Lignin degradation by a novel thermophilic and alkaline yellow laccase from Chitinophaga sp

Abstract

Laccases (EC 1.10.3.2) are oxidoreductases that belong to the multicopper oxidase subfamily and are classified as yellow/white or blue according to their absorption spectrum. Yellow laccases are more useful for industrial processes since they oxidize nonphenolic compounds in the absence of a redox mediator and stand out for being more stable and functional under extreme conditions. This study aimed to characterize a new laccase that was predicted to be present in the genome of Chitinophaga sp. CB10 - Lac_CB10. Lac_CB10, with a molecular mass of 100.06 kDa, was purified and characterized via biochemical assays using guaiacol as a substrate. The enzyme demonstrated extremophilic characteristics, exhibiting relative activity under alkaline conditions (CAPS buffer pH 10.5) and thermophilic conditions (80-90°C), as well as maintaining its activity above 50% for 5 h at 80°C and 90°C. Furthermore, Lac_CB10 presented a spectral profile typical of yellow laccases, exhibiting only one absorbance peak at 300 nm (at the T2/T3 site) and no peak at 600 nm (at the T1 site). When lignin was degraded using copper as an inducer, 52.27% of the material was degraded within 32 h. These results highlight the potential of this enzyme, which is a novel yellow laccase with thermophilic and alkaline activity and the ability to act on lignin. This enzyme could be a valuable addition to the biorefinery process. In addition, this approach has high potential for industrial application and in the bioremediation of contaminated environments since these processes often occur at extreme temperatures and pH values.

Importance: The characterization of the novel yellow laccase, Lac_CB10, derived from Chitinophaga sp. CB10, represents a significant advancement with broad implications. This enzyme displays exceptional stability and functionality under extreme conditions, operating effectively under both alkaline (pH 10.5) and thermophilic (80-90°C) environments. Its capability to maintain considerable activity over extended periods, even at high temperatures, showcases its potential for various industrial applications. Moreover, its distinctive ability to efficiently degrade lignin-demonstrated by a significant 52.27% degradation within 32 h-signifies a promising avenue for biorefinery processes. This newfound laccase's characteristics position it as a crucial asset in the realm of bioremediation, particularly in scenarios involving contamination at extreme pH and temperature levels. The study's findings highlight the enzyme's capacity to address challenges in industrial processes and environmental cleanup, signifying its vital role in advancing biotechnological solutions.

Keywords: extremophile; lignin; yellow laccase.

Fig 1

Schematic representation of the pET-SUMO plasmid containing the Lac_CB10 insert. From this expression system, cloning was performed to enable expression in the E. coli ER2265 strain.

Fig 2

Extraction and purification of Lac_CB10. (M) Molecular mass marker. (A) Purified Lac_CB10 in 7% SDS-PAGE gel. (B) Native PAGE gel stained with 10 mM catechol in CAPS buffer pH 10.5. (C) Native PAGE gel stained with 0.02% guaiacol in CAPS buffer pH 10.5. (D) Chromatographic profile of purified laccase by gel filtration. Estimation of molecular mass of Lac_CB10 by gel filtration based on linear correlation. Elution of molecular mass standard of Lac_CB10 and protein standards versus their logarithmic molecular mass values. P1–P4: Proteins used as molecular mass standards: ρ-aminobenzoic acid (pABA) (0.13 kDa), ribonuclease A (13.7 kDa), albumin (43 kDa), bovine thyroglobulin (670 kDa), γ-globulin (150 kDa), and Protein Standard Mix 15 ± 600 kDa, Sigma, St. Louis, MO, USA.

Fig 3

UV-Vis spectrum of Lac_CB10 apoenzyme and holoenzyme. (A) UV-vis spectrum of Lac_CB10 in its holoenzyme form, with an absorption peak near 330 nm. (B) UV-vis spectrum of Lac_CB10 (black circles) in its apoenzyme form (without any absorption peak) and of the holoenzyme compared to the blue laccase LacMeta (gray circles), showing their absorption peaks at 330 nm and 600 nm.

Fig 4

Effect of pH on the activity of Lac_CB10. Enzymatic activity was performed using the following 20 mM buffers: MES (pH 5.5, 6.0, and 6.5); PIPES (pH 6.0, 6.5, 7.0, and 7.5); MOPS (pH 6.5, 7.0, 7.5, and 8.0); HEPES (pH 6.5, 7.0, 7.5, and 8.0); TAPS (pH 8.0 and 9.0); CAPS (pH 9.5, 10.0, 10.5, and 11.0); Glycine (pH 8.5, 9.0, 9.5, 10.0, and 10.5); sodium bicarbonate (pH 9.0, 9.5, 10.0, 10.5, 11.0, and 12.0); and potassium phosphate (pH 11.0, 11.5, and 12.0). The tests were conducted with three replicates. Values with the same letter do not differ statistically, according to the ANOVA and Tukey test, with a 5% probability.

Fig 5

Effects of temperature on Lac_CB10 Activity. (A) The enzymatic activity was assessed at temperatures ranging from 10°C to 90°C in a 20 mM ampol buffer (pH 10.0). (B and C) The thermostability tests were conducted in a 20 mM CAPS buffer (pH 10.5) over 5 h at 80°C and 90°C. The tests were carried out with three replicates. Values followed by the same letter do not differ statistically according to the ANOVA and Tukey test at a 5% probability level.

Fig 6

Scanning electron microscopy (SEM) images of alkalin lignin degradation. Treatment without inducer [(A) abiotic control and (B) treatment] and treatment with copper [(C) abiotic control and (D) treatment]. Experiments were conducted at 55°C for 32 h at 150 rpm.