The NCP (Neurospora crassa gene expressed in the periplasmic space) is a recombinant Escherichia coli expressing the Neurospora crassa metallothionein gene within the periplasmic space. The NCP has been shown to remove heavy metals from solutions containing low concentration of heavy metals. In this study, the ability of NCP to remove heavy metals from wastewater was compared to the commercial sorbents Duolite GT-73, IRC-718, and BIO-FIX. Three process wastewater streams, acid/alkali, chromate, and cyanide, were tested at their original pH and near neutral pH. In addition to other contaminants, the wastewaters contained metals including Cd, Cu, Ni, and Ag. NCP was most efficient in removing the heavy metals from wastewaters at near neutral pH. Chromium removal per gram of IRC-718 at pH 2.05 was an order of magnitude higher than any other sorbent in either pH-unadjusted or pH-adjusted samples. Duolite GT-73, IRC-718, and BIO-FIX had similar efficiency in removing heavy metals from the pH-adjusted wastewater. When the sorbents were tested for removal of part per billion levels of 109 Cd, NCP demonstrated the highest percent removal. These results suggest potential treatment strategies for metalcontaminated wastewaters involving pH adjustment and primary treatment with a commercial sorbent followed by polishing with NCP.

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The gene coding for the Neurospora crassa metallothionein protein was chemically synthesized and cloned into the fusion expression vectors pMal-c and pMal-p. Cell-fractionation experiments demonstrated the proper localization of the pMal-c and pMal-p- expressed proteins to the cytosol and periplasm of the bacteria respectively. Control bacteria as well as the recombinant bacteria producing the metallothionein protein were incubated with solutions of 109Cd at concentrations of 0.2 microM, 1 microM, and 10 microM. The recombinant bacteria were able to accumulate significantly more 109Cd than control bacteria at all concentrations tested. Cadmium accumulation was rapid and highly selective. Maximum uptake was achieved at a pH of 7.0, with lower accumulation at lower or higher pH values. The pH-dependent uptake of cadmium by the recombinant bacteria was exploited to strip off the bound cadmium from the recombinant bacteria and to regenerate most of the cadmium-binding sites. These observations suggest the potential for using a metallothionein-based biosorbent for certain heavy-metal removal applications.

Abstract. 3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, a key regulatory enzyme involved in cholesterol biosynthesis, has recently been reported to be present in rat liver peroxisomes (Keller, G. A., M. C. Barton, D. J. Shapiro, and S. J. Singer, 1985, Proc. Natl. Acad. Sci. USA, 82:770-774). Immunoelectron labeling of ultrathin frozen sections of normal liver, using two monoclonal antibodies to purified rat liver microsomal HMG-CoA reductase, indicated that the enzyme is present in the matrix of peroxisomes. This study is a quantitative biochemical and immunoelectron microscopical analysis of HMGCoA reductase in rat liver peroxisomes and microsomes of normal and cholestyramine-treated animals. Cholestyramine treatment produced a six-to sevenfold increase in the specific activity of peroxisomal HMGCoA reductase, whereas the microsomal HMG-CoA reductase specific activity increased by about twofold. Using a computer program that calculates optimal linear combinations of marker enzymes, it was determined that between 20 and 30% of the total reductase activity was located in the peroxisomes of cholestyramine-treated animals. Less than 5 % of the reductase activity was present in peroxisomes under control conditions. Quantitation of the immunoelectron microscopical data was in excellent agreement with the biochemical results. After cholestymmine treatment there was an eightfold increase in the density of gold particles per peroxisome, and we estimate about a threefold increase in the labeling of the ER.T HE key regulatory enzyme of cholesterol, dolichol, and isopentenyl adenosine biosynthesis, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) 1 reductase, is a 97-kD transmembrane glycoprotein that was believed until recently to reside exclusively in the endoplasmic reticulum (ER) of mammalian cells (7,8, 27,28). However, a recent publication showed that the enzyme in liver cells is present not only in the ER but also within peroxisomes (18). Immunoelectron labeling of ultrathin frozen sections of normal liver, using two monoclonal antibodies to purified rat liver microsomal HMG-CoA reductase, indicated that the enzyme is concentrated in the matrix of peroxisomes.This study was designed to determine what percentage of the total liver HMG-CoA reductase activity is attributable to the peroxisomal enzyme and to determine if the peroxisomal and the ER enzymes are dependently or independently regulated. Normal rat livers and livers obtained from animals in which the total HMG-CoA reductase activity was increased 1. Abbreviations used in this paper: ER, endoplasmic reticulum; HMGCoA, 3-hydroxy-3-methylglutaryl coenzyme A.by cholestyramine treatment were fractionated by differential and density gradient centrifugation.Two completely independent analyses of the fractions were performed: (a) immunoelectron microscopy with quantitation of the antigenic sites of HMG-CoA reductase; and (b) quantitative enzyme activity measurements of the subcellular fractions with computer-assisted analyses. Materials and Methods Anim...

This study investigated the potential to utilize phage-displayed peptides as reagents in sensor applications. A library of random 12-mers displayed on phage was panned against staphylococcal enterotoxin B (SEB), a causative agent of food poisoning. Nine SEB binding phage clones were isolated, all of which share the consensus sequence Trp His Lys at their amino terminus. Binding of several of these phage was shown to be inhibited when they were assayed in a competitive enzyme-linked immunosorbent assay (ELISA) format with synthesized peptide corresponding to the peptide-encoding region of one of the clones. Whole phage were labeled with the dye Cy5, and incorporated into fluoroimmunoassays. Labeled phage were able to detect SEB down to a concentration of 1.4 ng/well in a fluorescence-based immunoassay. When incorporated into an automated fluorescence-based sensing assay, Cy5-labeled phage bound to probes coated with SEB generated a robust signal of about 10,000 pA, vs a signal of 1,000 pA using a control fiber coated with streptavidin. These results demonstrate the potential for development of phage-based sensor reagents.