Expression of Melanocarpus albomyces laccase in Trichoderma reesei and characterization of the purified enzyme

Abstract

Previous studies on Melanocarpus albomyces laccase have shown that this enzyme is very interesting for both basic research purposes and industrial applications. In order to obtain a reliable and efficient source for this laccase, it was produced in the filamentous fungus Trichoderma reesei. Two approaches were used: production of a non-fused laccase and a hydrophobin-laccase fusion protein. Both proteins were expressed in T. reesei under the cbh1 promoter, and significantly higher activities were obtained with the non-fused laccase in shake-flask cultures (corresponding to about 230 mg l "1 ). Northern blot analyses showed rather similar mRNA levels from both expression constructs. Western analysis indicated intracellular accumulation and degradation of the hydrophobin-laccase fusion protein, showing that production of the fusion was limited at the post-transcriptional level. No induction of the unfolded protein response pathway by laccase production was detected in the transformants by Northern hybridization. The most promising transformant was grown in a fermenter in batch and fed-batch modes. The highest production level obtained in the fed-batch culture was 920 mg l "1 . The recombinant laccase was purified from the culture supernatant after cleaving the major contaminating protein, cellobiohydrolase I, by papain. The recombinant and wild-type laccases were compared with regard to substrate kinetics, molecular mass, pH optimum, thermostability, and processing of the N-and C-termini, and they showed very similar properties.

Application of Immobilized Laccase from Bacillus subtilis MTCC 2414 on Decolourization of Synthetic Dyes

“…SUPER-h168 was pH 6 (Yang et al, 2013) and laccase immobilized from P. sanguineus exhibited higher activity at pH 9 (Garcia et al, 2007). Moreover, laccase from M. albomyces showed the optimal pH range of 6-7.5 (Kiiskinen et al, 2004) and 8.5 for S. psammoticus (Niladevi and Prema, 2007). Also, B. subtilis MTCC 241 immobilized laccase showed optimum pH of 9 and pH 7 for free laccase which was similar to previously discussed results.…”

Comparative study of Trichoderma gene expression in interactions with tomato plants using high-density oligonucleotide microarrays

“…These data are consistent with the upregulation of genes involved in lipid biosynthesis and catabolism observed in the T34-tomato interaction (Chacó n et al, 2007). However, genes encoding laccases, which are enzymes with polyphenol oxidase activity related to melanin-like pigment synthesis in conidiospores, the induction of fruiting bodies and pathogenic interactions with plants (Hölker et al, 2002;Kiiskinen et al, 2004), were downregulated in T34.…”

Heterologous expression and characterization of a high redox potential laccase from Coriolopsis polyzona MUCL 38443

“…Many laccase genes and cDNAs were cloned from different microorganisms and heterologously expressed in yeasts [Saccharomyces cerevisiae (Cassland and Jönsson, 1999), Pichia pastoris , Pichia methalonica (Guo et al, 2006), Yarrowia lipolytica (Madzak et al, 2005), and Kluyveromyces lactis (Pezzella et al, 2009)], filamentous fungi [Trichoderma reesei (Kiiskinen et al, 2004) and Aspergillus niger (Téllez-Jurado Abstract: In this study, a novel laccase gene, named as Cplcc1, and its corresponding cDNA were isolated and characterized from the Coriolopsis polyzona MUCL 38443 strain. The Cplcc1 gene consists of a 1563-bp open reading frame encoding a protein of 520 amino acids with a 20-residue putative signal peptide.…”