巨大芽孢杆菌D01吸附金(Au3+)的研究

巨大芽孢杆菌（Ｂａｃｉｌｌｕｓ ｍｅｇａｔｅｒｉｕｍ）Ｄ０１菌体吸附ＡＵ＾３＋的最适ｐＨ值为３．０,其生物吸附作用是一种快速的过程,最初５ｍｉｎ 的吸附量可达到最大吸附量的９５％,温度不影响该吸附作用。在ｐＨ３．０和３０℃、起始金离子浓度与菌体浓度之比为３０５ｍｇ／ｇ的条件下,吸附３０ｍｉｎ,吸附率达９９．１％,吸附量为３０２．０ｍｇ／ｇ干菌体。Ｄ０１菌体能将浓度中的Ａｕ＾３＋还原成Ａｕ＾０,在细     

Binding of Escherichia coli to Functionalized Gold Nanoparticles

The molecular basis of the diversity of fimbrial lectins dictates the extent of adhesion in different types of Escherichia coli strains to mammalian cells. The mechanism of receptor binding by E. coli in eukaryotic cells differs based on the adhesin domains, patterns in the macromolecular structure and the ligand-binding groove. Current sensor technologies utilize biosensors that are based on the carbohydrate moieties that are involved in pathogen adhesion to host cells. Nanoparticles have been extensively used as carriers for pathogen detection. Gold nanoparticles (Au NPs) of 200?nm size were functionalized with two distinct glycoconjugates mannose (Mn?CAu NPs) and Neu??c(??2-3)-Gal-(??1-4)Glc?CPaa (Sg?CAu NPs) in order to investigate primary and fine sugar specificity of uropathogenic E. coli ORN178 and enterotoxigenic E. coli 13762, respectively. The UV-Vis measurement of pristine, 16-mercaptohexadecanoic acid (MHDA)/2-(2-aminoethoxy)ethanol (AEE)/sugar functionalized Au NPs showed a surface plasmon resonance band for Au. Dynamic light scattering analysis showed that the mean averages of the MHDA/AEE/Mn?CAu NP samples increased due to aggregation. The negative zeta potentials of the samples were indicative of aggregation. Fine sugar specificity was observed when Neu??c(??2-3)-Gal-(??1-4)Glc?CPaa functionalized Au NPs (Sg?CAu NPs) specifically showed binding with E. coli 13762 but not with E. coli ORN178. This specificity of E. coli strains to identify and bind to characteristic sugar moieties can be used in the development of biodiagnostic tools with Au NPs as carriers for diagnosis/treatment of human and veterinary diseases. In regards to the growing antibiotic resistance of microorganisms, gold nanoparticles can also be functionalized specifically to reverse adhesion of E. coli to host tissue and can be detected by their optical properties.     

The interaction of ribonuclease T 1 with DNA.

The interaction of RNase T1 with calf thymus DNA was studied using uv difference spectroscopy and the effect of the enzyme on DNA melting. There was no indication of RNase T1 binding with native DNA. A prominent difference spectrum for RNase T1 binding with denatured DNA (d-DNA) was observed at pH 5, 25 degrees and low ionic strength (mu = .01 M) which was depressed at higher ionic strength and pH. The normalized difference spectrum at mu = .01 M, pH 5 and 25 degrees can be interpreted as indicating an interaction of an exposed guanine residue directly with the enzyme and a coupling of this process with the "melting" of short folded segments of d-DNA. The apparent association constant calculated per M guanine residues was 2.4 X 10-4 M-1 under these conditions. The results are discussed in reference to comparable studies on the interaction of RNase T1 with RNA and small guanine ligands.     

Effects of pH, temperature, and calcium concentration on the stoichiometry of the calcium pump of sarcoplasmic reticulum

Coupling of Ca2+ transport to ATP hydrolysis by isolated skeletal muscle sarcoplasmic reticulum vesicles has been investigated by means of ATP pulse methods. The stoichiometric amounts of Ca2+ transported per pulse of ATP were measured by Ca2+-stat methods, using either a Ca2+ electrode or arsenazo III as end point detectors, or by means of 45CaCl2. Maximum coupling ratios (Ca2+/ATP), of 1.82 +/- 0.13 occurred at pH 6.8, 25 degrees C, and in the presence of saturating Ca2+ concentrations. Ca2+/ATP values decreased at alkaline pH, with an apparent pK alpha of 7.9. The coupling ratio was unaltered between 6 and 30 degrees C, but decreased to 0.4 at 42 degrees C. Uncoupling by alkaline pH and high temperatures was reversible. The coupling process was Ca2+-dependent, with a K0.5 value for Ca2+ of 0.12 microM and a Hill coefficient of 2.0. Ca2+ ions, which were transported into vesicles under conditions resulting in low coupling ratios, were retained as the calcium oxalate precipitate, following complete hydrolysis of substrate. Passive Ca2+ efflux and Ca2+ exchange, were independent of pH. The observed variations in Ca2+/ATP ratio cannot readily be explained on the basis of a pump-leak model. Rather, the Ca2+-ATPase appears to be capable of pumping Ca2+ ions, under physiological conditions, with variable stoichiometry that is dependent upon its thermodynamic loading.     

Enzymatic biotransformation of the azo dye Sudan Orange G with bacterial CotA-laccase

In the present study we show that recombinant bacterial CotA-laccase from Bacillus subtilis is able to decolourise, at alkaline pH and in the absence of redox mediators, a variety of structurally different synthetic dyes. The enzymatic biotransformation of the azo dye Sudan Orange G (SOG) was addressed in more detail following a multidisciplinary approach. Biotransformation proceeds in a broad span of temperatures (30-80 degrees C) and more than 98% of Sudan Orange G is decolourised within 7h by using 1 U mL(-1) of CotA-laccase at 37 degrees C. The bell-shape pH profile of the enzyme with an optimum at 8, is in agreement with the pH dependence of the dye oxidation imposed by its acid-basic behavior as measured by potentiometric and electrochemical experiments. Seven biotransformation products were identified using high-performance liquid chromatography and mass spectrometry and a mechanistic pathway for the azo dye conversion by CotA-laccase is proposed. The enzymatic oxidation of the Sudan Orange G results in the production of oligomers and, possibly polymers, through radical coupling reactions. A bioassay based on inhibitory effects over the growth of Saccharomyces cerevisiae shows that the enzymatic bioremediation process reduces 3-fold the toxicity of Sudan Orange G.     

The enzyme coupling process in urease immobilization on O-alkylated nylon tubes

Coupling of Jack bean urease (EC 3.5.1.5) to the inside surface of type 6 nylon tubes, activated by high-temperature O-alkylation with dimethyl sulphate and modified subsequently with lysine and glutaraldehyde, was investigated to establish optimal experimental conditions for the coupling process. For the system described, the most active immobilized urease derivatives were prepared with 2 mg/ml of the solubilized urease solution and use of higher enzyme concentrations proved wasteful. Although urease coupling without thermal denaturation of the solubilized enzyme was achieved at 20 degrees C, derivatives prepared at 37 degrees C yielded maximal activity over the 3 h coupling period. Also, longer incubations of the enzyme solution in the tube were unnecessary under these conditions. Optimal pH for the coupling process was 6.5, one at which the solubilized enzyme was most stable.     

A self-assembled monolayer-based piezoelectric immunosensor for rapid detection of Escherichia coli O157:H7

A piezoelectric immunosensor was developed for rapid detection of Escherichia coli O157:H7. It was based on the immobilization of affinity-purified antibodies onto a monolayer of 16-mercaptohexadecanoic acid (MHDA), a long-chain carboxylic acid-terminating alkanethiol, self-assembled on an AT-cut quartz crystal's Au electrode surface with N-hydroxysuccinimide (NHS) ester as a reactive intermediate. The binding of target bacteria onto the immobilized antibodies decreased the sensor's resonant frequency, and the frequency shift was correlated to the bacterial concentration. The stepwise assembly of the immunosensor was characterized by means of both quartz crystal microbalance (QCM) and cyclic voltammetry techniques. Three analytical procedures, namely immersion, dip-and-dry and flow-through methods, were investigated. The immunosensor could detect the target bacteria in a range of 10(3)-10(8)CFU/ml within 30-50 min, and the sensor-to-sensor reproducibility obtained at 10(3) and 10(5) colony-forming units (CFU)/ml was 18 and 11% R.S.D., respectively. The proposed sensor was comparable to Protein A-based piezoelectric immunosensor in terms of the amount of immobilized antibodies and detection sensitivity.     

Properties of immobilized fig alpha-galactosidase and effect on ceramide-3 content of plasma from patients with Fabry's disease.

The possibility of lowering the level of ceramide-3 (galactosyl-alpha(1 leads to 4)-galactosyl-beta(1 leads to 4)-glucosyl-beta(1 leads to 1)-ceramide) in the plasma of patients with Fabry's disease was investigated. An immobilized alpha-galactosidase (alpha-D-galactoside galactohydrolase, EC 3.2.1.22) was prepared by coupling purified fig alpha-galactosidase to Sepharose 4B. The pH optimum for the hydrolysis of the artificial substrate p-nitro-phenyl-alpha-D-galactopyranoside was shifted by approx. 0.5--1.0 pH unit to higher pH values upon coupling of the enzyme to Sepharose 4B. The immobilized enzyme was more stable than the native enzyme to incubation at 60 degrees C. The immobilized enzyme was able to hydrolyse ceramide-3 either at pH 4.5 or at pH 7.4 in an artificial system in which sodium taurocholate was used to solubilize the substrate. In contrast, when the immobilized enzyme was incubated with normal plasma or plasma from a patient with Fabry's disease, in which elevated levels of ceramide-3 occur, no hydrolysis of the glycosphingo-lipid could be detected. The results suggest that lowering of level of ceramide-3 in plasma from patients with Fabry's disease by enzymic means is not feasible.     

A new scheme of hybridization based on the Au(nano)-DNA modified glassy carbon electrode

DNA hybridization on the Au(nano)-DNA modified glassy carbon electrode (GCE) was investigated. The thiol modified probe oligonucleotides (SH-ssDNA) at the 5' phosphate end were assembled on the Au(nano)-DNA modified GCE surface. The electrochemical response of the probe immobilization and hybridization with target DNA was measured by differential pulse voltammetry (DPV) using methylene blue (MB) as the electroactive indicator. Gold nanoparticles can be dispersed effectively on the GCE surface in the presence of calf thymus DNA. Au(nano)-DNA modified GCE could greatly increase the active sites and enhance the response signal during immobilization and hybridization. The hybridization amount of target DNA could be greatly increased. The linear detection range of Au(nano)-DNA electrode for the complementary 21-mer oligonucleotide (cDNA) was achieved from 1.52 x 10(-10) to 4.05 x 10(-8) mol L(-1). The detection limit could reach the concentration of 10(-10) mol/L.     

Detection of cadmium by a fiber-optic biosensor based on localized surface plasmon resonance

A novel transmission-based localized surface plasmon resonance (LSPR) fiber-optic probe has been developed to determine the heavy metal cadmium ion (Cd(II)) concentration. The LSPR sensor was constructed by immobilizing phytochelatins (PCs), (gammaGlc-Cys)(8)-Gly, onto gold nanoparticle-modified optical fiber (NM(Au)OF). The optimal immobilizing conditions of PCs on to the NM(Au)OF are 71.6mug/ml PCs in pH 7.4 PBS for 2h. The absorbability (change of light absorption) of the PC-functionalized NM(Au)OF sensor increases to 9% upon changing the Cd(II) level from 1 to 8ppb with a sensitivity of 1.24ppb(-1) and a detection limit of 0.16ppb. The sensor retained 85% of its original activity after nine cycles of deactivation and reactivations. In addition, the sensor retains its activity and gives reproducible results after storage in 5% d-(+)-trehalose dehydrate solution at 4 degrees C for 35 days. The dissociation constant (K(d)) of the immobilized PCs with Cd(II) was about 6.77x10(-8)M. In conclusion, the PCs-functionalized NM(Au)OF sensor can be used to determine the concentration of Cd(II) with high sensitivity.     

Studies on the preparation of water-insoluble derivatives of rennin and chymotrypsin and their use in the hydrolysis of casein and the clotting of milk

1. Enzymically active insoluble derivatives of chymotrypsin and rennin were prepared by coupling each enzyme to agarose as described by Porath, Axén & Ernback (1967) and rennin to aminoethylcellulose by the method of Habeeb (1967). 2. Agarose-chymotrypsin was stable over the range pH2-9, but agarose-rennin released active enzyme into solution at above pH2 and aminoethylcellulose-rennin was similarly unstable at certain pH values. 3. Each derivative appeared to catalyse the clotting of milk at 30 degrees , but this was probably entirely due to enzyme released into solution from the carrier. 4. The presence of a competitive inhibitor of chymotrypsin during its coupling to agarose had no effect on the activity or stability of the resulting derivative. 5. The characteristics of agarose and cellulose render them not entirely suitable for use in a continuous system with milk.     

Accurate measurement of psoralen-crosslinked DNA: direct biochemical measurements and indirect measurement by hybridization

This paper evaluates methods to measure crosslinkage due to psoralen plus light in total DNA and in specific sequences. DNA exposed in cells or in vitro to a bifunctional psoralen and near ultraviolet light accumulates interstrand crosslinks. Crosslinkage is the DNA mass fraction that is attached in both strands to a crosslink. We show here biochemical methods to measure psoralen photocrosslinkage accurately in total DNA. We also describe methods to measure photocrosslinkage indirectly, in specific sequences, by nucleic acid hybridization. We show that a single 4,5',8-trimethylpsoralen (TMP) crosslink causes at least 50 kbp of alkali-denatured DNA contiguous in both strands with it to snap back into the duplex form when the denatured preparation is returned to neutral pH. This process was so efficient that the DNA was not nicked by the single-strand nuclease S1 at 100-fold excess after snapping back. Uncrosslinked DNA was digested to acid-soluble material by the enzyme. Crosslinkage therefore equals the fraction of S1-resistant nucleotide in this kind of experiment. We alkali-denatured DNA samples crosslinked to varying degrees by varying TMP concentration at constant light exposure. We then measured crosslinkage by ethidium bromide (EtBr) fluorometry at pH 11.8; by EtBr fluorometry at neutral pH of S1 digests of the DNA; and by the fraction of radioactivity remaining acid insoluble in S1-digests of DNA labeled uniformly with [3H]deoxythymidine. These assays measure distinct physical properties of crosslinked DNA. Numerical agreement is expected only when all three measurements are accurate. Under optimum conditions, the three methods yielded identical results over the range of measurement. Using alkaline EtBr fluorescence in crude cell lysates, we detected crosslinks at frequencies in the range of 1.6 X 10(-7) per base pair. These levels were compatible with cell survival, attesting to the sensitivity of the measurement system. Crosslinkage affected hybridization as well. One crosslink prevented all alkali-denatured DNA contiguous in both strands with it from hybridizing to complementary DNA either on solid supports or in solution. Strand-length effects on crosslinkage and on reassociation caused solution hybridization levels to exceed those predicted by simple theory. In a quantitative, dot-blotting assay hybridization was linear up to membrane saturation by denatured, uncrosslinked DNA of any strand length.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of pH, ionic strength, and temperature on activation by calmodulin an catalytic activity of myosin light chain kinase

The reversible association of Ca42+-calmodulin with the inactive catalytic subunit of myosin light chain kinase results in the formation of the catalytically active holoenzyme complex [Blumenthal, D. K., & Stull, J. T. (1980) Biochemistry 19, 5608--5614]. The present study was undertaken in order to determine the effects of pH, temperature, and ionic strength on the processes of activation and catalysis. The catalytic activity of myosin light chain kinase, when fully activated by calmodulin, exhibited a broad pH optimum (greater than 90% of maximal activity from pH 6.5 to pH 9.0), showed only a slight inhibition by moderate ionic strengths (less than 20% inhibition at mu = 0.22), and displayed a marked temperature dependence (Q10 congruent to 2; Ea = 10.4 kcal mol-1). Thermodynamic parameters calculated from Arrhenius plots indicate that the Gibb's energy barrier associated with the rate-limiting step of catalysis is primarily enthalpic. The process of kinase activation by calmodulin had a narrower pH optimum (pH 6.0--7.5) than did catalytic activity, was markedly inhibited by increasing ionic strength (greater than 70% inhibition at mu = 0.22), and exhibited nonlinear van't Hoff plots. Between 10 and 20 degrees C, activation was primarily entropically driven (delta S degrees congruent to 40 cal mol-1 deg-1; delta H degrees = -900 cal mol-1), but between 20 and 30 degrees C, enthalpic factors predominated in driving the activation process (delta S degrees congruent to 10 cal mol-1 deg-1; delta H degrees = -9980 cal mol-1). The apparent change in heat capacity (delta Cp) accompanying activation was estimated to be -910 cal mol-1 deg-1. On the basis of these data we propose that although hydrophobic interactions between calmodulin and the kinase are necessary for the activation of the enzyme, other types of interactions such as hydrogen bonding, ionic, and van der Waals interactions also make significant and probably obligatory contributions to the activation process.     

An optimized method for extraction and detection of Coconut cadang-cadang viroid(CCCVd) from oil palm

Coconut cadong-cadong viroid (CCCVd) causes the Lethal cadang-cadang disease of coconut palms in the Philippines and it is recently reported to be associated with the orange spotting disease on oil palm in Malaysia. The low concentration of the viroid RNA in oil palm as well as the high content of polyphenols and polysaccharides in this plant which interfere with the purification steps makes it difficult to extract and detect this viroid from oil palm. A previously described method was modified and optimized for extraction and detection of CCCVd from infected oil palms. Briefly, 7 g of leaf material was homogenized in a mortar or a blender using liquid nitrogen. 10 ml of extraction buffer (100 mM Tris-HCl pH 7.5, 100 mM NaCl, 10 mM EDTA) along with 100 mM 2-mercaptoethanol and 10 ml water saturated phenol was added to the frozen powder. After centrifuging at 4 degrees C, 4000 g for 30 min, the aqueous phase was extracted once more with phenol then once with chloroform-isoamyl alcohol (24:1). After adding sodium acetate, pH 5.6 to 200 mM, the mixture was precipitated with 2.5 vol ethanol overnight in -20 freezer and then the pellet was washed with 70% ethanol and air-dried. One milliliter of 8 M LiCl was added to the dried pellet and after shaking overnight at 4 degrees C and another centrifugation step the supernatant was collected and precipitated again with ethanol and then the resulting pellet was washed and air-dried. To carry out northern blotting, samples equivalent to 40 g of plant tissue were mixed with formamide buffer and loaded onto a 12% polyacrylamide gel containing 7 M urea and after separation by electrophoresis, were electroblotted onto membrane and fixed by UV cross-linking. Pre-hybridization and hybridization using hybridization buffer (50% formamide, 25%SSPE, 0.1% Ficol and PVP, 0.1 % SDS, 0.02 % DNA (5mg/ml)) was carried out at 45 degrees C for 90 min and 16 h, respectively followed by two low stringency washes (0.5 X SSC, 0.1% SDS, at room temperature for 5 min) and one high stringency wash (0.1X SSC, 0.1% SDS at 60 degrees C for 1 hour). In vitro synthesized DIG-labeled full-length CCCVd(-) RNA probe was used in hybridization step. DIG Nucleic Acid Detection Kit (Roche) instructions were followed for detection procedure and as a result the blue bands corresponding to the position of the viroid were appeared on the membrane. The result of this study showed the ability of DIG labeled probe in detection of the viroid and also provided a suitable extraction and hybridization method for the detection of CCCVd from oil palm.     

Synthesis of specific diastereomers of a DNA methylphosphonate heptamer, d(CpCpApApApCpA), and stability of base pairing with the normal DNA octamer d(TPGPTPTPTPGPGPC).

DNA methylphosphonates are candidate derivatives for use in antisense DNA therapy. Their efficacy is limited by weak hybridization. One hypothesis to explain this phenomenon holds that one configuration of the chiral methylphosphonate linkage, Rp, permits stronger base pairing than the other configuration, Sp. To test this hypothesis, four specific pairs of Rp and Sp diastereomers of the DNA methylphosphonate heptamer d(CpCpApApApCpA) were prepared by block coupling of different combinations of individual diastereomers of d(CpCpApA) and d(ApCpA). Each pair of the diastereomers of the heptamer was separated into individual diastereomes using affinity chromatography on a Lichrosorb-NH2 silica column with a covalently attached complementary normal DNA octamer, d(pTpGpTpTpTpGpGpC). The stabilities of complementary complexes of phosphodiester d(TpGpTpTpTpGpGpC) with 8 individual diastereomers of methylphosphonate d(CpCpApApApCpA) were studied by measuring their melting temperatures (Tm). A direct correlation of Tm values with the number of Rp methylphosphonate centers in the heptamer was found: the more Rp centers, the higher the stability of the complex. Tm values for the diastereomers with 6 all-Rp or all-Sp methylphosphonate centers were found to be 30.5 degrees and 12.5 degrees C, respectively, in 100 mM NaCl, 10 mM Na2HPO4, 1 mM EDTA, pH 7.0 with 15 microM of each oligomer. On the average, each substitution of one Rp-center to an Sp-center in the heptamer decreased the Tm by 3 degrees C. Under the same conditions, the Tm of the normal DNA heptamer with its complement was 21 degrees C. These results are consistent with the model that all-Rp methylphosphonate DNAs hybridize much more tightly to complementary normal DNA than do racemic methylphosphonate DNAs, and may therefore exhibit greater potency as antisense inhibitors.     

Chitosan scaffolds for biomolecular assembly: coupling nucleic acid probes for detecting hybridization

Chitosan, a naturally occurring biopolymer, was used as a scaffold for the covalent binding of single-stranded DNA oligonucleotide probes in a fluorescence-based nucleic acid hybridization assay. Chitosan's pH dependent chemical and electrostatic properties enable its deposition on electrodes and metal surfaces, as well as on the bottom of microtiter plates. A combinatorial 96-well microtiter plate format was used to optimize chemistries and reaction conditions leading to hybridization experiments. We found the coupling of oligonucleotides using relatively common glutaraldehyde chemistry was quite robust. Our hybridization results for complementary ssDNA oligonucleotides (E. coli dnaK sequences) demonstrated linear fluorescence intensity with concentration of E. coli dnaK-specific oligonucleotide from 0.73 microM to 6.6 microM. Moreover, hybridization assays were specific as there was minimal fluorescence associated with noncomplementary groEL oligonucleotide. Finally, these results demonstrate the portability of a DNA hybridization assay based on covalent coupling to chitosan, which, in turn, can be deposited onto various surfaces. More arduous surface preparation techniques involving silanizing agents and hazardous washing reagents are eliminated using this technique.     

A sodium ion-dependent A1AO ATP synthase from the hyperthermophilic archaeon Pyrococcus furiosus

The rotor subunit c of the A(1)A(O) ATP synthase of the hyperthermophilic archaeon Pyrococcus furiosus contains a conserved Na(+)-binding motif, indicating that Na(+) is a coupling ion. To experimentally address the nature of the coupling ion, we isolated the enzyme by detergent solubilization from native membranes followed by chromatographic separation techniques. The entire membrane-embedded motor domain was present in the preparation. The rotor subunit c was found to form an SDS-resistant oligomer. Under the conditions tested, the enzyme had maximal activity at 100 degrees C, had a rather broad pH optimum between pH 5.5 and 8.0, and was inhibited by diethystilbestrol and derivatives thereof. ATP hydrolysis was strictly dependent on Na(+), with a K(m) of 0.6 mM. Li(+), but not K(+), could substitute for Na(+). The Na(+) dependence was less pronounced at higher proton concentrations, indicating competition between Na(+) and H(+) for a common binding site. Moreover, inhibition of the ATPase by N',N'-dicyclohexylcarbodiimide could be relieved by Na(+). Taken together, these data demonstrate the use of Na(+) as coupling ion for the A(1)A(O) ATP synthase of Pyrococcus furiosus, the first Na(+) A(1)A(O) ATP synthase described.     

Optimizing the immobilization of single-stranded DNA onto glass beads

The attachment of single-stranded DNA to a solid support has many biotechnology and molecular biology applications. This paper compares different immobilization chemistries to covalently link single-stranded DNA (20 base pairs), oligo(1), onto glass beads via a 5'-amino terminal end. Immobilization methods included a one-step 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and a two-step EDC reaction to succinylated and PEG-modified glass beads. The third method used 1,4-phenylene diisothiocyanate to immobilize oligo(1) to aminopropyl glass beads. The influence of coupling buffer, oligo(1) concentration, and EDC concentration was also investigated. The one-step EDC-mediated procedure with succinylated or PEG-modified beads in 0.1 M MES buffer, pH 4.5, resulted in the highest immobilization efficiency, 82-89%. EDC concentrations greater than 50 mM and oligo(1) concentrations of 3 microg/g bead were required for effective immobilization. A complementary oligonucleotide, oligo(2), was able to hybridize to the immobilized oligo(1) with a 58% efficiency. This oligonucleotide was subsequently released at 70 degrees C. The relationship between the surface density of oligo(1) and the hybridization efficiency of the complementary oligonucleotide is described.     

Viricidal effects of Lactobacillus and yeast fermentation

The survival of selected viruses in Lactobacillus- and yeast-fermented edible waste material was studied to determine the feasibility of using this material as a livestock feed ingredient. Five viruses, including Newcastle disease virus, infectious canine hepatitis virus, a porcine picornavirus, frog virus 3, and bovine virus diarrhea, were inoculated into a mixture of ground food waste (collected from a school lunch program) containing Lactobacillus acidophilus. Mixtures were incubated at 20, 30, and 40 degrees C for 216 h. In a second trial, four viruses, including Newcastle disease virus, infectious canine hepatitis virus, frog virus 3, and a porcine picornavirus, were inoculated into similar edible waste material containing Saccharomyces cerevisiae. Mixtures were incubated at 20 and 30 degrees C for 216 h. Samples were obtained daily for quantitative (trial 1) and qualitative (trial 2) virus isolation. Temperature, pH, and redox potential were monitored. Controlled pH and temperature studies were also done and compared with the inactivation rates in the fermentation processes. In trial 1 (Lactobacillus fermentation), infectious canine hepatitis virus survived the entire test period in the fermentation process but was inactivated below pH 4.5 in the controlled studies. Newcastle disease virus was inactivated by day 8 in the fermentation process and appeared to be primarily heat sensitive and secondarily pH sensitive in the controlled studies. The porcine picornavirus survived the fermentation process for 8 days at 20 degrees C but was inactivated more rapidly at 30 and 40 degrees C. The controlled studies verified these findings. Frog virus 3 was inactivated by day 3 in the fermentation process and appeared to be sensitive to low pH in the controlled studies. Bovine virus diarrhea was rapidly inactivated in the fermentation process (less than 2 h) and was pH and temperature sensitive. In trial 2 (yeast fermentation), infectious hepatitis virus survived the entire test period in the fermentation process. Newcastle disease virus was inactivated by day 7 at 20 degrees C and day 6 at 30 degrees C. The porcine picornavirus was inactivated by day 7 at 30 degrees C but survived the entire test period at 20 degrees C. Frog virus 3 was inactivated by day 3 at 20 degrees C and day 2 at 30 degrees C.