Abstract:Laccases have gained significant attention due to their emerging applications including bioremediation, biomass degradation and biofuel cells. One of the prerequisites for the industrial application of laccases is their sufficient availability. However, expression levels of recombinantly expressed laccases are often low. In this study Mrl2, a new laccase from the basidiomycete Moniliophthora roreri, was cloned in Pichia pastoris and produced in an optimized fed-batch process at an exceptionally high yield of 1… Show more
“…These parameter values were 0.17 µM -1 s -1 and 0.14 µM -1 s -1 in the presence of ABTS and 2,6-DMP, respectively. The catalytic efficiency of TaLac1 towards 2,6-DMP was similar to those earlier reported for laccases from the basidiomycetes Moniliophthora roreri (Mrl2) (0.2 µM -1 s -1 )[50] and Trametes trogii BAFC 463 (LCC3) (0.16 µM -1 s -1 )[51]. For ABTS, close catalytic efficiencies of 0.267 µM -1 s -1 and 0.177 µM -1 s -1 have been reported for laccases from Physisporinus rivulosus strain T241i (Lac-3.5)[39]…”
“…These parameter values were 0.17 µM -1 s -1 and 0.14 µM -1 s -1 in the presence of ABTS and 2,6-DMP, respectively. The catalytic efficiency of TaLac1 towards 2,6-DMP was similar to those earlier reported for laccases from the basidiomycetes Moniliophthora roreri (Mrl2) (0.2 µM -1 s -1 )[50] and Trametes trogii BAFC 463 (LCC3) (0.16 µM -1 s -1 )[51]. For ABTS, close catalytic efficiencies of 0.267 µM -1 s -1 and 0.177 µM -1 s -1 have been reported for laccases from Physisporinus rivulosus strain T241i (Lac-3.5)[39]…”
“…Fed‐batch fermentations were performed as described earlier for the laccase Mrl2 from M. roreri , with the exception that the pH was set to 5 instead of 6, and no additional CuSO 4 was supplemented, but hemin was added after induction to a final concentration of 10 μ m …”
Section: Methodsmentioning
confidence: 86%
“…In our previous work, we showed that the laccase Mrl2 from Moniliophthora roreri , a ligninolytic enzyme exhibiting a relatively low redox potential (0.57 V), could be expressed under the AOX1 promoter in easy‐to‐cultivate Pichia pastoris at a remarkably high level of 1.05 g L −1 . M. roreri causes frosty pod rot in cacao, and thus, is supposed to produce a number of lignocellulose‐degrading enzymes.…”
Manganese peroxidases, lignin peroxidases, and versatile peroxidases secreted by white rot fungi are supposed to play an essential role in lignin degradation. Thus, these enzymes have attracted significant attention as potential biocatalysts. Versatile peroxidases are the most interesting ones, since they comprise activities of manganese and lignin peroxidases. Herein, we demonstrate how the properties of a new manganese peroxidase from Moniliophthora roreri, designated MrMnP1, were shifted towards those of a versatile peroxidase. MrMnP1 was cloned in Pichia pastoris X‐33 and highly expressed in a fed‐batch fermentation, yielding 132 mg L−1 of active enzyme. To extend the substrate spectrum of MrMnP1, a catalytically active tryptophan present in lignin and versatile peroxidases was first introduced. Additionally, the role of five amino acids at positions adjacent to the catalytic tryptophan was elucidated through their replacement by those found in a versatile peroxidase from Pleurotus eryngii. The resulting mutants demonstrated new activities towards high‐redox‐potential substrates, such as lignin dimers, veratryl alcohol, and the azo dye Reactive Black 5.
“…As indicated above, 15 putative N-glycosylation sites were predicted for the MsLAC1 protein sequence. Thus, the conspicuous difference between the calculated molecular mass for the MsLAC1 protein sequence (63 kDa) and the produced recombinant MsLAC1 (150 kDa) is likely due to N-glycosylation and, possibly, additional O-glycosylation or other post-translational modifications [24, 34, 35]. Fig.…”
Section: Resultsmentioning
confidence: 99%
“…Positive colonies were selected on YPD plates containing 50 μg/mL Zeocin™, followed by colony PCR using the α-factor and the 3′ AOX1 Sequencing Primers (Additional file 1: Table S1). Colonies were then screened on minimal methanol histidine medium and minimal dextrose histidine medium plates supplemented with Zeocin™ for fast methanol utilization (Mut+) phenotype [34].…”
BackgroundUnderstanding lignin biosynthesis and composition is of central importance for sustainable bioenergy and biomaterials production. Species of the genus Miscanthus have emerged as promising bioenergy crop due to their rapid growth and modest nutrient requirements. However, lignin polymerization in Miscanthus is poorly understood. It was previously shown that plant laccases are phenol oxidases that have multiple functions in plant, one of which is the polymerization of monolignols. Herein, we link a newly discovered Miscanthus laccase, MsLAC1, to cell wall lignification. Characterization of recombinant MsLAC1 and Arabidopsis transgenic plants expressing MsLAC1 were carried out to understand the function of MsLAC1 both in vitro and in vivo.ResultsUsing a comprehensive suite of molecular, biochemical and histochemical analyses, we show that MsLAC1 localizes to cell walls and identify Miscanthus transcription factors capable of regulating MsLAC1 expression. In addition, MsLAC1 complements the Arabidopsis lac4–2 lac17 mutant and recombinant MsLAC1 is able to oxidize monolignol in vitro. Transgenic Arabidopsis plants over-expressing MsLAC1 show higher G-lignin content, although recombinant MsLAC1 seemed to prefer sinapyl alcohol as substrate.ConclusionsIn summary, our results suggest that MsLAC1 is regulated by secondary cell wall MYB transcription factors and is involved in lignification of xylem fibers. This report identifies MsLAC1 as a promising breeding target in Miscanthus for biofuel and biomaterial applications.
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