N-Benzoyl-D-lysine

During shrimp larval development, changes occur in molecular components. Enzyme activity and mRNA expression of proteinases were assayed in Penaeus vannamei during larval development, which consists of 5 nauplius stages, 3 protozoeal stages, 3 mysis stages, and 12 postlarval stages. Trypsin activity reached a maximum at the beginning of postlarval stages 1 and 2, and significantly decreased in subsequent postlarval stages. Chymotrypsin activity increased at the third protozoeal stage, then significantly decreased in subsequent stages. Identification of proteinase by mass spectrometry and inhibitors allowed us to track their appearance in zymograms and to distinguish between isoenzymes. Chymotrypsin BI and BII had a distinguishing pattern of appearance during larval development, which could compensate for the reduction in trypsin activity. The mRNA content of isotrypsin 21, chymotrypsin 1, and zinc proteinase was differentially expressed in larvae. Zinc proteinase and chymotrypsin 1 mRNA were expressed at a basal content at the beginning of the protozoeal stages, increased by the end of the mysis stages and onward, while isotrypsin 21 mRNA had a peak at mysis stage 3. Transcript changes reflect transcriptional regulation of the proteinases tested. Proteinase mRNA in tissues, other than the digestive gland, suggests potentially different roles besides digestion during ontogeny.

Clostridium perfringens produces a homologue of clostripain (Clo), the arginine-specific endopeptidase of Clostridium histolyticum. To determine the biochemical and biological properties of the C. perfringens homologue (Clp), it was purified from the culture supernatant of a recombinant C. perfringens strain by cation-exchange chromatography and ultrafiltration. Analysis by SDS-PAGE, N-terminal amino acid sequencing and TOF mass spectrometry revealed that Clp consists of two polypeptides comprising heavy (38 kDa) and light (16 kDa or 15 kDa) chains, and that the two light chains differ in the N-terminal cleavage site. This difference in the light chain did not affect the enzymic activity toward N-benzoyl-l-arginine p-nitroanilide (Bz-l-arginine pNA), as demonstrated by assaying culture supernatants differing in the relative ratio of the two light chains. Although the purified Clp preferentially degraded Bz-dl-arginine pNA rather than Bz-dl-lysine pNA, it degraded the latter more efficiently than did Clo. Clp showed 2.3-fold higher caseinolytic activity than Clo, as expected from the difference in substrate specificity. Clp caused an increase in vascular permeability when injected intradermally into mice, implying a possible role of Clp in the pathogenesis of clostridial myonecrosis.

Pterygoplichthys disjunctivus viscera trypsin was purified by fractionation with ammonium sulphate, gel filtration, affinity and ion exchange chromatography (DEAE-Sepharose). Trypsin molecular weight was approximately 27.5kDa according to SDS-PAGE, shown a single band in zymography. It exhibited maximal activity at pH 9.5 and 40°C, using N-benzoyl-dl-arginine-p-nitroanilide (BAPNA) as substrate. Enzyme was effectively inhibited by phenyl methyl sulphonyl fluoride (PMSF) (100%), N-α-p-tosyl-l-lysine chloromethyl ketone (TLCK) (85.4%), benzamidine (80.2%), and soybean trypsin inhibitor (75.6%) and partially inhibited by N-tosyl-l-phenylalanine chloromethyl ketone (TPCK) (10.3%), ethylendiaminetetraacetic acid (EDTA) (8.7%) and pepstatin A (1.2%). Enzyme activity was slightly affected by metal ions (Fe(2+)>Hg(2+)>Mn(2+)>K(+)>Mg(2+)>Li(+)>Cu(2+)). Trypsin activity decreased continuously as NaCl concentration increased (0-30%). Km and kcat values were 0.13mM and 1.46s(-1), respectively. Results suggest the enzyme have a potential application where room processing temperatures (25-35°C) or high salt (30%) concentration are needed, such as in fish sauce production.