subsequent catalysis sensu stricto ( 6, 7 ). Most of the lipases are water-soluble enzymes acting on water insoluble substrates (super-substrates). The two-dimensional nature of enzyme catalysis by lipases does not obey MichaelisMenten kinetics and critically depends on the quality of the interface ( 3,8,9 ). Obtaining accurate, i.e., substratespecifi c, measurements of lipase activity as well as developing reliable lipase assay systems requires taking these unique features into account.Many biotechnological applications for lipases have been described in the food, cosmetic, detergent, and pharmaceutical industries ( 3, 10, 11 ), and an interest exists for new sources of this kind of enzyme in an industrial setting.Novel lipases can be obtained either by isolating them from various natural sources or by using classical protein engineering methods and/or directed evolution procedures (12)(13)(14). All these studies require convenient, sensitive, and specifi c assays for measuring lipase activity ( 15 ). In addition, screening procedures require continuous assays and substrate stability that are compatible with high sample throughput.Considerable progress has been made in the past few years in the development of high-throughput screening (HTS) methods for carboxyl-ester hydrolases. These HTS assays have been developed using chromogenic or fl uorogenic substrates such as butyrate, octanoate, and palmitate of nitrophenol or 4-methylumbelliferone. However, all of these synthetic esters are liable to undergo nonenzymatic alkaline hydrolysis as well as hydrolysis by the nonspecifi c carboxyl-ester hydrolases often present in biological Abstract A continuous assay is proposed for the screening of acidic, neutral, or alkaline lipases using microtiter plates, emulsifi ed short-and medium-chain TGs, and a pH indicator. The lipase activity measurement is based on the decrease of the pH indicator optical density due to protonation which is caused by the release of FFAs during the hydrolysis of TGs and thus acidifi cation. Purifi ed lipases with distinct pH optima and an esterase were used to validate the method. The rate of lipolysis was found to be linear with time and proportional to the amount of enzyme added in each case. Specific activities measured with this microplate assay method were lower than those obtained by the pH-stat technique. Nevertheless, the pH-dependent profi les of enzymatic activity were similar with both assays. In addition, the substrate preference of each enzyme tested was not modifi ed and this allowed discriminating lipase and esterase activities using tributyrin (low water solubility) and tricaprylin (not water soluble) as substrates. This continuous lipase assay is compatible with a high sample throughput and can be applied for the screening of lipases and lipase inhibitors from biological samples. Lipases (TG ester hydrolases, EC 3.1.1.3) are lipolytic carboxylester hydrolases which catalyze the hydrolysis of the ester bonds of TGs to form FFAs and glycerol in some cases . They are widely distr...

High-throughput screening (HTS) methods for lipases and esterases are generally performed by using synthetic chromogenic substrates (e.g., p-nitrophenyl, resorufin, and umbelliferyl esters) which may be misleading since they are not their natural substrates (e.g., partially or insoluble triglycerides). In previous works, we have shown that soluble nonchromogenic substrates and p-nitrophenol (as a pH indicator) can be used to quantify the hydrolysis and estimate the substrate selectivity of lipases and esterases from several sources. However, in order to implement a spectrophotometric HTS method using partially or insoluble triglycerides, it is necessary to find particular conditions which allow a quantitative detection of the enzymatic activity. In this work, we used Triton X-100, CHAPS, and N-lauroyl sarcosine as emulsifiers, β-cyclodextrin as a fatty acid captor, and two substrate concentrations, 1 mM of tributyrin (TC4) and 5 mM of trioctanoin (TC8), to improve the test conditions. To demonstrate the utility of this method, we screened 12 enzymes (commercial preparations and culture broth extracts) for the hydrolysis of TC4 and TC8, which are both classical substrates for lipases and esterases (for esterases, only TC4 may be hydrolyzed). Subsequent pH-stat experiments were performed to confirm the preference of substrate hydrolysis with the hydrolases tested. We have shown that this method is very useful for screening a high number of lipases (hydrolysis of TC4 and TC8) or esterases (only hydrolysis of TC4) from wild isolates or variants generated by directed evolution using nonchromogenic triglycerides directly in the test.

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In the present study, a novel laccase from ascomycete Gliomastix murorum was produced in agro-industrial wastes and entrapped in galactomannan beads for Reactive Blue 2 (Rb-2) decolorization. The maximum laccase production in agave bagasse-based medium occurred at 72 h (1798.6 UL−1). Entrapped laccase decolorized ˃80% of 0.5 mM Rb-2 in 2 h without the addition of redox mediator. Km for Rb-2 substrate was 1.42 mM, with a Vmax of 1.19 µmol min−1 for entrapped laccase. Galactomannan matrices produce stability to acid pH (2–5) and temperatures from 20–70 °C. Reusability assays showed that entrapped laccase could retain efficient Rb-2 decolorization of ˃80% six times. In general, galactomannan used for entrapment of laccase provides economic advantages in large-scale wastewater treatment due to its natural origin and efficient results.

Modelado de la biodegradación en biorreactores de lodos de hidrocarburos totales del petróleo intemperizados en suelos y sedimentos (Biodegradation modeling of sludge bioreactors of total petroleum hydrocarbons weathering in soil and sediments)