Carbohydrate recognition is an integral part of normal biological processes. It is critical for host-pathogen interactions, biological development, and increasingly important for disease-state biomarker detection. Due to the importance of carbohydrate recognition and variation in host glycosylation, glycans are obvious targets for detection, diagnostic, and therapeutic applications. Not only do glycans serve as important disease biomarkers, they also impact the pharmacological properties of therapeutic biologics. For example, glycan heterogeneity can impact the batch-to-batch consistency, immunogenicity, pharmacokinetics, activity, and biological clearance of recombinant glycoproteins. Given that more than two thirds of therapeutic biologics are glycosylated recombinant proteins, new tools for glycosylation analysis during bioprocess monitoring are required.Reported here is the development of a novel reagent for detecting the core chitobiose component common to all N-linked glycans. Through a combination of computationally guided biocombinatorial library design and in vitro directed evolution, the N-glycan processing enzyme, PNGase F from Flavobacterium meningosepticum, has been engineered into a catalytically inactive protein with enhanced affinity for the substrates of the wild-type enzyme. The engineering of a lectin-like carbohydrate-recognizing biomolecule from a carbohydrate-processing enzyme (a Lectenz) was initiated in silico to determine optimal carbohydrate-enzyme interactions using molecular dynamics simulations. In silico structure/function analyses guided the design of focused biocombinatorial libraries for in vitro directed evolution via yeast-displayed selection of Lectenz candidates. The selected clone, R911, was observed to have a 10x affinity enhancement (KD = 0.26 uM) relative to a non-affinity enhanced control clone (D60A). In addition, enrichment of the N-glycan bearing glycoprotein, Ribonuclease B, and N-glycopeptides was demonstrated via Lectenz affinity chromatography. Furthermore, successful enrichment of glycoproteins from the cell extract of a human breast cancer cell line, MCF7, demonstrated the utility of R911 Lectenz as a capture reagent for the enrichment of glycoproteins from complex mixtures. Molecular modeling of R911 provided insights into mutations critical for affinity and specificity, thus rationalizing experimental observations.The successful creation of the R911 Lectenz reagent presents not only a unique solution to the challenge of glycopeptide and glycoprotein sample enrichment, but also demonstrates a novel strategy for engineering glycan-targeting reagents for glycans and glycoconjugates of biological relevance.