Ronaldo O. Cavalli

Purification and partial characterisation of a trypsin from the processing waste of the silver mojarra (Diapterus rhombeus)

An alkaline peptidase was purified from the viscera of the silver mojarra (Diapterus rhombeus) in a three-step process: heat treatment, ammonium sulphate fractionation and molecular exclusion chromatography (Sephadex® G-75), with final specific activity 86-fold higher than the enzyme extract and yield of 22.1%. The purified enzyme had an estimated molecular mass of 26.5kDa and NH2-terminal amino acid sequence IVGGYECTMHSEAHE. Higher enzyme activity was observed at pH 8.5 and between 50 and 55°C. The enzyme was completely inactivated after 30min at 55°C and it was significantly more stable at alkaline pH. Km, Kcat and Kcat·Km(-1) values, using BApNA as substrate, were 0.266mM, 0.93s(-1) and 3.48mM(-1)s(-1), respectively. Enzyme activity increased in the presence of the ions (1mM) K(+), Li(+) and Ca(2+), but was inhibited by Fe(2+), Cd(2+), Cu(2+), Al(3+), Hg(2+), Zn(2+) and Pb(2+) as well as by the trypsin inhibitors TLCK and benzamidine.

Métodos de extrusão manual e elétrica dos espermatóforos de reprodutores selvagens do camarão-rosa Farfantepenaeus paulensis (Decapoda: Penaeidae)

The present study compared manual and electrical methods to extrude the spermatophore of the pink shrimp Farfantepenaeus paulensis aiming to analyze their influence on the number of spermatic cells and spermatophore regeneration. The males were extruded in the beginning (day zero) and in the end (43rd day) of the experiment to evaluate the efficiency of these methods in the regeneration process. For the extrusion, a gentle pressure was applied manually in the fifth pair of pereiopods or electrically by a 9 volt pulse in the same area. Both methods were efficient in removing the spermatophore and no significant differences were found (P>0.05) in the number of sperm cells. Nevertheless, significant decreases (P<0.05) in the body weight, spermatophore weight and spermatosomatic index (ESI) at the end of the experimental period were observed by using the electrical stimulation. The mean values (±SD) of the number of sperm cells were 1.46 (±0.84) and 3.25 (±2.12) millions for the electrical and manual treatments, respectively. Results indicate that both methods may be applied to collect initial samples of spermatophores as well as for sperm quality testing. However, when previously spermatophore-extruded males are to be used, the manual method is indicated as the number of spermatic cells, spermatophore weight, body weight, and ESI are maintained after regeneration.

Bovine pancreatic trypsin inhibitor immobilized onto sepharose as a new strategy to purify a thermostable alkaline peptidase from cobia (Rachycentron canadum) processing waste

A thermostable alkaline peptidase was purified from the processing waste of cobia (Rachycentron canadum) using bovine pancreatic trypsin inhibitor (BPTI) immobilized onto Sepharose. The purified enzyme had an apparent molecular mass of 24kDa by both sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry. Its optimal temperature and pH were 50°C and 8.5, respectively. The enzyme was thermostable until 55°C and its activity was strongly inhibited by the classic trypsin inhibitors N-ρ-tosyl-l-lysine chloromethyl ketone (TLCK) and benzamidine. BPTI column allowed at least 15 assays without loss of efficacy. The purified enzyme was identified as a trypsin and the N-terminal amino acid sequence of this trypsin was IVGGYECTPHSQAHQVSLNSGYHFC, which was highly homologous to trypsin from cold water fish species. Using Nα-benzoyl-dl-arginine ρ-nitroanilide hydrochloride (BApNA) as substrate, the apparent km value of the purified trypsin was 0.38mM, kcat value was 3.14s(-1), and kcat/km was 8.26s(-1)mM(-1). The catalytic proficiency of the purified enzyme was 2.75×10(12)M(-1) showing higher affinity for the substrate at the transition state than other fish trypsin. The activation energy (AE) of the BApNA hydrolysis catalyzed by this enzyme was estimated to be 11.93kcalmol(-1) while the resulting rate enhancement of this reaction was found to be approximately in a range from 10(9) to 10(10)-fold evidencing its efficiency in comparison to other trypsin. This new purification strategy showed to be appropriate to obtain an alkaline peptidase from cobia processing waste with high purification degree. According with N-terminal homology and kinetic parameters, R. canadum trypsin may gathers desirable properties of psychrophilic and thermostable enzymes.

Use of a mathematical model to estimate the impact of shrimp pen culture at Patos Lagoon estuary, Brazil

Ecological modeling has been used as a tool to estimate potential impacts caused by aquaculture to the surrounding environment. In this work, a mathematical model was applied to estimate the maximum amount of pink shrimp (Farfantepenaeus paulensis) culture units (3,100m2 pen enclosures) that could be installed at two shallow estuarine bays of Patos Lagoon (known as Coreia and Porto do Rei) with no significant effects on either water quality or viability of the culture system. To calibrate the model, information about the culture of Litopenaeus vannamei and F. paulensis as well as field data (influence of netting material, water current speed and nitrogen concentrations) were used. Under a bad scenario (water current velocity of 0.01m s-1 and a mesh clogging effect of 40%), it would be possible to install up to 29 pens at the Coreia bay, and 39 pens at the Porto do Rei bay. Results indicate that the model was useful in determining the maximum number of culture units that could be installed at these bays, and thus have the potential to become an important tool in the definition of environmental management strategies in relation to aquaculture development.