Cloning, sequence analysis, expression of Cyathus bulleri laccase in Pichia pastoris and characterization of recombinant laccase

Abstract

BackgroundLaccases are blue multi-copper oxidases and catalyze the oxidation of phenolic and non-phenolic compounds. There is considerable interest in using these enzymes for dye degradation as well as for synthesis of aromatic compounds. Laccases are produced at relatively low levels and, sometimes, as isozymes in the native fungi. The investigation of properties of individual enzymes therefore becomes difficult. The goal of this study was to over-produce a previously reported laccase from Cyathus bulleri using the well-established expression system of Pichia pastoris and examine and compare the properties of the recombinant enzyme with that of the native laccase.ResultsIn this study, complete cDNA encoding laccase (Lac) from white rot fungus Cyathus bulleri was amplified by RACE-PCR, cloned and expressed in the culture supernatant of Pichia pastoris under the control of the alcohol oxidase (AOX)1 promoter. The coding region consisted of 1,542 bp and encodes a protein of 513 amino acids with a signal peptide of 16 amino acids. The deduced amino acid sequence of the matured protein displayed high homology with laccases from Trametes versicolor and Coprinus cinereus. The sequence analysis indicated the presence of Glu 460 and Ser 113 and LEL tripeptide at the position known to influence redox potential of laccases placing this enzyme as a high redox enzyme. Addition of copper sulfate to the production medium enhanced the level of laccase by about 12-fold to a final activity of 7200 U L-1. The recombinant laccase (rLac) was purified by ~4-fold to a specific activity of ~85 U mg-1 protein. A detailed study of thermostability, chloride and solvent tolerance of the rLac indicated improvement in the first two properties when compared to the native laccase (nLac). Altered glycosylation pattern, identified by peptide mass finger printing, was proposed to contribute to altered properties of the rLac.ConclusionLaccase of C. bulleri was successfully produced extra-cellularly to a high level of 7200 U L-1 in P. pastoris under the control of the AOX1 promoter and purified by a simple three-step procedure to homogeneity. The kinetic parameters against ABTS, Guaiacol and Pyrogallol were similar with the nLac and the rLac. Tryptic finger print analysis of the nLac and the rLac indicated altered glycosylation patterns. Increased thermo-stability and salt tolerance of the rLac was attributed to this changed pattern of glycosylation.

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

“…For kinetic studies, the WtLcc and the mutant enzymes were purified according to the method of Garg et al (17). Briefly, laccase expressed in the culture filtrate of recombinant P. pastoris was concentrated by ammonium sulfate precipitation and then dialyzed in 20 mM Tris-HCl (pH 8).…”

“…Pichia pastoris X33 strain was grown in YPD medium containing 10 g/liter of yeast extract (Himedia, India), 20 g/liter of peptone, and 20 g/liter of dextrose. The cDNA of C. bulleri laccase (lcc) cloned in the pPICZ␣B vector (17), in frame with the ␣-mating type factor, was used for generating the mutant library in P. pastoris. The transformants were grown in buffered complex glycerol medium (BMGY medium) followed by buffered complex methanol medium (BMMY medium) (Invitrogen).…”

“…Phe at this position determines high potential, and this has been proven by site-di-rected mutagenesis and on the basis of several crystal structures (16). This position is occupied by Leu in laccase of Cyathus bulleri, making it a "medium redox" enzyme (17). Amino acids that line the substrate binding pocket near the T1 site also influence the redox potential (18) and, in turn, the ability to act on dyes and sterically large molecules (19).…”

Expression of CotA laccase in Pichia pastoris and its electrocatalytic sensing application for hydrogen peroxide

“…Pichia pastoris can serve as a potential host for heterologous protein production because of its efficient secretion of extracellular proteins, high expression levels, and ease of genetic manipulation (Damasceno et al 2012). The P. pastoris expression system has been widely used for heterologous production of fungal laccases, which indicates that this system is suitable for laccase expression (Garg et al 2012;Jolivalt et al 2005;Lu et al 2013;Otterbein et al 2000).…”

Functional expression ofTrametes versicolorthermotolerant laccase variant inPichia pastoris

“…[4] Many native fungi are slow growers and produce low amounts of laccase, which makes them difficult for studying and for large-scale application of their enzymes. [5] Meanwhile, the thermostability of a protein is both of fundamental and industrial importance. [6] Thermostable enzymes allow high process temperatures with associated higher reaction rates and less risk of microbial contamination.…”