Comparison of H2O2-producing enzymes in selected white rot fungi

Using fungi grown on synthetic agar medium, we evaluated and compared the concentration of various H2O2-producing enzymes. Our results showed that oxidase production in solid medium was better than that found in liquid medium and as high as that detected in wood samples. High yields of oxidases made it possible to compare different oxidases in the same culture extracts and under different conditions. Our results also indicated that H2O2 production is ubiquitous in the white rot fungi tested and that enzyme levels are influenced by the substrate composition.Articl

One-step enzymatic hydrolysis of starch using a recombinant strain of Saccharomyces cerevisiae producing α-amylase, glucoamylase and pullulanase

A recombinant strain of Saccharomyces cerevisiae was constructed that contained the genes encoding a bacterial α-amylase (AMY1), a yeast glucoamylase (STA2) and a bacterial pullulanase (pulA). The Bacillus amyloliquefaciens α-amylase and S. cerevisiae var. diastaticus glucoamylase genes were expressed in S. cerevisiae using their native promoters and the encoded enzymes secreted under direction of their native leader sequences. In contrast, the Klebsiella pneumoniae pullulanase gene was placed under the control of the Least alcohol dehydrogenase gene promoter (ADC1(P)) and secreted using the yeast mating pheromone α-factor secretion signal (MFα1(S)). Transcription termination of the pullulanase gene was effected by the yeast tryptophan synthase gene terminator (TRP5(T)), whereas termination of the glucoamylase and α-amylase genes was directed by their native terminators. Pullulanase (PUL1) produced by recombinant yeasts containing ADC1(P) MFα1(S) pulA TRP5(T) (designated PUL1) was further characterized and compared to its bacterial counterpart (PulA). The different genes were introduced into S. cerevisiae in different combinations and the various amylolytic Saccharomyces transformants compared to Schwanniomyces occidentalis. Introduction of PUL1 into a S. cerevisiae strain containing both STA2 and AMY1, resulted in 99% assimilation of starch.Articl

Expression of the Klebsiella pneumoniae pullulanase-encoding gene in Saccharomyces cerevisiae

A 3800-base pair (bp) DNA fragment encoding the mature pullulanase from Klebsiella pneumoniae was inserted between two different yeast expression-secretion cassettes and an yeast gene terminator. These cassettes were cloned into an yeast centromeric plasmid YCplacIII and transformed into laboratory strains of Saccharomyces cerevisiae. Transcription initiation signals were derived from the mating pheromone α-factor (MFα1(p)) and alcohol dehydrogenase (ADC1(p)) gene promoters. Secretion of pullulanase was directed by the leader sequence of the yeast mating pheromone α-factor (MFα1(s)). Transcription termination was effected by the yeast tryptophan synthase gene terminator (TRP5(r)). Southernblot analysis confirmed the presence of pulA in transformed yeasts and Northern-blot analysis revealed the presence of PUL1 mRNA. A pullulan agarose assay indicated the extracellular production of biologically active pullulanase by S. cerevisiae.Articl

Expression of the Klebsiella pneumoniae pullulanase-encoding gene in Saccharomyces cerevisiae

A 3800-base pair (bp) DNA fragment encoding the mature pullulanase from Klebsiella pneumoniae was inserted between two different yeast expression-secretion cassettes and an yeast gene terminator. These cassettes were cloned into an yeast centromeric plasmid YCplacIII and transformed into laboratory strains of Saccharomyces cerevisiae. Transcription initiation signals were derived from the mating pheromone α-factor (MFα1(p)) and alcohol dehydrogenase (ADC1(p)) gene promoters. Secretion of pullulanase was directed by the leader sequence of the yeast mating pheromone α-factor (MFα1(s)). Transcription termination was effected by the yeast tryptophan synthase gene terminator (TRP5(r)). Southernblot analysis confirmed the presence of pulA in transformed yeasts and Northern-blot analysis revealed the presence of PUL1 mRNA. A pullulan agarose assay indicated the extracellular production of biologically active pullulanase by S. cerevisiae.Articl

Utilising Grape Pomace for Ethanol Production

Chemical analyses of grape pomace revealed the presence of significant amounts of fermentable sugars that are retained in the pomace after pressing of the grapes. Furthermore, treatment of the pomace with purified hydrolases indicated that the enzymatic biodegradation of the pomace could release additional fermentable sugars. We isolated and evaluated yeast strains associated with grape pomace for their ability to hydrolyse the complex polysaccharides found in grape pomace and to utilise the fermentable sugars for the production of ethanol. Two Pichia rhodanensis isolates were able to partially hydrolyse the pomace polysaccharides, but fermentation of the pomace resulted only in a small increase in the amount of ethanol produced. The study revealed that significant amounts of ethanol could be obtained from the residual sugars associated with grape pomace. However, the complex structure of the pomace polysaccharides apparently renders it unsusceptible to efficient hydrolysis under fermentative conditions

Comparative studies of lignin peroxidases and manganese-dependent peroxidases produced by selected white rot fungi in solid media

The effect of various incubation conditions and media composition on ligninolytic activity by selected strains of white-rot fungi was determined in solid media. When compared to conventional methods using liquid media or woody substrates, this method is fast, simple and also quantitative. Manganese-dependent peroxidase was easily detected in all strains studied. However, detection of lignin peroxidase required optimisation of both growth medium and enzyme assay conditions. Using this method, we showed that the role of nitrogen and oxygen in ligninolytic activity varies and that conditions must be optimised for each individual even within the same species. Furthermore, several white rot fungi produced manganese-dependent peroxidase during the primary growth phase.Articl