Highly selective purification of three lipases from Geotrichum candidum 4013 and their characterization and biotechnological applications

Simple purification steps were used to purify two lipases (Lip1 and Lip2) and one novel lipase (Lip3) produced by Geotrichum candidum 4013. The octyl-Sepharose at low ionic strength was used to adsorbe Lip2 and novel Lip3, by addition of octadecyl-Sepabeads the Lip1 was also adsorbed from the crude mixture. Desorption of proteins with a Triton X-100 gradient or 1% (v/v) Triton X-100 permitted to fully purify Lip1, Lip2 and Lip3 lipases. The identification of Lip1 and Lip2 was confirmed by using mass spectrometry approach and their characterization by molecular modeling approach. CD spectra and fluorescence showed differences in the secondary and tertiary structure; the apparent difference was the presence of 4 extra loops in Lip2 in contrast to Lip1 structure. The Edman degradation together with mass spectrometry revealed the presence of unique peptide AVGGGATLPEK in the novel lipase Lip3. The catalytic properties of the lipases were tested through hydrolysis of p-nitrophenyl esters, triacylglycerols and peracetylated thymidine esters. The enantioselectivity potential of purified lipases was studied for the hydrolyses of trans-2-(4-methoxybenzyl)-1-cyclohexyl acetate, where Lip3 was the only active lipase with an excellent selectivity (eep > 99%, E = 372). The all three purified lipases from G. candidum 4013 have shown promising catalytic properties with biotechnological application.This work has been sponsored by the Spanish National Research Council (CSIC). Jana Brabcová is grateful to the Academy of Science of the Czech Republic (project No. M200551203).Peer Reviewe

Molecular mechanism of the two-component suicidal weapon of neocapritermes taracua old workers

In termites, as in many social insects, some individuals specialize in colony defense, developing diverse weaponry. As workers of the termite Neocapritermes taracua (Termitidae: Termitinae) age, their efficiency to perform general tasks decreases, while they accumulate defensive secretions and increase their readiness to fight. This defensive mechanism involves self-sacrifice through body rupture during which an enzyme, stored as blue crystals in dorsal pouches, converts precursors produced by the labial glands into highly toxic compounds. Here, we identify both components of this activated defense system and describe the molecular basis responsible for the toxicity of N. taracua worker autothysis. The blue crystals are formed almost exclusively by a specific protein named BP76. By matching N. taracua transcriptome databases with amino acid sequences, we identified BP76 to be a laccase. Following autothysis, the series of hydroquinone precursors produced by labial glands get mixed with BP76, resulting in the conversion of relatively harmless hydroquinones into toxic benzoquinone analogues. Neocapritermes taracua workers therefore rely on a two-component activated defense system, consisting of two separately stored secretions that can react only after suicidal body rupture, which produces a sticky and toxic cocktail harmful to opponents.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Characterization of Neutral Lipase BT-1 Isolated from the Labial Gland of <i>Bombus terrestris</i> Males

<div><p>Background</p><p>In addition to their general role in the hydrolysis of storage lipids, bumblebee lipases can participate in the biosynthesis of fatty acids that serve as precursors of pheromones used for sexual communication.</p> <p>Results</p><p>We studied the temporal dynamics of lipolytic activity in crude extracts from the cephalic part of <i>Bombus terrestris</i> labial glands. Extracts from 3-day-old males displayed the highest lipolytic activity. The highest lipase gene expression level was observed in freshly emerged bumblebees, and both gene expression and lipase activity were lower in bumblebees older than 3 days. Lipase was purified from labial glands, further characterized and named as BT-1. The <i>B. terrestris</i> orthologue shares 88% sequence identity with <i>B. impatiens</i> lipase HA. The molecular weight of <i>B. terrestris</i> lipase BT-1 was approximately 30 kDa, the pH optimum was 8.3, and the temperature optimum was 50°C. Lipase BT-1 showed a notable preference for C8-C10 <i>p</i>-nitrophenyl esters, with the highest activity toward <i>p</i>-nitrophenyl caprylate (C8). The Michaelis constant (K_m) and maximum reaction rate (V_max) for <i>p</i>-nitrophenyl laurate hydrolysis were K_m = 0.0011 mM and V_max = 0.15 U/mg.</p> <p>Conclusion</p><p>This is the first report describing neutral lipase from the labial gland of <i>B. terrestris</i>. Our findings help increase understanding of its possible function in the labial gland.</p> </div