Abstract
Protein extraction techniques are absolutely required for the research of biological catalysts. The present study compared four universal protein extraction methodologies (ammonium sulfate precipitation, TCA/acetone precipitation, and two commercial kits) to provide practical information on protein extraction in order to discover a novel lipase in agricultural products. Yields of protein extraction from 24 domestic agricultural products and their specific activities were evaluated and compared with each other. TCA/acetone precipitation showed a relatively higher extraction yield (on average, 3.41 ± 1.08 mg protein/0.1 g sample) in crude protein extraction, whereas the Pierce™ Plant Total Protein Extraction Kit showed the highest specific lipase activity on average in both spectrophotometric (266.61 ± 235.78 μU/mg protein) and fluorometric (41.52 ± 32.63 μU/mg protein) assays. Our results suggest that commercial kits for the rapid extraction of soluble functional proteins would be a better choice than conventional precipitation techniques to perform the high-throughput screening of enzyme activity from plant sources. Finally, several agricultural products such as cordyceps, pepper, bracken, and hemp, all of which exhibited an excellent specific lipase activity, were proposed as promising candidates for a source of novel lipases.
Highlights
Lipases are a group of carboxylic-ester hydrolases that catalyze the hydrolysis as well as the esterification of triacylglycerol species as one of its major substrates [1]
From the 9th revision of Korean National Standard Food Composition Table [16], 87 kinds of domestic agricultural products containing both more than 10.0 g of different proteins and 1.0 g of lipids were chosen, and their crude protein extracts were screened for lipase activity using both spectrophotometric and fluorometric assays (Supplementary Data S1)
In the standardized conditions of 100% total proteins, the results should theoretically be identical in both spectrophotometric and fluorometric assays, and the precipitated proteins should be totally soluble [28]; fluorescence-interfering substances such as remnants of precipitated proteins and other chemicals could remain in rehydrated precipitant solution. These results eventually suggested that the PierceTM Plant Total Protein Extraction Kit could be the best choice for the lipase screening of agricultural products in both assays
Summary
Lipases (i.e., triacylglycerol hydrolase, EC 3.1.1.3) are a group of carboxylic-ester hydrolases that catalyze the hydrolysis (forward reaction) as well as the esterification (backward reaction) of triacylglycerol species as one of its major substrates [1]. These enzymes have unique substrate specificities (e.g., typoselectivity, regioselectivity, and stereoselectivity) [2] unlike other hydrolases, and on that basis, are able to catalyze specialized reactions suited to the needs of various industries such as the chemical, food, and pharmaceutical industries [3,4]. To find novel and unique lipases in agricultural products, it is of primary importance to obtain a single protein with as much target catalytic activity as possible while using the appropriate protein extraction and purification methodologies to overcome these problems
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