Browsing by Subject "Pichia pastoris"

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  • Tuomiranta, Petra (Helsingin yliopisto, 2018)
    Glucuronoyl esterase (GE) is an enzyme produced by plant biomass degrading basidiomycete and ascomycete fungi. GEs have been found to hydrolyse a specific ester bond between lignin alcohols and xylan hemicellulose, which is one of the crosslinks in plant biomass. GEs are the only known enzymes capable of hydrolysing both glycosidically bound and free methyl esters of 4-O-methyl-D-glucuronic acid. In biodegradation of plant biomass, fungal GEs are assumed to cleave the bonds between aromatic lignin alcohols and 4-O-methyl-D-glucuronic acid of hemicellulose. Due to these characteristics, GEs are promising candidates for future industrial applications, such as biofuel production. The genome of white-rot bacidiomycete Dichomitus squalens contains two GE encoding genes. The aim of this work was to express one wild-type GE from D. squalens, DsGE1, and its site-directed mutants as recombinant proteins in the yeast Pichia pastoris. The catalytic activity of the wild-type and mutated recombinant DsGE1 enzymes was examined in optimum conditions with synthetic benzyl glucuronate as a substrate. Recombinant GEs were then exposed to varying of temperature and pH values as well as organic solvents. The aim was to assess whether the mutated DsGE1 enzymes retained a higher GE activity than the wild type enzyme in the tested conditions. Hypothesis of this work was that mutated recombinant DsGE1 enzymes catalyse the hydrolysis of a synthetic benzyl glucuronase. The mutated enzymes were expected to have improved thermal resistance in temperatures higher to the optimum when compared to the wild-type GE. In addition, another hypothesis was that the DsGE1 mutants retain their specific activity better than the wild-type enzyme when pH changes from optimum as well as when the enzymes are exposed to organic solvents. The first research hypothesis was confirmed, while the results of the work disproved the following two hypotheses.
  • Zhang, Angel (Helsingfors universitet, 2016)
    The enzymatic degradation of plant cell walls has being extensively explored during the past decade chiefly due to the current and potential uses of plant biomass found in several industries. In this process, the effective removal of the hemicellulosic portion is of paramount importance. For this purpose, hemicellulases have been intensively studied and produced from bacterial and fungal sources. Within this group of enzymes, mannanases, which are mostly used during the processing of softwood, have been found in several industrial applications. Currently, white rot basidiomycetes have gained increasing attention due to their ability to fully decompose wood. One of these fungi, Dichomitus squalens, has been regarded as an effective wood degrader; nonetheless, its enzymatic arsenal and catalytic potential have only recently been explored. In the present study, six mannan acting enzymes were selected from the genome of D. squalens and heterologously produced using the methylotrophic yeast Pichia pastoris. Four of these enzymes, consisting of two β-1,4-endomannanases, one β-1,4-mannosidase and one α-galactosidase, were successfully produced. The biochemical characterization of the concentrated enzymes using synthetic p-nitrophenyl substrates corresponding to their predicted activities showed optimal pH ranging from 3 to 5 and optimal temperatures ranging from 50 to 60 °C. In addition, three of the enzymes, namely MAN1, MND1 and GAL2, showed high thermostability retaining the majority of their enzyme activities after an incubation of 60 minutes at 60 °C. In contrast, MAN2 lost all of its activity after an incubation of 30 minutes at 60 °C. Furthermore, MAN1 and GAL2 showed optimal enzyme activities of 1922.2 (± 26.6) and 903.7 (± 7.4) U/mL respectively, with specific activities of 11.1 (± 1.0) and 27.9 (± 0.5) U/mg respectively. The four enzymes had a molecular mass ranging from 47 to 110 kDa, without showing evidence that any of them formed dimers or more complex structures. The results of the present study revealed the function of four putative mannan degrading enzymes from D. squalens and provided evidence of its mannolytic capacity. Future studies with other cell wall degrading enzymes or the synergistic effects of characterized enzymes of this species may further reveal its potential as a source of industrial enzymes.
  • Jiang, Xinyu (Helsingin yliopisto, 2015)
    Biofilms constitute a successful protection mechanism for planktonic bacterial cells to survive in hostile environments. To date, biofilm-associated infections in medical devices represent a major cause of morbidity and mortality among patients. As a potential candidate for anti-biofilm therapy, fungal hydrophobins provide new solutions to manipulate the physical and chemical properties of surfaces, which in turn may give protection against bacterial colonization. However, in practice, native hydrophobin coatings generally have no impact on bacterial surface colonization, because of the lack of being antibacterial by these fungal proteins themselves. The aim of this study was to explore the feasibility of using recombinant fusion hydrophobins to control bacterial growth. In this study, the class I hydrophobin hgfI gene isolated from the edible mushroom Grifola frondosa was in frame fused with two antimicrobial peptide genes (bac8c and p11-5), respectively, and subsequently cloned into the corresponding expression vectors with a view to obtain two recombinant fusion hydrophobins, Bac8c-HGFI and P11-5-linker-HGFI. These two chimeric genes were separately expressed in Pichia pastoris under the regulation of alcohol oxidase 1 promoter. SDS-PAGE and immunoblot analyses confirmed that these two fusion proteins were successfully expressed and secreted into the culture medium. Minimum inhibitory concentration (MIC) test demonstrated that the highly active antimicrobial peptide Bac8c became inactivated when it was fused with the hydrophobin HGFI. Interestingly, the hydrophobin HGFI gained an acquired antibacterial nature when it was fused with the antimicrobial peptide P11-5 through a 10-mer flexible polypeptide linker, with the MIC of 100 μg/ml against Escherichia coli. To the best of my knowledge, this study presents the first heterologous expression of an antibacterial fusion hydrophobin in P. pastoris. This finding in combination with surface modification mediated by hydrophobin may broaden the current approaches used for anti-biofilm therapies.
  • Sun, Mengyi (Helsingfors universitet, 2016)
    As the sustainable energy is becoming increasingly important, utilization of lignocellulosic biomass for biofuel production is the central part of this area. Fungal enzymes play an important role in lignocellulose degradation. Glucuronoyl esterase (GE) is a less studied fungal enzyme which degrades the ester linkage between lignin alcohol and hemicellulose side chain 4-O-methyl D-glucuronic acids. Genes encoding GE have been identified from various fungal species and they have been expressed in different production systems to be able to study their biochemical properties in detail. The gene encoding GE from the basidiomycete litter-decomposing fungus Stropharia coronilla was cloned and heterologous expressed in Pichia pastoris yeast. The expression and secretion of GE was induced by growing S. coronilla on lignocellulose supplemented cultivations. ScGE activity can be detected after the fifth day cultivation and it peaked on the 14th day. The heterologous expression of ScGE in P. pastoris showed that ScGE was produced as an enzymatically active protein. The commercial K-URONIC kit supplemented with a GE specific substrate benzyl-D-glucuronate was used to determine GE activity.
  • Salumäe, Astrid (Helsingin yliopisto, 2020)
    In biotechnological protein production and metabolic engineering, regulating the expression of genes is essential. For this, expression systems composed of promoters, terminators and transcription factors are essential. So far, majority of these systems use native promoters and transcription factors. That however rises two problems: 1) these systems usually work in only a set of closely related species, 2) native regulatory components can cause unintended expression levels due to the complexity of cellular regulation. Recently, a synthetic expression system (SES) was established for a wide range of fungal species. The transcription factor used in this system comprises an activation domain that originates from a virus. However, in the field of biotechnology and especially food industry, viral DNA constructs are not favorable because of customer concerns. In this paper, plant-derived activation domains were screened in Trichoderma reesei and Pichia pastoris using mCherry as a target gene for measuring the expression levels. The best expression systems were also tested for protein production in T. reesei and P. pastoris. We tested the production of two different proteins – a bacterial xylanase and a phytase. Two of the novel activation domains provided similar expression levels to the viral activation domain in both fungi. In addition, we developed optimized expression systems for an unconventional yeast from Zygosaccharomyces spp. using the novel transcription factors. The best SES version was used for secretion signal sequence screening for xylanase protein production. To further improve the use of T. reesei as a production host, the CRISPR-Cas9 system with the Cas9 D10A nickase version was tested for transformation of T. reesei. Here, we demonstrated the genomic integration and expression of Cas9 D10A nickase in T. reesei using the SES system with the novel plant-derived activation domain. Furthermore, we successfully transformed the T. reesei Cas9 D10A nickase expressing strain using only guide-RNAs and a donor DNA.
  • Cononi Linares, Nancy; Dilokpimol, Adiphol; Stålbrand, Henrik; Mäkelä, Miia; de Vries, Ronald (2020)
    alpha-Galactosidases are important industrial enzymes for hemicellulosic biomass degradation or modification. In this study, six novel extracellular alpha-galactosidases from Penicillium subrubescens were produced in Pichia pastoris and characterized. All alpha-galactosidases exhibited high affinity to pNP alpha Gal, and only AglE was not active towards galacto-oligomers. Especially AglB and AglD released high amounts of galactose from guar gum, carob galactomannan and locust bean, but combining alpha-galactosidases with an endomannanase dramatically improved galactose release. Structural comparisons to other alpha-galactosidases and homology modelling showed high sequence similarities, albeit significant differences in mechanisms of productive binding, including discrimination between various galactosides. To our knowledge, this is the first study of such an extensive repertoire of extracellular fungal alpha-galactosidases, to demonstrate their potential for degradation of galactomannan-rich biomass. These findings contribute to understanding the differences within glycoside hydrolase families, to facilitate the development of new strategies to generate tailor-made enzymes for new industrial bioprocesses.