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Expression of Melanocarpus albomyces laccase in Trichoderma reesei and characterization of the purified enzyme
- Source :
- Kiiskinen, L-L, Kruus, K, Bailey, M, Ylösmäki, E, Siika-aho, M & Saloheimo, M 2004, ' Expression of Melanocarpus albomyces laccase in Trichoderma reesei and characterization of the purified enzyme ', Microbiology, vol. 150, pp. 3065-3074 . https://doi.org/10.1099/mic.0.27147-0
- Publication Year :
- 2004
- Publisher :
- Microbiology Society, 2004.
-
Abstract
- Previous studies onMelanocarpus albomyceslaccase 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 fungusTrichoderma reesei. Two approaches were used: production of a non-fused laccase and a hydrophobin–laccase fusion protein. Both proteins were expressed inT. reeseiunder thecbh1promoter, 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.
- Subjects :
- Trichoderma reesei
Recombinant Fusion Proteins
enzymes
Sordariales
Gene Expression
Biology
Microbiology
laccase
Melanocarpus albomyces laccase
Substrate Specificity
law.invention
Fungal Proteins
chemistry.chemical_compound
law
Enzyme Stability
Papain
Cellulose 1,4-beta-Cellobiosidase
RNA, Messenger
Cloning, Molecular
Promoter Regions, Genetic
Thermostability
Trichoderma
Laccase
chemistry.chemical_classification
Molecular mass
filamentous fungi
Temperature
RNA, Fungal
Hydrogen-Ion Concentration
biology.organism_classification
Fusion protein
Recombinant Proteins
Culture Media
Molecular Weight
Enzyme
Biochemistry
chemistry
Fermentation
gene expression
Recombinant DNA
Subjects
Details
- ISSN :
- 14652080 and 13500872
- Volume :
- 150
- Database :
- OpenAIRE
- Journal :
- Microbiology
- Accession number :
- edsair.doi.dedup.....ab44ef3c3812e45ed1177a2763a96b83