Random mutants of a Pleurotus ostreatus laccase as new biocatalysts for industrial effluents bioremediation

Abstract

Aims: To select better performing laccase variants among the 2300 randomly mutated variants of Pleurotus ostreatus POXA1b laccase to develop improved laccase‐based biocatalysts. Methods and Results: Screening of collections of 2300 randomly mutated variants of POXA1b was performed by assaying activity towards the phenolic substrate 2,6‐dimethoxyphenol. Two new variants endowed with higher enzyme activity than the wild‐type laccase were characterized, and their ability to decolourize industrial dyes with complex trisazo‐, polyazo‐ and stilbene‐type structures, in the absence of mediators, was demonstrated. One of the mutants (2L4A) was also proved to be highly stable at both acidic and alkaline pH values (displaying a half‐life of around 1 month at the pH levels of both 5 and 10). Conclusions: In comparison with the wild‐type laccase, the new selected 2L4A mutant shows a significant increase in stability at acidic pH, whilst storing its high stability at alkaline pH. This variant also represents a more versatile enzyme with respect to both the variety of xenobiotics degraded and the operative conditions. Significance and Impact of the Study: This work represents the first example of improvement of a basidiomycete laccase for industrial effluents bioremediation by directed evolution.

Fungal decolouration and degradation of azo dyes: A review

“…The purified recombinant Lac obtained from the heterologous production of Pleurotus sanguineus Lac in P. pastoris could efficiently decolourise synthetic dyes in the absence of mediators (Lu et al, 2009). Among 2300 randomly mutated variants of P. ostreatus POXA1b Lac, two mutants showed higher stability in a variety of environmental conditions and higher ability to decolourise azo dyes than the wild-type Lac; the mutant 2L4A also proved to be highly stable at both acidic and alkaline pHs (Miele et al, 2010). In contrast, fungal Lacs show optimal dye decolouration at acidic pHs and in the presence of redox mediators (Pereira et al, 2009;Martins et al,2002).…”

Structural Insights into 2,2′-Azino-Bis(3-Ethylbenzothiazoline-6-Sulfonic Acid) (ABTS)-Mediated Degradation of Reactive Blue 21 by Engineered Cyathus bulleri Laccase and Characterization of Degradation Products

“…In the present study, an attempt was made to extend the oxidizing ability of a "medium redox" laccase to the PC category of dyes. The strategy adopted to develop the library and screen for RB21-degrading variants was based on the following observations and inferences from the literature: (i) the amino acids toward the C terminus of laccase affect the redox potential of laccases (15,18,24) and thereby its reactivity; (ii) in general, higher catalytic activity on a laccase substrate, such as ABTS or 2,6-dimethoxy phenol, can be used as a screen for detection of altered catalytic rates or higher dye decolorization efficiencies (31)(32)(33); (iii) a high-throughput screening method would allow detection of clones that degrade the PC dye; and (iv) easy monitoring of the degradation process by qualitative TLC. Low-error-rate (0 to 4 mutations/kb) mutagenesis was used for introducing random mutations in the 816-bp segment of the lcc gene from C. bulleri.…”

The good, the bad and the tasty: The many roles of mushrooms

“…Beyond its role as a culinary basidiomycete, being a white rot fungus P. ostreatus plays an important role in decomposition and carbon cycling and has garnered significant attention for its potential use as a bioremediation agent (Eggen and Majcherczyk, 1998, Hirano et al., 2000, Byss et al., 2008, Miele et al., 2010, Purnomo et al., 2010). Genome sequencing has recently revealed the wood-degrading machinery of P. ostreatus to be typical of white rot fungi (Riley et al.…”