Foam generated by vigorous stirring of a nonionic detergent, Triton X-114, was used for the recovery of recombinant cutinase expressed by Saccharomyces cerevisiae. The enzyme with a hydrophobic fusion tag, (Trp-Pro)(4), was recovered with a higher yield as compared to the wild-type cutinase, indicating the involvement of hydrophobic interactions in protein isolation with the foam. The influence of various factors including volume, dilution, pH, different additives, and cell concentration in the medium on enzyme recovery was investigated. Interaction of the enzyme with detergent was monitored using fluorescence spectroscopy. No significant changes in protein conformation after the isolation procedure were observed using circular dichroism.

Selective isolation of lactate dehydrogenase (LDH) from porcine muscle extract was studied using foam generated from the vigorous stirring of a non-ionic surfactant, Triton X-114 derivatized with Cibacron blue. The cloud point of the surfactant-dye conjugate was higher than that of the native Triton X-114, and also the foam prepared from the affinity surfactant was more rigid taking a longer time to collapse. The equilibrium dissociation constant between pure LDH and surfactant-dye conjugate was 5.0 microM as compared to the value of 2.2 microM for the enzyme and free dye as measured by differential spectroscopy. The isolation procedure involved mixing of the porcine muscle extract with the affinity foam, separating and collapsing the foam, and warming the solution formed to 37 degrees C to yield the surfactant-dye phase and an aqueous phase containing the enzyme. The effect of surfactant concentration and protein load on enzyme recovery and purification was investigated. Under optimal conditions, LDH was quantitatively recovered with high purification factor in a very short time. Both recovery and purification were higher when foam prepared from an equivalent mixture of surfactant-dye conjugate and unmodified surfactant was used. The selectivity of interaction between LDH and detergent-dye conjugate was confirmed by lowered recovery when NADH was included during the binding step.

Purification of recombinant wild-type cutinase from the culture supernatant of Saccharomyces cerevisiae by extraction in aqueous two-phase system was investigated. The partition of the enzyme in a polyethylene glycol (PEG)-potassium phosphate system to the top phase was increased with lower molecular weight PEG. Enzyme partition in a 20% PEG/15% phosphate two-phase system was studied in the presence of detergents, fatty acids, and alcohols, respectively. Addition of 0.5% (w/w) butyrate increased the partition coefficient from 17 to 135 and the purification factor from 10 to 23. The effect of butyrate was also confirmed by using the countercurrent mode of extraction. Recovery of cutinase from the top phase was achieved by a secondary extraction into a new salt phase at a lower pH or a lower temperature. A specific interaction of butyrate to the active site of the enzyme was demonstrated by fluorescence spectroscopy. Size exclusion chromatography showed the cutinase-butyrate complex to be over two times the size of the free enzyme.

Influência da temperatura e do tempo de armazenamento nas dosagens bioquímicas de uréia e creatinina em soro ou plasma caninos RESUMOO trabalho teve como objetivos avaliar algumas condições de estocagem de soro e de plasma com EDTA 10% utilizados para determinação dos valores de uréia e creatinina em cães, e avaliar a possibilidade de substituir o soro pelo plasma conservado com EDTA 10%. Foram realizadas 3600 análises (1800 amostras de soro e 1800 de plasma) com amostras que permaneceram em temperaturas ambiente, de refrigeração e de congelamento. As análises foram realizadas imediatamente após a obtenção da amostra (tempo 0) e após 2, 6, 12, 24, 72 horas e 30 e 60 dias após a coleta do sangue. Os resultados permitiram concluir que a uréia conservou-se por mais tempo na amostra sérica refrigerada até 30 dias e a creatinina na amostra sérica congelada até 72h. O soro mostrou mais estabilidade que o plasma em todas as condições de estoque estabelecidas.Palavras-chave: Cão, uréia, creatinina, conservação ABSTRACTAiming to evaluating some conditions of EDTA 10% serum and plasma storage for urea and creatinine values determinations in canines as well as analyzing the possibility of serum substitution by plasma, a total of 3600 samples (1800 serum and 1800 of plasma) were analyzed at room, refrigeration and freezing temperatures. Different time intervals were used in the analysis: immediately after sampling (time 0), and at 2, 6, 12, 24, and 72 hours, and 30 and 60