Yuan-Tih Ko

Separation and quantification of neoagaro- and agaro-oligosaccharide products generated from agarose digestion by β-agarase and HCl in liquid chromatography systems

A series of neoagaro-oligosaccharides (NAOS) were separated and isolated by beta-agarase digestion and agaro-oligosaccharides (AOS) by HCl hydrolysis from agarose with defined quantity and degree of polymerization (DP). Profiles of the oligomer length in the crude product mixtures were monitored by two high-performance liquid chromatography (HPLC) systems: size-exclusion chromatography (SEC) and NH2-column chromatography (NH2-HPLC), coupled with an evaporative light-scattering detector (ELSD). Calibration curves were established separately to identify the DP and quantify the amount of the oligomer products analyzed in the two systems. Each system was optimized to generate a spectrum of saccharide oligomers with various DP, where the reaction yield for NAOS was 52.7% by 4U/mg beta-agarase and for AOS was 45.6% by 0.4M HCl. SEC resolved the product in size ranges consisting of DP 1-22 for NAOS and DP 1-14 for AOS. NH2-HPLC clearly resolved both distinct saccharide product sizes within DP 12. The optimized system was connected with a fraction collector to isolate and quantify these individually separated products. The total product yields of the recovered NAOS of DP 1-22 and AOS of DP 1-14 by the SEC system were 84.7% and 82.9%, respectively. NH2-HPLC recovered NAOS and AOS, both with a DP of 1-10 with total product yields of 48.9% and 90.0%, respectively. Isolated NAOS and AOS product fractions were inspected by (1)H NMR spectroscopy and ESIMS spectrometry to confirm structure, molecular mass, and purity. This study established feasible systems for the preparation and qualitative and quantitative measurements, as well as for the isolation of various sizes of oligomers generated from agarose.

Identification and roles of proteins for seed development in mungbean (Vigna radiata L.) seed proteomes.

Proteomic analysis of developing mungbean (Vigna radiata L.) seeds has not yet been investigated in detail. Fifty-seven proteins were separated by 2-DE, identified by nanoelectrospray mass spectrometry from the present protein databases, and categorized according to their functions. Many of the identified enzymes were involved in central carbon metabolism; thus, a pathway illustrating starch synthesis/breakdown, sugar conversion for glycolysis, and tricarboxylic acid (TCA) cycle was proposed. Quantitative comparison of the protein expression revealed that during developmental process (11-21 days after flowering, DAF), proteins involved in glycolysis, TCA cycle, and alcoholic fermentation showed a trend to be down-regulated, whereas storage proteins were generally up-regulated. The downward tendency of central carbon metabolic proteins suggests a reduction in ATP and oxygen consumption associated with accumulation of storage compounds. UDP-glucose-1-pyrophosphorylase, an upstream enzyme in the starch ADP-Glc pathway, was found as a stably expressed protein throughout the growth stage, demonstrating its importance in mungbean starch biosynthesis. The temporal expression of metabolic enzymes suggests the coordination of an acclimation mechanism and cellular processes associated with accumulation of storage compounds in seed development.

Monitoring and preparation of neoagaro- and agaro-oligosaccharide products by high performance anion exchange chromatography systems

A series of neoagaro-oligosaccharides (NAOS) were prepared by β-agarase digestion and agaro-oligosaccharides (AOS) by HCl hydrolysis from agarose with defined quantity and degree of polymerization (DP). Chain-length distribution in the crude product mixtures were monitored by two high performance anion exchange chromatography systems coupled with a pulsed amperometric detector. Method 1 utilized two separation columns: a CarboPac(™) PA1 and a CarboPac(™) PA100 connected in series and method 2 used the PA100 alone. Method 1 resolved the product in size ranges consisting of DP 1-46 for NAOS and DP 1-32 for AOS. Method 2 clearly resolved saccharide product sizes within DP 26. The optimized system utilizing a semi-preparative CarboPac(™) PA100 column was connected with a fraction collector to isolate and quantify individually separated products. This study established systems for the preparation and qualitative and quantitative measurements as well as for the isolation of various sizes of oligomers generated from agarose.

Evaluation of HPSEC-ELSD method for precise measurement of β-agarase activity.

β-agarase activity was monitored by traditional reducing sugar content methods: Somogyi-Nelsons arsenomolybdate, Millers dinitrosalicylic acid and Kidby and Davidsons ferricyanide methods, as well as by high-performance size exclusion chromatography coupled with a refractive index detector and an evaporative light scattering detector (ELSD). Calibration curves were established separately for each method to measure the amounts of the neoagaro-oligosaccharides (NAOS) in the reaction mixtures, which are the products from 1-10 units (U) of β-agarase cleavage activity on agarose. Product quantities from each monitoring method were compared with the isolated NAOS products. The graphs plotted by agarase activity unit and product concentration clearly displayed that the ELSD method closely followed the results of the isolated products. The percentage deviation of results measured by the five methods away from those of the isolated NAOS product mixture amounted to -13.1-35.1, -21.1-25.5, -27.1-23.81, 6.1-24.3 and 16.2-22.8%, respectively. When the loss during product isolation, about 15-17%, was taken into account, the high precision of the ELSD method was confirmed. HPSEC-ELSD methods also accurately measured the enzyme kinetics as well as enabling partial identification of oligosaccharides assembled in the NAOS product mixture. This study established the HPSEC-ELSD system as an alternative method for monitoring agarase activity.

Molecular characterization of mungbean (Vigna radiata L.) starch branching enzyme I

Mungbean (Vigna radiata L. cv. Tainan no. 5) starch branching enzyme I (SBE, EC 2.4.1.18) cDNA, VrsbeI, was cloned, and its expression was characterized. Conserved regions of the family B SBE were used to amplify a full length cDNA of 2208 bp. Phylogeny was analyzed, and the partial 3D structure and functional features were predicted. Catalytic residues were identified in the (α/β)(8)-fold, and a unique loop from F365 to F376 between β3/α3 was located. Gene expression of VrsbeI in seeds during growth showed that the transcript appeared from week 1 and increased substantially at week 3-4. It was cloned into the pET30 vector and expressed in E. coli BL21(DE3) pLysS cells as a soluble recombinant protein. The affinity-purified recombinant VrSBEI exhibited a specific activity of 314.6 U/mg as an active enzyme with 114-fold activity enrichment from the crude extract.

Reaction of phosphorylase-a with α-d-glucose 1-phosphate and maltodextrin acceptors to give products with degree of polymerization 6–89

A series of linear glucan saccharides (GS) with defined quantity and degree of polymerization (DP) were synthesized from α-d-glucose 1-phosphate (α-d-Glc 1-P) by phosphorylase-a. The GS product fractions with average DP 11, 22, 38, 52, 60, 70, and 79 were measured by HPSEC-ELSD system. Then the same seven fractions were resolved into individual peaks with DP: 6-14, 10-32, 27-55, 37-67, 44-75, 49-83 and 53-89 by HPAEC-PAD system. Results showed that measurement of α-d-Glc 1-P amount consuming during GS synthesis by both systems enable calculation of reaction yield. The reaction yield for the 24h biosynthesis of the GS product was 25.3% (measured by HPSEC-ELSD) or 29.1% (measured by HPAEC-PAD). The HPSEC-ELSD and HPAEC-PAD systems were also successfully used for phosphorylase-a activity measurement in order to perform its kinetic characterization. This study established feasible systems for preparation of various sizes of the GS with defined DP and quantity as well as characterization of phosphorylase-a kinetics.