Marine Lactobacillus pentosus H16 protects Artemia franciscana from Vibrio alginolyticus pathogenic effects.

Vibrio alginolyticus is an opportunistic pathogen which may affect different aquatic organisms. The aim of this study was to assess the probiotic properties and the protective mode of action of Lactobacillus pentosus H16 against V. alginolyticus 03/8525, through in vitro and in vivo studies using Artemia franciscana (hereafter Artemia). This strain showed antimicrobial activity against V. alginolyticus 03/8525 and Aeromonas salmonicida subsp. salmonicida ATCC33658 possibly related to lactobacilli organic acid production. It was able to survive at high rainbow trout bile concentrations and showed high selective adhesion to rainbow trout mucus (1.2×10(5)±8.0×10(3) cells cm(-2)). H16 outcompeted V. alginolyticus 03/8525 and A. salmonicida subsp. salmonicida ATCC33658, greatly reducing their adherence to rainbow trout mucus (64.8 and 74.1%, respectively). Moreover, H16 produced a cell-bound biosurfactant which caused an important decrease in the surface tension. H16 also protected Artemia nauplii against mortality when it was administered previous to V. alginolyticus 03/8525 inoculation. Furthermore, H16 bioencapsulated in Artemia, suggesting that it is possible to use live carriers in its administration. We conclude that the ability of L. pentosus H16 to selectively adhere to mucosal surfaces and produce cell-bound biosurfactants, displacing pathogenic strains, in addition to its antimicrobial activity, confer H16 competitive advantages against pathogens as demonstrated in in vivo challenge experiments. Thus, L. pentosus H16, a marine bacterium from the intestinal tract of hake, is an interesting probiotic for Artemia culture and also has the potential to prevent vibriosis in other aquaculture activities such as larvae culture and fish farming.

Philibertain g I, the most basic cysteine endopeptidase purified from the latex of Philibertia gilliesii Hook. et Arn. (Apocynaceae).

A new papain-like cysteine peptidase isolated from latex of Philibertia gilliesii Hook. et Arn., Apocynaceae (formerly Asclepiadaceae) has been purified and characterized. The enzyme, named philibertain g I, is the most basic component present in latex extracts and was purified by acetone fractionation followed by cation exchange chromatography (SP-Sepharose HR) using FPLC system. Homogeneity was confirmed by SDS-PAGE and mass spectroscopy (MS). Molecular mass of the enzyme was 23,530 Da (MALDI-TOF MS), its isoelectric point was >10.25, and maximum proteolytic activity (casein) was achieved at pH 7-8. The new protease was inhibited by E-64 a cysteine peptidases inhibitor. Km was 0.15 mM, using PFLNA as substrate. The N-terminal sequence of philibertain g I (LPASVDWRKEGAVLPIRHQGQCG) was compared with those of twenty plant proteases. Philibertain g I showed the higher degree of identity (73%) with caricain, one of the Carica papaya endopepetidases.

Comparison of two cysteine endopeptidases from Pseudananas macrodontes (Morr.) Harms (Bromeliaceae).

The properties of two cysteine peptidases (macrodontain I and II) isolated from fruits of Pseudananas macrodontes have been compared. The enzymes showed optimum pH ranges near neutrality and were inhibited by E-64 and other cysteine peptidase inhibitors. Molecular masses were 23459 and 23703 kDa, the isoelectric points were 6.1 and 5.9, and the Km values were 13.4 and 8.9 microM (Bz-Phe-Val-Arg-AMC) for macrodontain I and II, respectively. N-alpha-CBZ-L-amino acid p-nitrophenyl esters were tested for both enzymes. The N-terminal sequences of both proteases differed slightly and showed high sequence similarity to other pineapple stem-derived cysteine endopeptidases.

Purification and characterization of macrodontain I, a cysteine peptidase from unripe fruits of Pseudananas macrodontes (Morr.) harms (Bromeliaceae).

A new papain-like cysteine peptidase isolated from fruits of Pseudananas macrodontes (Morr.) Harms, a species closely related to pineapple (Ananas comosus L.), has been purified and characterized. The enzyme, named macrodontain I, is the main proteolytic component present in fruit extracts and was purified by acetone fractionation followed by anion-exchange chromatography. Separation was improved by selecting both an adequate pH value and a narrow saline gradient. Optimum pH range (more than 90% of maximum activity with casein) was achieved at pH 6.1-8.5. Homogeneity of the enzyme was confirmed by bidimensional electrophoresis and mass spectroscopy (MS). Molecular mass of the enzyme was 23,459 (MS) and its isoelectric point was 6.1. The alanine, glutamine, and tyrosine derivatives were strongly preferred when the enzyme was assayed on N-alpha-CBZ-l-amino acid p-nitrophenyl esters. The N-terminal sequence of macrodontain (by comparison with the N-terminus of 30 plant proteases with more than 50% homology) showed a great deal of sequence similarity to the other pineapple-stem-derived cysteine endopeptidases, being 85.7, 85. 2, and 77.8% identical to comosain, stem bromelain, and ananain, respectively. It seems clear that the Bromeliaceae endopeptidases are more closely related to each other than to other members of the papain family, suggesting relatively recent divergence.