SBIR Phase I: A Novel Probe for Biofilms

这项小企业创新研究（SBIR）第一阶段项目的更广泛影响将是改善纸浆和造纸厂的业绩，特别是农村地区的小型造纸厂，并提高其产品的质量。木材的主要成分是纤维素（用于造纸）、半纤维素和木质素（随着时间的推移，纸张会变黄变脆）。去除木质素是造纸的关键，但它通常附着在半纤维素上。该项目推进了一项测量木浆中木质素残留量的技术。从长远来看，探针还可以检测生物膜，生物膜是由碳水化合物和其他物质组成的层，细菌和真菌可以在其中生长。水处理设施和植物源材料的加工者经常发现，微生物自然存在于其原料中，形成生物膜，可以积聚在机器上或污染产品。这项技术有广泛的应用。提出的项目创建一个肽探针非糖苷键到低聚或多糖的木糖残基。这些键已知存在于木材中，也可能存在于其他木本植物和生物膜中。该项目采用了已知的唯一能破坏这些键的酶，XLE（木聚糖：木质素醚酶），以创建肽探针来指示它们的存在。XLE与已知木糖苷酶的同源性使得鉴定很可能参与催化活性的残基成为可能。这些残基将成为去除催化活性的修饰目标，同时保留与XLE的非糖苷底物结合的能力。这些修饰将使用分子生物学的标准技术进行，并使用一种新的指示底物对XLE活性进行评估。XLE与已知糖苷酶的同源性也允许鉴定酶的底物的潜在结合位点。将合成具有与该序列对应的可见标记的肽，但包括去除催化活性的序列变化。生成的肽将被测试，看它是否与木浆和生物膜发生反应。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

The broader impact of this Small Business Innovation Research (SBIR) Phase I project will be to improve performance of pulp and paper mills, especially small mills in rural areas, and to improve the quality of their products. The main components of wood are cellulose (used for paper), hemicellulose, and lignin (which makes paper turn yellow and brittle with age). Removal of lignin is key to papermaking, but it is typically attached to the hemicellulose. The project advances a technology to measure how much lignin is left in wood pulp. In the longer term, the probe may also detect biofilms which are layers of carbohydrates and other materials in which bacteria and fungi can grow. Water treatment facilities and processors of plant-derived materials routinely find that microorganisms naturally present in their starting materials form biofilms that can accumulate on machinery or contaminate the products. This technology has wide-ranging applications. The proposed project creates a peptide probe for non-glycosidic bonds to xylose residues of oligo- or polysaccharides. These bonds are known to exist in wood, and may exist in other woody plants and biofilms. The project employs the only enzyme known to break these bonds, XLE (xylan:lignin etherase) in order to create a peptide probe to indicate their presence. XLE’s homology to known xylosidases has permitted the identification of residues very likely to participate in the catalytic activity. These residues will be targets of modification that remove catalytic activity while retaining the ability to bind to XLE’s non-glycosidic substrate. The modifications will be performed using standard techniques of molecular biology and evaluated using with a novel indicating substrate for XLE activity. XLE’s homology with known glycosidases has also allowed identification of the enzyme’s potential binding site for its substrate. A peptide with an attached visible marker corresponding to this sequence but including the sequence changes that remove the catalytic activity will be synthesized. The resulting peptide will be tested to see if it reacts with wood pulp and biofilms.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.