Yu-Chun Huang

Foam Properties and Detergent Abilities of the Saponins from Camellia oleifera

The defatted seed meal of Camellia oleifera has been used as a natural detergent and its extract is commercially utilized as a foam-stabilizing and emulsifying agent. The goal of this study was to investigate the foam properties and detergent ability of the saponins from the defatted seed meal of C. oleifera. The crude saponin content in the defatted seed meal of C. oleifera was 8.34 and the total saponins content in the crude saponins extract was 39.5% (w/w). The foaming power of the 0.5 crude saponins extract solution from defatted seed meal of C. oleifera was 37.1 of 0.5 SLS solution and 51.3% to that of 0.5% Tween 80 solution. The R5 value of 86.0% represents good foam stability of the crude saponins extracted from the defatted seed meal of the plant. With the reduction of water surface tension from 72 mN/m to 50.0 mN/m, the 0.5% crude saponins extract solution has wetting ability. The sebum-removal experiment indicated that the crude saponins extract has moderate detergency. The detergent abilities of the saponins from C. oleifera and Sapindus mukorossi were also compared.

Production of ferulic acid from lignocellulolytic agricultural biomass by Thermobifida fusca thermostable esterase produced in Yarrowia lipolytica transformant

A gene (axe) encoding the AXE thermostable esterase in Thermobifida fusca NTU22 was cloned into a Yarrowia lipolytica P01g host strain. Recombinant expression resulted in extracellular esterase production at levels as high as 70.94 U/ml in Hinton flask culture broth, approximately 140 times higher than observed in a Pichia pastoris expression system. After 72 h of fermentation by the Y. lipolytica transformant in the fed-batch fermentor, the fermentation broth accumulated 41.11 U/ml esterase activity. Rice bran, wheat bran, bagasse and corncob were used as hydrolysis substrates for the esterase, with corncob giving the best ferulic acid yield. The corncob was incubated with T. fusca xylanase (Tfx) for 12h and then with the AXE esterase for an additional 12h. Ferulic acid accumulated to 396 μM in the culture broth, a higher concentration than with esterase alone or with Tfx and esterase together for 24 h.

Constitutive expression of Thermobifida fusca thermostable Acetylxylan Esterase gene in Pichia pastoris.

A gene encoding the thermostable acetylxylan esterase (AXE) in Thermobifida fusca NTU22 was amplified by PCR, sequenced and cloned into the Pichia pastoris X-33 host strain using the vector pGAPZαA, allowing constitutive expression and secretion of the protein. Recombinant expression resulted in high levels of extracellular AXE production, as high as 526 U/mL in the Hinton flask culture broth. The purified enzyme showed a single band at about 28 kDa by SDS-polyacrylamide gel electrophoresis after being treated with endo-β-N-acetylglycosaminidase H; this agrees with the predicted size based on the nucleotide sequence. About 70% of the original activity remained after heat treatment at 60 °C for three hours. The optimal pH and temperature of the purified enzyme were 8.0 and 60 °C, respectively. The properties of the purified AXE from the P. pastoris transformant are similar to those of the AXE from an E. coli transformant.

Degradation of konjac glucomannan by Thermobifida fusca thermostable β-mannanase from yeast transformant.

Native konjac glucomannan was used as the substrate for thermophilic actinomycetes, Thermobifida fusca BCRC19214, to produce β-mannanase. The β-mannanase was purified and five internal amino acid sequences were determined by LC-MS/MS. These sequences had high homology with the β-mannanase from T. fusca YX. The tfm gene which encoded the β-mannanase was cloned, sequenced and heterologous expressed in Yarrowia lipolytica P01 g expression system. Recombinant heterologous expression resulted in extracellular β-mannanase production at levels as high as 3.16 U/ml in the culture broth within 48 h cultivation. The recombinant β-mannanase from Y. lipolytica transformant had superior thermal property. The optimal temperature of the recombinant β-mannanase from Y. lipolytica transformant (pYLSC1-tfm) was 80°C. When native konjac glucomannan was incubated with the recombinant β-mannanase from Y. lipolytica transformant (pYLSC1-tfm) at 50°C, there was a fast decrease of viscosity happen during the initial phase of reaction. This viscosity reduction was accompanied by an increase of reducing sugars. The surface of konjac glucomannan film became smooth. After 24h of treatment, the DPw of native konjac glucomannan decreased from 6,435,139 to 3089.

Synthesis and structure-activity relationships of fenbufen amide analogs.

The previous discoveries of butyl fenbufen amide analogs with antitumor effects were further examined. The amide analogs with 1, 3, 4 and 8 carbons chains were prepared in 70–80% yield. Fenbufen had no cytotoxic effects at concentrations ranging from 10 to 100 μM. Methyl fenbufen amide had significant cytotoxic effects at a concentration of 100 μM. As the length of the alkyl amide side chain increased, the cytotoxic effects increased, and the octyl fenbufen amide had the greatest cytotoxic effect. After treatment with 30 μM octyl fenbufen amide, nearly seventy percent of the cells lost their viability. At the concentration of 10 μM, fenbufen amide analogs did not show cytotoxicity according to the MTT assay results. The NO scavenging activities of the fenbufen amide analogs were not significantly different from those of fenbufen.

Locust bean gum galactomannan hydrolyzed by thermostable β-d-mannanase may reduce the secretion of pro-inflammatory factors and the release of granule constituents

Locust bean gum (LBG) galactomannan has been claimed to have applications in the biopharmaceutical field. However, the effects of LBG galactomannan on immunomodulatory aspects are not yet clear. The purpose of this study was to over-express thermostable β-d-mannanase from the thermophilic actinomycete Thermobifida fusca BCRC 19214 using a Pichia pastoris expression system. The maximum intracellular β-d-mannanase activity obtained from the cell-free extract was approximately 40.0U/mL after 72h of cultivating a P. pastoris transformant (pPICZ-man) induced with methanol. Hydrolysis of native LBG galactomannan with 8U/mL β-d-mannanase for 24h significantly decreased the weight-average molecular weight of LBG galactomannan from 5,580,010 to 3188. Native and hydrolyzed LBG galactomannan in a range of 0-0.2% did not trigger significant cytotoxicity after 24h of treatment compared with the control. The native LBG galactomannan stimulated RAW 264.7 cells to produce cytokine TNF-α dose-dependently, but there was no significant IL-1β or nitric oxide production. The native LBG galactomannan also stimulated β-hexosaminidase secretion in RBL-2H3 cells. After the native LBG galactomannan was hydrolyzed with β-d-mannanase, all of the immunological properties disappeared. These results suggest the possible immunomodulatory effects of native LBG galactomannan.