Physical and chemical differences between one-stage and two-stage hydrothermal pretreated hardwood substrates for use in cellulosic ethanol production.

BACKGROUND: There are many different types of pretreatment carried out to prepare cellulosic substrates for fermentation. In this study, one- and two-stage hydrothermal pretreatment were carried out to determine their effects on subsequent fermentations. The two substrates were found to behave differently during fermentation. The two substrates were then characterized using physical and chemical parameters. RESULTS: The one-stage substrate was found to have higher carbohydrate content and lower lignin content. It exhibited a higher level of viscosity, a larger settled volume, and a slower settling time than the two-stage substrate. It also showed higher polarity and reduced crystallinity. Glycome profiling showed physical differences between the two substrates, specifically pointing toward higher levels of pectin and hemicellulose in the one-stage substrate (MS1112) as compared to the two-stage substrate (MS1107). CONCLUSIONS: We hypothesize that these physical and chemical differences between the substrates contribute to the differences seen during fermentation including: ethanol yield, ethanol titer, fermentation rate, fermentation completion time, mixing, and substrate solubilization. These findings can be used in optimizing pretreatment parameters to maximize ethanol conversion and overall process yield for hardwood substrates.

Computationally driven deletion of broadly distributed T cell epitopes in a biotherapeutic candidate.

Biotherapeutics are subject to immune surveillance within the body, and anti-biotherapeutic immune responses can compromise drug efficacy and patient safety. Initial development of targeted antidrug immune memory is coordinated by T cell recognition of immunogenic subsequences, termed "T cell epitopes." Biotherapeutics may therefore be deimmunized by mutating key residues within cognate epitopes, but there exist complex trade-offs between immunogenicity, mutational load, and protein structure-function. Here, a protein deimmunization algorithm has been applied to P99 beta-lactamase, a component of antibody-directed enzyme prodrug therapies. The algorithm, integer programming for immunogenic proteins, seamlessly integrates computational prediction of T cell epitopes with both 1- and 2-body sequence potentials that assess protein tolerance to epitope-deleting mutations. Compared to previously deimmunized P99 variants, which bore only one or two mutations, the enzymes designed here contain 4-5 widely distributed substitutions. As a result, they exhibit broad reductions in major histocompatibility complex recognition. Despite their high mutational loads and markedly reduced immunoreactivity, all eight engineered variants possessed wild-type or better catalytic activity. Thus, the protein design algorithm is able to disrupt broadly distributed epitopes while maintaining protein function. As a result, this computational tool may prove useful in expanding the repertoire of next-generation biotherapeutics.