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Biofilm, a microbial community embedded in extracellular polymeric substance, fortifies bacteria against the immune system. In biofilms, the expression of functional amyloids is linked with highly aggregative, multi-resistant strains, and chronic infections. Serrapeptase (SPT), a protease possessing similar or superior anti-microbial properties with many antibiotics, presents anti-amyloid potential. However, studies on\u00a0the employment of SPT against <jats:italic>Pseudomonas<\/jats:italic> biofilms and Fap amyloid, or the possible mechanisms of action are scarce. Here,\u00a0SPT inhibited biofilm formation of <jats:italic>P. aeruginosa<\/jats:italic> ATCC 27853 on both plastic and glass surfaces, with an IC<jats:sub>50<\/jats:sub> of 11.26\u00a0\u00b5g\/mL\u00a0and\u00a00.27\u00a0\u00b5g\/mL, respectively.\u00a0The inhibitory effect of SPT on biofilm was also verified with optical microscopy of crystal violet-stained biofilms and with confocal microscopy. Additionally, SPT caused a dose-dependent decrease of bacterial viability (IC<jats:sub>50<\/jats:sub> of 3.07\u00a0\u00b5g\/mL) as demonstrated by MTT assay. Reduction of bacterial functional amyloids\u00a0was also demonstrated, employing both fluorescence microscopy with thioflavin\u00a0T and photometrical determination of Congo-red-positive compounds. Both viability and functional amyloids correlated significantly with biofilm inhibition. Finally, in silico molecular docking studies provided a mechanistic insight into the interaction of SPT\u00a0with FapC or FapD, proving that both peptides are possible targets of SPT. These results offer new insights into the biofilm formation of <jats:italic>P. aeruginosa<\/jats:italic> and potentiate the involvement of SPT in the prevention\u00a0and eradication of <jats:italic>Pseudomonas<\/jats:italic> biofilms.<\/jats:p>\n              <\/jats:sec><jats:sec>\n                <jats:title>Graphical abstract<\/jats:title>\n                \n              <\/jats:sec><jats:sec>\n                <jats:title>Key points<\/jats:title>\n                <jats:p>\u2022 <jats:italic>Serrapeptase inhibits biofilm formation of P. aeruginosa on plastic and glass.<\/jats:italic><\/jats:p>\n                <jats:p>\u2022 <jats:italic>Biofilm inhibition correlated with reduced viability and functional amyloid levels.<\/jats:italic><\/jats:p>\n                <jats:p>\u2022 <jats:italic>In silico studies indicated that serrapeptase may target FapC and FapD peptides.<\/jats:italic><\/jats:p>\n              <\/jats:sec>","DOI":"10.1007\/s00253-023-12772-1","type":"journal-article","created":{"date-parts":[[2023,9,23]],"date-time":"2023-09-23T16:01:32Z","timestamp":1695484892000},"page":"7269-7285","update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":12,"title":["In vitro and in silico evaluation of the serrapeptase effect on biofilm and amyloids of Pseudomonas aeruginosa"],"prefix":"10.1007","volume":"107","author":[{"given":"Georgios","family":"Katsipis","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dimitrios I.","family":"Avgoulas","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"George D.","family":"Geromichalos","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Maria","family":"Petala","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3791-6955","authenticated-orcid":false,"given":"Anastasia A.","family":"Pantazaki","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2023,9,23]]},"reference":[{"key":"12772_CR1","doi-asserted-by":"publisher","first-page":"264","DOI":"10.1016\/j.ijom.2007.11.011","volume":"37","author":"TH Al-Khateeb","year":"2008","unstructured":"Al-Khateeb TH, Nusair Y (2008) Effect of the proteolytic enzyme serrapeptase on swelling, pain and trismus after surgical extraction of mandibular third molars. 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