Optimization of batch processes involving simultaneous enzymatic and microbial reactions

Two general models for batch simultaneous enzymatic and microbial reaction (SEMR) processes are presented, the second derived from and simpler than the first and accounting for enzyme denaturation. Using the second model and parameter values from the literature, simulation was used to examine a range of enzyme addition rate strategies (in which the rate was a linear function of time) for a relatively fast ethanol fermentation and for a longer duration citric acid fermentation, both using cellulose as the substrate. For the ethanol process it is optimal (for a specific objective function which accounts for product value and enzyme cost) to add all the enzyme at the beginning of the process. But for the citric acid process a linearly decreasing enzyme addition rate, coupled with the addition of a small fraction of the enzyme at time zero, is better than pure batch operation or operation with the best constant enzyme feed rate.     

Protection against acute paraquat toxicity by ambroxol

An expression vector for G-CSF, pASLB3-3, was constructed and introduced into Namalwa KJM-1 cells (Hosoi et al., 1988), and cells resistant to 100 nM of methotrexate (MTX) were obtained. Among them, the highest producer, clone SC57, was selected and the productivity of this clone was further characterized. The maximal production of G-CSF was at the most 1.8 g/ml/day using a 25 cm² tissue culture flask, even though the cell number was above 7×10⁵ cells/ml. The limiting factors at high density were analyzed as the deficiency of nutrients, such as glucose, cysteine and serine, and pH control. The depression of specific G-CSF productivity per cell under the batch culture conditions was overcome by using a perfusion culture system, Biofermenter^TM (Sato, 1983) with modifications of nutrients supplementation by a dialysis membrane and/or dissolved oxygen (DO) supplementation by microsilicone fibers. ITPSGF medium was modified to elevate concentrations of amino acids and glucose by 2.0- and 2.5-times, respectively. Under the control of pH at 7.4 and DO at 3 ppm, the specific G-CSF productivity was not depressed even at high cell density (above 1×10⁷ cells/ml), and the amount of G-CSF reached 41 g/ml. These results indicated the possibility of finding the optimum culture conditions for the production of recombinant proteins by Namalwa KJM-1 cells.Abbreviations ABTS 2,2-Azino-di-(3-ethylbenzothiazoline)-6-sulfonic acid - BSA Bovine Serum Albumin - BSA-PBS Phosphate-buffered Saline without Ca²⁺ and Mg²⁺ containing Bovine Serum Albumin - dhfr Dihydrofolate Reductase - DO Dissolved Oxygen - G-CSF Granulocyte Colony-stimulating Factor - HEPES 4-(2-Hydroxyethyl)-1-piperazineethansulfonic Acid - IFN Interferon - MTX Methotrexate - PBS(-) Phosphate-buffered saline without Ca²⁺ and Mg²⁺ - Tween-PBS Phosphate-buffered saline without Ca²⁺ and Mg²⁺ containing 0.05% of Tween 20     

Isolation and characterization of human glucose-6-phosphate isomerase isoforms containing two different size subunits

Previously undetected isoforms of human glucose-6-phosphate isomerase (GPI) have been isolated utilizing substrate-induced elution of the enzyme from spherical cross-linked phosphocellulose as an affinity ligand and subjected to a series of physical and chemical studies. The two major isoforms (1, 48%, pI 9.13; 2, 36%, pI 9.00) are homodimers of subunits of 63.2 kDa (Type-A) and are charge isomers, probably representing deamidation of specific Asn-Gly sequences as in other species. Isoform 3 (13%, pI 8.84) is a heterodimer composed of the Type-A subunit and a previously unreported larger subunit of 69.8 kDa (Type-B). Isoform 4 (3%, pI 8.62) is a BB-homodimer. Structural differences in the two types of subunits are also apparent from CNBr fragmentation patterns. Carbohydrate analyses show that, even though potential N- and O-linked glycosylation sites exist, the isoforms are not due to glycosylation. Recently recognized sequence similarities between GPI and the neurotropic lymphokine, neuroleukin (NLK) suggest that GPI and NLK are either derived from the same gene or represent modifications of the same protein. The possibility of NLK-GPI dimers exists, but the new isoforms identified in this study do not appear to represent hybrids of GPI subunits with mature NLK.     

Lipid conjugates of antiretroviral agents. II. Disodium palmityl phosphonoformate: anti-HIV activity, physical properties, and interaction with plasma proteins

Disodium palmityl phosphonoformate, a novel lipid phosphoester of the anti HIV agent phosphonoformate (foscarnet), inhibits HIV replication in H9 cells and syncytia formation in MOLT-3 cells as effectively as foscarnet itself, as shown by dose-response data from assays for expression of p17 and p24 viral antigens and syncytia formation. Protein binding studies indicate that in serum, the derivative exists bound to albumin and the lipoproteins, and would therefore be likely to exhibit improved serum lifetime in vivo.     

Large scale purification and immunolocalization of bovine uroplakins I, II, and III. Molecular markers of urothelial differentiation

The differentiation of mammalian urothelium culminates in the formation of asymmetrical unit membrane (AUM). Using gradient centrifugation and detergent wash, we purified milligram quantities of AUMs which, interestingly, contained three major proteins (15, 27, and 47 kDa) that appeared to be identical to the three immunoaffinity purified, putatively AUM-associated proteins that we described earlier (Yu, J., Manabe, M., Wu, X.-R., Xu, C., Surya, B., and Sun, T.-T. (1990) J. Cell Biol., 111, 1207-1216). Peptide mapping and immunoblotting established that these three proteins were distinct molecules. Using monospecific antibodies to these three proteins, we showed that they were all restricted to the superficial urothelial cells and were AUM-associated. The 27- and 15-kDa proteins were detected exclusively on the luminal side of mature, apical AUMs. In contrast, epitopes of the 47-kDa protein were detected on both sides of apical AUMs suggesting a transmembranous configuration. These results (i) provide the strongest evidence thus far that AUM contains three major proteins (the 27-kDa uroplakin I, 15-kDa uroplakin II, and 47-kDa uroplakin III) which form an extremely insoluble complex, (ii) suggest that uroplakin II, like uroplakin I (Yu, J., Manabe, M., Wu, X.-R., Xu, C., Surya, B., and Sun, T.-T. (1990) J. Cell. Biol. 111, 1207-1216), translocates from one side of the membrane to another during AUM maturation, (iii) indicate that uroplakin III may play a different structural role than uroplakins I and II in AUM formation, and (iv) establish the three uroplakins as markers for an advanced stage of urothelial differentiation.     

Selective-Differential Medium for Isolation and Differentiation of Pectinatus from Other Brewery Microorganisms

An agar medium, LL-agar (lactate-lead acetate) was designed to selectively differentiate members of the genus Pectinatus (S. Y. Lee, M. S. Mabee, and N. O. Jangaard, Int. J. Syst. Bacteriol. 28:582-594, 1978; S. Y. Lee, M. S. Mabee, N. O. Jangaard, and E. K. Horiuchi, J. Inst. Brew. 86:28-30, 1980) from other brewery microorganisms. Selectivity was achieved by the use of sodium lactate as the sole source of carbon and phenylethyl alcohol as an inhibitor for aerobic gram-negative bacteria and yeast. Differentiation was established by the introduction of lead acetate into the medium, which reacted with the H₂S liberated by Pectinatus and resulted in a blackening of the Pectinatus colonies while the other brewery organisms, when present, remained white. In combination with the Lee tube (J. E. Ogg, S. Y. Lee, and B. J. Ogg, Can. J. Microbiol. 25:987-990, 1979) and this medium, isolation of Pectinatus organisms from beer samples was accomplished with convenience and simplicity.     

Preparation of two dinor-PGI2 metabolites from 6-keto-PGF1α by mycobacterium rhodochrous

The transformation of 6-keto-PGF_1α to two prostacyclin metabolites, 2,3-dinor-6-keto-PGF_1α (I) and 2,3-dinor-6,15-diketo-13,14-dihydro-PGF_1α (II) by $\text{Mycobacterium rhodochrous}$ UC-6176 is described. The finding that the bacterium oxidized 6-keto-PGF_1α to the 6,15-diketo metabolite II shows that it contains 15-hydroxy prostaglandin dehydrogenase and Δ¹³ reductase enzyme systems.     

Influence of clay minerals on sorption of bacteriolytic enzymes

Myxobacteria presumably produce extracellular bacteriolytic enzymes when they are growing in soil. In order to study their ecological significance, adsorption experiments were performed with lytic enzymes produced byMyxococcus virescens in casitone media. Different soils as well as montmorillonite and kaolinite can rapidly adsorb the bacteriolytic but not the proteolytic enzymes. About 1 gm of montmorillonite per liter of cell-free culture solution is enough for the adsorption of 97% of the bacteriolytic enzymes. The adsorption per unit weight is about 100 times greater on montmorillonite than on kaolinite. About 40% of the adsorbed enzymes can be eluted with solutions of high pH or high ionic strength. The only desorbed bacteriolytic enzyme is the alanyl-∈-N-lysine endopeptidase.