Isolation and characterization of a novel thermostable and catalytically efficient laccase from Peniophora sp. strain UD4
Improved enzyme properties are constantly being sought to enhance the range of applications for enzymes. Biodiversity provides a wide variety of enzymes. Several researchers have concentrated on extremophiles as their primary source of superior enzymes, consequently neglecting temperate environments in their search for these enzymes. The relatively neglected fungal biodiversity of South Africa provided an opportunity to test the hypothesis that potentially important industrial enzymes with unusual properties could be isolated from mesophilic basidiomycetous fungi.
Subsequent screening of Eastern Cape biodiversity for thermostable ligninolytic enzymes from basidiomycetes resulted in the isolation of a novel laccase enzyme from a basidiomycetous species. This fungus was identified as Peniophora sp. UD4 by phylogenetic analysis of rDNA ITS sequences. Initial studies indicated a superior optimum temperature of 70°C and thermostability, indicated by no loss in activity at 60°C over nine hours. Further characterization of the laccase revealed a broader than usual substrate range through its unusual ability to oxidatively couple DMAB and MBTH. The laccase also exhibited a broad pH oxidation range for ABTS (pH 2 – 6.8), and a relatively high affinity (K_m_ = 0.0123 mM) and catalytic efficiency (63 252 mM^(-1)^s^(-1)^) for ABTS as a substrate. The laccase activity from Peniophora sp. UD4 was shown to be comprised of three isozymes with a molecular weight of 62 kDa and pI’s of 6.33, 6.45 and 6.50.
Investigation of the nutrient and physical factors affecting ligninolytic enzyme production and growth of Peniophora sp. UD4 indicated that the wild-type organism was unsuitable for large scale production of the thermostable laccase due to the low levels of laccase production.
The thermostable laccase was applied to defouling of ultrafiltration membranes, bioremediation of industrial waste streams, biocatalysis, and biosensor technology as potential applications. Application of the Peniophora sp. UD4 laccase to defouling of membranes used for ultrafiltration of brown water showed large flux recoveries of 31, 21 and 21% after the first three defouling recycles respectively, compared to 3% for the control without immobilized enzyme. The novel laccase showed potential for the bioremediation of industrial waste streams, the most successful being that of bleach plant effluent, where a reduction of 66% of the phenolic load was achieved. Application of the novel laccase to biocatalytic oxidation of ferulic acid and (±)-?-pinene showed higher product yield as compared to oxidation of these compounds by Trametes versicolor laccase in mediated and non-mediated systems. The major products of (±)-?-pinene oxidation were identified as verbenol and trans-sorberol. The Peniophora sp. UD4 laccase was successfully applied to biosensor technology, which benchmarked significantly better than Trametes versicolor laccase for the detection of 4-chlorophenol. The biosensor developed with laccase from UD4 by covalent binding to a glassy carbon electrode exhibited the best combination of sensitivity and stability.
This thesis shows that a laccase with superior properties was obtained from a mesophilic South African basidiomycete. The catalytic properties displayed by the novel laccase from Peniophora sp. UD4 all contribute to the increased industrial applicability of laccases, and may be the most industrially feasible enzyme of its class isolated to date.
School Location:South Africa
Source Type:Master's Thesis
Keywords:biochemistry microbiology biotechnology
Date of Publication:01/01/2005