{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,3]],"date-time":"2026-06-03T07:29:28Z","timestamp":1780471768522,"version":"3.54.1"},"reference-count":22,"publisher":"Springer Science and Business Media LLC","issue":"1","license":[{"start":{"date-parts":[[2024,2,2]],"date-time":"2024-02-02T00:00:00Z","timestamp":1706832000000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"},{"start":{"date-parts":[[2024,2,2]],"date-time":"2024-02-02T00:00:00Z","timestamp":1706832000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"}],"funder":[{"name":"National Key Research and Development Program of China","award":["2021YFF0600703"],"award-info":[{"award-number":["2021YFF0600703"]}]}],"content-domain":{"domain":["link.springer.com"],"crossmark-restriction":false},"short-container-title":["Bioresour. Bioprocess."],"abstract":"<jats:title>Abstract<\/jats:title><jats:p>Chitooligosaccharides (COS) find numerous applications due to their exceptional properties. Enzymatic hydrolysis of chitosan by chitosanase is considered an advantageous route for COS production. Heterologous expression of chitosanase holds significant promise, yet studies using commonly employed <jats:italic>Escherichia coli<\/jats:italic> and <jats:italic>Pichia pastoris<\/jats:italic> strains encounter challenges in subsequent handling and industrial scalability. In this investigation, we opted for using the safe yeast strain Saccharomyces cerevisiae (GRAS), obviating the need for methanol induction, resulting in successful expression. Ultimately, utilizing the GTR-CRISPR editing system, shake flask enzyme activity reached 2 U\/ml. The optimal chitosanase activity was achieved at 55\u2103 and pH 5, with favorable stability between 30 and 50\u00a0\u00b0C. Following a 2-h catalytic reaction, the product primarily consisted of chitobiose to chitotetraose, predominantly at the chitotriose position, with a slight increase in chitobiose content observed during the later stages of enzymatic hydrolysis. The results affirm the feasibility of heterologous chitosanase expression through Saccharomyces cerevisiae, underscoring its significant industrial potential.<\/jats:p>\n                <jats:p><jats:bold>Graphical Abstract<\/jats:bold><\/jats:p>","DOI":"10.1186\/s40643-023-00718-4","type":"journal-article","created":{"date-parts":[[2024,2,2]],"date-time":"2024-02-02T10:02:38Z","timestamp":1706868158000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":6,"title":["Expression of chitosanase from Aspergillus fumigatus chitosanase in Saccharomyces cerevisiae by CRISPR-Cas9 tools"],"prefix":"10.1186","volume":"11","author":[{"given":"Qingshuai","family":"Zhang","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hui","family":"Cao","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2024,2,2]]},"reference":[{"key":"718_CR1","doi-asserted-by":"publisher","first-page":"2074","DOI":"10.1271\/bbb.80175","volume":"72","author":"A Ando","year":"2008","unstructured":"Ando A, Saito A, Arai S, Usuda S, Nagata Y (2008) Molecular characterization of a novel family-46 Chitosanase from Pseudomonas sp. A-01. Biosci Biotechnol Biochem 72:2074\u20132081","journal-title":"Biosci Biotechnol Biochem"},{"issue":"3","key":"718_CR2","doi-asserted-by":"publisher","first-page":"197","DOI":"10.1042\/BA20000063","volume":"32","author":"CY Cheng","year":"2000","unstructured":"Cheng CY, Li YK (2000) An Aspergillus chitosanase with potential for large-scale preparation of chitosan oligosaccharides. Biotechnol Appl Biochem 32(3):197","journal-title":"Biotechnol Appl Biochem"},{"key":"718_CR3","doi-asserted-by":"publisher","first-page":"3137","DOI":"10.1074\/jbc.M512506200","volume":"281","author":"CY Cheng","year":"2006","unstructured":"Cheng CY, Chang CH, Wu YJ, Li YK (2006) Exploration of glycosyl hydrolase family 75, a chitosanase from Aspergillus fumigatus. J Biol Chem 281:3137\u20133144","journal-title":"J Biol Chem"},{"key":"718_CR4","doi-asserted-by":"publisher","first-page":"4522","DOI":"10.1128\/AEM.70.8.4522-4531.2004","volume":"70","author":"YJ Choi","year":"2004","unstructured":"Choi YJ, Kim EJ, Piao Z, Yun YC, Shin YC (2004) Purification and Characterization of Chitosanase from Bacillus sp. Strain KCTC 0377BP and Its Application for the Production of Chitosan Oligosaccharides. Appl Environ Microbiol 70:4522\u20134531","journal-title":"Appl Environ Microbiol"},{"key":"718_CR5","doi-asserted-by":"publisher","first-page":"696","DOI":"10.1016\/j.foodchem.2019.02.056","volume":"286","author":"N Guo","year":"2019","unstructured":"Guo N, Sun J, Wang W, Gao L, Liu J, Liu Z, Xue C, Mao X (2019) Cloning, expression and characterization of a novel chitosanase from Streptomyces albolongus ATCC 27414. Food Chem 286:696\u2013702","journal-title":"Food Chem"},{"issue":"1","key":"718_CR6","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1186\/1475-2859-9-5","volume":"9","author":"MG Johnsen","year":"2010","unstructured":"Johnsen MG, Hansen OC, Stougaard P (2010) Isolation, characterization and heterologous expression of a novel chitosanase from Janthinobacterium sp. strain 4239. Microbial Cell Factories 9(1):1\u20139","journal-title":"Microbial Cell Factories"},{"key":"718_CR7","doi-asserted-by":"publisher","first-page":"125","DOI":"10.1016\/j.pep.2005.06.016","volume":"45","author":"WJ Jung","year":"2006","unstructured":"Jung WJ, Kuk JH, Kim KY, Jung KC, Park RD (2006) Purification and characterization of exo-beta-D-glucosaminidase from Aspergillus fumigatus S-26. Protein Expr Purif 45:125\u2013131","journal-title":"Protein Expr Purif"},{"key":"718_CR8","doi-asserted-by":"publisher","first-page":"37","DOI":"10.1016\/j.carres.2012.01.025","volume":"352","author":"LX Kang","year":"2012","unstructured":"Kang LX, Chen XM, Fu L, Ma LX (2012) Recombinant expression of chitosanase from Bacillus subtilis HD145 in Pichia pastoris. Carbohyd Res 352:37\u201343","journal-title":"Carbohyd Res"},{"key":"718_CR9","doi-asserted-by":"publisher","first-page":"1100","DOI":"10.1248\/bpb.26.1100","volume":"26","author":"HW Lee","year":"2003","unstructured":"Lee HW, Park YS, Choi JW, Yi SY, Shin WS (2003) Antidiabetic effects of chitosan oligosaccharides in neonatal streptozotocin-induced noninsulin-dependent diabetes mellitus in rats. Biol Pharm Bull 26:1100\u20131103","journal-title":"Biol Pharm Bull"},{"key":"718_CR10","doi-asserted-by":"publisher","first-page":"617","DOI":"10.1016\/0922-338X(94)90142-2","volume":"77","author":"S Okajima","year":"1994","unstructured":"Okajima S, Ando A, Shinoyama H, Fujii T (1994) Purification and characterization of an extracellular chitosanase produced by Amycolatopsis sp. CsO-2. J Ferment Bioeng 77:617\u2013620","journal-title":"J Ferment Bioeng"},{"key":"718_CR11","doi-asserted-by":"publisher","first-page":"6566","DOI":"10.3390\/md13116566","volume":"13","author":"V Pascal","year":"2015","unstructured":"Pascal V, Marie-\u00e8ve L-H, Ryszard B (2015) Chitosanases from family 46 of glycoside hydrolases: from proteins to phenotypes. Mar Drugs 13:6566\u20136587","journal-title":"Mar Drugs"},{"key":"718_CR12","doi-asserted-by":"publisher","first-page":"57","DOI":"10.1016\/j.carbpol.2005.10.025","volume":"64","author":"J Rhoades","year":"2006","unstructured":"Rhoades J, Gibson G, Formentin K, Beer M, Rastall R (2006) Inhibition of the adhesion of enteropathogenic Escherichia coli strains to HT-29 cells in culture by chito-oligosaccharides. Carbohyd Polym 64:57\u201359","journal-title":"Carbohyd Polym"},{"key":"718_CR13","doi-asserted-by":"publisher","unstructured":"San-Lang, Wang Pe-Yi, Yeh (2008) Purification and characterization of a chitosanase from a nattokinase producing strain Bacillus subtilis TKU007 Process Biochemistry 43(2):132-138 https:\/\/doi.org\/10.1016\/j.procbio.2007.11.002.","DOI":"10.1016\/j.procbio.2007.11.002"},{"key":"718_CR14","doi-asserted-by":"publisher","first-page":"1864","DOI":"10.1016\/j.fct.2009.04.044","volume":"47","author":"KT Shen","year":"2009","unstructured":"Shen KT, Chen MH, Chan HY, Jeng JH, Wang YJ (2009) Inhibitory effects of chitooligosaccharides on tumor growth and metastasis. Food Chem Toxicol 47:1864\u20131871","journal-title":"Food Chem Toxicol"},{"key":"718_CR15","first-page":"231","volume":"57","author":"M Shimosaka","year":"1993","unstructured":"Shimosaka M, Nogawa M, Ohno Y, Okazaki M (1993) Chitosanase from the Plant pathogenic fungus, Fusarium solani f. sp. Phaseoli\u2014purification and some properties. J Agric Chem Soc Jpn 57:231\u2013235","journal-title":"J Agric Chem Soc Jpn"},{"key":"718_CR16","doi-asserted-by":"publisher","first-page":"1316","DOI":"10.1016\/j.carres.2008.03.030","volume":"343","author":"SL Wang","year":"2008","unstructured":"Wang SL, Peng JH, Liang TW, Liu KC (2008) Purification and characterization of a chitosanase from Serratia marcescens TKU011\u2014ScienceDirect. Carbohyd Res 343:1316\u20131323","journal-title":"Carbohyd Res"},{"key":"718_CR17","doi-asserted-by":"publisher","first-page":"297","DOI":"10.1023\/B:JMSM.0000015491.91918.64","volume":"15","author":"Y You","year":"2004","unstructured":"You Y, Park WH, Ko BM, Min BM (2004) Effects of PVA sponge containing chitooligosaccharide in the early stage of wound healing. J Mater Sci Mater Med 15:297\u2013301","journal-title":"J Mater Sci Mater Med"},{"issue":"21","key":"718_CR18","doi-asserted-by":"publisher","first-page":"3915","DOI":"10.3390\/molecules24213915","volume":"24","author":"Yang Yue","year":"2019","unstructured":"Yue Yang, Zhou Zheng, Yifei Xiao, Jiaojiao Zhang, Shangyong Huiqing (2019) Cloning and characterization of a cold-adapted chitosanase from marine bacterium Bacillus sp. BY01. Molecules 24(21):3915","journal-title":"Molecules"},{"issue":"1","key":"718_CR19","doi-asserted-by":"publisher","first-page":"1053","DOI":"10.1038\/s41467-019-09005-3","volume":"10","author":"Z Yueping","year":"2019","unstructured":"Yueping Z, Juan W, Zibai Y, Shuobo S, Jens N (2019) A gRNA-tRNA array for CRISPR-Cas9 based rapid multiplexed genome editing in Saccharomyces cerevisiae. Nat Commun 10(1):1053","journal-title":"Nat Commun"},{"key":"718_CR20","doi-asserted-by":"publisher","first-page":"71","DOI":"10.1007\/BF01578757","volume":"8","author":"BL Zamost","year":"1991","unstructured":"Zamost BL, Nielsen HK, Starnes RL (1991) Thermostable enzymes for industrial applications. J Ind Microbiol 8:71\u201381","journal-title":"J Ind Microbiol"},{"key":"718_CR21","doi-asserted-by":"publisher","first-page":"1221","DOI":"10.1007\/s10529-007-9373-y","volume":"29","author":"J Zhang","year":"2007","unstructured":"Zhang J, Sun Y (2007) Molecular cloning, expression and characterization of a chitosanase from Microbacterium sp. Biotech Lett 29:1221\u20131225","journal-title":"Biotech Lett"},{"key":"718_CR22","doi-asserted-by":"publisher","DOI":"10.1007\/s10529-007-9373-y","author":"L Zhiqiang","year":"2008","unstructured":"Zhiqiang L, Xiaoyu L, Zhenming C, Lin W, Jing LX (2008) Cloning, characterization and expression of the extracellular lipase gene from Aureobasidium pullulans HN2\u20133 isolated from sea saltern. Antonie Van Leeuwenhoek. https:\/\/doi.org\/10.1007\/s10529-007-9373-y","journal-title":"Antonie Van Leeuwenhoek"}],"container-title":["Bioresources and Bioprocessing"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/link.springer.com\/content\/pdf\/10.1186\/s40643-023-00718-4.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/link.springer.com\/article\/10.1186\/s40643-023-00718-4\/fulltext.html","content-type":"text\/html","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/link.springer.com\/content\/pdf\/10.1186\/s40643-023-00718-4.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2024,2,5]],"date-time":"2024-02-05T14:11:41Z","timestamp":1707142301000},"score":1,"resource":{"primary":{"URL":"https:\/\/bioresourcesbioprocessing.springeropen.com\/articles\/10.1186\/s40643-023-00718-4"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2024,2,2]]},"references-count":22,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2024,12]]}},"alternative-id":["718"],"URL":"https:\/\/doi.org\/10.1186\/s40643-023-00718-4","relation":{},"ISSN":["2197-4365"],"issn-type":[{"value":"2197-4365","type":"electronic"}],"subject":[],"published":{"date-parts":[[2024,2,2]]},"assertion":[{"value":"27 September 2023","order":1,"name":"received","label":"Received","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"18 December 2023","order":2,"name":"accepted","label":"Accepted","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"2 February 2024","order":3,"name":"first_online","label":"First Online","group":{"name":"ArticleHistory","label":"Article History"}},{"order":1,"name":"Ethics","group":{"name":"EthicsHeading","label":"Declarations"}},{"value":"Not applicable.","order":2,"name":"Ethics","group":{"name":"EthicsHeading","label":"Ethics approval and consent to participate"}},{"value":"Not applicable.","order":3,"name":"Ethics","group":{"name":"EthicsHeading","label":"Consent for publication"}},{"value":"The authors declare that they have no competing interests.","order":4,"name":"Ethics","group":{"name":"EthicsHeading","label":"Competing interests"}}],"article-number":"20"}}