Metal-sensitive and thermostable trypsin from the crevalle jack (<Emphasis Type="Italic">Caranx hippos</Emphasis>) pyloric caeca: purification and characterization

Background

Over the past decades, the economic development and world population growth has led to increased for food demand. Increasing the fish production is considered one of the alternatives to meet the increased food demand, but the processing of fish leads to by-products such as skin, bones and viscera, a source of environmental contamination. Fish viscera have been reported as an important source of digestive proteases with interesting characteristics for biotechnological processes. Thus, the aim of this study was to purify and to characterize a trypsin from the processing by-products of crevalle jack (Caranx hippos) fish.

Results

A 27.5 kDa trypsin with N-terminal amino acid sequence IVGGFECTPHVFAYQ was easily purified from the pyloric caeca of the crevalle jack. Its physicochemical and kinetic properties were evaluated using N-α-benzoyl-_DL-arginine-p-nitroanilide (BApNA) as substrate. In addition, the effects of various metal ions and specific protease inhibitors on trypsin activity were determined. Optimum pH and temperature were 8.0 and 50°C, respectively. After incubation at 50°C for 30 min the enzyme lost only 20% of its activity. K _m , k_cat, and k _cat /K _m values using BApNA as substrate were 0.689 mM, 6.9 s^-1, and 10 s^-1 mM^-1, respectively. High inhibition of trypsin activity was observed after incubation with Cd²⁺, Al³⁺, Zn²⁺, Cu²⁺, Pb²⁺, and Hg²⁺ at 1 mM, revealing high sensitivity of the enzyme to metal ions.

Conclusions

Extraction of a thermostable trypsin from by-products of the fishery industry confirms the potential of these materials as an alternative source of these biomolecules. Furthermore, the results suggest that this trypsin-like enzyme presents interesting biotechnological properties for industrial applications.

     

Additivity of atomic static polarizabilities and dispersion coefficients

A new empirical method is proposed to evaluate the average molecular polarizabilities assuming the additivity of atomic static polarizability. Atomic static polarizability for each atom in a particular valence state is obtained. Calculated molecular polarizabilities of 94 non-halogenated compounds and of the bases in nucleic acids show the excellent agreement with experimental data.To check the further validity of this method, dispersion coefficients for CH₄, C₂H₆, C₃H₈,n–C₄H₁₀,n–C₅H₁₂,n–C₆H₁₄,n–C₇H₁₆,n–C₈H₁₈, H₂, H₂O and NH₃ are obtained from a sum of atomic terms using a London-type formula, and are compared with the accurate values of dipole oscillator strength distribution (DOSD) method. The results show the excellent agreement between theory and experiment.     

Conformational preferences of proline analogues with different ring size

The conformational study on L-azetidine-2-carboxylic acid (Ac-Aze-NHMe, the Aze dipeptide) and (S)-piperidine-2-carboxylic acid (Ac-Pip-NHMe, the Pip dipeptide) is carried out using ab initio HF and density functional methods with the self-consistent reaction field method to explore the differences in conformational preferences and cis-trans isomerization for proline residue and its analogues with different ring size in the gas phase and in solution (chloroform and water). The change of ring size by deleting a CH2 group from or adding a CH2 group to the prolyl ring results the remarkable changes in backbone and ring structures compared with those of the Pro dipeptide, especially in the C'-N imide bond length and the bond angles around the N-C(alpha) bond. The four-membered azetidine ring can have either puckered structure depending on the backbone structure because of the less puckered structure. The six-membered piperidine ring can adopt chair and boat conformations, but the chair conformation is more preferred than the boat conformation. These calculated preferences for puckering are consistent with experimental results from analysis of X-ray structures of Aze- and Pip-containing peptides. On going from Pro to Aze to Pip, the axiality (i.e., a tendency to adopt the axial orientation) of the NHMe group becomes stronger, which can be ascribed to reduce the steric hindrances between 1,2-substituted Ac and NHMe groups. As the solvent polarity increases, the polyproline II-like conformation becomes more populated and the relative stability of conformation tC with a C7 hydrogen bond between C'=O of the amino group and N-H of the carboxyl group decreases for both the Aze and Pip dipeptides, as seen for the Pro dipeptide. The cis population and rotational barriers for the imide bond increase with the increase of solvent polarity for both the Aze and Pip dipeptides, as seen for the Pro dipeptide. In particular, the cis-trans isomerization proceeds in common through only the clockwise rotation with omega' approximately +120 degrees about azetyl and piperidyl peptide bonds in the gas phase and in solution, as seen for alanyl and prolyl peptide bonds. The pertinent distance d(N...H-N(NHMe)) and the pyramidality of imide nitrogen can describe the role of this hydrogen bond in stabilizing the transition state structure, but the lower rotational barriers for the Aze and Pip dipeptides than those for the Pro dipeptide, which is observed from experiments, cannot be rationalized.     

Memetic cooperative models for the tool switching problem

This work deals with memetic-computing agent-models based on the cooperative integration of search agents endowed with (possibly different) optimization strategies, in particular memetic algorithms. As a proof-of-concept of the model, we deploy it on the tool switching problem (ToSP), a hard combinatorial optimization problem that arises in the area of flexible manufacturing. The ToSP has been tackled by different algorithmic methods ranging from exact to heuristic methods (including local search meta-heuristics, population-based techniques and hybrids thereof, i.e., memetic algorithms). Here we consider an ample number of instances of this cooperative memetic model, whose agents are adapted to cope with this problem. A detailed experimental analysis shows that the meta-models promoting the cooperation among memetic algorithms provide the best overall results compared with their constituent parts (i.e., memetic algorithms and local search approaches). In addition, a parameter sensitivity analysis of the meta-models is developed in order to understand the interplay among the elements of the proposed topologies.     

Rheomagnetic properties of mixed magnetic particle suspensions

Single-domain magnetic particles are the essential ingredient of magnetic tapes, particulate recording disks and magnetic stripes. The particles are single-domain γ-Fe₂O₃, CrO₂ or barium ferrite, and non-magnetic α-Fe₂O₃ mixture. Each of these particles has intrinsic coercivity, which should be matched with the magnetic field strength of the writing element of a particular device. In this study a magnetic inductance measurement with low field strength was employed to obtain the magnetic permeability of suspensions containing two of the particle types mixed together as a function of composition and volume fraction of particles. The bulk magnetic property B is a linear combination of the contributions from each particle type such that the “excess” inductance is L − L_s = Σφ_iB_i where φ_i is the volume fraction and B_i, is the magnetic property of particle type i. For the non-magnetic α-Fe₂O₃, B_i = 0. This allows the formulation of mixed particle suspensions to obtain a desired property for custom-designed magnetic particle coatings. However, mixing magnetic particle types will broaden or produce a bimodal switching field distribution. This may affect the squareness of the magnetic hysteresis loop. These properties should be taken into account for the design of a practical magnetic coating with mixed particle suspension. Another requirement of the magnetic particle suspensions is that they remain well dispersed, even though strong magnetic forces between the particles promote flocculation. An extension of the inductance measurement technique is employed to study the flocculation of a suspension containing magnetic γ-Fe₂O₃ and non-magnetic α-Fe₂O₃. The presence of the α-Fe₂O₃ decreases the flocculation state of the suspension. Thus the suspension stability is enhanced by incorporating a small amount of non-magnetic particles in addition to surfactant.     

Synthesis and electrorheological properties of polyaniline-Na+-montmorillonite suspensions

We synthesized polyaniline-Na⁺-montmorillonite nanocomposite particles using an emulsion intercalation method and prepared electrorheological (ER) fluids by dispersing the synthesized nanocomposite particles in an electrically insulating silicone oil. The conducting polymer (polyaniline) was inserted into the layers of clay, and this insertion of polyaniline was confirmed by X-ray diffraction. For the first time, ER properties were determined via a rotational rheometer equipped with a high voltage generator.