COBRE P3: HUMANIZING ALGINATE DEPOLYMERASE

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 囊性纤维化的过早死亡通常是由于患者呼吸道的慢性铜绿假单胞菌感染所致。与囊性纤维化相关的铜绿假单胞菌黏液样菌株的特征是产生海藻糖胞外多糖，这是一种已知的致病因素。海藻酸裂解酶是一种降解海藻酸聚合物的酶，在囊性纤维化的对症治疗中显示出巨大的前景。然而，这些生物催化剂的微生物来源使其易于获得高水平的免疫原性，并使其在临床环境中的有效性受到质疑。不幸的是，就像许多微生物生物治疗候选者的情况一样，藻酸盐裂解酶没有确切的人类对应物。然而，人类基因组编码了许多具有相似催化功能的酶，即降解基于碳水化合物的生物聚合物。具有相似催化功能但底物选择性不同的人酶的存在表明，可以通过工程人碳水化合物水解酶作用于海藻酸底物来灭活海藻酸裂解酶的治疗活性。具体地说，通过改变底物选择性来定向进化人类酶可以产生一种有效降解藻酸盐治疗靶点的生物催化剂，但由于它来自人类，因此不太可能在人类患者身上产生有害的免疫反应。定向进化将被用来改变人溶菌酶的底物选择性，以产生人源化的藻酸盐解聚酶。这些酶的活性将与真正的细菌藻酸盐裂解酶进行比较，工程酶的免疫原性将相对于野生型人类溶菌酶模板进行评估。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Premature mortality in cystic fibrosis typically results from chronic P. aeruginosa infection of the patient's airways. The mucoid strains of P. aeruginosa associated with cystic fibrosis are characterized by their production of the exopolysaccharide alginate, a known pathogenic factor. Alginate lyases are enzymes that degrade the alginate polymer, and have shown great promise for symptomatic treatment of cystic fibrosis. However, the microbial origin of these biocatalysts predisposes them towards high level immunogenicity, and brings into question their utility in a clinical setting. Unfortunately, as is the case with numerous microbial biotherapeutic candidates, alginate lyases have no exact human counterpart. However, the human genome encodes numerous enzymes with similar catalytic functions, i.e. degradation of carbohydrate-based biopolymers. The existence of human enzymes exhibiting similar catalytic functions but different substrate selectivities suggests that the alginate lyase therapeutic activity could be deimmunized by engineering a human carbohydrate hydrolase to act on the alginate substrate. Specifically, directed evolution of a human enzyme with altered substrate selectivity could yield a biocatalyst that efficiently degrades the alginate therapeutic target, but because of its human origin would be less likely to illicit a deleterious immune response in human patients. Directed evolution will be employed to alter the substrate selectivity of human lysozyme to generate a humanized alginate depolymerase. The activity of these enzymes will be compared to an authentic bacterial alginate lyase, and the immunogenicity of the engineered enzymes will be assessed relative to the wild type human lysozyme template.