Measurement of amyloglucosidase using P-nitrophenyl β-maltoside as substrate.
McCleary, B. V., Bouhet, F. & Driguez, H. (1991). Biotechnology Techniques, 5(4), 255-258.
An enzyme-linked assay for the measurement of amyloglucosidase in commercial enzyme mixtures and crude culture filtrates is described. A method for the synthesis of the substrate employed, p-nitrophenyl β-D-maltoside, is also described. The substrate is used in the presence of saturating levels of β-glucosidase. With a range of Aspergillus sp. culture filtrates, an excellent correlation was found for values obtained with this assay and a conventional assay employing maltose as substrate with measurement of released glucose.
Functionality of selected strains of moulds and yeasts from Vietnamese rice wine starters.
Dung, N. T. P., Rombouts, F. M. & Nout, M. J. R. (2006). Food Microbiology, 23(4), 331-340.
The role of starch-degrading mycelial fungi, and the alcohol production and ethanol tolerance of the yeasts isolated from selected Vietnamese traditional rice wine starters were examined, and optimum conditions for these essential steps in rice wine fermentation were determined. Of pure isolates from Vietnamese rice wine starters, mould strains identified as Amylomyces rouxii, Amylomyces aff. rouxii, Rhizopus oligosporus and Rhizopus oryzae, were superior in starch degradation, glucose production and amyloglucosidase activity during the saccharification of purple glutinous rice. A. rouxii was able to produce up to 25%w/w glucose with an amyloglucosidase activity up to 0.6 U g-1 of fermented moulded mass. Five yeast isolates identified as Saccharomyces cerevisiae were selected for their superior alcohol productivity. They were able to deplete a relatively high initial percentage of glucose (20% w/v), forming 8.8% w/v ethanol. The ethanol tolerance of S. cerevisiae in challenge tests was 9–10% w/v, and 13.4% w/v as measured in fed-batch fermentations. Optimum conditions for the saccharification were: incubation for 2 d at 34°C, of steamed rice inoculated with 5 log cfu g-1; for the alcoholic fermentation 4 d at 28.3°C, of saccharified rice liquid inoculated with 5.5 log cfu mL-1.
Biomimetic silica encapsulation of enzymes for replacement of biocides in antifouling coatings.
Kristensen, J. B., Meyer, R. L., Poulsen, C. H., Kragh, K. M., Besenbacher, F. & Laursen, B. S. (2010). Green Chemistry, 12(3), 387-394.
Current antifouling technologies for ship hulls are based on metals such as cuprous oxide and co-biocides like zinc pyrithione. Due to the persistent adverse environmental effects of these biocides, enzyme-based antifouling paints are proposed as a bio-based, non-accumulating alternative. Here, a hydrogen peroxide-producing system composed of hexose oxidase (HOX, EC 18.104.22.168), glucoamylase (GA, EC 22.214.171.124) and starch is tested for the chemical and physical functionalities necessary for successful incorporation into a marine coating. The activity and stability of the enzymes in seawater was evaluated at different temperatures, and paint compatibility was assessed by measuring the distribution and activity of the enzymes incorporated into prototype coating formulations. We used a biomimetic encapsulation procedure for HOX through polyethylenimine-templated silica co-precipitation. The co-precipitation and formulation of a powder for mixing into a marine paint was performed in a one-step economical and gentle formulation process, in which silica co-precipitated HOX was combined with GA and starch to form the antifouling system. Silica co-precipitation significantly improved the stability and performance of the antifouling system in marine-like conditions. For example, encapsulation of HOX resulted in 46% higher activity at pH 8, and its stability in artificial seawater increased from retaining only 3.5% activity after 2 weeks to retaining 55% activity after 12 weeks. A coating comprising the full enzyme system released hydrogen peroxide at rates exceeding a target of 36 nmol cm-2 d-1 for 3 months in a laboratory assay, and had potential for prolonged action through incorporation in a self-polishing coating.
Development of defined mixed-culture fungal fermentation starter granulate for controlled production of rice wine.
Dung, N. T. P., Rombouts, F. M. & Nout, M. J. R. (2005). Innovative Food Science & Emerging Technologies, 6(4), 429-441.
As a first step in the development of defined fungal starter granules for controlled winemaking from purple glutinous rice, the interaction of moulds and yeasts isolated from Vietnamese rice wine starters and the effect of some representative oriental herbs on the growth of moulds and yeasts were examined. Amylomyces rouxii and Saccharomyces cerevisae were shown to be compatible in mixed cultures, and the herbs “Tieu Hoi” (Fennel: Foeniculum vulgare Miller) and “Dinh Huong” (Clove: Syzygium aromaticum L.) which are used as supplementary ingredients by some local starter producers, were observed to stimulate the mould and yeast growth. Based on traditional starter manufacturing methods and modified on the basis of optimization experiments, a laboratory-scale manufacturing process for defined mixed-culture starter granules was established. In accordance with the national standard method, the wine produced with new experimental starter granules was found to have superior flavour and overall acceptability, compared with local commercial rice wines. Industrial relevance. One of the major problems faced by commercial brewers of rice wine in Vietnam, is the variable quality and performance of the traditional starter tablets that are commonly used. The relevance of the present paper is that a stable, granulated starter has been developed, containing a defined mixture of mould and yeast cultures. This has proven to be shelf stable for more than 3 months, producing a very well accepted quality of wine.
Comparative assessment of amylolytic and cellulolytic enzyme activity of malts prepared from tropical cereals.
Dziedzoave, N. T., Graffham, A. J., Westby, A. & Komlaga, G. (2010). Food Control, 21(10), 1349-1353.
The activity levels of amylolytic enzymes and β-glucanase in malts prepared from four tropical cereal grains were assessed to establish the relative usefulness of these malts for production of glucose syrups. Rice malt showed the highest activity for the amylolytic enzymes, whilst millet and sorghum malts were richest in β-glucanase activity. Optimum amylolytic enzyme development in rice malts occurred between 9–13 days; and 11 days for optimum β-glucanase development in millet malt. β-Amylase was the predominant enzyme in all the cereal malts except maize, for which the predominant enzyme was α-amylase. Options for optimising the production of enzymes from the three cereals, and their potential for use in the production of glucose syrups are discussed.
Microfluidic screening and whole-genome sequencing identifies mutations associated with improved protein secretion by yeast.
Huang, M., Bai, Y., Sjostrom, S. L., Hallström, B. M., Liu, Z., Petranovic, D., Uhlén, M., Joensson, H. N., Andersson-Svahn, H. & Nielsen, J. (2015). Proceedings of the National Academy of Sciences, 112(34), E4689-E4696.
There is an increasing demand for biotech-based production of recombinant proteins for use as pharmaceuticals in the food and feed industry and in industrial applications. Yeast Saccharomyces cerevisiae is among preferred cell factories for recombinant protein production, and there is increasing interest in improving its protein secretion capacity. Due to the complexity of the secretory machinery in eukaryotic cells, it is difficult to apply rational engineering for construction of improved strains. Here we used high-throughput microfluidics for the screening of yeast libraries, generated by UV mutagenesis. Several screening and sorting rounds resulted in the selection of eight yeast clones with significantly improved secretion of recombinant α-amylase. Efficient secretion was genetically stable in the selected clones. We performed whole-genome sequencing of the eight clones and identified 330 mutations in total. Gene ontology analysis of mutated genes revealed many biological processes, including some that have not been identified before in the context of protein secretion. Mutated genes identified in this study can be potentially used for reverse metabolic engineering, with the objective to construct efficient cell factories for protein secretion. The combined use of microfluidics screening and whole-genome sequencing to map the mutations associated with the improved phenotype can easily be adapted for other products and cell types to identify novel engineering targets, and this approach could broadly facilitate design of novel cell factories.