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Overproduction of pro-transglutaminase from Streptomyces hygroscopicus in Yarrowia lipolytica and its biochemical characterization
BACKGROUND: Transglutaminases (TGase), synthesized as a zymogen (pro-TGase) in Streptomyces sp., are important enzymes in food industry. Due to the important applications of TGase in food industry, obtaining robust and food-safe TGase-producing strains has attracted much attention during the past de...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2015
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4535380/ https://www.ncbi.nlm.nih.gov/pubmed/26272462 http://dx.doi.org/10.1186/s12896-015-0193-1 |
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author | Liu, Song Wan, Dan Wang, Miao Madzak, Catherine Du, Guocheng Chen, Jian |
author_facet | Liu, Song Wan, Dan Wang, Miao Madzak, Catherine Du, Guocheng Chen, Jian |
author_sort | Liu, Song |
collection | PubMed |
description | BACKGROUND: Transglutaminases (TGase), synthesized as a zymogen (pro-TGase) in Streptomyces sp., are important enzymes in food industry. Due to the important applications of TGase in food industry, obtaining robust and food-safe TGase-producing strains has attracted much attention during the past decade. In this study, Streptomyces hygroscopicus pro-TGase was efficiently expressed and secreted by a food-grade host, Yarrowia lipolytica, without antibiotic markers. RESULTS: The pro-TGase gene was cloned into integrative vectors pINA1296 (monocopy) and pINA1297 (multicopy), and was used to transform the Y. lipolytica Po1g or Po1h strain, respectively. Expression was driven by a recombinant hp4d promoter and secretion obtained using a XPR2 pre-sequence as a signal peptide. The highest yield of extracellular pro-TGase produced by the recombinant Po1h strain corresponded to 5.3 U/mL of TGase, a level 8.8 fold higher than that obtained using the recombinant Po1g strain. Asparagines in two potential Asn-linked glycosylation sites (Asn160 and Asn355) from pro-TGase were mutated to glutamine individually or simultaneously, yielding the deglycosylated variants N160Q, N355Q, and N160Q/N355Q. The activities of N160Q, N355Q and N160Q/N355Q constructs were respectively 5.3 U/mL, 7.8 U/mL, and 3.0 U/mL, equivalent to 100 %, 147 %, and 57 % of that from wild-type pro-TGase. The TGase yield of N355Q variant was raised to 35.3 U/mL of by using a glycerol feeding strategy in a 3 L fermenter. The optimal pH and temperature of the activated pro-TGase, and of its deglycosylated variants, were in the range of 5.0-6.0 pH and 40-45 °C, respectively. The half-life of the recombinant wild-type pro-TGase at 37 °C reached 34.0 min, and those of the variants were from 24.2 min to 11.5 min. In contrast to the wild-type pro-TGase, all of the variants had decreased specific activities, and both the K(m) and k(cat) values of the variants decreased accordingly. CONCLUSIONS: This study constitutes the first report of the heterologous expression of a pro-TGase in Y. lipolytica, and provides new possibilities for the efficient production of TGases used in food processing. |
format | Online Article Text |
id | pubmed-4535380 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2015 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-45353802015-08-14 Overproduction of pro-transglutaminase from Streptomyces hygroscopicus in Yarrowia lipolytica and its biochemical characterization Liu, Song Wan, Dan Wang, Miao Madzak, Catherine Du, Guocheng Chen, Jian BMC Biotechnol Research Article BACKGROUND: Transglutaminases (TGase), synthesized as a zymogen (pro-TGase) in Streptomyces sp., are important enzymes in food industry. Due to the important applications of TGase in food industry, obtaining robust and food-safe TGase-producing strains has attracted much attention during the past decade. In this study, Streptomyces hygroscopicus pro-TGase was efficiently expressed and secreted by a food-grade host, Yarrowia lipolytica, without antibiotic markers. RESULTS: The pro-TGase gene was cloned into integrative vectors pINA1296 (monocopy) and pINA1297 (multicopy), and was used to transform the Y. lipolytica Po1g or Po1h strain, respectively. Expression was driven by a recombinant hp4d promoter and secretion obtained using a XPR2 pre-sequence as a signal peptide. The highest yield of extracellular pro-TGase produced by the recombinant Po1h strain corresponded to 5.3 U/mL of TGase, a level 8.8 fold higher than that obtained using the recombinant Po1g strain. Asparagines in two potential Asn-linked glycosylation sites (Asn160 and Asn355) from pro-TGase were mutated to glutamine individually or simultaneously, yielding the deglycosylated variants N160Q, N355Q, and N160Q/N355Q. The activities of N160Q, N355Q and N160Q/N355Q constructs were respectively 5.3 U/mL, 7.8 U/mL, and 3.0 U/mL, equivalent to 100 %, 147 %, and 57 % of that from wild-type pro-TGase. The TGase yield of N355Q variant was raised to 35.3 U/mL of by using a glycerol feeding strategy in a 3 L fermenter. The optimal pH and temperature of the activated pro-TGase, and of its deglycosylated variants, were in the range of 5.0-6.0 pH and 40-45 °C, respectively. The half-life of the recombinant wild-type pro-TGase at 37 °C reached 34.0 min, and those of the variants were from 24.2 min to 11.5 min. In contrast to the wild-type pro-TGase, all of the variants had decreased specific activities, and both the K(m) and k(cat) values of the variants decreased accordingly. CONCLUSIONS: This study constitutes the first report of the heterologous expression of a pro-TGase in Y. lipolytica, and provides new possibilities for the efficient production of TGases used in food processing. BioMed Central 2015-08-14 /pmc/articles/PMC4535380/ /pubmed/26272462 http://dx.doi.org/10.1186/s12896-015-0193-1 Text en © Liu et al. 2015 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. |
spellingShingle | Research Article Liu, Song Wan, Dan Wang, Miao Madzak, Catherine Du, Guocheng Chen, Jian Overproduction of pro-transglutaminase from Streptomyces hygroscopicus in Yarrowia lipolytica and its biochemical characterization |
title | Overproduction of pro-transglutaminase from Streptomyces hygroscopicus in Yarrowia lipolytica and its biochemical characterization |
title_full | Overproduction of pro-transglutaminase from Streptomyces hygroscopicus in Yarrowia lipolytica and its biochemical characterization |
title_fullStr | Overproduction of pro-transglutaminase from Streptomyces hygroscopicus in Yarrowia lipolytica and its biochemical characterization |
title_full_unstemmed | Overproduction of pro-transglutaminase from Streptomyces hygroscopicus in Yarrowia lipolytica and its biochemical characterization |
title_short | Overproduction of pro-transglutaminase from Streptomyces hygroscopicus in Yarrowia lipolytica and its biochemical characterization |
title_sort | overproduction of pro-transglutaminase from streptomyces hygroscopicus in yarrowia lipolytica and its biochemical characterization |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4535380/ https://www.ncbi.nlm.nih.gov/pubmed/26272462 http://dx.doi.org/10.1186/s12896-015-0193-1 |
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