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The effects of surface type (stainless steel, acetal resin, and fiberglass reinforced plastic wall paneling [FRP]), soil, and temperature on the survival of Listeria monocytogenes, Salmonella spp., and Yersinia enterocolitica, in the presence of condensate were evaluated. Surface coupons--half soiled with sterile porcine serum--were exposed to cell suspensions made from individual five-strain cocktails composed of organisms from the same genus (10(7) CFU/ml) in Butterfield's phosphate buffer and incubated for 2 h at 25 degrees C allowing attachment of cells to coupon surfaces. Coupons were rinsed to remove unattached cells, incubated at either 4 or 10 degrees C under condensate-forming conditions, and sampled at six time intervals over a 15-day period. For enumeration, cells were removed from the coupons by vigorous shaking in 100 ml of Butterfield's phosphate buffer with 3 g of glass beads and plated on tryptic soy agar with 0.6% yeast extract. Stainless steel did not support the survival of Listeria as well as acetal resin or FRP. Acetal resin and stainless steel were less supportive of Salmonella than FRP. All surfaces supported the survival of Yersinia over the 15-day trial equally. Temperature had little effect on survival of all organisms across all surfaces with one exception. However, Yersinia displayed growth on FRP at 10 degrees C. but death at 4 degrees C. Serum had a protective effect on L. monocytogenes on all surfaces, with populations sustained at significantly (P < or = 0.05) higher numbers over time than unsoiled coupons. Serum didnot effect survival of Salmonella or Yersinia on stainless steel, acetal resin, or FRP.
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PMID:Surface material, temperature, and soil effects on the survival of selected foodborne pathogens in the presence of condensate. 1563 69

To develop an effective fermentation system for producing Escherichia coliphytase AppA2, we expressed the enzyme in three inducible yeast systems: Saccharomyces cerevisiae (pYES2), Schizosaccharomyces pombe (pDS472a), and Pichia pastoris (pPICZ alphaA), and one constitutive system: P. pastoris (pGAPZalphaA). All four systems produced an extracellular functional AppA2 phytase with apparent molecular masses ranging from 51.5 to 56 kDa. During 8-day batch fermentation in shaking flasks, the inducible Pichia system produced the highest activity (272 units ml(-1) medium), whereas the Schizo. pombe system produced the lowest activity (2.8 units ml(-1)). The AppA2 phytase expressed in Schizo. pombe had 60-75% lower K(m)for sodium phytate and 28% higher heat-stability at 65 degrees C than that expressed in other three systems. However, all four recombinant AppA2 phytases had pH optimum at 3.5 and temperature optimum at 55 degrees C and similar efficacy in hydrolyzing phytate-phosphate from soybean meal.
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PMID:Expression of Escherichia coli AppA2 phytase in four yeast systems. 1583 94

Removing arsenic from contaminated groundwater in Bangladesh is challenging due to high concentrations of As(III), phosphate and silicate. Application of zero-valent iron as a promising removal method was investigated in detail with synthetic groundwater containing 500 microg/L As(III), 2-3mg/L P, 20mg/L Si, 8.2mM HCO3-, 2.5mM Ca2+, 1.6mM Mg2+ and pH 7.0. In a series of experiments, 1L was repeatedly passed through a mixture of 1.5 g iron filings and 3-4 g quartz sand in a vertical glass column (10mm diameter), allowing the water to re-aerate between each filtration. At a flow rate of 1L/h, up to 8 mg/L dissolved Fe(II) was released. During the subsequent oxidation of Fe(II) by dissolved oxygen, As(III) was partially oxidized and As(V) sorbed on the forming hydrous ferric oxides (HFO). HFO was retained in the next filtration step and was removed by shaking of the sand-iron mixture with water. Rapid phosphate removal provided optimal conditions for the sorption of As(V). Four filtrations lead to almost complete As(III) oxidation and removal of As(tot) to below 50 microg/L. In a prototype treatment with a succession of four filters, each containing 1.5 g iron and 60 g sand, 36 L could be treated to below 50 microg/L in one continuous filtration, without an added oxidant.
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PMID:Oxidation and removal of arsenic (III) from aerated groundwater by filtration through sand and zero-valent iron. 1589 71

Tetracaine hydrochloride (TH) is nominated as the prohibited ingredients in cosmetics in Japanese Pharmaceutical Affairs Act. So the analytical method for TH was investigated by HPLC. After adding 5 ml of TH solution at 10 microg/ml and 2 ml of salicylic acid solution at 75 microg/ml as the internal standard to 0.5 g of the lotion, the mixture was made up to 10 ml with a mixture of water and methanol (1:1) as the testing solution. Milky lotion was procedured as follows: After adding 5 ml of TH solution at 10 microg/ml and 2 ml of internal standard solution to 0.5 g of the milky lotion, the mixture was made up to 10 ml with a mixture of water and methanol (1:1). Two milliliter of this mixture was placed into a centrifuging tube with a cap and 2 ml of hexane was added. After shaking vigorously and centrifuging, the lower layer was used as the testing solution. In the case of the cream, the other procedures were used: 0.5 g of cream was placed into a 10-ml volumetric flask and 1 ml of tetrahydrofuran was added. After dissolving, the mixture of methanol and water (1:1) was added to make up 10.0 ml. Two milliliter of this mixture was placed into a centrifuging tube with a cap and 2.0 ml of hexane was added. After shaking vigorously and centrifuging, the lower layer was used as the testing solution. The testing solution of 20 microl was analyzed by HPLC using the ODS column (CAPCELL PAK C18 column, 4.6 x 250 mm), the mixture of acetonitrile and 50 mmol/l phosphate buffer(pH 2.0)(7:3) and the detection wavelength of 303 nm. The working curves from 0.5 to 6.0 microg/ml showed a linear line between the concentrations of TH and the peak area ratio. There was no interference of peak of TH from the lotion, milky lotion and cream.
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PMID:[Studies for analyzing prohibited ingredients such as tetracaine hydrochloride in cosmetics]. 1594 Sep

Considering Alcaligenes faecalis pencillin G acylase(AfPGA), which possesses the attractive characteristics for beta-lactam antibiotics conversions, the gene of PGA was cloned into an expressing vector pKKFPGA. The recombinant plasmid contained multicopy replicon(COLE 1), trc promoter, AfPGA gene, rrnB transcript terminator and ampicillin marker transformed Escherichia coli DH5alpha. As both the recombinant plasmid and the host DH5alpha had no laclq gene, the trc promoter was always active and the AfPGA could be constitutively expressed without IPTG induction in the host DH5alpha. In the shaking flask, the recombinant cell was inoculated into the fermentation medium (tryptone 10g/L, yeast extract 5g/L, MgSO4 x 7 H2O 1g, KH2 PO4 2g/L, K2HPO4 x 3H2O 5g/L, Na2HPO4 x 12H2O 7g/L, (NH4)2SO4 1.2g/L, NH4Cl 0.2 g/L, NaCl 0.1g/L, dextrin 30g/L) and cultured at 28 degrees C for 20h. The production of AfPGA reached 2,590u/L(NIPAB method), with a cell-density-specific activity of more than 300(u/L)/A600, this yield increased 432 fold higher than the native expression of Alcaligenes faecalis . Without ammonium sulphate fractionation and dialysis, the supernatant of crude extract was directly loaded on DEAE-Sepharose CL 6B column equilibrated by phosphate buffer (50mmol/L, pH7.8), and the enzyme fraction was not absorbed on the column but impurities were absorbed. Subsequently the effluent was added ammonium sulphate to 1mol/L and loaded on Butyl-Sepharose CL 4B column equilibrated by 50mmol/L phosphate buffer pH7.8-1mol/L ammonium sulphate. The enzyme was eluted as concentration of ammonium sulphate in phosphate buffer decreased to 0, PGA was eluted. After these two column chromatography, the enzyme was enriched 20 times with a 91% activity recovery. The purified enzyme had a specific activity of 68.6u/mg protein. However, the overproduction of PGA was often limited by translocation and/or periplasmic processing steps, subsequently resulted in intracellular accumulation of various types of PGA precursors and then formed inclusion bodies in the cytoplasm and/or periplasm. In this study, 5% PGA precursors formed as inclusion bodies in the cytoplasm while no inclusion bodies formed in the periplasm. It suggested most PGA precursors were transported to the periplasm and matured to active PGA and also explained why PGA gene was highly expressed in the host DH5alpha. On the other hand, inclusion bodies in the cytoplasm indicated that the maturation of PGA in the host DHSalpha was limited by the translocation step.
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PMID:[Constitutive expression and purification of Alcaligenes faecalis penicillin G acylase in Escherichia coli]. 1597

A highly selective immunoaffinity column was obtained by coupling anti-papaverine polyclonal antibodies to CNBr-activated Sepharose 4B. It was found that the coupling efficiency and performance of the immunoaffinity column were greatly improved by prolonging the coupling reaction time from 3 h at 20 degrees C with shaking to incubation overnight at 4 degrees C after the 3 h shaking reaction. The pH and ionic strength were observed to be the most important factors that influence the binding of papaverine to the immunoaffinity column. Using 0.01 mol/L phosphate buffered saline (PBS, pH 8.3) and methanol-water (80:20, v/v) as the loading and eluting solutions, respectively, papaverine was first retained on the column and then quantitatively eluted out with a mean recovery of 86% at a loading concentration of 1 microg/mL. When applied to real samples of pericarpium papaveris and food products, the established immunoaffinity column showed high efficiency in removing the matrix interferences in the samples and satisfactory recovery results were obtained. The method was useful for extraction and purification of papaverine from related samples.
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PMID:An efficient immunoaffinity chromatographic method for extraction and purification of papaverine from samples of pericarpium papaveris and food products. 1611 93

A protocol for direct extraction of mRNA from soil samples was developed. Soil samples (10 g) were washed twice with 120 mM phosphate buffer (pH 5.2). The lysis of cells, fixation of RNA, and hydrolysis of DNA were achieved by vigorously shaking the washed soil in a 4 M guanidine thiocyanate solution containing 25 mM sodium citrate, 0.5% sarcosyl, and 0.1 M 2-mercaptoethanol. The pH of the homogenized mixture was adjusted with 2 M sodium acetate (pH 4.0); the mRNA was then extracted with phenol and chloroform. Total RNA was precipitated with isopropanol. This method extracts up to 17 mug of total RNA per g (wet weight) of soil containing 8.0 x 10 cells of Pseudomonas aeruginosa PU21, and mRNA has been detected in 160-ng total RNA fractions. This method has been used for the detection of mRNA transcribed from specific biodegradative genes, including the nah and mer operons, in contaminated soils. This extraction method can be completed within a few hours and has tremendous potential for ecological studies of in situ gene expression among soil microbiotas.
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PMID:Rapid method for direct extraction of mRNA from seeded soils. 1634 41

Acetohexamide (AH) is nominated as the prohibited ingredients in cosmetics in Japanese Pharmaceutical Affairs Act. So the analytical method for AH was investigated by HPLC. The lotion or milky lotion of 0.5g was put into a 10-ml volumetric flask. After adding 1.0ml of AH solution at 50 microg/ml into the volumetric flask, the mixture was made up to 10ml with methanol as the testing solution. Creams were procedured as follows; 0.5 g of cream was put into a 10-ml volumetric flask. After adding 1.0ml of tetrahydrofuran into the volumetric flask, the mixture was stirred for several minutes and the ingredients of the creams were dissolved. After adding 1.0ml of AH solution at 50 microg/ml into the volumetric flask, the mixture was made up to 10ml with methanol. One milliliter of the mixture including AH at 5 microg/ml was exactly put into a test tube with a cap and then 1 ml of water and 1 ml of hexane were added. After shaking vigorously, stand for several minutes. After centrifuging, the hexane layer was eliminated and the residual mixture was used as the test solution. The testing solution of 20 microl was analyzed by HPLC using the ODS column (CAPCELL PAK C18 column, 4.6 x 250mm), the mixture of acetonitrile and 50 mmol/l phosphate buffer(pH 5.3)(3:1) and the detection wavelength of 247 nm. The working curve from 0.5 to 6.0 microg/ml showed a linear line between the concentrations of AH and the peak areas. There was no interference of peak of AH with the ingredients such as methylparaben, ethylparaben in the lotions, milky lotion and creams.
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PMID:[Studies for analyzing the prohibited ingredients such as acetohexamide in cosmetics]. 1654 46

Immobilized Candida antarctica lipase B (Novozym 435)-catalyzed enantioselective hydrolysis of D,L-phenylglycine methyl ester to enatiopure D-phenylglycine was successfully conducted in the systems with ionic liquids (ILs). Novozym 435 exhibited excellent activity and enantioselectivity in the system containing the IL BMIMxBF(4) compared to several typical organic solvents tested. It has been found that the cations and, particularly, the anions of ILs have a significant effect on the reaction, and the IL BMIMxBF(4), which shows to be the most suitable for the reaction, gave the highest initial rate and enantioselectivity among various ILs examined. The reaction became much less active and enantioselective in the systems with BMIMxHSO(4). Also, it was noticed that the enzymatic hydrolysis was strongly dependent on BMIMxBF(4) content in the co-solvent systems and the favorable content of the IL was 20% (v/v). Of the assayed four co-solvents and phosphate buffer, the lowest apparent K(m) and activation energy, and the highest V(max) of the reaction were achieved using 20% (v/v) BMIMxBF(4) co-solvent with phosphate buffer. Additionally, various influential variables were investigated. The optimum pH, substrate concentration, reaction temperature and shaking rate were 8.0, 80mM, 25-30 degrees Celsius and 150rpm, respectively, under which the initial rate, the residual substrate e.e. and the enantioselectivity were 2.46mM/min, 93.8% (at substrate conversion of 53.0%) and 38, respectively. When the hydrolysis was performed under reduced pressure, the initial rate (2.64mM/min) and the enantioselectivity (E=43) were boosted.
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PMID:Efficient enantioselective hydrolysis of D,L-phenylglycine methyl ester catalyzed by immobilized Candida antarctica lipase B in ionic liquid containing systems. 1656 44

Hypersaline wastewater, generated by many industrial activities, is difficult to treat through conventional biological processes. In this kind of hypersaline environment, complex nutrients are needed for the normal growth of many microorganisms. For this paper, the organisms were taken from a municipal wastewater treatment plant and acclimated to 15% salinity in a biofilm treatment process (Sequencing Batch Biofilm Reactor) during the treatment of phenol-containing synthetic wastewater. They are used to evaluate the effects of nutrient addition on the phenol biodegradation rate. Adding yeast extract, glucose, KCl and four mineral nutrients into the shaking flasks containing wastewater and cultivating organisms, revealed phosphate as the crucial nutrient stimulating phenol biodegradation at 15% salinity. The operation results of the sequencing batch biofilm reactor indicates that phosphate content increased up to five times the original level can increase the phenol removal rate by 150%. A 99% phenol removal efficiency could be achieved by shortening the reaction time in the biofilm reactor from 40 h to 16 h, compared with basic nutrients added. In this article we applied an applicable and effective shaking-culture method to determine nutrient requirements in biotreatment processes without stopping the running reactors.
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PMID:Effects of nutrient addition on phenol biodegradation rate in biofilm reactors for hypersaline wastewater treatment. 1674 19


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