{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,18]],"date-time":"2026-03-18T10:09:21Z","timestamp":1773828561301,"version":"3.50.1"},"reference-count":32,"publisher":"American Society for Microbiology","issue":"5","license":[{"start":{"date-parts":[[2000,5,1]],"date-time":"2000-05-01T00:00:00Z","timestamp":957139200000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["Appl Environ Microbiol"],"published-print":{"date-parts":[[2000,5]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n          <jats:p>\n            A fully enzymatic process employing two sequential enzymes,\n            <jats:sc>d<\/jats:sc>\n            -hydantoinase and\n            <jats:italic>N<\/jats:italic>\n            -carbamylase, is a typical case requiring combined enzyme activity for the production of\n            <jats:sc>d<\/jats:sc>\n            -amino acids. To test the possibility of generating a bifunctional fusion enzyme, we constructed a fusion protein via end-to-end fusion of a whole gene that encodes an intact protein at the N terminus of the\n            <jats:sc>d<\/jats:sc>\n            -hydantoinase. Firstly, maltose-binding protein (MBP) gene of\n            <jats:italic>E. coli<\/jats:italic>\n            was fused with\n            <jats:sc>d<\/jats:sc>\n            -hydantoinase gene from\n            <jats:italic>Bacillus stearothermophilus<\/jats:italic>\n            SD1, and the properties of the resulting fusion protein (MBP-HYD) were compared with those of native\n            <jats:sc>d<\/jats:sc>\n            -hydantoinase. Gel filtration and kinetic analyses clearly demonstrated that the typical characteristics of\n            <jats:sc>d<\/jats:sc>\n            -hydantoinase are maintained even in a fusion state. Based on this result, we constructed an artificial fusion enzyme composed of the whole length of\n            <jats:italic>N<\/jats:italic>\n            -carbamylase (304 amino acids [aa]) from\n            <jats:italic>Agrobacterim radiobacter<\/jats:italic>\n            NRRL B11291 and\n            <jats:sc>d<\/jats:sc>\n            -hydantoinase (471 aa). The fusion enzyme (CAB-HYD) was functionally expressed with an expected molecular mass of 86 kDa and efficiently converted exogenous hydantoin derivatives to the\n            <jats:sc>d<\/jats:sc>\n            -amino acids. A related\n            <jats:sc>d<\/jats:sc>\n            -hydantoinase (HYD1) gene from\n            <jats:italic>Bacillus thermocatenulatus<\/jats:italic>\n            GH2 was also fused with the\n            <jats:italic>N<\/jats:italic>\n            -carbamylase gene at its N terminus. The resulting enzyme (CAB-HYD1) was bifunctional as expected and showed better performance than the CAB-HYD fusion enzyme. The conversion of hydantoin derivatives to corresponding amino acids by the fusion enzymes was much higher than that by the separately expressed enzymes, and comparable to that by the coexpressed enzymes. Thus, the fusion enzyme might be useful as a potential biocatalyst for the production of nonnatural amino acids.\n          <\/jats:p>","DOI":"10.1128\/aem.66.5.2133-2138.2000","type":"journal-article","created":{"date-parts":[[2002,7,27]],"date-time":"2002-07-27T10:03:59Z","timestamp":1027764239000},"page":"2133-2138","update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":24,"title":["Construction and Evaluation of a Novel Bifunctional\n            <i>N<\/i>\n            -Carbamylase\u2013\n            <scp>d<\/scp>\n            -Hydantoinase Fusion Enzyme"],"prefix":"10.1128","volume":"66","author":[{"given":"Geun-Joong","family":"Kim","sequence":"first","affiliation":[{"name":"<!--label omitted: 1-->Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 373-1, Kusung-dong, Yusung-gu, Taejon 305-701, Korea"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Dong-Eun","family":"Lee","sequence":"additional","affiliation":[{"name":"<!--label omitted: 1-->Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 373-1, Kusung-dong, Yusung-gu, Taejon 305-701, Korea"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Hak-Sung","family":"Kim","sequence":"additional","affiliation":[{"name":"<!--label omitted: 1-->Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 373-1, Kusung-dong, Yusung-gu, Taejon 305-701, Korea"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"235","reference":[{"key":"e_1_3_1_2_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.95.12.6613"},{"key":"e_1_3_1_3_2","doi-asserted-by":"publisher","DOI":"10.1021\/bi00019a015"},{"key":"e_1_3_1_4_2","doi-asserted-by":"publisher","DOI":"10.1016\/0003-9861(83)90316-8"},{"key":"e_1_3_1_5_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0167-4781(97)00097-3"},{"key":"e_1_3_1_6_2","doi-asserted-by":"publisher","DOI":"10.1007\/BF01811705"},{"key":"e_1_3_1_7_2","doi-asserted-by":"publisher","DOI":"10.1038\/nbt0898-769"},{"key":"e_1_3_1_8_2","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.271.16.9326"},{"key":"e_1_3_1_9_2","doi-asserted-by":"publisher","DOI":"10.1099\/00221287-144-4-947"},{"key":"e_1_3_1_10_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0378-1119(96)00445-3"},{"key":"e_1_3_1_11_2","first-page":"1937","article-title":"Possible prediction of adverse reactions to pyrimidine chemotherapy from urinary pyrimidine levels and a case of asymptomatic adult dihydropyrimidinuria.","volume":"2","author":"Hayashi K.","year":"1996","unstructured":"Hayashi K. 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