Enhancement of 2,3-butanediol production from Jerusalem artichoke tuber extract by a recombinant Bacillus sp. strain BRC1 with increased inulinase activity.

A Bacillus sp. strain named BRC1 is capable of producing 2,3-butanediol (2,3-BD) using hydrolysates of the Jerusalem artichoke tuber (JAT), a rich source of the fructose polymer inulin. To enhance 2,3-BD production, we undertook an extensive analysis of the Bacillus sp. BRC1 genome, identifying a putative gene (sacC) encoding a fructan hydrolysis enzyme and characterizing the activity of the resulting recombinant protein expressed in and purified from Escherichia coli. Introduction of the sacC gene into Bacillus sp. BRC1 using an expression vector increased enzymatic activity more than twofold. Consistent with this increased enzyme expression, 2,3-BD production from JAT was also increased from 3.98 to 8.10 g L-1. Fed-batch fermentation of the recombinant strain produced a maximal level of 2,3-BD production of 28.6 g L-1, showing a high theoretical yield of 92.3%.

Characterization of a novel manganese dependent endoglucanase belongs in GH family 5 from Phanerochaete chrysosporium.

The cDNA encoding a putative glycoside hydrolase family 5, which has been predicted to be an endoglucanase (PcEg5A), was cloned from Phanerochaete chrysosporium and expressed in Pichia pastoris. PcEg5A contains a carbohydrate-binding domain and two important amino acids, E209 and E319, playing as proton donor and nucleophile in substrate catalytic domain. SDS-PAGE analysis indicated that the recombinant endoglucanase 5A (rPcEg5A) has a molecular size of 43 kDa which corresponds with the theoretical calculation. Optimum pH and temperature were found to be 4.5-6.0, and 50°C-60°C, respectively. Moreover, rPcEg5A exhibited maximal activity in the pH range of 3.0-8.0, whereas over 50% of activity still remained at 20°C and 80°C. rPcEg5A was stable at 60°C for 12 h incubation, indicating that rPcEg5A is a thermostable enzyme. Manganese ion enhanced the enzyme activity by 77%, indicating that rPcEg5A is a metal dependent enzyme. The addition of rPcEg5A to cellobiase (cellobiohydrolase and ß-glucosidase) resulted in a 53% increasing saccharification of NaOH-pretreated barley straw, whereas the glucose release was 47% higher than that cellobiase treatment alone. Our study suggested that rPcEg5A is an enzyme with great potential for biomass saccharification.

Manganese peroxidase h4 isozyme mediated degradation and detoxification of triarylmethane dye malachite green: optimization of decolorization by response surface methodology.

A cDNA encoding for manganese peroxidase isozyme H4 (MnPH4), isolated from Phanerochaete chrysosporium, was expressed in Pichia pastoris, under the control of alcohol oxidase I promoter. The recombinant MnPH4 was efficiently secreted onto media supplemented with hemin at a maximum concentration of 500 U/L, after which purified rMnPH4 was used to decolorize the triarylmethane dye malachite green (MG). Response surface methodology (RSM) was employed to optimize three different operational parameters for the decolorization of MG. RSM showed that the optimized variables of enzyme (0.662 U), MnSO4 (448 µM), and hydrogen peroxide (159 µM) decolorized 100 mg/L of MG completely at 3 h. Additionally, UV-VIS spectra, high-performance liquid chromatography, gas chromatography-mass spectrometry, and liquid chromatography-electrospray ionization/mass spectrometry analysis confirmed the degradation of MG by the formation of main metabolites 4-dimethylamino-benzophenone hydrate, N, N-dimethylaniline (N,N-dimethyl-benzenamine), and methylbenzaldehyde. Interestingly, it was found that rMnPH4 mediates hydroxyl radical attack on the central carbon of MG. Finally, rMnPH4 degraded MG resulted in the complete removal of its toxicity, which was checked under in vitro conditions.

Anti-inflammatory potential of Phaseolus calcaratus Roxburgh, a oriental medicine, on LPS-stimulated RAW 264.7 macrophages.

OBJECTIVES: The seed of Phaseolus calcaratus Roxburgh (PHCR) has traditionally been used as a herbal medicine, considered to have anti-inflammatory potential. Here we examined the ability of PHCR seed extract to inhibit inflammatory responses of macrophages to bacterial toxin and the mechanism involved. METHODS: In the present study, we prepared four fractions from an ethanol extract of PHCR seed and investigated their effects on the production of nitric oxide and cytokines, and the expression of inducible nitric oxide synthase (iNOS) in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells. KEY FINDINGS: The fractions inhibited LPS-induced nitric oxide production and cyclooxygenase-2 (COX-2) expression in the cells. The ethyl acetate fraction at 100 µg/ml almost completely suppressed NO production, iNOS and COX-2 expression, and TNF-α and IL-6 secretion in cells stimulated with LPS. The fraction also inhibited phosphorylation of extracellular signal-regulated kinase (ERK) and p38 in LPS-stimulated cells with the attendant suppression of IκBα nuclear translocation and nuclear factor (NF)-κB activation. Furthermore, PHCR seed extracts contained a large number of phenolic compounds having antioxidant potentials against 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals and hydroxyl radicals. We identified catechin-7-O-ß-D-glucopyranoside as one of the active compounds responsible for the biological activity of PHCR seed extract. CONCLUSIONS: These results suggest for the first time that ethanol extracts from PHCR seed have anti-inflammatory potential on LPS-stimulated macrophages through the down-regulation of ERK/p38- and NF-κB-mediated signalling pathways.

Chemoprevention of a flavonoid fraction from Rhus verniciflua Stokes on aflatoxin B1-induced hepatic damage in mice.

Since aflatoxin B(1) (AFB(1))-mediated hepatic damage is related to the production of AFB(1)-8,9-epoxide and reactive oxygen species, bioactive compounds having antioxidant potentials are suggested to be capable of reducing AFB(1)-induced toxicity. We previously purified a mixture of flavonoids that we named RCMF (Rhus verniciflua Stokes chloroform-methanol fraction), from a traditional Korean food additive and herbal medicine. RCMF exhibited various biological effects, including antioxidant and antitumor activities. In this study, we examined whether RCMF protects against AFB(1)-induced liver injury using in vitro and in vivo systems. Pretreatment of HepG2 cells with RCMF significantly reduced AFB(1)-stimulated production of ROS and malondialdehyde (MDA) to the control levels. RCMF also prevented the reduction in HepG2 cell viability caused by AFB(1). Oral administration of RCMF to mice significantly suppressed an AFB(1)-induced increase in serum levels of alanine aminotransferase, alkaline phosphatase and lactate dehydrogenase. It also prevented MDA formation and blocked decreases in glutathione levels and superoxide dismutase activities in the livers of AFB(1)-treated mice. In addition, RCMF supplementation prevented an AFB(1) -induced decrease in serum titers of IgA and IgG1. Collectively, these results suggest that RCMF attenuates AFB(1)-mediated damage to the liver, and that this effect is at least partially related to the restoration of antioxidant defense systems and an increase in AFB(1)-GSH conjugate formation.

Production of a heat-labile enterotoxin B subunit-porcine epidemic diarrhea virus-neutralizing epitope fusion protein in transgenic lettuce (Lactuca sativa).

Plant-based vaccines have been produced in transgenic plants including tobacco, potatoes, corn, and rice. However, these plants are not suitable for administration without cooking. To overcome this obstacle, a fusion gene encoding the synthetic enterotoxigenic Escherichia coli heat-labile enterotoxin B subunit genetically fused with a synthetic neutralizing epitope of porcine epidemic diarrhea virus (sLTB-sCOE) was introduced into lettuce cells (Lactuca sativa) by Agrobacterium-mediated transformation methods. The integration and expression of the sLTB-sCOE fusion gene was confirmed in transgenic lettuce by genomic DNA PCR amplification and Northern blot analysis, respectively. Synthesis and assembly of the LTB-COE fusion protein into oligomeric structures with pentamer size were observed in transgenic plant extracts by Western blot analysis with anti-LTB or anti-COE antibodies. The binding of plantproduced LTB-COE to intestinal epithelial cell membrane glycolipid receptors was confirmed by GM1-ganglioside enzyme-linked immunosorbent assay (GM1-ELISA). Based on the ELISA results, LTB-COE fusion protein made up about 0.026∼0.048% of the total soluble protein in the transgenic lettuce leaf tissues. The synthesis and assembly of LTB-COE monomers into biologically active oligomers in transgenic lettuce leaf tissues demonstrates the feasibility of using uncooked edible plant-based vaccines for mucosal immunization.