Fermentation ofCandida utilis for uricase production

Summary Conditions for the production of microbial uricase byCandida utilis were studied. For the selected strain, hypoxanthine proved to be the most effective inducer of uricase formation. The highest values of biomass as well as uricase activity in the mechanically agitated fermentor were obtained under the following conditions: 50 h, rotation impeller speed 7 s^–1, air flow rate 1.25×10^–5 m³s^–1, concentration of inducer 0.1%.List of symbols b width of baffle, m - c length of baffle, m - D diameter of cylindrical fermentor, m - d diameter of impeller, m - d ₁ diameter of impeller disc, m - Fr _m impeller Froud number - g gravitional acceleration, ms^–2 - H height of batch surface above bottom, m - H ₂ height of impeller disc above bottom, m - h height of impeller blade, m - Kp _g flow rate number - L length of impeller blade, m - N rotational speed of impeller, s^–1 - Re _m impeller Reynolds number - T time, h - V volume of batch, m³ - V _g air (gas) flow rate, m³s^–1 - x mass fraction of the dry matter of cells - x ₀ initial value of the mass fraction of the dry matter of cells - _r volume fraction of the dry matter of cells - <eta<₁ viscosity of pure liquid, Pa s - viscosity of batch (suspension of microbial suspension), Pa s - density of batch, kg m^–3     

Neuro-fuzzy structural classification of proteins for improved protein secondary structure prediction

Fourier transform infrared (FTIR) spectroscopy is a very flexible technique for characterization of protein secondary structure. Measurements can be carried out rapidly in a number of different environments based on only small quantities of proteins. For this technique to become more widely used for protein secondary structure characterization, however, further developments in methods to accurately quantify protein secondary structure are necessary. Here we propose a structural classification of proteins (SCOP) class specialized neural networks architecture combining an adaptive neuro-fuzzy inference system (ANFIS) with SCOP class specialized backpropagation neural networks for improved protein secondary structure prediction. Our study shows that proteins can be accurately classified into two main classes "all alpha proteins" and "all beta proteins" merely based on the amide I band maximum position of their FTIR spectra. ANFIS is employed to perform the classification task to demonstrate the potential of this architecture with moderately complex problems. Based on studies using a reference set of 17 proteins and an evaluation set of 4 proteins, improved predictions were achieved compared to a conventional neural network approach, where structure specialized neural networks are trained based on protein spectra of both "all alpha" and "all beta" proteins. The standard errors of prediction (SEPs) in % structure were improved by 4.05% for helix structure, by 5.91% for sheet structure, by 2.68% for turn structure, and by 2.15% for bend structure. For other structure, an increase of SEP by 2.43% was observed. Those results were confirmed by a "leave-one-out" run with the combined set of 21 FTIR spectra of proteins.     

Mucor hiemalis: a new source for uricase production

Summary One hundred and sixty-five strains of microorganisms with the ability to grow in a medium containing uric acid as a major source of nitrogen were isolated from soil samples during a screening program. Among them, a zygomycete fungus with well-developed columellae was recognized to produce high levels of the enzyme in a short time. Classification of the isolated fungus was carried out according to the morphological and culture characteristics of the organism, and it was identified as Mucor hiemalis. The fungus was able to produce an intracellular urate oxidase in a fermentation medium mainly containing uric acid. Optimized composition of the medium consisted of (l⁻¹ of distilled water) uric acid, 7.0 g; maltose, 6.0 g; Vogel stock solution, 20 and 1 ml of 0.5 M copper sulphate. The optimum pH and temperature for uricase production in the optimized medium were pH 6 and 30 °C, respectively.     

Neuro-fuzzy decision trees

Fuzzy decision trees are powerful, top-down, hierarchical search methodology to extract human interpretable classification rules. However, they are often criticized to result in poor learning accuracy. In this paper, we propose Neuro-Fuzzy Decision Trees (N-FDTs); a fuzzy decision tree structure with neural like parameter adaptation strategy. In the forward cycle, we construct fuzzy decision trees using any of the standard induction algorithms like fuzzy ID3. In the feedback cycle, parameters of fuzzy decision trees have been adapted using stochastic gradient descent algorithm by traversing back from leaf to root nodes. With this strategy, during the parameter adaptation stage, we keep the hierarchical structure of fuzzy decision trees intact. The proposed approach of applying backpropagation algorithm directly on the structure of fuzzy decision trees improves its learning accuracy without compromising the comprehensibility (interpretability). The proposed methodology has been validated using computational experiments on real-world datasets.     

Neuro-fuzzy model of homocysteine metabolism

In view of well-documented association of hyperhomocysteinaemia with a wide spectrum of diseases and higher incidence of vitamin deficiencies in Indians, we proposed a mathematical model to forecast the role of demographic and genetic variables in influencing homocysteine metabolism and investigated the influence of life style modulations in controlling homocysteine levels. Total plasma homocysteine levels were measured in fasting samples using reverse phase HPLC. Multiple linear regression (MLR) and neuro-fuzzy models were developed. The MLR model explained 64% variability in homocysteine, while the neuro-fuzzy model showed higher accuracy in predicting homocysteine with a mean absolute error of 0.00002 \(\mu \hbox {mol}/\hbox {L}\). Methylene tetrahydrofolate reductase (MTHFR) C677T, 5-methyltetrahydrofolate homocysteine methyltransferase (MTR) A2756G and 5-methyltetrahydrofolate homocysteine methyltransferase reductase (MTRR) A66G were shown to be positively associatiated with homocysteine, while nonvegetarian diet, serine hydroxymethyltransferase 1 (SHMT1) C1420T and TYMS \(5^\prime \)-UTR 28 bp tandem repeat exhibited negative association with homocysteine. The protective role of SHMT1 C1420T was attributed to more H-bonding interactions in the mutant modelled compared to the wild type, as shown through in silico analysis. To conclude, polymorphisms in genes regulating remethylation of homocysteine strongly influence homocysteine levels. The restoration of one-carbon homeostasis by SHMT1 C1420T or increased flux of folate towards remethylation due to TYMS \(5^\prime \)-UTR 28 bp tandem repeat or nonvegetarian diet can lower homocysteine levels.     

Mechanism of uricase action.

Uricase (urate:oxygen oxidoreductase, EC 1.7.3.3) exposes a positional and steric specificity in the enzymic conversion of urate to allantoin. C-2 of urate was recovered as C-2 of allantoin. By the consecutive oxidation and hydrolysis reactions a levorotatory intermediate was formed, presumably (-)-2-oxo-4-hydroxy-4-carbohydroxy-5-ureido-imidazoline. The absorption and optical rotation dispersion spectra of the intermediate were established. In the presence of borate buffer, the intermediate was transformed to (+)-alloxanate. The decay of the former compound depends on general base and acid catalysis. RS-(+/-)-allantoin was formed by chemical decarboxylation and S-(+)-allantoin by enzymic decarboxylation.     

Effect of surfactants and inducers on increased uricase production under submerged fermentations by Bacillus cereus

Uricase is a clinical enzyme used for the oxidation of uric acid crystals in gout disease. The present study aimed to increase the suitable surfactant-mediated uricase production on induction by different concentrations of inducers. The efficiency of Bacillus cereus to produce extracellular uricase enzyme was studied in uric acid-containing agar plates. Among the studied inducers, uric acid is the potential inducer for uricase production under submerged fermentations (SMF), which induced 19.41?U/ml uricase in medium containing 2.0?g/L of uric acid, however further increase in the uric acid concentration decreased uricase production, which could be because of substrate inhibition. The physical parameters including agitation speed (rpm) and time duration (h) of uricase production were optimized and found to produce optimum uricase at 150?rpm in 26?h of SMF. Among the studied surfactants, nonionic surfactant, polyvinyl alcohol has shown a remarkable increase in the uricase production of 31.58?U/ml, which is a 61% increase under optimized conditions in SMF. The stability of produced uricase was found at pH 7.5 and temperature 30°C. Also the effects of various metal ions (1?mM) on the uricase activity were studied and observed to be inhibitory in nature in the descending order K⁺?>?Ca²⁺?>?Zn²⁺?>?Fe³⁺?>?Ni²⁺?>?Mg²⁺?>?Mn²⁺?>?Cu²⁺.     

Purification of uricase by biospecific adsorption-desorption

A simple procedure for the purification of uricase from bovine kidney is described. The procedure involves the following steps: 1) processing of kidney mince by borate/butanol, 2) ammonium sulphate precipitation, and 3) biospecific adsorption-desorption. The adsorbents were prepared by chemical attachment of urate or xanthine to agarose gel beads. The desorption was performed by a xanthine solution. The adsorption-desorption procedure resulted in an 11 000-12 000-fold purification. The specific activity of the purified uricase was 19.8 U/mg using either "urate" adsorbent. The recovery was about 70%. The adsorbents were also used for the purification of commercial uricase preparations from hog liver. In this case the purified uricase also possessed a specific activity of 19.8 U/mg. The products were homogenous as judged by gradipore electrophoresis and gel filtration.     

Extraction of uricase from Candida utilis

Summary Six methods of uricase extraction from Candida utilis were tested and compared. X-press disintegration and sonic oscillation gave the best results.     

Rapid purification of uricase from Bacillus fastidiosus

Summary A simple, rapid procedure was developed for the purification of uricase from Bacillus fastidiosus. The enzyme was purified to homogeneity in a two-step procedure with the aid of fast-flow column chromatography. In this way high yields (37 %) of pure uricase with a specific activity of 78.3 U/mg were obtained in a short time.     

Purification of uricase from mammalian tissue.

A simple, rapid procedure for the purification of uricase from mammalian tissue is reported. The procedure is based on the precipitation of mammalian uricase under certain dialysis conditions, and on its low solubility near neutral pH. Exceptionally high yields of homogeneous enzyme are obtained.     

Non-classical inhibition of uricase by cyanide.

The existence of unoccupied nuclear oestradiol-receptor sites in normal human endometrium was investigated. Nuclei were prepared from endometrial samples obtained by curettage and exposed to [3H]oestradiol, which became maximmaly bound at 0 degrees C within 1 h. This result contrasted with the binding kinetics of oestradiol--receptor complexes, since the exchange of hormone took at least 3 h at 30 degrees C and no displacement occurred at 0 degrees C. Before concluding that the nuclear sites were unoccupied, the presence of endogenous low-affinity ligands was excluded, because the association rate of oestradiol was unchanged after nuclei were stripped from their putative ligands, and the displacement of oestrone bound to nuclear receptor by oestradiol was very slow at 0 degrees C. The available sites had high affinity for oestradiol (KD 1.3 nM) and binding-specificity characteristics of oestradiol receptors. Similar results were observed with crude and purified nuclear preparations. It was concluded that a significant proportion of nuclear oestradiol receptors in normal human endometrium is unoccupied by endogenous hormones.