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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.80, #1)
Complete mineralization of methylparathion by Pseudomonas sp. A3 by Mathura P. Ramanathan; Damodharan Lalithakumari (pp. 1-12).
Organophosphorus insecticides are widely used in agriculture. Despite their biodegradable nature, some are highly toxic and their residues are found in the environment. Reports on the mineralization of a spectrum of these insecticides by a single potential strain are scarce. We have isolated a soil isolate, Pseudomonas sp. A3, through enrichment technique, able to degrade methylparathion (MP), malathion, monocrotophos, and Diazinon. The potential of this strain to mineralize MP as a carbon and/or phosphorus source has been evaluated. On hydrolysis of MP, the aromatic portion (p-nitrophenol) was used as a carbon and energy source whereas the alkyl moiety (dithiomethylphosphorothioate) was broken down for the phosphorus source. The results from the experiments involving [U-14C]p-nitrophenol provided the evidence for incorporation of carbon into the cellular constituents and release of CO2 from this insecticide. During the breakdown of MP, nitrite was released as a catabolic by-product.
Keywords: Biodegradation; methylparathion; mineralization; organophosphates; Pseudomonas sp. A3
Production and characterization of an antibody specific for a novel protein serine/threonine kinase, MPK38, highly expressed in hematopoietic cells by Young Yang; Minchan Gil; Younghoon Lee; Hyunjung Ha (pp. 13-22).
We report an antibody that selectively recognizes MPK38, a new protein serine/threonine kinase closely related to the SNF1 serine/threonine kinase family. This antibody recognized a region of the N-terminal kinase catalytic domain and part of the remaining C-terminal portion and was sensitive enough to detect a 72-kDa recombinant MPK38 in insect cells by Western blotting. Immunoblot analysis showed that the recombinant MPK38 was expressed in a time-dependent manner and reached a maximum after 48 h postinfection. In addition, the immune complex kinase assay revealed that the recombinant and endogenous MPK38 protein autophosphorylated in vitro. Phosphoamino acid analysis of autophosphorylated MPK38 protein showed that the phosphorylation was exclusively on serine and threonine residues, suggesting that MPK38 is a protein serine/threonine kinase. Thus, this antibody could be helpful for elucidating the biological functions of MPK38 in the MPK38-expressing cells.
Keywords: Anti-MPK38 antibody; serine/threonine kinase; autophosphorylation; signal transduction; recombinant protein
Heat-shock and stringent responses have overlapping protease activity in Escherichia coli by Sarah W. Harcum; William E. Bentley (pp. 23-37).
The cellular response of a heat-shocked controlled chemostat of Escherichia coli JM105 [pSH101] was characterized and compared to that of a similar culture induced by isopropyl-β-d-thiogalactopyranoside (IPTG). The proteases elicited by the IPTG pulse were previously shown to be upregulated by the stringent stress response and were shown here to be upregulated by heat shock, although to a lesser extent. Owing to the apparent overlap between these responses, a relaxed mutant (rel −, devoid of the stringent response; JM109) was examined for its response to both a chemically imposed stringent response and to IPTG induction in controlled chemostats. There was no significant upregulation of protease activity under either imposed stress. More important, a nine-fold increase of chloramphenicol acetyltransferase (CAT) activity was found for the IPTG-induced relaxed mutant culture. Additionally, the responses from heat shock and IPTG induction were examined in batch cultures. The culture that was simultaneously IPTG-induced and heat-shocked was observed to have the highest CAT activity as well as the most rapid loss in activity after a maximum. Control experiments indicated that the heat shock did not affect loss of CAT activity; instead, the loss of activity correlated with the amount of CAT synthesized. Furthermore, an increase in CAT expression was found during heat shock. Results indicated that heat shock and, alternatively, the use of stringent response-mutant hosts could both be used to facilitate increased recombinant protein yields in the E. coli expression system.
Keywords: Recombinant; Escherichia coli ; heat-shock response; stringent response; protein yield
A simple structured model for continuous production of a hybrid antibiotic by Streptomyces lividans pellets in a fluidized-bed bioreactor by Montserrat Sarrà; Carlos Casas; Manel Poch; Francesc Gòdia (pp. 39-50).
A simple structured model is developed for the description of the experiments of continuous production of a hybrid antibiotic by Streptomyces lividans TK21 pellets in a fluidized-bed reactor. The model is based on the effect of internal and external phosphate concentrations on antibiotic production during cyclic feeding. These concentrations can be calculated on the basis of the equations postulated by the model. The model also considers the cell growth, reflected in changes of the pellet size along the culture. The model parameters are evaluated sequentially by performing experiments at different operational conditions. The validity of the model and its corresponding parameters is corroborated further by the satisfactory modeling of the bioreactor operation during an extended period of time at various operation conditions.
Keywords: Hybrid antibiotic; continuous cultivation; fluidized-bed; mathematical model; immobilized cells; Streptomyces
Development and application of a system for analysis of mixed cultures of microorganisms by Hideo Tanaka; Tomoharu Ebata; Isamu Kuwahara; Masaru Matsuo; James C. Ogbonna (pp. 51-64).
Development and application of a system for real-time quantitative assessment of individual cell activities in a mixed culture system was investigated. This was based on a concept that the activities of individual cells in a mixed culture can be assessed if the cells are physically separated (in separate compartments) in a vessel while the culture conditions, including the broth components, are maintained the same in all the compartments during the cultivation. On this basis, three different apparatus (M-1, M-2, and M-3) were constructed using various types of membranes. In terms of mass transfer characteristics and membrane fouling, the M-3 apparatus was the most effective system for analysis of mixed cultures at high cell densities. With the M-3 apparatus, the interrelationships between two alcohol-producing strains (Saccharomyces cerevisiae and Zymomonas mobilis) under anaerobic and aerobic conditions were studied. Under anaerobic condition, except for possible competition for nutrients, there were no significant effects of the activities of one microorganism on the other. However, under aerobic condition, amensalism was observed because acetaldehyde that was produced by Z. mobilis inhibited the growth of S. cerevisiae.
Keywords: Mixed cultures; cell activities; on-line monitoring; alcohol fermentation; acetaldehyde; aerobic cultures; anaerobic cultures
Effect of fermentation conditions in the enzymatic activity and stereoselectivity of crude lipase from Candida rugosa by Antoni Sánchez; Rosa Maria De la Casa; Jose Vicente Sinisterra; Francisco Valero; Jose Maria Sánchez-Montero (pp. 65-75).
Different fed-batch cultures of Candida rugosa were carried out using oleic acid as the only carbon source. The crude lipases obtained under several operational conditions and downstream processes showed different catalytic activity and isoenzymes ratio. This fact implied that the performance of the lipase produced could be modulated by using different operational fermentation conditions. These powders were compared with commercial lipase from Sigma (St. Louis, MO) in hydrolysis and synthesis reactions. Especially interesting was the fact that the enantioselectivity of a crude lipase was higher than that observed with commercial lipase in the resolution of recemic Ketoprofen. In addition, response of both lipases in the presence of water was different.
Keywords: Candida rugosa lipase; downstream; isoenzymes; lipolytic activity; esterification; Ketoprofen
Effect of the shear rate on pullulan production from beet molasses by Aureobasidium pullulans in an airlift reactor by T. Roukas; G. Serris (pp. 77-89).
The effect of the shear rate on pullulan production from beet molasses by Aureobasidium pullulans P56 in an airlift reactor was investigated. A maximum polysaccharide concentration (18.5 g/L), biomass dry weight (14.0 g/L), polysaccharide yield (38.5%), and sugar utilization (96%) was achieved at a shear rate of 42 s−1. A. pullulans grown on beet molasses produced a mixture of pullulan and other polysaccharides. The highest value of pullulan proportion (30% of total polysaccharide) was obtained at a low shear rate (42 s−1). The apparent viscosity of the fermentation broth increased as the shear rate increased up to 42 s−1 and then decreased. On the other hand, the dissolved oxygen concentration and the volumetric mass transfer coefficient increased with the increase of the shear rate from 21 to 84 s−1. The external addition of L-glutamic acid, olive oil, and Tween-80 improved significantly the production of crude polysaccharide (27.0 g/L), but the pullulan content of the polysaccharide was low (20%).
Keywords: Beet molasses; pullulan; Aureobasidium pullulans ; batch culture; airlift reactor