Purification and characterization of a lysine-p-nitroanilide hydrolase, a broad specificity aminopeptidase, from the cytoplasm of Lactococcus lactis subsp. cremoris AM2

peer-reviewedSummary. A hydrolase activity that cleaves lysyl-p-nitroanilide (Lys-pNA) has been purified from the cytoplasm of Lactococcus lactis subsp. cremoris AM2 by chromatography on DE52, DEAE Affi-Gel Blue Gel, Hydroxyapatite Bio-Gel HTP and Phenyl Sepharose. The purified aminopeptidase was found to have a native Mr of 50000-55000 by gel filtration chromatography and by FPLC gel filtration on Superose 12 and to be composed of a single polypeptide chain following SDS-PAGE. Enzyme activity was almost completely inhibited by EDTA, amastatin, puromycin and bestatin, while the sulphydryl-reactive agents p-chloromercuribenzoate and iodoacetamide were inhibitory. The enzyme was found to be very unstable during the puriification procedures at 4 °C and its stability was greatly improved when 10 ml glycerol/l and 2 mm-dithiothreitol were included in the puri®cation buffers. The puriified enzyme was found to hydrolyse a wide range of dipeptides, tripeptides and longer peptides provided that proline was not present in the penultimate position from the N-terminus or that a pyroglutamyl residue was not present at the N-terminus. While neither Asp-pNA nor Pro-pNA was hydrolysed by the purified enzyme, the release of N-terminal acidic residues from peptides was observed in addition to the release of N-terminal proline from Pro-Leu-Gly-NH2, Pro-Leu- Gly-Gly and Pro-His-Pro-Phe-His-Leu-Phe-Val-Tyr. This ability of Lys-pNA hydrolase to release N-terminal proline residues was employed in concert with a puriified aminopeptidase P preparation to release alternate N-terminal amino acids from Tyr-Pro-Phe-Pro-Gly. The complementary action of these enzymes represents an alternative mechanism to that of post-proline dipeptidyl aminopeptidase for metabolism of proline-containing peptides

Hydrolysis of Ks1- and L-casein-derived peptides with a broad specifcity aminopeptidase and proline specific aminopeptidases from Lactococcus lactis subsp. cremoris AM2

peer-reviewedAminopeptidase hydrolysis of αs1- and β-casein-derived synthetic peptides containing non-consecutive and consecutive proline residues was characterised. Aminopeptidase P (Pep P) (EC 3.4.11.9) or post-proline dipeptidyl aminopeptidase (PPDA) (EC 3.4.14.5) along with lysine-paranitroanilide hydrolase (KpNA-H) (EC 3.4.11.1) activities are required in the degradation of peptides containing non-consecutive proline residues. However, both Pep P and PPDA along with KpNA-H are required for hydrolysis of peptides containing consecutive proline residues. The results demonstrate the mechanism by which combinations of purified general and proline specific aminopeptidases from Lactococcus lactis subsp. cremoris AM2 hydrolyse peptides containing proline residues

End-to-End Document Classification and Key Information Extraction using Assignment Optimization

We propose end-to-end document classification and key information extraction (KIE) for automating document processing in forms. Through accurate document classification we harness known information from templates to enhance KIE from forms. We use text and layout encoding with a cosine similarity measure to classify visually-similar documents. We then demonstrate a novel application of mixed integer programming by using assignment optimization to extract key information from documents. Our approach is validated on an in-house dataset of noisy scanned forms. The best performing document classification approach achieved 0.97 f1 score. A mean f1 score of 0.94 for the KIE task suggests there is significant potential in applying optimization techniques. Abation results show that the method relies on document preprocessing techniques to mitigate Type II errors and achieve optimal performance.Comment: 10 pages, 5 figure