A Semi-Rational Approach to Engineering Laccase Enzymes

Abstract

In order to develop improved laccase-based bio-catalysts, semi-rational mutagenesis of the laccase POXA1b from Pleurotus ostreatus was performed through a combination of directed evolution with elements of rational enzyme modification. The R4 laccase was prepared by joining mutations of previously selected POXA1b random variants. An enhancement of stability features was thus obtained, making the novel enzyme R4 more appropriate as scaffold for directed evolution. A library of 1000 randomly mutated variants of R4 was prepared and screened for the ability of oxidising 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS). One of the variants selected (V148L) for improved activity was also proved to show higher stability than R4 at pH 5, and to retain its high stability at pH 7 and 10. In comparison with the POXA1b wild-type laccase, the semi-rational approach allowed us to develop a more efficient bio-catalyst, rising specific activity on ABTS up to around 5-fold. The new variant was also proved to be both more versatile and more durable than the wild-type enzyme, exhibiting higher activity in wide temperature and pH ranges and higher stability at acidic (t (1/2) at pH 5 = 35 days), neutral (t (1/2) at pH 7 = 38 days) and alkaline (t (1/2) at pH 10 = 62 days) pH values.

A Novel Lentinula edodes Laccase and Its Comparative Enzymology Suggest Guaiacol-Based Laccase Engineering for Bioremediation

“…, 1×McIlvaine buffer, pH 4, 30°C) to allow fair comparisons. Indeed, the correlations had never been addressed despite engineers continuing to employ ABTS and/or DMP as a “direction” for molecular evolution of laccases [6], [7], [8], [10]. The appropriateness of using these screening substrates remained questionable unless clear correlations were shown [13].…”

“…This simple requirement and versatile catalysis have resulted in an exponential growth of interest both in academia and industry [1], [6]. Novel and engineered laccases have thus been emerging to find an outstanding candidate with superior performance on “green” applications [6], [7], [8].…”

“…Among them, ABTS is a non-phenolic substrate which is most commonly recruited for assaying laccase activity due to its intrinsic advantages of pH independence and the high molar extinction coefficient of its oxidized product (ABTS +• , ε 420nm  = 36,000 M −1 cm −1 ) [12], [13]. Oxidation of ABTS, in the absence of H 2 O 2 , thus has become a common screening criterion in the molecular evolution of laccases [6], [7], [8]. Either DMP or SGZ, two phenolic substrates, were frequently employed to supplement the screening even though similar trends with ABTS were usually observed [6], [10], [14].…”

Fungal Laccases Degradation of Endocrine Disrupting Compounds

“…Three out of four selected enzymes were high redox potential laccases from Pleurotus ostreatus : POXC [20, 21], POXA1b [21, 22] heterologously expressed in the filamentous fungus Aspergillus niger [23], 1H6C, a POXA1b variant obtained through random mutagenesis [24] and produced in A. niger [23]. Finally, a commercial laccase, the Novoprime Base 268 (Novozymes), was also used for enzymatic treatment.…”

Directed Evolution of Fungal Laccases

“…Several directed evolution experiments have been attempted by error prone PCR using HRPLs from Pleurotus ostreatus [46-48]. The results confirmed a general improvement in the total activity but poor secretion limits their practical engineering for other purposes.…”