Biotechnology Letters

A novel antifungal protein, Mr = ca. 40 kDa, was isolated from pumpkin rind and designated Pr-1. When purified by anion exchange chromatography and HPLC, it inhibited growth of several fungi including Botrytis cinerea, Fusarium oxysporum, Fusarium solani and Rhizoctonia solani, as well as the yeast, Candida albicans, at 10–20 μM. It did not inhibit growth of Escherichia coli or Staphylococcus aureus even at 200 μM. Laser scanning microscopy of fungal cells exposed to rhodamine-labeled Pr-1 revealed that the protein accumulated and was localized on the cell surface. Uptake of the vital stain, SYTOX Green, was enhanced when fungal conidia were treated with Pr-1 suggesting that the protein has membrane permeabilization activity. Pr-1 was thermostable at 70°C and did not lyse human red blood cells at 128 μM suggesting that the protein may be useful as an antifungal agent with little, if any human cytotoxicity.

A Technicon AutoAnalyzer has been adapted to analyse lysine of fermentation broths from lysine-producer mutants, with a photometric method with ninhydrin. Linearity and accuracy have been tested, with errors lower than 5.5%. The great advantadge is its speed, analysing one sample every 5 minutes, automatically.

The aim of this article is to review how yeast has contributed to contemporary biotechnology and to seek underlying principles relevant to its future exploitation for human benefit. Recent advances in systems biology combined with new knowledge of genome diversity promise to make yeast the eukaryotic workhorse of choice for production of everything from probiotics and pharmaceuticals to fuels and chemicals. The ability to engineer new capabilities through introduction of controlled diversity based on a complete understanding of genome complexity and metabolic flux is key. Here, we briefly summarise the history that has led to these apparently simple organisms being employed in such a broad range of commercial applications. Subsequently, we discuss the likely consequences of current yeast research for the future of biotechnological innovation.

While the most accurate method for analysis of sugars in biomass is based on gas chromatography of trimethylsilane or alditol acetate derivatives of sugars, the derivation method is time consuming and laborious. In comparison, sample preparation for sugar analysis of hydrolyzed biomass samples using liquid chromatography is a simple dilution procedure with water. A gradient HPLC method using a anion-exchange column and pulsed-amperometric detection modified to reduce analysis time from 75 to 40 min was further improved. The new method no longer requires post-column addition to stablilize the baseline using a pulsed-amperometric detector with the mobile phase gradient. The method provides good resolution of arabinose, rhamnose, galactose, xylose, glucose, fructose, sucrose, cellobiose, and galacturonic acid in both standards and hydrolyzed citrus waste materials. By changing the waveform used with the PAD detector, the requirement for post-column addition was eliminated while maintaining a stable baseline.

The 3C-like protease (3CLpro) of severe acute respiratory syndrome associated coronavirus (SARS-CoV) is vital for SARS-CoV replication and is a promising drug target. Recombinant 3CLpro was expressed in Pichia pastoris GS115 as a 42 kDa protein that displayed a K m of 15 ± 2 μM with Dabcyl-KTSAVLQSGFRKME-Edans as substrate. Purified 3CLpro was used for inhibition and kinetic assays with seven flavonoid compounds. The IC50 of six flavonoid compounds were 47–381 μM. Quercetin, epigallocatechin gallate and gallocatechin gallate (GCG) displayed good inhibition toward 3CLpro with IC50 values of 73, 73 and 47 μM, respectively. GCG showed a competitive inhibition pattern with K i value of 25 ± 1.7 μM. In molecular docking experiments, GCG displayed a binding energy of −14 kcal mol−1 to the active site of 3CLpro and the galloyl moiety at 3-OH position was required for 3CLpro inhibition activity.

Chinese hamster ovary (CHO) cells are the leading cell factories for producing recombinant proteins in the biopharmaceutical industry. In this regard, constraint-based metabolic models are useful platforms to perform computational analysis of cell metabolism. These models need to be regularly updated in order to include the latest biochemical data of the cells, and to increase their predictive power. Here, we provide an update to iCHO1766, the metabolic model of CHO cells. We expanded the existing model of Chinese hamster metabolism with the help of four gap-filling approaches, leading to the addition of 773 new reactions and 335 new genes. We incorporated these into an updated genome-scale metabolic network model of CHO cells, named iCHO2101. In this updated model, the number of reactions and pathways capable of carrying flux is substantially increased. The present CHO model is an important step towards more complete metabolic models of CHO cells.

Based on DNA-cloning analysis, the microbial community of a phototrophic sludge producing H2 from an acidified wastewater was composed of 81% of a species resembling Rhodobacter capsulatus (with 99.2% similarity) and two unidentified species of the Bacillus/Clostridium group. The sludge produced a biogas comprising 82 ± 2% H2, 13 ± 2% CO2, 4.5 ± 0.5% N2, and 0.5 ± 0.2% H2S.