NDLI: Heterologous expression and structural characterization of two low pH laccases from a biopulping white-rot fungus Physisporinus rivulosus

Content Provider	Springer Nature Link
Author	Hildén, Kristiina Mäkelä, Miia R. Lundell, Taina Kuuskeri, Jaana Chernykh, Alexey Golovleva, Ludmila Archer, David B. Hatakka, Annele
Copyright Year	2012
Abstract	The lignin-degrading, biopulping white-rot fungus Physisporinus rivulosus secretes several laccases of distinct features such as thermostability, extremely low pH optima and thermal activation for oxidation of phenolic substrates. Here we describe the cloning, heterologous expression and structural and enzymatic characterisation of two previously undescribed P. rivulosus laccases. The laccase cDNAs were expressed in the methylotrophic yeast Pichia pastoris either with the native or with Saccharomyces cerevisiae α-factor signal peptide. The specific activity of rLac1 and rLac2 was 5 and 0.3 μkat/μg, respectively. However, mutation of the last amino acid in the rLac2 increased the specific laccase activity by over 50-fold. The recombinant rLac1 and rLac2 enzymes demonstrated low pH optima with both 2,6-dimethoxyphenol (2,6-DMP) and 2,2′-azino-bis(3-ethylbenzathiazoline-6-sulfonate). Both recombinant laccases showed moderate thermotolerance and thermal activation at +60 °C was detected with rLac1. By homology modelling, it was deduced that Lac1 and Lac2 enzymes demonstrate structural similarity with the Trametes versicolor and Trametes trogii laccase crystal structures. Comparison of the protein architecture at the reducing substrate-binding pocket near the T1-Cu site indicated the presence of five amino acid substitutions in the structural models of Lac1 and Lac2. These data add up to our previous reports on laccase production by P. rivulosus during biopulping and growth on Norway spruce. Heterologous expression of the novel Lac1 and Lac2 isoenzymes in P. pastoris enables the detailed study of their properties and the evaluation of their potential as oxidative biocatalysts for conversion of wood lignin, lignin-like compounds and soil-polluting xenobiotics.
Starting Page	1589
Ending Page	1599
Page Count	11
File Format	PDF
ISSN	01757598
Journal	Applied Microbiology and Biotechnology
Volume Number	97
Issue Number	4
e-ISSN	14320614
Language	English
Publisher	Springer-Verlag
Publisher Date	2012-04-12
Publisher Place	Berlin, Heidelberg
Access Restriction	Subscribed
Subject Keyword	Laccase Phenol oxidase Thermotolerance White-rot fungi Physisporinus rivulosus Basidiomycete 3-D homology modelling Wood decay Lignin degradation Biotechnology Microbiology Microbial Genetics and Genomics
Content Type	Text
Resource Type	Article
Subject	Medicine Applied Microbiology and Biotechnology Biotechnology

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