Cold-adapted RTX lipase from antarctic Pseudomonas sp. strain AMS8: isolation, molecular modeling and heterologous expression

A new strain of psychrophilic bacteria (designated strain AMS8) from Antarctic soil was screened for extracellular lipolytic activity and further analyzed using molecular approach. Analysis of 16S rDNA showed that strain AMS8 was similar to Pseudomonas sp. A lipase gene named lipAMS8 was successfully isolated from strain AMS8, cloned, sequenced and overexpressed in Escherichia coli. Sequence analysis revealed that lipAMS8 consist of 1,431 bp nucleotides that encoded a polypeptide consisting of 476 amino acids. It lacked an N-terminal signal peptide and contained a glycine- and aspartate-rich nonapeptide sequence at the C-terminus, which are known to be the characteristics of repeats-in-toxin bacterial lipases. Furthermore, the substrate binding site of lipAMS8 was identified as S207, D 255 and H313, based on homology modeling and multiple sequence alignment. Crude lipase exhibited maximum activity at 20 C and retained almost 50 % of its activity at 10 C. The molecular weight of lipAMS8 was estimated to be 50 kDa via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimal expression level was attained using the recombinant plasmid pET32b/BL21(DE3) expressed at 15 C for 8 h, induced by 0.1 mM isopropyl β-D thiogalactoside (IPTG) at E. coli growth optimal density of 0.5

Batch and continuous extraction of bromelain enzyme by reversed micelles

The main aim of this study was to optimize the conditions for bromelain extraction by reversed micelles from pineapple juice (Ananas comosus). The purification was carried out in batch extraction and a micro-column with pulsed caps for continuous extraction. The cationic micellar solution was made of BDBAC as a surfactant, isooctane as a solvent and hexanol as a co-solvent. For the batch process, a purification factor of 3 times at the best values of surfactant agent, co-solvent and salt concentrations, pH of the back and forward extractions were, 100 mM, 10% v/v, 1 M, 3.5 and 8, respectively. For the continuous operation, independent variables optimal point was determined: ratio between light phase flow rate and total flow rate equal to 0.67 and 1 second for the time interval between the pulses. This optimal point led to a productivity of 1.29 mL/min and a purification factor of 4.96.<br>Este trabalho teve como objetivo principal otimizar as condições para extração da bromelina do suco do abacaxi (Ananas comosus) por micelas reversas. A purificação foi feita usando o processo de extração em batelada e contínuo, este último em uma micro-coluna de campânulas pulsantes. A solução micelar catiônica foi preparada com o surfactante BDBAC, i-octano como solvente e hexanol como co-solvente. Na extração em batelada encontrou-se um fator de purificação de 3 vezes, e seus melhores valores de concentração do agente surfactante, co-solvente e sal, de pH da re-extração e extração, foram respectivamente iguais a: 100 mM, 10% v/v, 1 M, 3,5 e 8. Para a operação contínua, as variáveis independentes ótimas foram: 0,67 para a razão entre as taxas de fluxos da fase leve e a total e 1 s para o intervalo de tempo entre pulsos das campânulas. Este ponto ótimo leva a uma produtividade de 1,29 mL/min e a um fator de purificação igual a 4,96