, pH 7.0, 2 h	86.1% (D)	[72]
	0.002	Myceliophthora thermophila	200	SA [0.001]	15 °C, pH 8.0, 2 h	100% (D)	[74]
Doxycycline	100	Bacillus amyloliquefaciens	500	-	30 °C, pH 7.0, 2 h	96.5% (D)	[72]
	0.01	Trametes versicolor	n.s.	-	25 °C, pH 7.0, 24 h	30.4% (n.s.)	[66]
				SYR [0.01]		89.1% (n.s.)
Tigecycline	100	Bacillus amyloliquefaciens	500	-	30 °C, pH 7.0, 2 h	81.0% (D)	[72]
Norfloxacin	0.01	Trametes versicolor	n.s.	-	25 °C, pH 7.0, 24 h	58.1% (n.s.)	[66]
				SYR [1.0]		82.4% (n.s.)
Enrofloxacin	0.01	Trametes versicolor	n.s.	-	25 °C, pH 7.0, 24 h	50.1% (n.s.)	[66]
				SYR [1.0]		76.6% (n.s.)
Ciprofloxacin	10	Trametes versicolor	n.s.	p-CA [3.0]	45 °C, pH 6.8, 6 h	57% (n.s.)	[71]
				HBT [3.0]		81% (n.s.)
Amoxicillin	0.01	Trametes versicolor	n.s.	-	25 °C, pH 7.0, 24 h	96.6% (n.s.)	[66]
				SYR [1.0]		94.7% (n.s.)
Ampicillin	0.01	Trametes versicolor	n.s.	-	25 °C, pH 7.0, 24 h	88.6% (n.s.)	[66]
				SYR [1.0]		99.9% (n.s.)
	100	Bacillus subtilis	34.3	-	25 °C, pH 5.5, 2 h	100% (D)	[69]
Trimethoprim	5.0	Trametes versicolor	430–460	-	25 °C, pH 6.8–6.9, 24 h	95% (n.s.)	[68]
	0.01		n.s.	-	25 °C, pH 7.0, 24 h	26.6% (n.s.)	[66]
				SYR [1.0]		66.8% (n.s.)