| Summary: | The increasing interest in the development of more competitive biotechnological processes is demanding the development of new downstream strategies to maximize product recovery and foster the economic feasibility and robustness of any desired process. From a biotechnological point of view, lipase production is considered one of the three most important bioprocesses in terms of enzyme sales.
During the last years, lipolytic enzymes applications have been broaden to sectors ranging from the petrochemical, pharmaceutical, food and paper to waste management industries, as a result of a close collaboration between academics and industry (Houde et al., 2004). The interest on triacylglycerol hydrolases or lipases (EC 3.1.1.3) lies in the fact that they play a crucial role in biocatalysis of a plethora of chemical reactions, such as hydrolysis, interesterification, esterification, alcoholysis, acidolysis and aminolysis. Their reputation is built largely on their distinctive features, namely, they are quite stable and active in organic solvents, they do not require cofactors, they exhibit a high degree of chemo-, enantio- and regioselectivity, and they possess a wide range of substrate specificity. These features make these enzymes trade to be a well-known billion dollar business (Jaeger and Reetz, 1998; Hasan <i>et al</i>., 2006). However, there are concerns related to the stability of these enzymes at the operating conditions usually employed in biocatalysis. This problem can be circumvented by using extremozymes, whose naturally developed resistance to drastic reaction conditions (like resistance to denaturalization by chemical agents and by extreme values of temperature, pH and salinity) turns out to be their main appeal.
One of the main limitations observed for the industrial implementation of the processes to produce this kind of enzymes lies in the high costs of downstream operations which represent more than 50-80% of the total processing cost. Surprisingly, downstream strategies are usually neglected until a later phase of process development. This is a major reason for increased processing cost and eventually the reason for process implementation failures at real scale (Datar and Rosen, 1990). In this line, one of the bottlenecks faced in this kind of biological process is placed in the recovery of the desired intracellular metabolites, which makes it necessary to apply cell lysis methods. With this purpose, conventional methods fall into two main categories: physical (agitation in the presence of glass and ceramic beads, ultrasonication, high pressure homogenization, cryogenic grinding) and chemical (acid, alkali) (Ulloa <i>et al.</i>, 2012).
In this sense, another chemical method that can be proposed could be the addition of ionic liquids. These are liquid salts at or close to room temperature that have posed a new inspiration for Green Chemistry. These neoteric solvents have been getting increasing attention from the scientific community and industry not only due to their green potential but also to their unique combination of properties (Earle <i>et al.</i>, 2006; Rebelo <i>et al.</i>, 2005; Smiglak <i>et al.</i>, 2006; Baranyai <i>et al.</i>, 2004; Wasserscheid and Welton, 2007). The most distinctive feature of these compounds is their negligible vapour pressure, which may reduce the air pollution with respect to the conventional volatile organic solvents. Due to this, ionic liquids have opened up new fields at the interface of many conventional disciplines such as green chemistry, electrochemistry, analytical chemistry, surface science, catalysis and nanotechnology (Plechkova and Seddon, 2008). Another appealing aspect of ionic liquids lies in their tunability, which eases the existence of a plethora of combinations cation-anion, leading to thousands of new compounds with unknown properties and environmental risks. Due to all these properties, they are already used in extant processes and their production per annum surpasses the tonne magnitude (BASF Patent, 2005; Institute Français Du Pétrole, 2001).
In this work, we have exploited their structural modularity to apply them as thermophiles cell wall disruptor, hypothesizing that the variation in polarity or hydrophobicity are crucial to interact with the lipidic bilayer of the cell membranes, even up to a complete solubilization. We have bet in this study in the bacterium Thermus thermophilus HB27, whose lipolytic enzymes are mostly located intracellularly and attached to membrane. The interest of this new approach is supported by the fact that these enzymes are already being commercialized by top-companies such as Sigma-Aldrich but with very low levels of activity, as a consequence of the low levels of extracellular enzyme expression.
First, flask cultures of <i>Thermus thermophilus</i> HB27 were cultivated for 24h at 70°C. Then, six different ionic liquids (C2MIMCl, C6MIMCl, C10MIMCl, C2MIMC2SO4, C2MIMHSO4 and C2MIM(C2H5)2PO4) were added to the culture medium at concentrations of 1g/L, and the effect of these molten salts pressure was analyzed by monitoring both the cell growth and the lipolytic enzyme distribution for 6 hours (from 24 to 30 h). The enzyme activity data were obtained from spectrometric measurement of the enzymatic hydrolysis of p-nitrophenyl laurate (Deive <i>et al.</i>, 2009), and allowed concluding a great lytic effect of C10MIMCl, since a drastic increase of the extracellular activity of 308% was observed, to the detriment of the intracellular and membrane bound enzyme. In parallel, the cell growth was monitored by turbidimetric measurements at 600 nm and showed 43% of decay, thus confirming the excellent lytic effect provided by the ionic liquid.
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