Analysis of unsaturated alginate oligosaccharides using high-performance anion exchange chromatography coupled with mass spectrometry.

Alginate is a commercially important polysaccharide composed of mannuronic acid and its C5 differential isomer guluronic acid. Comprehensive research on alginate and alginate lyases requires efficient and precise analytical methods for alginate oligosaccharides. In this research, high-performance anion exchange chromatography (HPAEC) in parallel with pulsed amperometric detection (PAD) and mass spectrometry (MS) was applied to the analysis of oligosaccharides obtained by alginate lyase. By optimizing the chromatographic conditions including mobile phase concentration, flow rate, and elution gradient, the analysis of a single sample could be completed in 30 min. Seven unsaturated alginate oligosaccharides were separated and identified through their analysis time observed with PAD, including all structurally different unsaturated disaccharides and trisaccharides. The quantitative analysis of seven oligosaccharides was performed based on the quantitative capability of PAD. The method exhibited adequate linearity and precision parameters. All the calibration curves showed good linearity at least in the concentration range of 0.002 to 0.1 mg/mL. The HPAEC-PAD/MS method provides a general and efficient online method to analyze alginate oligosaccharides.

Characterization and elucidation of a novel M-specific alginate lyase Aly7Aq with strict recognition at subsites ±2.

A novel alginate lyase Aly7Aq was cloned and heterologous expressed by a combination of bioinformatics and molecular biology. Aly7Aq was an M-specific alginate lyase, exhibiting optimum reaction conditions at 50 °C and pH 10.0. Aly7Aq was determined to degrade polysaccharides in a random endo-acting manner. The minimum reaction substrate was tetrasaccharide, and Aly7Aq mainly attacked the third glycosidic linkage from the reducing end of oligosaccharide substrates. The disaccharide product of Aly7Aq was ΔM and the trisaccharide products were ΔMM and ΔMG, which differed from all previously characterized M-specific alginate lyases. The degradation products demonstrated that the ±2 subsites of Aly7Aq strictly recognized M units, while the -1 subsite accommodated both M and G units. Therefore, the substrate specificity of Aly7Aq was derived from the specificity of ±2 subsites. This is the first report on the specificity at subsite ±2 of M-specific alginate lyase. The novel M-specific Aly7Aq could serve as a potential tool in the specific degradation of alginate and targeted preparation of oligosaccharide.

Action Pattern of a Novel G-Specific Alginate Lyase: Determination of Subsite Specificity by HPAEC-PAD/MS.

G-specific alginate lyases are important tools for alginate fragment biodegradation and oligosaccharide production, which have great potential in alginate refining research. In this research, a novel G-specific alginate lyase Aly7Ce was cloned, expressed, and characterized, with the optimal reaction conditions at 30 °C and pH 8.0. By employing the UPSEC-VWD-MS method, Aly7Ce was confirmed as a random endoacting alginate lyase. Its minimum substrate was tetrasaccharide, and the final product majorly consisted of disaccharide to tetrasaccharide. HPAEC-PAD/MS method was employed to investigate the structurally different unsaturated alginate oligosaccharides. The substrate recognition and subsite specificity of Aly7Ce were revealed by detecting the oligosaccharide pattern in the enzymatic products with oligosaccharides or polysaccharides as substrates. Aly7Ce mainly attacked the second glycosidic linkage from the nonreducing end of oligosaccharide substrates. The subsite specificity of Aly7Ce was revealed as -2 (M/G), - 1 (G), + 1 (M/G), and +2 (M/G). The regular oligosaccharide products of Aly7Ce could be applied for the efficient preparation of ΔG, ΔGG, and ΔGGG with high purity. The G-specific alginate lyase Aly7Ce with a well-defined product composition and action pattern provided a novel tool for the modification and structural elucidation of alginate, as well as for the targeted preparation of oligosaccharides.

Characterization and Structural Analysis of a Novel Carbohydrate-Binding Module from Family 96 with Chondroitin Sulfate-Specific Binding Capacity.

Chondroitin sulfate (CS) is the predominant glycosaminoglycan within the human body and is widely applied in various industries. Carbohydrate-binding modules (CBMs) possessing the capacity for carbohydrate recognition are verified to be important tools for polysaccharide investigation. Only one CS-specific CBM, PhCBM100, has hitherto been characterized. In the present study, two CBM96 domains present in the same putative PL8_3 chondroitin AC lyase were discovered and recombinantly expressed. The results of microtiter plate assays and affinity gel electrophoresis assays showed that the two corresponding proteins, DmCBM96-1 and DmCBM96-2, bind specifically to CSs. The crystal structure of DmCBM96-1 was determined at a 2.20 Å resolution. It adopts a ß-sandwich fold comprising two antiparallel ß-sheets, showing structural similarities to TM6-N4, which is the founding member of the CBM96 family. Site mutagenesis analysis revealed that the residues of Arg27, Lys45, Tyr51, Arg53, and Arg157 are critical for CS binding. The characterization of the two CBM96 proteins demonstrates the diverse ligand specificity of the CBM96 family and provides promising tools for CS investigation.

Establishment of a carbohydrate binding module-based lateral flow immunoassay method for identifying hyaluronic acid.

Hyaluronic acid is a commercially important polysaccharide with wide applications. Along with the rapid development of hyaluronic acid-based products, their authenticity has aroused considerable attention from consumers. In the present study, a carbohydrate-binding module (CBM) SrCBM70 was cloned and expressed. The protein could specifically bind to hyaluronic acid with a strong affinity. A novel method for the identification of hyaluronic acid was subsequently established by integrating SrCBM70 into the lateral flow immunoassay (LFIA). Its detection limit for hyaluronic acid was approximately 0.1 µg/mL, and the assay could be completed in 5 min. The feasibility of this method in the authenticity identification of commercialized products containing hyaluronic acid was confirmed. The establishment of the SrCBM70-based LFIA method provided a solution for the on-site authenticity identification and would facilitate the market supervision of hyaluronic acid-based products.

[Propensity score matching in SPSS].

OBJECTIVE: To realize propensity score matching in PS Matching module of SPSS and interpret the analysis results. METHODS: The R software and plug-in that could link with the corresponding versions of SPSS and propensity score matching package were installed. A PS matching module was added in the SPSS interface, and its use was demonstrated with test data. RESULTS: Score estimation and nearest neighbor matching was achieved with the PS matching module, and the results of qualitative and quantitative statistical description and evaluation were presented in the form of a graph matching. CONCLUSION: Propensity score matching can be accomplished conveniently using SPSS software.