Modeling the specific growth rate of Lactobacillus plantarum in cucumber extract

Extensive empirical research has been published on the fermentation of vegetables, but little predictive modeling of the process is available. The objectives of this study were to assess the effects of key variables involved in cucumber fermentation and to develop models for predicting the growth of Lactobacillus plantarum in pure and mixed culture fermentations. The growth medium for the studies was cucumber juice. The effects of various concentrations of lactic, acetic, and hydochloric acids and sodium chloride on growth at 30° C were determined in batch culture. Limiting conditions for growth were pH 3.37 (lower limit), 69 mm undissociated lactic acid, 150 mm undissociated acetic acid, or 11.8% NaCl. Acetic acid was stimulatory to growth at low concentrations (up to 40 mm) but inhibitory at higher concentrations. Lactic acid was more inhibitory than acetic acid, whether total or undissociated concentrations were used as the basis of comparison. A predictive equation for specific growth rate was developed, tested, and shown to predict growth of L. plantarum in batch processes reasonably well.Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the U. S. Department of Agriculture or North Carolina Agricultural Research Service, nor does it imply approval to the exclusion of other products that may be suitable Correspondence to: H. P. Fleming     

Activity and specificity of covalently immobolized wheat germ agglutinin toward cell surfaces

Wheat germ agglutinin protein, which is able to agglutinate tumor cells better than normal cells, was covalently bound to polyacrylamide gel beads. The specific binding activity of the protein was preserved on these beads and was expressed heterogeneously by the binding of mouse leukemia cells (L1210) to the protein coupled gels. The selective activity of the immobilized protein was maximal when the number of sites available to covalently couple the protein was lowest. The application of this observation to the general field of covalent immobilization of proteins and enzymes may be of considerable utility.     

Lysis ofMicrococcus lysodeikticus by lysozyme covalently immobilized on cellulose and polyacrylamide

Lysozyme immobilized on polyacrylamide beads or cellulose fibers is found to retain activity for hydrolysis of the cell walls of Micrococcus lysodeikticus. The immobilization on cellulose is somewhat reversible; the polyacrylamide immobilized lysozyme does not release any enzyme upon washing as evidenced by UV and lytic activity tests. The specific catalytic activity of the lysozyme-polyacrylamide system is found to decline as the density of derivatized surface groups is increased; a model of protein deactivation due to excess surface coupling is presented as a possible rationale for such specific activity variations.     

Duartin,an isoflavan from Machaerium opacum

The heartwood of Machaerium opacum contains 2,3-dimethoxyphenol, the stilbenes pinosylvin monomethyl ether and dimethyl ether, and the isoflavans (?)-duartin and (?)-mucronulatol. The structure of (3S)-7,3′-dihydroxy-8,2′,4′-trimethoxyisoflavan was deduced for duartin and the constitution confirmed by synthesis of the racemate.     

Rheological,Mass Transfer,and Mixing Characterization of Cellulase-Producing Trichoderma reesei Suspensions

Steady and dynamic shear measurements are utilized to characterize the rheological behavior of Trichoderma reesei RUT-C30 fungal suspensions during batch growth on xylose (soluble substrate) or cellulose (particulate solid substrate) at three different fermentor impeller speeds (250, 400, and 550 rpm). Biomass concentrations versus time were unimodal on xylose and bimodal on cellulose. This behavior is consistent with relatively rapid, early growth on easily metabolized growth medium components (yeast extract), followed by a second, slower growth phase due to hydrolysis of recalcitrant cellulose by increasing cellulase concentrations. Critical dissolved oxygen (DO) concentration for T. reesei growth on cellulose was found to be 0.073 mmol/L. The DO was kept above this level by supplementing the air feed with pure oxygen, implying that mass transfer limitations were not the cause of bimodal cell growth. Steady shear rheological data showed shear thinning behavior and a yield stress for all broth samples regardless of substrate. Casson and Herschel−Bulkley constitutive equations fit steady shear data well. Dynamic shear measurements on broth suspensions indicated “gel-like” behavior at low strains, with microstructural breakdown at larger displacements. Time variations of the Casson model parameters (yield stress and Casson viscosity) and dynamic moduli (elastic and viscous modulus) followed both cell mass and morphology: a single maximum in all rheological variables resulted when cells were grown on xylose or on cellulose at impeller speeds of 400 or 550 rpm, and dual maxima were observed for cellulose-grown cells at 250 rpm.     

Functional effects of amino acid substitutions within the large binding pocket of the phosphotriesterase OpdA from Agrobacterium sp. P230

The phosphotriesterase OpdA from Agrobacterium sp. P230 has about 10-fold higher activity for dimethyl organophosphate (OP) insecticides, than its homologue from Flavobacterium sp. ATCC27551, organophosphate hydrolase (OPH). OpdA shows about 10% amino acid sequence divergence from OPH and also has a 20 residue C-terminal extension. Here we show that the difference in kinetics is largely explained by just two amino acid differences between the two proteins. A truncated form of OpdA demonstrated that the C-terminal extension has no effect on its preference for dimethyl organophosphate substrates. Chimeric proteins of OPH and OpdA were then analysed to show that replacement of a central region of OpdA sequence, which encodes the residues in the large subsite of the active site, with the homologous region in OPH decreased the activity of OpdA towards dimethyl OPs, to values close to those for OPH. Site-directed mutagenesis in this region identified two differences between the proteins, Y257H and F272L (with the OpdA residues first) as being responsible for this reduction. These two differences were also responsible for the increased activity of OpdA towards the diisopropyl organophosphate, diisopropyl fluorophosphate, relative to OPH. Molecular modelling of triethyl phosphate in the active site of OpdA confirmed a reduction in the size of the large subsite relative to OPH.     

Anomalous scattering analysis of Agrobacterium radiobacter phosphotriesterase: the prominent role of iron in the heterobinuclear active site

Bacterial phosphotriesterases are binuclear metalloproteins for which the catalytic mechanism has been studied with a variety of techniques, principally using active sites reconstituted in vitro from apoenzymes. Here, atomic absorption spectroscopy and anomalous X-ray scattering have been used to determine the identity of the metals incorporated into the active site in vivo. We have recombinantly expressed the phosphotriesterase from Agrobacterium radiobacter (OpdA) in Escherichia coli grown in medium supplemented with 1 mM CoCl2 and in unsupplemented medium. Anomalous scattering data, collected from a single crystal at the Fe-K, Co-K and Zn-K edges, indicate that iron and cobalt are the primary constituents of the two metal-binding sites in the catalytic centre (alpha and beta) in the protein expressed in E. coli grown in supplemented medium. Comparison with OpdA expressed in unsupplemented medium demonstrates that the cobalt present in the supplemented medium replaced zinc at the beta-position of the active site, which results in an increase in the catalytic efficiency of the enzyme. These results suggest an essential role for iron in the catalytic mechanism of bacterial phosphotriesterases, and that these phosphotriesterases are natively heterobinuclear iron-zinc enzymes.     

Electronic and geometric structures of the organophosphate-degrading enzyme from <Emphasis Type="Italic">Agrobacterium radiobacter</Emphasis> (OpdA)

The organophosphate-degrading enzyme from Agrobacterium radiobacter (OpdA) is a highly efficient catalyst for the degradation of pesticides and some nerve agents such as sarin. OpdA requires two metal ions for catalytic activity, and hydrolysis is initiated by a nucleophilic hydroxide that is bound to one of these metal ions. The precise location of this nucleophile has been contentious, with both a terminal and a metal-ion-bridging hydroxide as likely candidates. Here, we employed magnetic circular dichroism to probe the electronic and geometric structures of the Co(II)-reconstituted dinuclear metal center in OpdA. In the resting state the metal ion in the more secluded α site is five-coordinate, whereas the Co(II) in the solvent-exposed β site is predominantly six-coordinate with two terminal water ligands. Addition of the slow substrate diethyl 4-methoxyphenyl phosphate does not affect the α site greatly but lowers the coordination number of the β site to five. A reduction in the exchange coupling constant indicates that substrate binding also triggers a shift of the μ-hydroxide into a pseudoterminal position in the coordination sphere of either the α or the β metal ion. Mechanistic implications of these observations are discussed.     

Discriminating lymphomas and reactive lymphadenopathy in lymph node biopsies by gene expression profiling

Background

Diagnostic accuracy of lymphoma, a heterogeneous cancer, is essential for patient management. Several ancillary tests including immunophenotyping, and sometimes cytogenetics and PCR are required to aid histological diagnosis. In this proof of principle study, gene expression microarray was evaluated as a single platform test in the differential diagnosis of common lymphoma subtypes and reactive lymphadenopathy (RL) in lymph node biopsies.

Methods

116 lymph node biopsies diagnosed as RL, classical Hodgkin lymphoma (cHL), diffuse large B cell lymphoma (DLBCL) or follicular lymphoma (FL) were assayed by mRNA microarray. Three supervised classification strategies (global multi-class, local binary-class and global binary-class classifications) using diagonal linear discriminant analysis was performed on training sets of array data and the classification error rates calculated by leave one out cross-validation. The independent error rate was then evaluated by testing the identified gene classifiers on an independent (test) set of array data.

Results

The binary classifications provided prediction accuracies, between a subtype of interest and the remaining samples, of 88.5%, 82.8%, 82.8% and 80.0% for FL, cHL, DLBCL, and RL respectively. Identified gene classifiers include LIM domain only-2 (LMO2), Chemokine (C-C motif) ligand 22 (CCL22) and Cyclin-dependent kinase inhibitor-3 (CDK3) specifically for FL, cHL and DLBCL subtypes respectively.

Conclusions

This study highlights the ability of gene expression profiling to distinguish lymphoma from reactive conditions and classify the major subtypes of lymphoma in a diagnostic setting. A cost-effective single platform "mini-chip" assay could, in principle, be developed to aid the quick diagnosis of lymph node biopsies with the potential to incorporate other pathological entities into such an assay.