Immobilization of Laccase Enzyme on Chitosan and MOF Supports for Bioremediation Applications

Abstract

The detection and removal of organic emerging pollutants (EPs) in water bodies have become one of the pressing concerns and issues of our time. The effects of these pollutants on aquatic life and human health, in addition to their recalcitrant nature have made developing efficient remediation strategies an imperative necessity. Laccases are enzymes that have been shown to have a great capability at degrading a wide range of pollutants. However, despite their efficiency, their non-reusability and instability have hindered their wide-spread industrial applications for water treatment. Consequently, enzyme immobilization on solid supports have emerged as a reliable approach that enables enzyme recyclability and, in some cases, has been shown to enhance enzymes’ stabilities. However, there is a dearth of information and correlation between different immobilization supports and approaches and their impact on enzyme activity. Hence, in this study, laccase was immobilized on two supports of very different nature (by different immobilization techniques) to investigate the effects of these two factors on the overall performance of the immobilized enzyme. The first support was chitosan: a sustainable biopolymer, with which the enzyme was chemically crosslinked. And the second was NH2-MIL-125: a crystalline and porous Metal Organic Framework (MOF) on which the enzyme was physically adsorbed. The two biocatalytic systems were subsequently designated as Lac@chitosan and Lac@NH2-MIL-125 and were assessed for their stability and efficiency at degrading a panel of 15 diverse emerging pollutants. Pollutants degradation was studied using sensitive Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) analysis. Both of the biocatalytic systems exhibited remarkable recyclability; maintaining approximately 80% and 70% of their activity after ten cycles for Lac@chitosan and Lac@NH2-MIL-125, respectively. Interesting differences were observed on the enzyme’s stability, when immobilized on these two different supports. Laccase when immobilized on chitosan (Lac@chitosan) exhibited enhanced thermal stability compared to free laccase. In addition, it enhanced laccase stability at acidic pH and decreased it for pH values higher than five. On the other hand, Lac@NH2-MIL-125 exhibited decreased thermal stability and similar pH stability compared to free laccase. Both free and immobilized laccase efficiently degraded three pollutants (2mercaptobenzothiazole, paracetamol, and caffeic acid) out of the 15 pollutants tested. Lac@chitosan exhibited a much superior performance at degrading the EPs as it achieved almost complete degradation of paracetamol at much lower concentrations compared to free laccase and Lac@NH2-MIL-125. Additionally, it was able to degrade sulfamethoxazole which did not degrade by free laccase and Lac@NH2-MIL125 at the low enzyme concentration tested. The effect of using 1hydroxybenzotriazole (HOBT) and 2'azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as redox mediators on the degradation efficiency was also examined. It was found that HOBT slightly enhanced the degradation of paracetamol while ABTS enhanced the degradation of paracetamol and salicylic acid but decreased the degradation of caffeic acid. The transformation products (TPs) of 2mercaptobenzothiazole pollutant degradation were analyzed using LC-MSMS. Two of the TPs identified were previously reported in literature, however four new TPs were detected. The current study presented an example of the applicability of laccases for degrading 2-mercaptobenzothiazole, paracetamol, caffeic acid, salicylic acid (for the first time), and sulfamethoxazole. Additionally, we showed that differently immobilized laccases show significant differences in their stabilities, as well as bioremediation potential, thus highlighting the need for more research in this exciting area of enzymatic bioremediation of emerging pollutants.

Date of Award	9 May 2024
Original language	American English
Supervisor	Syed Ashraf (Supervisor)