Structural differences of commercial and recombinant lipase B from Candida antarctica: An important implication on enzymes thermostability.

Lipase B from Candida antarctica (CalB) is the most widely used lipase, including in many industrial sectors, such as in biodiesel and pharmaceuticals production. CalB has been produced by heterologous expression using Pichia pastoris under PGK constitutive promoter (named LipB). Here, we have studied the structural features of commercial CalB and LipB enzymes using circular dichroism and fluorescence under different conditions. In the presence of denaturing agents CalB was more stable than LipB, in contrast, at increasing temperatures, LipB was more thermostable than CalB. Mass spectrometry data indicates that both enzymes have an insertion of amino acids related to α-factor yeast signal, however LipB enzyme showed the addition of nine residues at the N-terminal while CalB showed only four residues. Molecular modeling of LipB showed the formation of an amphipathic α-helix in N-terminal region that was not observed in CalB. This data suggests that this new α-helix possess could be involved in LipB thermostability. These results associated with new structural studies may provide information to the design of novel biocatalysts.

Inorganic polyphosphate inhibits an aspartic protease-like activity in the eggs of Rhodnius prolixus (Stahl) and impairs yolk mobilization in vitro.

Inorganic polyphosphate (poly P) is a polymer of phosphate residues that has been shown to act as modulator of some vertebrate cathepsins. In the egg yolk granules of Rhodnius prolixus, a cathepsin D is the main protease involved in yolk mobilization and is dependent on an activation by acid phosphatases. In this study, we showed a possible role of poly P stored inside yolk granules on the inhibition of cathepsin D and arrest of yolk mobilization during early embryogenesis of these insects. Enzymatic assays detected poly P stores inside the eggs of R. prolixus. We observed that micromolar poly P concentrations inhibited cathepsin D proteolytic activity using both synthetic peptides and homogenates of egg yolk as substrates. Poly P was a substrate for Rhodnius acid phosphatase and also a strong competitive inhibitor of a pNPPase activity. Fusion events have been suggested as important steps towards acid phosphatase transport to yolk granules. We observed that poly P levels in those compartments were reduced after in vitro fusion assays and that the remaining poly P did not have the same cathepsin D inhibition activity after fusion. Our results are consistent with the hypothesis that poly P is a cathepsin D inhibitor and a substrate for acid phosphatase inside yolk granules. It is possible that, once activated, acid phosphatase might degrade poly P, allowing cathepsin D to initiate yolk proteolysis. We, therefore, suggest that degradation of poly P might represent a new step toward yolk mobilization during embryogenesis of R. prolixus.

Calcium-regulated fusion of yolk granules is important for yolk degradation during early embryogenesis of Rhodnius prolixus Stahl.

This study examined the process of membrane fusion of yolk granules (YGs) during early embryogenesis of Rhodnius prolixus. We show that eggs collected at days 0 and 3 after oviposition contain different populations of YGs, for example day-3 eggs are enriched in large YGs (LYGs). Day-3 eggs also contain the highest free [Ca(2+)] during early embryogenesis of this insect. In vitro incubations of day-0 YGs with [Ca(2+)] similar to those found in day-3 eggs resulted in the formation of LYGs, as observed in vivo. Fractionation of LYGs and small YGs (SYGs) and their subsequent incubation with the fluorescent membrane marker PKH67 showed a calcium-dependent transference of fluorescence from SYGs to LYGs, possibly as the result of membrane fusion. Acid phosphatase and H(+)-PPase activities were remarkably increased in day-3 LYGs and in calcium-treated day-0 LYGs. Both fractions were found to contain vitellins as major components, and incubation of YGs with calcium induced yolk proteolysis in vitro. Altogether, our results suggest that calcium-induced membrane fusion events take part in yolk degradation, leading to the assembly of the yolk mobilization machinery.

Characterization of a tyrosine phosphatase activity in the oogenesis of Periplaneta americana.

In this work, phosphatase activity was characterized in the ovary and the haemolymph of Periplaneta americana. The optimum pH for these activities was 4.0, and a temperature of 44 degrees C was ideal for the maximal enzyme activity. The phosphatase activities were inhibited by NaF, sodium tartrate, Pi, sodium orthovanadate, and ammonium molybdate. The ovarian phosphatase activity at pH 4.0 was almost exclusive against phosphotyrosine, with little or no effect on the residues of phosphoserine or phosphothreonine. These results indicate that this phosphatase activity is due to the presence of an acid tyrosine phosphatase. The phosphatase activities of acid extracts from P. americana ovaries (OEX) and an acid extract from P. americana haemolymph (HEX) were analyzed in non-denaturant gel electrophoresis using an analog substrate beta-naphtyl phosphate. The gel revealed two bands with phosphatase activity in the ovary and one band in the haemolymph; these bands were excised and submitted to a 10% SDS-PAGE showing a single 70-kDa polypeptide in both samples. Histochemistry of the ovary with alpha-naphtyl phosphate for localization of acid phosphatase activity showed mainly labeling associated to the oocyte peripheral vesicles, basal lamina, and between follicle cells. Electron microscopy analysis showed that acid phosphatase was localized in small peripheral vesicles in the oocyte, but not inside yolk granules. The possible role of this phosphatase during oogenesis and embryogenesis is also discussed in this article.

A new model for proton pumping in animal cells: the role of pyrophosphate.

The H+-PPase activity was characterized in membrane fractions of ovary and eggs of Rhodnius prolixus. This activity is totally dependent on Mg2+, independent of K+ and strongly inhibited by NaF, IDP and Ca2+. The membrane proteins of eggs were analyzed by western blot using antibodies to the H+-PPase from Arabidopsis thaliana. The immunostain was associated with a single 65-kDa polypeptide. This polypeptide was immunolocalized in yolk granule membranes by optical and transmission electron microscopy. We describe the acidification of yolk granules in the presence of PPi and ATP. This acidification is inhibited in the presence of NAF, Ca2+ and antibodies against H+-PPase. These data show for the first time in animal cells that acidification of yolk granules involves an H+-PPase as well as H+-ATPase.