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 ß-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.

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.

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.

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

ß-agarase activity was monitored by traditional reducing sugar content methods: Somogyi-Nelson's arsenomolybdate, Miller's dinitrosalicylic acid and Kidby and Davidson's 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 ß-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/ß)(8)-fold, and a unique loop from F365 to F376 between ß3/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.

Morphology, associated protein analysis, and identification of 58-kDa starch synthase in mungbean (Vigna radiata L. cv. KPS1) starch granule preparations.

Raw starch granules of mature mungbean (Vigna radiata L. cv. KPS1) seeds were prepared by two methods into crude and cesium chloride (CsCl)-washed forms. The purity, shape, size distribution, and associated protein profiles were examined. The appearance of raw starch granules showed a bimodal type distribution in which average granules had typical ovoid shapes, whereas the small ones were spherical. Abnormal granule surface with distinct tumor-like or dented hole features were also observed in raw starch granules. CsCl-washed granules had a smooth surface compared to that of the crude form. The granule size distribution ranged from 6-35 µm; most 15-25 µm (∼53%), followed by 25-35 µm (∼26%). Small granules (<15 µm) amounted to ∼18%, and granules >35 µm consisted of ∼3%. The two forms were further refined by trichloroacetic (TCA) treatment to reveal surface proteins on the crude granules or tightly bound proteins on CsCl-washed granules. In the washed-refined granules, only a few integral proteins were retained. The major 58-kDa protein was identified to be granule-bound starch synthase I by sequence homology with that in cowpea (Vigna unguiculata) and maize (Zea mays) using MALDI-TOF mass and Mascot search.

Cloning, characterization, and expression of mungbean ( Vigna radiata L.) starch branching enzyme II cDNA in Escherichia coli.

Full-length starch branching enzyme II (SBE, EC 2.4.1.18) cDNA from mungbean ( Vigna radiata L. cv. Tainan no. 5), VrsbeII, was cloned, characterized, and expressed as an active enzyme in Escherichia coli . Gene-specific primers first amplified an internal cDNA by reverse transcriptase Polymerase Chain Reaction (RT-PCR), followed by obtaining 5' and 3' fragments by RT-PCR and rapid amplification of cDNA ends (RACE). VrsbeII possesses a complete open reading frame (ORF) of 2571 bp, and the deduced polypeptide includes the common catalytic (beta/alpha)(8)-barrel domain and conserved regions of the alpha-amylase family. Phylogenetic analysis classified VrsbeII into SBE family A. Its partial 3D structure and functional features were predicted. VrsbeII has a shorter N-terminal among SBEs; however, two 6 bp (CCAGTT) direct repeat sequences (DRS) were found. A 24 bp shortened VrsbeII at the 3' end, skipping one DRS, was ligated into pET21b vector and expressed as His(6)-rVrSBEII in E. coli BL21 (DE3) cells. The optimal expression condition for rVrSBEII was evaluated and detected by Western blot with a molecular size of 108 kDa and activity of 6.4 U/mg.

Separation and quantification of neoagaro- and agaro-oligosaccharide products generated from agarose digestion by beta-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.

The receptor of an oyster juice-borne coliphage OJ367 in the outer membrane of Salmonella derby.

The objective of this study was to identify the receptor of OJ367, an oyster juice-borne bacteriophage, in Salmonella derby ATCC 6960. The crude receptor outer membrane (OM) fraction was prepared and examined from the total cell envelope (TCE) by differential extraction with N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid (HEPES)-MgCl2 and then with Triton-HEPES-ethylenediamine tetra-acetic acid buffers. The OM proteins (Omps) were isolated by diethylaminoethyl column chromatography to screen for receptor activity. A 45-kDa protein belonging to a minor Omp species, with phage neutralization ability, was eluted in a homogeneous form. It was a non-peptidoglycan-associated protein which was digestible by trypsin. Lipopolysaccharide had no influence on its receptor activity when coexistent in the diethylaminoethyl column fractions. An S. derby mutant resistant to lysis by phage OJ367 was isolated. The mutant not only showed decreased receptor activity in vitro when its TCE was tested but had an altered Omp profile. This implied that the 45-kDa Omp is involved as a receptor in coliphage binding; however, this role is affected by the expression of other Omps.

The identification of starch phosphorylase in the developing mungbean (Vigna radiata L.).

Starch phosphorylase (SP) in immature mungbean (Vigna radiata L. cv KPS1) seed soluble extract was detected by in situ activity staining and identified by MALDI-TOF mass analysis. After in situ SP assay on native-PAGE, a major starch-enzyme complex was located on the gel zymogram in a dose-dependent manner. This complex depicted two major SP-activity related proteins, 105 kDa and 55 kDa, by SDS-PAGE. The mass and predicted sequence of the tryptic fragments of the isolated 105 kDa protein, analyzed by MALDI-TOF spectroscopy and bioinformatic analysis, confirmed it to be mungbean SP as a result of high similarity to the L-SP of known plant. Polyclonal antibodies raised from the 55 kDa recognized both the 105 kDa and the 55 kDa proteins on the Western blot and neutralized partial SP activity, indicating that the two proteins were immunologically related. The 55 kDa protein possess high similarity to the N-terminal half of the 105 kDa SP was further confirmed. The SP activity and the activity stained protein density in mungbean soluble extract decreased as the seed size increased during early seed growth. These data indicate that mungbean 105 kDa SP and SP activity-related 55 kDa were identified in the developing mungbean.

Detection of proteins related to starch synthase activity in the developing mungbean (Vigna radiata L.).

Proteins associated with starch synthase (SS) activities were identified in immature mungbeans (Vigna radiata L. cv KPS1). Seed soluble extract was separated by native-PAGE and subjected to in situ activity staining. The gel zymogram located starch-enzyme complex bands. The soluble extract was also partitioned by preparative-IEF and screened for SS activity using radioactive assay. IEF fractions eluted within pH 4-6 revealed enriched SS activity of 145-fold. Parallel comparison of the protein profiles among the activity stained enzyme complex and the active isoelectric focused fractions on SDS-PAGE depicted three SS-activity-related proteins with molecular size of 32, 53, and 85 kDa. The 85 kDa protein, however, was identified to be methionine synthase by MALDI-TOF analysis and should be a protein physically associated with the active SS. Polyclonal antibodies raised from eluted native enzyme complex neutralized up to 90% activity and antigenically recognize the other 53 and 32 kDa proteins on Western blot. Antibodies raised from the two individual denatured proteins were able to neutralize SS activities near 60% separately, indicating that the 53 kDa and 32 proteins associated with SS activity are potentially involved in starch biosynthesis during mungbean seed development.

1,3-beta-glucan quantification by a fluorescence microassay and analysis of its distribution in foods.

The objective of this study was to establish analytical approaches to quantify 1,3-beta-glucan (1,3-beta-G) in foods. Six food categories including legumes, cereals, tubers, vegetables, fruits, and mushrooms and 17 total items were tested. An extraction procedure was designed to prepare food cold-water soluble, hot-water soluble, cold-alkaline soluble, and hot-alkaline soluble fractions. A fluorescence microassay based on aniline blue dye, which bound specifically to 1,3-beta-G, was developed to measure its content in the food fractions. Curdlan was used as standard to construct the 1,3-beta-G calibration curve, and a linear correlation within a 14 microg/mL concentration range was obtained. This microassay displayed selectivities among various 1,3-beta-G species. Biologically active ones such as pachyman and yeast glucan possessed much stronger fluorescent signals than others such as laminarin and barley glucan. Possible fluorescent interference from food proteins was estimated. This assay tolerated up to 50% of bovine serum albumin in 10 microg/mL curdlan. Analysis of the four food fractions showed that besides the well-known lentinan-containing shiitake, popular foods such as celery, chin-chian leaves, carrot, and radish contained nearly 20% 1,3-beta-G in their total sugar. Soybean dry weight contained 0.8% 1,3-beta-G, which was twice the amount compared to shiitake. Snow mushroom dry weight had the highest 1,3-beta-G content, at 2.5%, and was rich in both water (0.67%) and alkaline soluble (1.87%) forms. In conclusion, this dye-binding fluorescence microassay in conjunction with the extraction procedure can be applied in the prescreening of potential foods rich in functional 1,3-beta-G.