Identification of N‐glycosylation in prolyl endoprotease from Aspergillus niger and evaluation of the enzyme for its possible application in proteomics

Abstract

An acidic prolyl endoprotease from Aspergillus niger was isolated from the commercial product Brewers Clarex to evaluate its possible application in proteomics. The chromatographic purification yielded a single protein band in sodium dodecyl sulfate polyacrylamide gel electrophoresis providing an apparent molecular mass of 63 kDa and a broad peak (m/z 58,061) in linear matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) indicating the glycoprotein nature of the enzyme. Indeed, a colorimetric assessment with phenol and sulfuric acid showed the presence of neutral sugars (9% of weight). The subsequent treatment with N-glycosidase F released a variety of high-mannose type N-glycans, which were successfully detected using MALDI-TOF MS. MALDI-TOF/TOF tandem MS analysis of glycopeptides from a tryptic digest of prolyl endoprotease unraveled the identity of the N-glycosylation site in the primary structure. The data obtained also show that the enzyme is present in its processed form, i.e. without putative signal and propeptide parts. Spectrophotometric measurements demonstrated optimal activity at pH 4.0-4.5 and also high thermostability for the cleavage at the C-terminal part of proline residues. In-solution digestion of standard proteins (12-200 kDa) allowed to evaluate the cleavage specificity. The enzyme acts upon proline and alanine residues, but there is an additional minor cleavage at some other residues like Gly, Leu, Arg, Ser and Tyr. The digestion of a honeybee peptide comprising six proline residues (apidaecin 1A) led to the detection of specific peptides terminated by proline as it was confirmed by MALDI postsource decay analysis.

Aspergillus niger Prolyl Endoprotease for Hydrogen–Deuterium Exchange Mass Spectrometry and Protein Structural Studies

“… 21 , 26 Therefore, we reasoned that the use of a full protein, in this case FTC-casein, would be a much more suitable probe for An-PEP activity than Pro-ending peptides. To our surprise and in contrast to previous reports using Pro-ending peptides, 21 , 26 we observed that An-PEP activity shows two optima at pH ∼2 and ∼6. This pattern of double pH optima has been previously reported also for neutral PEPs 31 but not for an acidic PEP.…”

Malt hydrolysates for gluten-free applications: Autolytic and proline endopeptidase assisted removal of prolamins from wheat, barley and rye

“…During the past decade, several studies have demonstrated the capacity of a specific group of microbial proline-specific proteases (EC 3.4.21.26) to eliminate such prolamin-derived toxic epitopes Piper et al, 2004;Shan et al, 2002). Among these prolinespecific endopeptidases, the enzyme from Aspergillus niger (AN-PEP; Edens et al, 2005;Lopez and Edens, 2005;Sebela et al, 2009;Stepniak et al, 2006) is of particular interest as it represents the only industrial food-grade proline-specific enzyme with an acidic pH optimum. No attempts to utilize such proline specific proteases in gluten-free food applications, for instance in the elimination of residual gluten, have this far been made.…”

ProAlanase is an Effective Alternative to Trypsin for Proteomics Applications and Disulfide Bond Mapping

“…Proteases with unique cleavage specificity may have significant value for proteomics since the resultant peptides are likely to come from different regions in the proteome. In 2009, an acidic prolyl-endopeptidase An-PEP from Aspergillus niger fungi was tested by Šebela et al for possible proteomic applications, showing potential for in-solution protein digestion 28 .…”