Papers by Helena Nevalainen
Current Advances in Fungal Biotechnology (Part II)
Expression of a shark antibody using Trichoderma reesei as a heterologous host
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Pyranose oxidase (pyranose 2-oxidase, P2O; EC 1.1.3.10) is an enzyme that widely occurs in basidi... more Pyranose oxidase (pyranose 2-oxidase, P2O; EC 1.1.3.10) is an enzyme that widely occurs in basidiomycetous fungi. It catalyzes the C-2 oxidation of several aldopyranoses to form the respective 2-keto derivatives, transferring electrons to molecular oxygen to yield hydrogen peroxide. There is indication of an involvement of P2O in lignocellulose degradation; the exact physiological role of P2O, however, is not clearly understood to date. P2O is a potentially interesting enzyme for biotechnological applications, its reaction products (2-keto sugars) can be attractive intermediates in the production of food additives, such as fructose, tagatose, or isomaltulose, and can easily be produced in high yields. Trametes multicolor is a potent producer of P2O activity and forms P2O constitutively during growth on a number of carbon sources. Although high levels of the enzyme can be produced by fermentation using lactose or whey powder as substrates, production by heterologous expression of the respective gene is an even more attractive alternative. We therefore isolated a clone from a genomic library encoding P2O, including 5´ regulatory regions, and used the sequence information to amplify a cDNA clone by RT-PCR. Delimitation of introns and exons was determined by comparison of the cDNA and genomic sequences. This is the first report of a genomic sequence of a pyranose oxidase. The cDNA was re-amplified with primers containing suitable restriction sites, inserted into the bacterial expression vector pET 21d+ and successfully expressed in E. coli. Purification of the enzyme was done either by an established two-step purification method or by utilizing the poly-His-tag that was fused to the protein via the expression vector. Properties of the heterologous protein and its use in carbohydrate transformations will be discussed.
α-amylase from a filamentous fungus - Ophiostoma floccosum
Ophiostoma floccosum, an ascomycete, is being developed as a new expression system for the produc... more Ophiostoma floccosum, an ascomycete, is being developed as a new expression system for the production of foreign proteins. Enzymes for starch degradation and several proteases are amongst the most efficiently secreted proteins of Ophiostoma. The organism secretes only a few proteins into the culture medium, which provides a considerable advantage for the purification of any recombinant gene product. Several mutants of O. floccosum derived by UV mutagenesis have been isolated and the total the amount of secreted protein was increased by 4 to 6 times. The amylase activity of the best mutant was improved 240-fold compared to the parental strain. The proteinase profiles in the culture supernatants of several key mutants have been characterised for the selection of a suitable expression host for a particular gene product. The regulatory sequences and the protein encoding region of α-amylase, one of the dominant secreted proteins, have been isolated. A series of expression vectors containing the α-amylase regulatory sequences and sequences encoding the mature α-amylase enzyme gene have been constructed. The expression system is being tested using dsRed as a reporter gene.1 page(s
The molecular biology of Trichoderma reesei and its application to biotechnology
Heterologous Gene Expression in Filamentous Fungi: A Holistic View
Applied Mycology and Biotechnology, 2005
... Studies addressing the effect of overexpression of irel (gene involved in activation of trans... more ... Studies addressing the effect of overexpression of irel (gene involved in activation of translation of the transcription factor Haclp) on growth and protein production in T. reesei transformants expressing Phlebia radiata laccase indicated that even though UPR was strongly ...
Molecular Biology of Cellulolytic Fungi
Springer eBooks, 2004
The synthesis, modification and hydrolysis of carbohydrates by glycosidase enzymes are some of th... more The synthesis, modification and hydrolysis of carbohydrates by glycosidase enzymes are some of the fundamental activities in nature. Enzymes responsible for these processes are produced across different organisms, genera and species including the kingdom fungi. Together with bacteria, fungi are responsible for the recycling of nature’s recalcitrant polymers such as lignocellulose which is mainly stored in the plant cell walls. The three main components of a plant cell wall are cellulose, hemicellulose and lignin in a percent ratio of about 40:30:30 (Sjostrom 1981). White rot fungi are capable of degrading all three polymeric substances, including the polyphenolic lignin, whereas brown rot and soft rot fungi prefer the carbohydrate polymers of cellulose, formed of 13-1,4-linked D-glucopyranose units with no side branches and hemicellulose, of which the backbone structure consists of s-1,4-linked D-xylopyranosyl units (xylan) or 13-1,4-linked D-mannose and D-glucose units (mannan) with sugar side chains that may be acetylated and/or methylated (reviewed in Tenkanen 1995). Earlier studies of lignocellulose hydrolysis have mainly concentrated on the biochemistry and molecular biology of cellulose degradation. More recently, the enzymology of lignin degradation (reviewed in Leonowicz et al. 1999) and especially molecular studies on the hydrolysis of hemicellulose have advanced considerably (e.g. de Vries et al. 2002). Xylan degradation has been studied in detail with genes and enzymes from Aspergillus (reviewed in de Vries et al. 2002) and lignin degradation with Phanerochaete chrysosporium (reviewed in Cameron et al. 2000). At present, some 20 enzymes involved in the degradation of lignocellulose have been described. In this chapter, we will concentrate on molecular aspects relating to cellulose hydrolysis.
Application of Genetic Engineering for Strain Improvement in Filamentous Fungi
Mycology [electronic resource], Dec 17, 2003
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Strategies for extending the uses of Trichoderma for enzyme production - an overview
Integration of protein Interaction and Metabolic Data for Subcellular Localization Prediction
... Data for Subcellular Localization Prediction Gaurav Kumar1,2 Adrian P. Cootes1,2 Helena Neval... more ... Data for Subcellular Localization Prediction Gaurav Kumar1,2 Adrian P. Cootes1,2 Helena Nevalainen2 ... [2] Mishra, GR, Suresh, M., Kumaran, K., et al., Human protein reference database-2006 update, Nucleic Acids Res., 34(Database issue):411-414, 2006. ...
Sources, Properties, and Modification of Lignocellulolytic Enzymes for Biomass Degradation
Springer eBooks, 2023
Correlative microscopy: application of electron microscopy finder-grids in quantitative scanning cytometry
Molecular biology of celluloytic fungi
Since publication of the first edition of Volume II in 1995, several developments in fungal molec... more Since publication of the first edition of Volume II in 1995, several developments in fungal molecular biology - such as fungal genome projects - have progressed tremendously. This in turn has affected fundamental genetics as well as biotechnology. To accommodate these developments, the second edition has been completely updated and all chapters have been revised. In addition, the volume contains five new chapters dealing with different aspects of fungal molecular genetics. Topics include: Nuclear and extranuclear genetics; functional genomics; biotechnical genetics; yeasts and filamentous fungi.22 page(s
Improvement of the enzyme producing properties of fungal strains using mutagenesis
Nanoparticles doped with rare earth and transition metal ions: from physics to applications
Current protocols in protein science, Apr 1, 2018
Filamentous fungi are lower eukaryotes increasingly used for expression of foreign proteins rangi... more Filamentous fungi are lower eukaryotes increasingly used for expression of foreign proteins ranging from industrial enzymes originating from other fungi and bacteria to proteins of mammalian origin, such as antibodies and growth factors. Their strengths include an excellent capacity for protein secretion and their eukaryotic protein processing machinery. Proteins secreted from filamentous fungi are modified in the secretory pathway, with folding, proteolytic processing, and addition of glycans being the main modifications. Unlike from many other expression systems, however, plasmids and host strains for expression of gene products in filamentous fungi are not readily available commercially, and the expression system must thus be stitched together in the laboratory. In this overview, the key elements of fungal expression systems are discussed from a practical point of view and with a view towards the future. The principles and considerations presented here can be applied to a range of filamentous fungi.
Rapid Communications in Mass Spectrometry, Apr 18, 2017
RATIONALE: High protein production and secretion with eukaryotic glycosylation machinery make T. ... more RATIONALE: High protein production and secretion with eukaryotic glycosylation machinery make T. reesei RUT-C30 a suitable expression host for recombinant proteins. The N-glycosylation of secreted proteins of RUT-C30 is known to vary depending on culture nutrients but O-glycosylation has been less extensively studied. METHODS: O-Glycans and glycopeptides from secreted proteins were separated by porous graphitised carbon and C-18 liquid chromatography, respectively. O-Glycans were analysed in negative ion mode by electrospray ionisation linear ion trap mass spectrometry and glycopeptides in positive ion mode by electrospray ionisation hybrid quadrupole-orbitrap mass spectrometry. Tandem mass spectrometry was used on O-glycans and glycopeptides including ion trap higher energy collision-induced dissociation (tHCD) to detect glycan fragments not detectable with standard ion trap fragmentation. tHCD allowed targeted MS 3 experiments to be performed on structures containing hexuronic acid, which was not possible with ion trap CID, validating this novel O-glycan composition. Positive mode C18-LC/ESI-MS/MS was used to identify and characterise glycopeptides found to be modified with this class of O-glycans, identifying cellobiohydrolase I as a carrier of these novel O-glycans. RESULTS: Negative mode ion trap higher energy collision-induced dissociation allowed detection and targeted MS 3 experiments to be performed on the hexuronic acid substituent of O-glycan structures, which was not possible with ion trap CID, validating the novel O-glycan composition to include hexuronic acid. Using glycopeptide analysis, this novel O-glycan composition was found to be present on the catalytic domain of cellobiohydrolase I, the most abundant secreted protein by T. reesei. CONCLUSIONS: These are the first reported O-glycans to contain acidic sugars in fungi and they could have significant implications for cellobiohydrolase I structure and activity as well as the activity of recombinant proteins expressed in this host system.
Carbohydrate Polymers, 2018
A new combined physical and enzymatic approach for the hydrolysis of paramylon was developed. ... more A new combined physical and enzymatic approach for the hydrolysis of paramylon was developed. Microwave pretreatment enhance enzymatic hydrolysis of paramylon. Microwave pretreatment of paramylon can be monitored with a new dye-based assay. Soluble β-1,3-glucans with immunostimulatory activity were obtained.
Algal Research, 2017
Euglena gracilis produces several important health-enhancing metabolites including ascorbate, α-t... more Euglena gracilis produces several important health-enhancing metabolites including ascorbate, α-tocopherol and free amino acids (faa). The yield of metabolites is dependent on the strain of E. gracilis and the metabolic growth condition. Here we investigated the effects of photoautotrophic (PT), mixotrophic (MT) and heterotrophic (HT) cultivation on the synthesis of ascorbate, α-tocopherol and faa in E. gracilis var. saccharophila, using label-free shotgun proteomics, and metabolite analysis using colourimetric assay, high-performance and ultra-performance liquid chromatography (HPLC/UPLC). PT cultivation resulted in the production of more antioxidants (up to 4.13 mg g -1 ascorbate and 2.52 mg g -1 α-tocopherol) than the MT and HT growth conditions (up to 0.97 and 0.50 mg g -1 ascorbate, and 1.40 and 0.21 mg g -1 α-tocopherol, respectively). The relative abundance of several faa varied between mid-log and initial stationary growth phases, but the total amount of faa remained about the same, with arginine as the most abundant amino acid. Proteomic analysis revealed a total of 3843 nonredundant proteins in E. gracilis var. saccharophila, of which 1890 were common among all cultivations. Gene ontology annotations suggested derivatisation of metabolic pathways from different organisms, such as lysine biosynthesis from fungi and serine biosynthesis from plants, while a few pathways were unique to Euglena, such as those of ascorbate and arginine. Some enzymes exhibited several isoforms that were influenced by the metabolic growth condition. For example, one of the isozymes of threonine aldolase was expressed in HT/MT cultures only, and one of the isozymes of phosphoglycerate dehydrogenase was expressed in PT cultures only. This is the first proteomic study of E. gracilis var. saccharophila, which provides a mechanistic insight into the biosynthetic pathway dynamics of primary metabolites (antioxidants and faa). This new information can serve as a framework for further development of Euglena as a producer of nutraceuticals.
Expression of a Humicola grisea var. thermoidea xylanase in Trichoderma reesei
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Papers by Helena Nevalainen