Reduction in mercury bioavailability to Asian clams (Corbicula fluminea) and changes in bacterial communities in sediments with activated carbon amendment.

Activated carbon (AC) amendment is considered as one of the alternatives for managing and remediating mercury (Hg) contaminated sediments because of its high sorptive capacity and potential to immobilize the contaminant. For this study, the underlying mechanisms that control the reduction of Hg bioavailability in AC-amended estuarine sediments were investigated in box microcosm set-ups with 28-day Asian clam bioassay experiments. The application of diffusive gradients in thin film technique (DGT) revealed that the total mercury and methylmercury levels in sediment pore water decreased by 60%-75% in 1%-3% AC-amended sediments. This decrease subsequently led to a linear reduction in the Hg body burden in Asian clams, even at 1% sorbent mixing. These observations implied that AC amendment reduced the net flux of Hg into the pore water and overlying water, resulting in reduced Hg bioaccumulation in benthic organisms. The addition of AC to sediment also led to reduced dissolved organic carbon and several biogeochemical indicators (HS-, Mn, and Fe) in the pore water. Furthermore, the 16 S rRNA gene amplicon sequencing analysis revealed noticeable alterations in the microbial communities after AC amendment. The predominant phylum was Firmicutes in control sediment, Bacteroidetes in 1% AC-amended sediment, and Proteobacteria in both 2% and 3% AC-amended sediment samples. The genera-level analysis showed that the relative abundance of the Hg-methylators decreased as the level of AC amendment increased. These observations suggested that AC amendment decreased Hg bioavailability not only by physicochemical sorption but also by changing geochemical species and shifting the microbial community composition.

Production and characterization of low-calorie turanose and digestion-resistant starch by an amylosucrase from Neisseria subflava.

This study was intended to produce turanose and resistant starch (RS) using recombinant amylosucrase from Neisseria subflava (NsAS). Turanose production yield maximally reached to 76% of sucrose substrate at 40 °C by NsAS treatment. To evaluate turanose as a low-calorie functional sweetener, its hydrolysis pattern was investigated in continuous artificial digestion system. When turanose was consecutively exposed through small intestinal phase, only 8% of disaccharide was hydrolyzed. Structural modification of gelatinized corn or rice starch was carried out by NsAS with sucrose as a glucosyl donor. Non-digestibility of enzyme-modified starches increased to 47.3% maximally through branch-chain elongation, enough for chain-chain association and recrystallization. Obviously, NsAS-modified starches had higher gelatinization peak temperatures than native counterparts, and their paste viscosity was inversely related to their digestibility due to elongated-chain induced retrogradation. These results suggested that NsAS could be a vital biocatalyst candidate in food industry to produce next generation low-calorie carbohydrate food materials.

Expression, purification, and characterization of a novel amylosucrase from Neisseria subflava.

Amylosucrase (ASase) is a glucosyltransferase, which catalyzes the de novo synthesis of amylose-like polymers from sucrose. In the present study, ASase from Neisseria subflava (NsAS) was cloned, sequenced, and expressed in Escherichia coli. The production of NsAS was achieved by inducting gene expression with 0.2 mM isopropyl-ß-d-thiogalactopyranoside. The molecular mass of the Ni-NTA column purified NsAS analyzed by SDS-PAGE was determined to be 72 kDa. NsAS exhibited maximal activity at 45 °C and pH 8.0, and showed strong thermal stability at 40 °C with a half-life of 385 h. The reaction pattern of NsAS at [sucrose] range of 0.1-1.0 M showed that at 0.7 M of [sucrose], the production yield of insoluble linear α-(1,4)-glucans reached 24% maximum, and any further increase in [sucrose] resulted in a slight decrease in yield. Meanwhile, the production yield of turanose significantly increased from 16 to 29% by increasing [sucrose] from 0.1 to 1.0 M. The synthesized glucan had degrees of polymerization (DP); for 0.1, 0.4, 0.7, and 1.0 M sucrose, the DP values were 77, 49, 39, and 31 respectively. These results suggested that NsAS would be a promising candidate for food industrial production of linear α-(1,4)-glucans and turanose as a next generation sweetener.

Efficient Biocatalytic Production of Cyclodextrins by Combined Action of Amylosucrase and Cyclodextrin Glucanotransferase.

A novel enzymatic process for cyclodextrin (CD) production was developed by utilizing sucrose as raw material instead of corn starch. Cyclodextrin glucanotransferase (CGTase) from Bacillus macerans was applied to produce the CDs from linear α-(1,4)-glucans, which were obtained by Neisseria polysaccharea amylosucrase (NpAS) treatment on sucrose. The greatest CD yield (21.1%, w/w) was achieved from a one-pot dual enzyme reaction at 40 °C for 24 h. The maximum level of CD production (15.1 mg/mL) was achieved with 0.5 M sucrose in a simultaneous mode of dual enzyme reaction, whereas the reaction with 0.1 M sucrose was the most efficient with regard to conversion yield. Consequently, dual enzyme synthesis of CDs was successfully carried out with no need of starch material. This result can be applied as a novel efficient bioconversion process that does not require the high temperature necessary for starch liquefaction by thermostable α-amylase in conventional industrial processing.

Enzymatic Process for High-Yield Turanose Production and Its Potential Property as an Adipogenesis Regulator.

Turanose is a sucrose isomer naturally existing in honey and a promising functional sweetener due to its low glycemic response. In this study, the extrinsic fructose effect on turanose productivity was examined in Neisseria amylosucrase reaction. Turanose was produced, by increasing the amount of extrinsic fructose as a reaction modulator, with high concentration of sucrose substrate, which resulted in 73.7% of production yield. In physiological functionality test, lipid accumulation in 3T3-L1 preadipocytes in the presence of high amounts of pure glucose was attenuated by turanose substitution in a dose-dependent manner. Turanose treatments at concentrations representing 50%, 75%, and 100% of total glucose concentration in cell media significantly reduced lipid accumulation by 18%, 35%, and 72%, respectively, as compared to controls. This result suggested that turanose had a positive role in controlling adipogenesis, and enzymatic process of turanose production has a potential to develop a functional food ingredient for controlling obesity and related chronic diseases.