An unstudied β-N-acetylhexosaminidase (SnHex) from the soil bacterium Stackebrandtia nassauensis was successfully cloned and subsequently expressed as a soluble protein in Escherichia coli. Activity tests and the biochemical characterization of the purified protein revealed an optimum pH of 6.0 and a robust thermal stability at 50 °C within 24 h. The addition of urea (1 M) or sodium dodecyl sulfate (1% w/v) reduced the activity of the enzyme by 44% and 58%, respectively, whereas the addition of divalent metal ions had no effect on the enzymatic activity. PUGNAc (O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate) strongly inhibited the enzyme in sub-micromolar concentrations. The β-N-acetylhexosaminidase was able to hydrolyze β1,2-linked, β1,3-linked, β1,4-linked, and β1,6-linked GlcNAc residues from the non-reducing end of various tested glycan standards, including bisecting GlcNAc from one of the tested hybrid-type N-glycan substrates. A mutational study revealed that the amino acids D306 and E307 bear the catalytically relevant side acid/base side chains. When coupled with a chitinase, the β-N-acetylhexosaminidase was able to generate GlcNAc directly from colloidal chitin, which showed the potential of this enzyme for biotechnological applications.

Sialic acids are a family of acidic monosaccharides often found on the termini of cell surface proteins or lipid glycoconjugates of higher animals. Herein we describe the enzymatic synthesis of the two isotopically labeled sialic acid derivatives d₃-X-Gal-α-2,3-Neu5Ac and d₃-X-Gal-α-2,3-Neu5Gc. Using deuterium oxide as the reaction solvent, deuterium atoms could be successfully introduced during the enzymatic epimerization and aldol addition reactions when the sialosides were generated. NMR and mass spectrometric analyses confirmed that the resulting sialosides were indeed tri-deuterated. These compounds may be of interest as internal standards in liquid chromatography/mass spectrometric assays for biochemical or clinical studies of sialic acids. This was further exemplified by the use of this tri-deuterated sialosides as internal standards for the quantification of sialic acids in meat and egg samples.

Reductive alkylation of the carbonyl group of carbohydrates with fluorescence or ionizing labels is a prerequisite for the sensitive analysis of carbohydrates by chromatographic and mass spectrometric techniques. Herein, 1-(2-aminoethyl)-3-methyl-1H-imidazol-3-ium tetrafluoroborate ([MIEA][BF₄]) was successfully synthesized using tert-butyl N-(2-bromoethyl)carbamate and N-methylimidazole as starting materials. MIEA⁺ was then investigated as a multifunctional oligosaccharide label for glycan profiling and identification using LC-ESI-ToF and by MALDI-ToF mass spectrometry. The reductive amination of this diazole with carbohydrates was exemplified by labeling N-glycans from the model glycoproteins horseradish peroxidase, RNase B, and bovine lactoferrin. The produced MIEA⁺ glycan profiles were comparable to the corresponding 2AB labeled glycan derivatives and showed improved ESI-MS ionization efficiency over the respective 2AB derivatives, with detection sensitivity in the low picomol to the high femtomol range.

Fibroblast growth factor 21 (FGF21), a hormone-like protein, plays a crucial role in enhancing glucose and lipid metabolism, offering promising therapeutic avenues for conditions such as nonalcoholic steatohepatitis and severe hypertriglyceridemia. Despite its potential, this protein’s limited stability and brief half-life pose significant challenges for its use in clinical settings. In this study, we created an FGF21 analog (named FGF21-164) that is a mutant of FGF21 and fused it with the tandem repeat sequence of human CD164. FGF21-164, characterized by extensive glycosylation and sialylation, exhibits enhanced pharmacokinetic properties, particularly in terms of its significantly longer half-life compared to its native form. The in vitro efficacy of FGF21-164 was evaluated using 3T3-L1-induced adipocytes. The protein demonstrated a dose-dependent increase in glucose uptake and effectively decreased lipid droplet accumulation surrounding the adipocytes. The in vivo activity of FGF21-164 was evaluated in leptin-deficient (ob/ob) and diet-induced obesity (DIO) mice. A single subcutaneous dose of FGF21-164 led to a rapid decrease in blood glucose levels and sustained normal fasting glucose levels for up to 28 days. Additionally, repeated dosing of FGF21-164 significantly curbed weight gain and reduced hepatic fat accumulation in DIO mice.

In this study, a novel protein glutaminase derived from Chryseobacterium lactis CGMCC 33780 (CLPG) was successfully purified via a one-step cell surface display approach, yielding its mature form. Subsequently, the enzymatic properties of CLPG were characterized. It exhibited optimal activity at a pH of 5 and a reaction temperature of 50 °C, and retained over 70% of its activity after a 12 h incubation at 50 °C. The study further investigated the impact of CLPG-mediated deamidation on the structural and functional attributes of krill protein isolate (KPI). A comprehensive analysis was conducted on the deamidation extent, conformational alterations, and microstructural morphology of KPI, employing techniques such as FTIR, CD, DSC, and SEM. After deamidation treatment with CLPG, the foaming and emulsifying properties of KPI were moderately shifted. When the CLPG dosage was 1.0 U/g with a corresponding deamidation degree of 15.18%, the emulsifying property of KPI reached the maximum value of 23%. These property enhancements were possibly primarily attributed to the increased electrostatic repulsion and hydrophobicity induced during the deamidation process. This work not only pioneers a novel method for the expression and purification of protein glutaminase but also applies it to the modification of krill protein, offering fresh insights for the development and application of protein glutaminases.