Identification of a Novel Thermostable Alkaline Protease from Bacillus megaterium-TK1 for the Detergent and Leather Industry

An increased need by the green industry for enzymes that can be exploited for eco-friendly industrial applications led us to isolate and identify a unique protease obtained from a proteolytic Bacillus megaterium-TK1 strain from a seawater source. The extracellular thermostable serine protease was processed by multiple chromatography steps. The isolated protease displayed a relative molecular weight (MW) of 33 kDa (confirmed by zymography), optimal enzyme performance at pH 8.0, and maximum enzyme performance at 70 °C with 100% substrate specificity towards casein. The proteolytic action was blocked by phenylmethylsulfonyl fluoride (PMSF), a serine hydrolase inactivator. Protease performance was augmented by several bivalent metal cations. The protease tolerance was studied under stringent conditions with different industrial dispersants and found to be stable with Surf Excel, Tide, or Rin detergents. Moreover, this protease could clean blood-stained fabrics and showed dehairing activity for cow skin with significantly reduced pollution loads. Our results suggest that this serine protease is a promising additive for various eco-friendly usages in both the detergent and leather industries.

Molecular Dynamics Simulations of the Interaction of Mouse andTorpedoAcetylcholinesterase with Covalent Inhibitors Explain Their Differential Reactivity: Implications for Drug Design

Although the three-dimensional structures of mouse andTorpedo californicaacetylcholinesterase are very similar, their responses to the covalent sulfonylating agents benzenesulfonyl fluoride and phenylmethylsulfonyl fluoride are qualitatively different. Both agents inhibit the mouse enzyme effectively by covalent modification of its active-site serine. In contrast, whereas theTorpedoenzyme is effectively inhibited by benzenesulfonyl fluoride, it is completely resistant to phenylmethylsulfonyl fluoride. A bottleneck midway down the active-site gorge in both enzymes restricts access of ligands to the active site at the bottom of the gorge. Molecular dynamics simulations revealed that the mouse enzyme is substantially more flexible than theTorpedoenzyme, suggesting that enhanced ‘breathing motions’ of the mouse enzyme relative to theTorpedoenzyme might explain why phenylmethylsulfonyl fluoride can reach the active site in mouse acetylcholinesterase, but not in theTorpedoenzyme. Accordingly, we performed docking of the two sulfonylating agents to the two enzymes, followed by molecular dynamics simulations. Whereas benzenesulfonyl fluoride closely approached the active-site serine in both mouse andTorpedoacetylcholinesterase in such simulations, phenylmethylsulfonyl fluoride was able to approach the active-site serine of mouse acetylcholinesterase - but remained trapped above the bottleneck in the case of theTorpedoenzyme. Our studies demonstrate that reliance on docking tools in drug design can produce misleading information. Docking studies should, therefore, also be complemented by molecular dynamics simulations in selection of lead compounds.Author summaryEnzymes are protein molecules that catalyze chemical reactions in living organisms, and are essential for their physiological functions. Proteins have well defined three-dimensional structures, but display flexibility; it is believed that this flexibility, known as their dynamics, plays a role in their function. Here we studied the neuronal enzyme acetylcholinesterase, which breaks down the neurotransmitter, acetylcholine. The active site of this enzyme is deeply buried, and accessed by a narrow gorge. A particular inhibitor, phenylmethylsulfonyl fluoride, is known to inhibit mouse acetylcholinesterase, but not that of the electric fish,Torpedo, even though their structures are very similar. A theoretical technique called molecular dynamics (MD) shows that the mouse enzyme is more flexible than theTorpedo enzyme. Furthermore, when the movement of the inhibitor down the gorge towards the active site is simulated using MD, the phenylmethylsulfonyl fluoride can reach the active site in the mouse enzyme, but not in theTorpedoenzyme, in which it remains trapped midway down the gorge. Our study emphasizes the importance of taking into account not only structure, but also dynamics, in designing drugs targeted towards proteins.

Epoxy-α-Lapachone HasIn VitroandIn VivoAnti-Leishmania (Leishmania) amazonensis Effects and Inhibits Serine Proteinase Activity in This Parasite

ABSTRACTLeishmania(Leishmania)amazonensisis a protozoan that causes infections with a broad spectrum of clinical manifestations. The currently available chemotherapeutic treatments present many problems, such as several adverse side effects and the development of resistant strains. Natural compounds have been investigated as potential antileishmanial agents, and the effects of epoxy-α-lapachone onL. (L.)amazonensiswere analyzed in the present study. This compound was able to cause measurable effects on promastigote and amastigote forms of the parasite, affecting plasma membrane organization and leading to death after 3 h of exposure. This compound also had an effect in experimentally infected BALB/c mice, causing reductions in paw lesions 6 weeks after treatment with 0.44 mM epoxy-α-lapachone (mean lesion area, 24.9 ± 2.0 mm2), compared to untreated animals (mean lesion area, 30.8 ± 2.6 mm2) or animals treated with Glucantime (mean lesion area, 28.3 ± 1.5 mm2). In addition, the effects of this compound on the serine proteinase activities of the parasite were evaluated. Serine proteinase-enriched fractions were extracted from both promastigotes and amastigotes and were shown to act on specific serine proteinase substrates and to be sensitive to classic serine proteinase inhibitors (phenylmethylsulfonyl fluoride, aprotinin, and antipain). These fractions were also affected by epoxy-α-lapachone. Furthermore,in silicosimulations indicated that epoxy-α-lapachone can bind to oligopeptidase B (OPB) ofL. (L.)amazonensis, a serine proteinase, in a manner similar to that of antipain, interacting with an S1 binding site. This evidence suggests that OPB may be a potential target for epoxy-α-lapachone and, as such, may be related to the compound's effects on the parasite.

Production and Characterization of Alkaline Protease from a High Yielding and Moderately Halophilic Strain of SD11 Marine Bacteria

A marine bacterium SD11, which was isolated from sea muds (Geziwo Qinhuangdao Sea area, China), was used to produce thermostable alkaline serine nonmetal protease in the skim milk agar plate medium with 10% NaCl. The optimal temperature about the manufacture of the extracellular protease was ~60°C. The crude enzyme was stable at 20–50°C. The activity was retained to 60% and 45% after heating for 1 h at 60 and 70°C, respectively. The protease was highly active in a wide pH scope (8.0–10.0) and maximum protease activity exhibited at pH 10.0. The activity was restrained by phenylmethylsulfonyl fluoride (PMSF) but mildly increased (~107%) in the presence of ethylenediaminetetraacetic acid (EDTA), indicating that the production contains serine-protease(s) and nonmetal protease(s). Moreover, the crude alkaline protease was active with the 5 mM Ca2+, Mn2+, Zn2+, Cu2+, Na+, and K+that existed separately. In addition, the protease showed superduper stability when exposed to an anionic surfactant (5 mM SDS), an oxidizing agent (1% H2O2), and several organic solvents (methanol, isopropanol, and acetone). These results suggest that the marine bacterium SD11 is significant in the industry from the prospects of its ability to produce thermally stable alkaline protease.

Effect of protease inhibitors on thermal gelation of squid (Illex argentinus). mantle paste

The characteristics of the thermal gelation of squid mantle paste and the effect of protease inhibitors on them were investigated. Pastes in the absence and presence the protease inhibitors, ethylendiaminetetracetic acid (EDTA) and phenylmethylsulfonyl fluoride (PMSF), were formulated. Pastes were made by the respective one or two step thermal treatments: direct heating at 85°C for 20 min and preincubation at 27 or 40 °C for 3 or 2 hours, followed by heating at 85 °C for 20 min. The gel strength, water holding capacity (WHC) and whiteness of gelled pastes were analyzed. The tricloroacetic acid (TCA) soluble peptides in homogenate of the muscle were determined. Gel strength decreased when heating was made in two steps. EDTA and PMSF were effective in avoiding that decrease when pre-incubation was made at 40 °C. Maximum gel strength was observed for the gels in presence of EDTA, giving values of 255 and 219 g x cm for the samples made by direct heating and pre-incubated at 40 °C respectively. TCA soluble peptides increased between 20 and 60 °C, with maximum values reached at 30 and 60 °C. No significant differences (p>0.05) were observed in gel whiteness, neither with the thermal treatment nor with the inhibitors. The WHC was higher (p<0.05) in the gelated paste formulated with EDTA. These results show a good gelation capacity of I argentinus pastes and improvements with protease inhibitors.

Effect of protease inhibitors on thermal gelation of squid (Illex argentinus). mantle paste

The characteristics of the thermal gelation of squid mantle paste and the effect of protease inhibitors on them were investigated. Pastes in the absence and presence the protease inhibitors, ethylendiaminetetracetic acid (EDTA) and phenylmethylsulfonyl fluoride (PMSF), were formulated. Pastes were made by the respective one or two step thermal treatments: direct heating at 85°C for 20 min and preincubation at 27 or 40 °C for 3 or 2 hours, followed by heating at 85 °C for 20 min. The gel strength, water holding capacity (WHC) and whiteness of gelled pastes were analyzed. The tricloroacetic acid (TCA) soluble peptides in homogenate of the muscle were determined. Gel strength decreased when heating was made in two steps. EDTA and PMSF were effective in avoiding that decrease when pre-incubation was made at 40 °C. Maximum gel strength was observed for the gels in presence of EDTA, giving values of 255 and 219 g x cm for the samples made by direct heating and pre-incubated at 40 °C respectively. TCA soluble peptides increased between 20 and 60 °C, with maximum values reached at 30 and 60 °C. No significant differences (p>0.05) were observed in gel whiteness, neither with the thermal treatment nor with the inhibitors. The WHC was higher (p<0.05) in the gelated paste formulated with EDTA. These results show a good gelation capacity of I argentinus pastes and improvements with protease inhibitors.