{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,14]],"date-time":"2026-10-14T01:01:49Z","timestamp":1791939709374,"version":"4.3.4"},"reference-count":74,"publisher":"American Society for Microbiology","issue":"8","license":[{"start":{"date-parts":[[2026,8,4]],"date-time":"2026-08-04T00:00:00Z","timestamp":1785801600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2026,8,4]],"date-time":"2026-08-04T00:00:00Z","timestamp":1785801600000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"funder":[{"DOI":"10.13039\/501100003093","name":"Malaysian Ministry of Higher Education","doi-asserted-by":"crossref","award":["FRGS\/1\/2023\/STG02\/USM\/02\/7"],"award-info":[{"award-number":["FRGS\/1\/2023\/STG02\/USM\/02\/7"]}],"id":[{"id":"10.13039\/501100003093","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["Microbiol Spectr"],"published-print":{"date-parts":[[2026,8,4]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:sec>\n                    <jats:title\/>\n                    <jats:p>\n                      The increasing demand for high-performance proteases in industrial applications necessitates strategies to enhance enzyme yield, stability, and functional robustness under extreme conditions. This study investigates a serine protease from\n                      <jats:italic toggle=\"yes\">Bacillus toyonensis<\/jats:italic>\n                      , isolated from fermented kitchen waste, and its co-expression with short peptides derived from late embryogenesis abundant (LEA) proteins, LEA-I, and LEA-K. The protease gene was cloned into a pRSFDuet-1 vector and expressed in\n                      <jats:italic toggle=\"yes\">Escherichia coli<\/jats:italic>\n                      BL21(DE3), followed by purification using immobilized metal affinity chromatography. Co-expression with LEA peptides significantly enhanced protease production and stability, with ProLEA-K achieving the highest purification fold (1,638.00) and specific activity (16.38 U\/mg) compared to 0.01 U\/mg in the crude extract. Functionally, ProLEA-K exhibited a 2.5-fold increase in activity at 60\u00b0C and retained substantial activity up to 70\u00b0C, whereas the protease expressed alone (ProWL) declined sharply at elevated temperatures and maintained high activity across a broad pH range spanning acidic to alkaline conditions, a rare feature for acidic serine proteases. Evaluation under detergent-containing conditions revealed a differential response, whereby detergents enhanced the activity of ProWL but reduced the activity of LEA-co-expressed variants, indicating a context-dependent effect of LEA peptides on enzyme performance.\n                      <jats:italic toggle=\"yes\">In silico<\/jats:italic>\n                      analysis supported these experimental findings. Multiple sequence alignment revealed strong conservation of the catalytic triad (Asp171, His203, and Ser225) and an amino acid profile consistent with secreted bacterial enzymes. Structural modeling confirmed stable folding, and molecular docking with azocasein indicated stronger substrate binding in LEA-containing variants (\u22126.9 kcal\/mol for ProLEA-K vs \u22125.8 kcal\/mol for ProWL). These results identify LEA peptides, particularly LEA-K, as effective molecular stabilizers, improving both the yield and functional robustness of industrial proteases.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>IMPORTANCE<\/jats:title>\n                    <jats:p>This work highlights a simple and effective approach to improve industrial proteases using short late embryogenesis abundant peptides as natural stabilizers. The method enhanced yield, thermal stability, and pH adaptability across acidic and alkaline conditions without genetic redesign of the protease itself, offering a broadly applicable and economical strategy for enzyme improvement.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.1128\/spectrum.03010-25","type":"journal-article","created":{"date-parts":[[2026,6,22]],"date-time":"2026-06-22T12:00:42Z","timestamp":1782129642000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":1,"title":["Functional and structural characterization of a serine protease from\n                    <i>Bacillus toyonensis<\/i>\n                    co-expressed with short peptides from late embryogenesis abundant (LEA) proteins"],"prefix":"10.1128","volume":"14","author":[{"ORCID":"https:\/\/orcid.org\/0009-0003-1854-4575","authenticated-orcid":false,"given":"Ammar Khazaal Kadhim","family":"Almansoori","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/02rgb2k63","id-type":"ROR","asserted-by":"publisher"}],"name":"School of Biological Sciences, Universiti Sains Malaysia","place":["Gelugor, Malaysia"]},{"id":[{"id":"https:\/\/ror.org\/02rgb2k63","id-type":"ROR","asserted-by":"publisher"}],"name":"Centre for Chemical Biology (CCB), Universiti Sains Malaysia","place":["Bayan Lepas, Malaysia"]},{"id":[{"id":"https:\/\/ror.org\/023a3xe97","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Medical Laboratory Techniques, Al-Mustaqbal University","place":["Hillah, Iraq"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nurul Ain","family":"Syukriyah","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/02rgb2k63","id-type":"ROR","asserted-by":"publisher"}],"name":"School of Biological Sciences, Universiti Sains Malaysia","place":["Gelugor, Malaysia"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Choo Yi","family":"Hang","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/02rgb2k63","id-type":"ROR","asserted-by":"publisher"}],"name":"School of Biological Sciences, Universiti Sains Malaysia","place":["Gelugor, Malaysia"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mustafa","family":"Abdulfattah","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/02rgb2k63","id-type":"ROR","asserted-by":"publisher"}],"name":"School of Biological Sciences, Universiti Sains Malaysia","place":["Gelugor, Malaysia"]},{"id":[{"id":"https:\/\/ror.org\/02rgb2k63","id-type":"ROR","asserted-by":"publisher"}],"name":"Centre for Chemical Biology (CCB), Universiti Sains Malaysia","place":["Bayan Lepas, Malaysia"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shinya","family":"Ikeno","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/02278tr80","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Biological Functions and Engineering, Graduate School of Life Science and System Engineering, Kyushu Institute of Technology","place":["Kitakyushu, Japan"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lo Hui","family":"Yu","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/02rgb2k63","id-type":"ROR","asserted-by":"publisher"}],"name":"School of Biological Sciences, Universiti Sains Malaysia","place":["Gelugor, Malaysia"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Haider Mahmod","family":"Jasim","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/03877wr45","id-type":"ROR","asserted-by":"publisher"}],"name":"College of Pharmacy, Al-Muthanna University","place":["Samawah, Iraq"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8945-4428","authenticated-orcid":true,"given":"Rashidah","family":"Abdul Rahim","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/02rgb2k63","id-type":"ROR","asserted-by":"publisher"}],"name":"School of Biological Sciences, Universiti Sains Malaysia","place":["Gelugor, Malaysia"]},{"id":[{"id":"https:\/\/ror.org\/02rgb2k63","id-type":"ROR","asserted-by":"publisher"}],"name":"Centre for Chemical Biology (CCB), Universiti Sains Malaysia","place":["Bayan Lepas, Malaysia"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"235","reference":[{"key":"e_1_3_5_2_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.tibtech.2018.11.005"},{"key":"e_1_3_5_3_2","doi-asserted-by":"publisher","DOI":"10.1186\/s13568-018-0611-6"},{"key":"e_1_3_5_4_2","doi-asserted-by":"publisher","DOI":"10.1038\/jidsymp.2011.2"},{"key":"e_1_3_5_5_2","doi-asserted-by":"publisher","DOI":"10.3389\/fmicb.2023.1236368"},{"key":"e_1_3_5_6_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0141-0229(02)00293-4"},{"key":"e_1_3_5_7_2","doi-asserted-by":"publisher","DOI":"10.1016\/B978-0-12-800217-9.00009-5"},{"key":"e_1_3_5_8_2","doi-asserted-by":"publisher","DOI":"10.1016\/s0968-0004(98)01254-7"},{"key":"e_1_3_5_9_2","doi-asserted-by":"publisher","DOI":"10.1016\/B978-0-08-100022-9.00011-6"},{"key":"e_1_3_5_10_2","doi-asserted-by":"publisher","DOI":"10.1016\/B978-012373944-5.00172-3"},{"key":"e_1_3_5_11_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.bcab.2021.102051"},{"key":"e_1_3_5_12_2","doi-asserted-by":"publisher","DOI":"10.1007\/s00018-013-1355-8"},{"key":"e_1_3_5_13_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.jgeb.2025.100459"},{"key":"e_1_3_5_14_2","doi-asserted-by":"publisher","DOI":"10.1080\/21505594.2021.1871822"},{"key":"e_1_3_5_15_2","doi-asserted-by":"publisher","DOI":"10.1111\/j.1745-4514.2001.tb00721.x"},{"key":"e_1_3_5_16_2","doi-asserted-by":"publisher","DOI":"10.17113\/ftb.60.02.22.7301"},{"key":"e_1_3_5_17_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0032-9592(98)00110-1"},{"key":"e_1_3_5_18_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.heliyon.2021.e08148"},{"key":"e_1_3_5_19_2","doi-asserted-by":"publisher","DOI":"10.3390\/fermentation8110628"},{"key":"e_1_3_5_20_2","doi-asserted-by":"publisher","DOI":"10.15414\/jmbfs.3721"},{"key":"e_1_3_5_21_2","doi-asserted-by":"publisher","DOI":"10.1021\/bi00517a033"},{"key":"e_1_3_5_22_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.colsurfb.2005.07.017"},{"key":"e_1_3_5_23_2","doi-asserted-by":"publisher","DOI":"10.1007\/s00253-022-12080-0"},{"key":"e_1_3_5_24_2","doi-asserted-by":"publisher","DOI":"10.1007\/s12192-017-0781-1"},{"key":"e_1_3_5_25_2","doi-asserted-by":"publisher","DOI":"10.1186\/1471-2164-9-118"},{"key":"e_1_3_5_26_2","doi-asserted-by":"publisher","DOI":"10.4161\/psb.6.4.15042"},{"key":"e_1_3_5_27_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.ijbiomac.2024.137641"},{"key":"e_1_3_5_28_2","doi-asserted-by":"publisher","DOI":"10.3389\/fpls.2016.01011"},{"key":"e_1_3_5_29_2","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0082824"},{"key":"e_1_3_5_30_2","doi-asserted-by":"publisher","DOI":"10.1002\/jmr.2658"},{"key":"e_1_3_5_31_2","doi-asserted-by":"publisher","DOI":"10.1007\/3-540-33774-1_8"},{"key":"e_1_3_5_32_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.mimet.2021.106207"},{"key":"e_1_3_5_33_2","doi-asserted-by":"publisher","DOI":"10.1002\/2211-5463.13448"},{"key":"e_1_3_5_34_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.bbrc.2025.152224"},{"key":"e_1_3_5_35_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.bbrc.2018.06.095"},{"key":"e_1_3_5_36_2","doi-asserted-by":"publisher","DOI":"10.1007\/s12010-020-03262-5"},{"key":"e_1_3_5_37_2","doi-asserted-by":"publisher","DOI":"10.1128\/spectrum.01422-21"},{"key":"e_1_3_5_38_2","doi-asserted-by":"publisher","unstructured":"Almansoori AKK Yu KS Mohamed F Ikeno S Patil RB G R-P Rahim RA. 2024. Enhancing thermostability of Bacillus licheniformis lipase with LEA peptide co-expression system. Res Square. doi:10.21203\/rs.3.rs-4160767\/v1","DOI":"10.21203\/rs.3.rs-4160767\/v1"},{"key":"e_1_3_5_39_2","doi-asserted-by":"publisher","DOI":"10.1016\/bs.mim.2023.11.003"},{"key":"e_1_3_5_40_2","doi-asserted-by":"publisher","DOI":"10.1385\/0-89603-471-2:45"},{"key":"e_1_3_5_41_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.74.11.5088"},{"key":"e_1_3_5_42_2","doi-asserted-by":"publisher","DOI":"10.1016\/1046-5928(92)90001-d"},{"key":"e_1_3_5_43_2","doi-asserted-by":"publisher","DOI":"10.1016\/0003-2697(76)90527-3"},{"key":"e_1_3_5_44_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0021-9258(17)41059-3"},{"key":"e_1_3_5_45_2","doi-asserted-by":"publisher","DOI":"10.1653\/024.103.00406"},{"key":"e_1_3_5_46_2","first-page":"19","volume-title":"Methods in enzymology","author":"Rawlings ND","year":"1994","unstructured":"Rawlings ND, Barrett AJ. 1994. Families of serine peptidases, p 19\u201361. In Methods in enzymology. Vol. 224. Academic Press."},{"key":"e_1_3_5_47_2","doi-asserted-by":"publisher","DOI":"10.1101\/gr.849004"},{"key":"e_1_3_5_48_2","doi-asserted-by":"publisher","DOI":"10.1093\/molbev\/msy096"},{"key":"e_1_3_5_49_2","doi-asserted-by":"publisher","DOI":"10.1038\/nprot.2015.053"},{"key":"e_1_3_5_50_2","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gky427"},{"key":"e_1_3_5_51_2","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkac240"},{"key":"e_1_3_5_52_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-021-03819-2"},{"key":"e_1_3_5_53_2","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkp323"},{"key":"e_1_3_5_54_2","doi-asserted-by":"publisher","DOI":"10.1002\/pro.5560020916"},{"key":"e_1_3_5_55_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0076-6879(97)77022-8"},{"key":"e_1_3_5_56_2","doi-asserted-by":"publisher","DOI":"10.1107\/S0021889892009944"},{"key":"e_1_3_5_57_2","doi-asserted-by":"publisher","DOI":"10.1016\/B978-0-323-89775-4.00023-7"},{"key":"e_1_3_5_58_2","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkt458"},{"key":"e_1_3_5_59_2","unstructured":"DeLano WL Scientific D Carlos S. 2026. PyMOL: an open-source molecular graphics tool. Available from: http:\/\/www.pymol.org\/pymol"},{"key":"e_1_3_5_60_2","doi-asserted-by":"publisher","DOI":"10.1038\/nprot.2016.169"},{"key":"e_1_3_5_61_2","doi-asserted-by":"publisher","DOI":"10.1002\/jcc.21334"},{"key":"e_1_3_5_62_2","doi-asserted-by":"publisher","DOI":"10.1186\/1758-2946-4-17"},{"key":"e_1_3_5_63_2","unstructured":"Dassault Syst\u00e8mes. 2020. Free download: BIOVIA discovery studio visualizer. Dassault Syst\u00e8mes. Available from: https:\/\/discover.3ds.com\/discovery-studio-visualizer-download"},{"key":"e_1_3_5_64_2","doi-asserted-by":"publisher","DOI":"10.1021\/acs.jafc.5c03673"},{"key":"e_1_3_5_65_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.ijbiomac.2024.132286"},{"key":"e_1_3_5_66_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.molstruc.2024.139204"},{"key":"e_1_3_5_67_2","doi-asserted-by":"publisher","DOI":"10.3389\/fmicb.2025.1558419"},{"key":"e_1_3_5_68_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.bbrc.2017.08.091"},{"key":"e_1_3_5_69_2","doi-asserted-by":"publisher","DOI":"10.1021\/acs.chemrev.4c00707"},{"key":"e_1_3_5_70_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0141-0229(03)00004-8"},{"key":"e_1_3_5_71_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.ijbiomac.2016.06.025"},{"key":"e_1_3_5_72_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.heliyon.2024.e25084"},{"key":"e_1_3_5_73_2","doi-asserted-by":"publisher","DOI":"10.4014\/jmb.2108.08037"},{"key":"e_1_3_5_74_2","doi-asserted-by":"publisher","DOI":"10.1007\/s00203-020-01898-9"},{"key":"e_1_3_5_75_2","doi-asserted-by":"publisher","DOI":"10.1002\/cbic.202401013"}],"container-title":["Microbiology Spectrum"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/journals.asm.org\/doi\/pdf\/10.1128\/spectrum.03010-25","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/journals.asm.org\/doi\/pdf\/10.1128\/spectrum.03010-25","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,8,6]],"date-time":"2026-08-06T14:45:07Z","timestamp":1786027507000},"score":1,"resource":{"primary":{"URL":"https:\/\/journals.asm.org\/doi\/10.1128\/spectrum.03010-25"}},"subtitle":[],"editor":[{"given":"Wenli","family":"Li","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"editor"}]}],"short-title":[],"issued":{"date-parts":[[2026,8,4]]},"references-count":74,"journal-issue":{"issue":"8","published-print":{"date-parts":[[2026,8,4]]}},"alternative-id":["10.1128\/spectrum.03010-25"],"URL":"https:\/\/doi.org\/10.1128\/spectrum.03010-25","relation":{},"ISSN":["2165-0497"],"issn-type":[{"value":"2165-0497","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,8,4]]},"assertion":[{"value":"2025-09-19","order":0,"name":"received","label":"Received","group":{"name":"publication_history","label":"Publication History"}},{"value":"2026-04-07","order":2,"name":"accepted","label":"Accepted","group":{"name":"publication_history","label":"Publication History"}},{"value":"2026-06-22","order":3,"name":"published","label":"Published","group":{"name":"publication_history","label":"Publication History"}}],"article-number":"e03010-25"}}