A liivibrio fischeri L-Asparaginase production by engineered Bacillus subtilis: a potential new biopharmaceutical
Autor(a) principal: | |
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Data de Publicação: | 2022 |
Outros Autores: | , , , , , , |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Repositório Institucional da UNESP |
Texto Completo: | http://dx.doi.org/10.1007/s00449-022-02769-x http://hdl.handle.net/11449/240676 |
Resumo: | l-Asparaginase (l-ASNase) is an enzyme applied in the treatment of lymphoid malignancies. However, an innovative l-ASNase with high yield and lower side effects than the commercially available preparations are still a market requirement. Here, a new-engineered Bacillus subtilis strain was evaluated for Aliivibrio fischeril-ASNase II production, being the bioprocess development and the enzyme characterization studied. The pBS0E plasmid replicative in Bacillus sp and containing PxylA promoter inducible by xylose and its repressive molecule sequence (XylR) was used for the genetic modification. Initially, cultivations were carried out in orbital shaker, and then the process was scaled up to stirred tank bioreactor (STB). After the bioprocess, the cells were recovered and submitted to ultrasound sonication for cells disruption and intracellular enzyme recovery. The enzymatic extract was characterized to assess its biochemical, kinetic and thermal properties using l-Asparagine and l-Glutamine as substrates. The results indicated the potential enzyme production in STB achieving l-ASNase activity up to 1.539 U mL−1. The enzymatic extract showed an optimum pH of 7.5, high l-Asparagine affinity (Km = 1.2275 mmol L−1) and low l-Glutaminase activity (0.568–0.738 U mL−1). In addition, thermal inactivation was analyzed by two different Kinect models to elucidate inactivation mechanisms, low kinetic thermal inactivation constants for 25 ºC and 37 ºC (0.128 and 0.148 h−1, respectively) indicate an elevated stability. The findings herein show that the produced recombinant l-ASNase has potential to be applied for pharmaceutical purposes. |
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A liivibrio fischeri L-Asparaginase production by engineered Bacillus subtilis: a potential new biopharmaceuticalBacillus subtilisEnzymatic characterizationEnzymatic thermal inactivationl-AsparaginaseStirred tank bioreactorl-Asparaginase (l-ASNase) is an enzyme applied in the treatment of lymphoid malignancies. However, an innovative l-ASNase with high yield and lower side effects than the commercially available preparations are still a market requirement. Here, a new-engineered Bacillus subtilis strain was evaluated for Aliivibrio fischeril-ASNase II production, being the bioprocess development and the enzyme characterization studied. The pBS0E plasmid replicative in Bacillus sp and containing PxylA promoter inducible by xylose and its repressive molecule sequence (XylR) was used for the genetic modification. Initially, cultivations were carried out in orbital shaker, and then the process was scaled up to stirred tank bioreactor (STB). After the bioprocess, the cells were recovered and submitted to ultrasound sonication for cells disruption and intracellular enzyme recovery. The enzymatic extract was characterized to assess its biochemical, kinetic and thermal properties using l-Asparagine and l-Glutamine as substrates. The results indicated the potential enzyme production in STB achieving l-ASNase activity up to 1.539 U mL−1. The enzymatic extract showed an optimum pH of 7.5, high l-Asparagine affinity (Km = 1.2275 mmol L−1) and low l-Glutaminase activity (0.568–0.738 U mL−1). In addition, thermal inactivation was analyzed by two different Kinect models to elucidate inactivation mechanisms, low kinetic thermal inactivation constants for 25 ºC and 37 ºC (0.128 and 0.148 h−1, respectively) indicate an elevated stability. The findings herein show that the produced recombinant l-ASNase has potential to be applied for pharmaceutical purposes.School of Pharmaceutical Sciences Department of Bioprocess Engineering and Biotechnology São Paulo State University (UNESP), São PauloDepartment of Chemistry CICECO-Aveiro Institute of Materials University of AveiroLSRE-LCM-Laboratory of Separation and Reaction Engineering–Laboratory of Catalysis and Materials Faculty of Engineering University of Porto, Rua Dr. Roberto FriasFaculty of Engineering ALiCE - Associate Laboratory in Chemical Engineering University of Porto, Rua Dr. Roberto FriasSchool of Pharmaceutical Sciences Department of Bioprocess Engineering and Biotechnology São Paulo State University (UNESP), São PauloUniversidade Estadual Paulista (UNESP)University of AveiroUniversity of PortoBento, Heitor B. S. [UNESP]Paiva, Gabriela B. [UNESP]Almeida, Mafalda R.Silva, Claúdia G.Carvalho, Pedro J.Tavares, Ana P. M.Pedrolli, Danielle B. [UNESP]Santos-Ebinuma, Valéria C. [UNESP]2023-03-01T20:27:54Z2023-03-01T20:27:54Z2022-01-01info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articlehttp://dx.doi.org/10.1007/s00449-022-02769-xBioprocess and Biosystems Engineering.1615-76051615-7591http://hdl.handle.net/11449/24067610.1007/s00449-022-02769-x2-s2.0-85136207908Scopusreponame:Repositório Institucional da UNESPinstname:Universidade Estadual Paulista (UNESP)instacron:UNESPengBioprocess and Biosystems Engineeringinfo:eu-repo/semantics/openAccess2023-03-01T20:27:54Zoai:repositorio.unesp.br:11449/240676Repositório InstitucionalPUBhttp://repositorio.unesp.br/oai/requestopendoar:29462023-03-01T20:27:54Repositório Institucional da UNESP - Universidade Estadual Paulista (UNESP)false |
dc.title.none.fl_str_mv |
A liivibrio fischeri L-Asparaginase production by engineered Bacillus subtilis: a potential new biopharmaceutical |
title |
A liivibrio fischeri L-Asparaginase production by engineered Bacillus subtilis: a potential new biopharmaceutical |
spellingShingle |
A liivibrio fischeri L-Asparaginase production by engineered Bacillus subtilis: a potential new biopharmaceutical Bento, Heitor B. S. [UNESP] Bacillus subtilis Enzymatic characterization Enzymatic thermal inactivation l-Asparaginase Stirred tank bioreactor |
title_short |
A liivibrio fischeri L-Asparaginase production by engineered Bacillus subtilis: a potential new biopharmaceutical |
title_full |
A liivibrio fischeri L-Asparaginase production by engineered Bacillus subtilis: a potential new biopharmaceutical |
title_fullStr |
A liivibrio fischeri L-Asparaginase production by engineered Bacillus subtilis: a potential new biopharmaceutical |
title_full_unstemmed |
A liivibrio fischeri L-Asparaginase production by engineered Bacillus subtilis: a potential new biopharmaceutical |
title_sort |
A liivibrio fischeri L-Asparaginase production by engineered Bacillus subtilis: a potential new biopharmaceutical |
author |
Bento, Heitor B. S. [UNESP] |
author_facet |
Bento, Heitor B. S. [UNESP] Paiva, Gabriela B. [UNESP] Almeida, Mafalda R. Silva, Claúdia G. Carvalho, Pedro J. Tavares, Ana P. M. Pedrolli, Danielle B. [UNESP] Santos-Ebinuma, Valéria C. [UNESP] |
author_role |
author |
author2 |
Paiva, Gabriela B. [UNESP] Almeida, Mafalda R. Silva, Claúdia G. Carvalho, Pedro J. Tavares, Ana P. M. Pedrolli, Danielle B. [UNESP] Santos-Ebinuma, Valéria C. [UNESP] |
author2_role |
author author author author author author author |
dc.contributor.none.fl_str_mv |
Universidade Estadual Paulista (UNESP) University of Aveiro University of Porto |
dc.contributor.author.fl_str_mv |
Bento, Heitor B. S. [UNESP] Paiva, Gabriela B. [UNESP] Almeida, Mafalda R. Silva, Claúdia G. Carvalho, Pedro J. Tavares, Ana P. M. Pedrolli, Danielle B. [UNESP] Santos-Ebinuma, Valéria C. [UNESP] |
dc.subject.por.fl_str_mv |
Bacillus subtilis Enzymatic characterization Enzymatic thermal inactivation l-Asparaginase Stirred tank bioreactor |
topic |
Bacillus subtilis Enzymatic characterization Enzymatic thermal inactivation l-Asparaginase Stirred tank bioreactor |
description |
l-Asparaginase (l-ASNase) is an enzyme applied in the treatment of lymphoid malignancies. However, an innovative l-ASNase with high yield and lower side effects than the commercially available preparations are still a market requirement. Here, a new-engineered Bacillus subtilis strain was evaluated for Aliivibrio fischeril-ASNase II production, being the bioprocess development and the enzyme characterization studied. The pBS0E plasmid replicative in Bacillus sp and containing PxylA promoter inducible by xylose and its repressive molecule sequence (XylR) was used for the genetic modification. Initially, cultivations were carried out in orbital shaker, and then the process was scaled up to stirred tank bioreactor (STB). After the bioprocess, the cells were recovered and submitted to ultrasound sonication for cells disruption and intracellular enzyme recovery. The enzymatic extract was characterized to assess its biochemical, kinetic and thermal properties using l-Asparagine and l-Glutamine as substrates. The results indicated the potential enzyme production in STB achieving l-ASNase activity up to 1.539 U mL−1. The enzymatic extract showed an optimum pH of 7.5, high l-Asparagine affinity (Km = 1.2275 mmol L−1) and low l-Glutaminase activity (0.568–0.738 U mL−1). In addition, thermal inactivation was analyzed by two different Kinect models to elucidate inactivation mechanisms, low kinetic thermal inactivation constants for 25 ºC and 37 ºC (0.128 and 0.148 h−1, respectively) indicate an elevated stability. The findings herein show that the produced recombinant l-ASNase has potential to be applied for pharmaceutical purposes. |
publishDate |
2022 |
dc.date.none.fl_str_mv |
2022-01-01 2023-03-01T20:27:54Z 2023-03-01T20:27:54Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/article |
format |
article |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
http://dx.doi.org/10.1007/s00449-022-02769-x Bioprocess and Biosystems Engineering. 1615-7605 1615-7591 http://hdl.handle.net/11449/240676 10.1007/s00449-022-02769-x 2-s2.0-85136207908 |
url |
http://dx.doi.org/10.1007/s00449-022-02769-x http://hdl.handle.net/11449/240676 |
identifier_str_mv |
Bioprocess and Biosystems Engineering. 1615-7605 1615-7591 10.1007/s00449-022-02769-x 2-s2.0-85136207908 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
Bioprocess and Biosystems Engineering |
dc.rights.driver.fl_str_mv |
info:eu-repo/semantics/openAccess |
eu_rights_str_mv |
openAccess |
dc.source.none.fl_str_mv |
Scopus reponame:Repositório Institucional da UNESP instname:Universidade Estadual Paulista (UNESP) instacron:UNESP |
instname_str |
Universidade Estadual Paulista (UNESP) |
instacron_str |
UNESP |
institution |
UNESP |
reponame_str |
Repositório Institucional da UNESP |
collection |
Repositório Institucional da UNESP |
repository.name.fl_str_mv |
Repositório Institucional da UNESP - Universidade Estadual Paulista (UNESP) |
repository.mail.fl_str_mv |
|
_version_ |
1797790016873693184 |