Development of an enzyme membrane reactor for treatment of cyanide-containing wastewaters from the food industry.

Cyanidase, an immobilized enzyme preparation for hydrolyzing cyanide to ammonia and formate, was applied for the treatment of cyanide-containing waste waters from the food industry. Apricot seed extract was chosen as a model effluent. The enzymatic hydrolysis of pure amygdalin, the main cyanogenic glycoside in the extract, and the degradation of the cyanide formed was investigated and compared with the behavior of the real extract in a batch slurry reactor. A diffusional-type, flat-membrane reactor with immobilized cyanidase was developed, where the enzyme is effectively protected from adverse effects of high molecular components contained in the extract. For monitoring continuous-membrane reactor operation, a new unsegmented ammonia measurement system was developed and applied. In continuous operation the cyanidase retained its original activity for more than 400 hours on steam.

GTP-binding mutants of rab1 and rab2 are potent inhibitors of vesicular transport from the endoplasmic reticulum to the Golgi complex.

We have examined the role of ras-related rab proteins in transport from the ER to the Golgi complex in vivo using a vaccinia recombinant T7 RNA polymerase virus to express site-directed rab mutants. These mutations are within highly conserved domains involved in guanine nucleotide binding and hydrolysis found in ras and all members of the ras superfamily. Substitutions in the GTP-binding domains of rab1a and rab1b (equivalent to the ras 17N and 116I mutants) resulted in proteins which were potent trans dominant inhibitors of vesicular stomatitis virus glycoprotein (VSV-G protein) transport between the ER and cis Golgi complex. Immunofluorescence analysis indicated that expression of rab1b121I prevented delivery of VSV-G protein to the Golgi stack, which resulted in VSV-G protein accumulation in pre-Golgi punctate structures. Mutants in guanine nucleotide exchange or hydrolysis of the rab2 protein were also strong trans dominant transport inhibitors. Analogous mutations in rab3a, rab5, rab6, and H-ras did not inhibit processing of VSV-G to the complex, sialic acid containing form diagnostic of transport to the trans Golgi compartment. We suggest that at least three members of the rab family (rab1a, rab1b, and rab2) use GTP hydrolysis to regulate components of the transport machinery involved in vesicle traffic between early compartments of the secretory pathway.

Kinetics of enzymatic degradation of cyanide.

Cyanidase is a new enzyme preparation capable of degrading cyanide in industrial wastewaters to ammonia and formate in an apparently one-step reaction, down to very low concentrations. This enzyme has both a high selectivity and affinity toward cyanide. A granular form of the biocatalyst was used in a recirculation fixed bed reactor in order to characterize the new biocatalyst with respect to pH, ionic strength, common ions normally present in wastewaters, mass transfer effects, and temperature. Long term stability was investigated. The kinetics of the enzymatic degradation of cyanide were studied in a batch reactor using the powdered immobilized enzyme preparation and modeled using a simple Michaelis-Menten equation.

Rab1b regulates vesicular transport between the endoplasmic reticulum and successive Golgi compartments.

We report an essential role for the ras-related small GTP-binding protein rab1b in vesicular transport in mammalian cells. mAbs detect rab1b in both the ER and Golgi compartments. Using an assay which reconstitutes transport between the ER and the cis-Golgi compartment, we find that rab1b is required during an initial step in export of protein from the ER. In addition, it is also required for transport of protein between successive cis- and medial-Golgi compartments. We suggest that rab1b may provide a common link between upstream and downstream components of the vesicular fission and fusion machinery functioning in early compartments of the secretory pathway.

Isoprenoid modification of rab proteins terminating in CC or CXC motifs.

Mevalonate starvation of hamster fibroblasts resulted in a shift of rab1b from the membrane to the cytosolic fraction, suggesting that rab1b depends upon an isoprenoid modification for its membrane localization. rab1b and rab3a proteins expressed in insect cells incorporated a product of [3H]mevalonate, and gas chromatography analysis of material released by Raney nickel cleavage demonstrated that rab1b and rab3a are modified by geranylgeranyl groups. Additionally, in vitro prenylation analysis demonstrated farnesyl modification of H-ras but geranylgeranyl modification of five rab proteins (1a, 1b, 2, 3a, and 6). Together, these results suggest that the carboxyl-terminal CC/CXC motifs (X = any amino acid) specifically signal for addition of geranylgeranyl, but not farnesyl, groups. A rab1b mutant protein lacking the two carboxyl-terminal cysteine residues was not prenylated in vitro. However, since a mutant H-ras protein that terminates with tandem cysteine residues was also not modified, the CC motif may be essential, but not sufficient, to signal prenylation of rab1b. Finally, rab1b and rab3a proteins were not efficient substrates for either farnesyl- or geranylgeranyltransferase activities that modify CAAX-containing proteins (A = any aliphatic amino acid). Therefore, rab proteins may be modified by a prenyltransferase(s) distinct from the prenyltransferases that modify carboxyl-terminal CAAX proteins.

Farnesol modification of Kirsten-ras exon 4B protein is essential for transformation.

Oncogenic forms of ras proteins are synthesized in the cytosol and must become membrane associated to cause malignant transformation. Palmitic acid and an isoprenoid (farnesol) intermediate in cholesterol biosynthesis are attached to separate cysteine residues near the C termini of H-ras, N-ras, and Kirsten-ras (K-ras) exon 4A-encoded proteins. These lipid modifications have been suggested to promote or stabilize the association of ras proteins with membranes. Because preventing isoprenylation also prevents palmitoylation, examining the importance of isoprenylation alone has not been possible. However, the oncogenic human [Val12]K-ras 4B protein is not palmitoylated but is isoprenylated, membrane associated, and fully transforming. We therefore constructed mutant [Val12]K-ras 4B proteins that were not isoprenylated to examine the effects of isoprenylation in the absence of palmitoylation. The nonisoprenylated mutant proteins both failed to associate with membranes and did not transform NIH 3T3 cells. In addition, inhibition of isoprenoid and cholesterol synthesis with the drug compactin also decreased [Val12]K-ras 4B protein isoprenylation and membrane association. These results unequivocally demonstrate that isoprenylation, rather than palmitoylation, is essential for ras membrane binding and ras transforming activity. These findings clearly indicate the biological significance of ras protein modification by farnesol and suggest that this modification may be important for facilitating the processing, trafficking, and biological activity of other isoprenylated proteins. Because K-ras is the most frequently activated oncogene in a wide spectrum of human malignancies, study of this pathway could lead to important therapeutic treatments.

Formation of oligosaccharides during enzymatic lactose: Part I: State of art.

Enzymatic lactose hydrolysis by beta-galactosidase (lactase) was investigated with respect to the formation of oligosaccharides. An analysis of the formation of oligosaccharides and their control is important in the development of technical applications for enzymatic lactose hydrolysis. The available literature data on transfer reactions of lactase were reviewed, compared, and presented in a concise tabular form. Mechanisms and possible ways of modelling enzymatic lactose hydrolysis, including formation of oligosaccharides, are presented.

Formation of oligosaccharides during enzymatic lactose hydrolysis and their importance in a whey hydrolysis process: Part II: Experimental.

Enzymatic lactose hydrolysis using two yeast and two fungal lactases that are of current technical interest was studied. The enzymes were compared regarding their oligosaccharide production. Parameters influencing oligosaccharide formation, together with the effect of immobilization were examined and conditions minimizing oligosaccharide content in the hydrolysis product were proposed. Enzymatic whey hydrolysis was also considered. A possibility of enzymatic lactose recombination from its hydrolysis products was shown.

Biological denitrification of drinking water using autotrophic organisms with H(2) in a fluidized-bed biofilm reactor.

Biological denitrification of drinking water was studied in a fluidized sand bed reactor using a mixed culture. Hydrogen gas was used as the reaction partner. The reaction kinetics were calculated with a double Monod saturation function. The K(s) value for hydrogen was below 0.1% of saturation. No appreciable biofilm diffusion effects were detected. Reactor performance was a function of the culture's past history. Batch experiments always exhibited an accumulation of NO(2) (-), but continuous experiments with a sufficiently long residence time always resulted in complete nitrogen removal. Rates of up to 23 mg N/L h, 25 mg N/g DW h, and 7.9 mg H(2)/L h were achieved. Residence times of 4.5 h would be required for complete denitrification of water containing 25 mg NO(3) (-)-N/L or approximately 1 h for every 5 mg/L.

A microbial culture with oxygen-sensitive product distribution as a potential tool for characterizing bioreactor oxygen transport.

The dissolved oxygen (DO) level has been shown to have a profound effect on the product distribution of a Bacillus subtilis culture, with acetoin being excreted with DO above 100 parts per billion (ppb) and butanediol below 100 ppb. The product concentration ratio changed rapidly in the 80-90 ppb range. Switching from one oxygen level to another caused one already accumulated product to be converted to the other in a reversible manner. Rates of change of 0.5-1 g/L h enabled detection within 10 min. Detection sensitivity is enhanced because the ratio of two concentrations can be measured. Remarkably sensitive to mixing rates, the culture responded to changes in stirring speed during experiments in which the dissolved oxygen was controlled at a constant level with a novel control system. Thus, the culture is capable of detecting dead zones in relatively well-mixed reactors and oxygen gradients in column and tubular reactors. High-viscosity effects can also be investigated since the culture grows well in xanthan gum solutions. Preliminary kinetic model development indicates that a useful model for simulating reactor mixing and transport effects can be developed to aid in the planning of experiments.

Expert systems in medicine: a biomedical engineering perspective.

Knowledge-based expert systems for medical applications have received considerable attention in recent years. In this review, fundamental terms and notions of artificial intelligence techniques as applied to expert systems are introduced. The most well-known and influential medical expert systems are discussed in detail, and newer efforts are surveyed. A critical comparison of strengths and weaknesses of the systems is made, discussing depth and complexity of knowledge, acquisition of knowledge, user interaction and explanations, knowledge engineering tools, system evaluations, and user resistance. Long- and short-term trends are appraised.

Computer methods, uremic encephalopathy, and adequacy of dialysis.

It has been recognized for over 30 years that increased renal dysfunction results in increased slow wave activity in the EEG generally prior to the clinical appearance of disabling, dialysis-responsive encephalopathic symptoms of clinical uremia. This paper describes computerized methods that have been used to quantify this slow wave activity and the results of studies that have employed such computerized techniques. Practical information is furnished to guide those who wish to use these methods in their own research and practice. A survey of the limitations and pitfalls inherent in the various techniques is given. A prospectus outlines possible future directions in the field.

Solubility and selective crystallization of lactose from solutions of its hydrolysis products glucose and galactose.

A high degree of conversion is desired when lactose is hydrolyzed to glucose and galactose. This produces, however, a high concentration of galactose, which is inhibitory for the enzyme catalyst (beta-galactosidase). The inhibition can be reduced by limiting the conversion per pass over the enzyme (e.g. to ca. 50%), separating unconverted lactose from the reactor effluent, and recycling it to the reactor inlet. (This allows the overall conversion to be raised to ca. 80-90%). The solubilities of lactose, glucose, and galactose have been determined at various temperatures and for sugar mixtures having different concentrations and degrees of hydrolysis. Various cooling crystallizations have defined convenient and simple processes for the selective separation of lactose from its hydrolysis products.