Browsing by Subject "substrate specificity"

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  • Huang, Liyang (Helsingfors universitet, 2016)
    Plant biomass consists largely of polymeric compounds of which diverse polysaccharides are the main components. Ferulic acid is a ubiquitous phenolic phytochemical in plant cell wall and forms the linkage between plant cell wall polymers. Therefore it is a major aspect in the recalcitrance of cell wall against microbial attack. Ferulic acid esterases (FAEs) are hydrolytic enzymes which participate in plant biomass degradation by removing ferulic acid from the polysaccharides in order to weaken the integrity of the cell wall. By using phylogenetic gene prediction strategy, three putative FAE gene models have been detected from the genome of the ascomycete fungus Aspergillus niger. The codon optimized putative FAE encoding genes have been synthetized for heterologous production in Pichia pastoris GS115. In this work, these three FAEs of A. niger, i.e. FAE796, FAE807 and FAE809, were produced in P. pastoris and their biochemical properties were characterized. The properties included substrate profiling, thermostability, pH optimum and solvent tolerance of the recombinant FAEs. The three A. niger FAEs were successfully produced in P. pastoris resulting as approximately 57 kDa molecular mass proteins. Substrate profiling was performed by using a set of 11 synthetic FAE model substrates and substrates for tannase and lipase activity. FAE796 and FAE809 preferred methoxy substrates, and thus were likely to belong to type A class of FAEs. FAE807 had activity towards a wider range of substrates including methyl sinapate, methyl cinnamate, chlorogenic acid and para-nitrophenyl ferulate, suggesting it to belong to type C class of FAEs. In addition, FAE807 had tannase activity which is a novel property described among the FAEs studied so far. The optimal temperature for FAE796, FAE807 and FAE809 were +37 °C, +55 °C and +55 °C, respectively. FAE809 was the most thermostable enzyme, and retained half of its activity up to +60 °C for 60 min. The studied FAEs were most active at pH 4.0-5.0. FAE809 was relatively stable towards the studied solvents retaining 70%-91% of its activity after solvent treatment.
  • Oeemig, Jesper S.; Beyer, Hannes M.; Aranko, A. Sesilja; Mutanen, Justus; Iwai, Hideo (2020)
    Inteins catalyze self-excision from host precursor proteins while concomitantly ligating the flanking substrates (exteins) with a peptide bond. Noncatalytic extein residues near the splice junctions, such as the residues at the -1 and +2 positions, often strongly influence the protein-splicing efficiency. The substrate specificities of inteins have not been studied for many inteins. We developed a convenient mutagenesis platform termed "QuickDrop"-cassette mutagenesis for investigating the influences of 20 amino acid types at the -1 and +2 positions of different inteins. We elucidated 17 different profiles of the 20 amino acid dependencies across different inteins. The substrate specificities will accelerate our understanding of the structure-function relationship at the splicing junctions for broader applications of inteins in biotechnology and molecular biosciences.
  • Sultana, Dalia Mrs (Helsingin yliopisto, 2021)
    Anthocyanins are an important class of flavonoids under the class of phenolic compounds and contribute to flower color variation. Gerbera hybrida is a flowering plant of Asteraceae family having mainly two colors of flowers – orange and red. Dihydroflavonol 4-reductase (DFR) is a key enzyme catalyzing a reaction in anthocyanin biosynthesis, the reduction of dihydroflavonols to leucoanthocyanidins. GDFR1-2 and GDFR1-3 are two allelic forms of gerbera DFR differing in substrate specificity for the dihydroflavonols - dihydrokaempferol, dihydroquercetin and dihydromyricetin and also differ in 13 amino acids where eight are considered to be important for substrate specificity. GDFR1-2 has strong preference for dihydrokaempferol and GDFR1-3 doesn’t have any preference for the three substrates. In order to find out the amino acids responsible for substrate specificity, swap mutations were generated between GDFR1-2 and GDFR1-3 by two PCR methods– first, running separate PCR from the templates of GDFR1-2 and GDFR1-3, making a heteroduplex by mixing separate PCR where non-matching nucleotides are expected to be corrected by E. coli and, second, by running PCR from mixed templates with short extension time of PCR to make swaps by template switching. The second method was found more effective than the first method. 81 lines (named GDAT1-81) were sequenced and 35 unique swap mutants were found. In this work the DFR assay was done from six randomly picked GDAT lines where GDAT5 had a swap in one amino acid showing still a similar pattern of substrate specificity as the reference (GDFR1-3) indicating that the mutated amino acid doesn’t have any role in substrate specificity. GDAT14 had an extra mutation (S167P) along with 2 swaps showing incapability of reducing dihydrokaempferol, demonstrating that the mutated amino acids are important and other 4 lines were identical to either GDFR1-2 or to GDFR1-3. This was a preliminary test with 6 lines. In order to get more explanations about the roles of amino acids in substrate specificity, DFR assay was done for all the 81 lines in experiments outside of this thesis and five patterns of substrate specificity were identified indicating that substrate specificity of DFR can be altered by changing only three important amino acids. The amino acids at the position 85,135 and 181 in DFR coding sequence have been identified having important roles in substrate specificity. In addition, the amino acid at position 167 may have a function in making the gerbera DFR able to reduce dihydrokaempferol.