A broad pH range indicator-based spectrophotometric assay for true lipases using tributyrin and tricaprylin

Abstract

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...

Screening of phospholipase A activity and its production by new actinomycete strains cultivated by solid-state fermentation

“…PLA activity measurement was based on the decrease of pH indicator optical density due to protonation caused by the release of free fatty acids during the hydrolysis of substrate and thus acidification of the reaction media (Camacho-Ruiz et al, 2015). A lipase (rHPL) and enzymes with PLA activity were used to validate the method.…”

Advantages of Heterofunctional Octyl Supports: Production of 1,2-Dibutyrin by Specific and Selective Hydrolysis of Tributyrin Catalyzed by Immobilized Lipases

“…This was facilitated by the good solubility of triacetin that permitted a good competition of this compound versus the produced diacetin. The diacetin was rapidly hydrolyzed to 2‐monoacetin when no triacetin was presented in the reaction medium …”

“…In this new paper, we present a study on the possibility of getting a high yield of dibutyrin by hydrolysis of tributyrin. This is one of the most used substrates to determine lipase activity, and usually scarce attention has been payed to the possibility of getting a specific product . From a fundamental point of view, the problem is interesting.…”

“…The diacetin was rapidly hydrolyzed to 2-monoacetin when no triacetin was presented in the reaction medium. [43] In this new paper, we present a study on the possibility of getting a high yield of dibutyrin by hydrolysis of tributyrin. This is one of the most used substrates to determine lipase activity, and usually scarce attention has been payed to the possibility of getting a specific product.…”

“…This is one of the most used substrates to determine lipase activity, and usually scarce attention has been payed to the possibility of getting a specific product. [43][44][45] From a fundamental point of view, the problem is interesting. The situation to get 1,2-diacylglycerol is far more complex using tributyrin than using triacetin: solubility of tributyrin in aqueous media is low and thus the competition with dibutyrin will be more complex.…”