Construction and expression of two-copy engineered yeast of feruloyl esterase

Background Aspergillus niger has the ability to secrete feruloyl esterase. However, for economically viable industrial applications, it is necessary to increase their catalytic activities and/or protein yields to satisfy the increasing needs for feruloyl esterases. Results The gene AnFaeA that encod...

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Autores principales: Zhou,Min, Huang,Zhiqing, Zhou,Bo, Luo,Yanliu, Jia,Gang, Liu,Guangmang, Zhao,Hua, Chen,Xiaoling
Lenguaje:English
Publicado: Pontificia Universidad Católica de Valparaíso 2015
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Acceso en línea:http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582015000500002
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spelling oai:scielo:S0717-345820150005000022016-03-21Construction and expression of two-copy engineered yeast of feruloyl esteraseZhou,MinHuang,ZhiqingZhou,BoLuo,YanliuJia,GangLiu,GuangmangZhao,HuaChen,Xiaoling Aspergillus niger Feruloyl esterase Pichia pastoris Background Aspergillus niger has the ability to secrete feruloyl esterase. However, for economically viable industrial applications, it is necessary to increase their catalytic activities and/or protein yields to satisfy the increasing needs for feruloyl esterases. Results The gene AnFaeA that encodes a type A feruloyl esterase was successfully expressed in Pichia pastoris by a two-copy engineered yeast. After a screen in shaker flask, a one-copy strain GSKFA3 having the highest feruloyl esterase activity of 2.4 U/mL was obtained. Then, the pPICZaA-AnFaeA plasmid was transformed into GSKFA3 and the transformants were grown on YPDS plates with antibiotic Zeocin. After cultivation, a two-copy strain GSKZaFA20 with the highest feruloyl esterase activity of 15.49 U/mL was obtained. The expressed protein (recombinant AnFaeA) may be a glycoprotein with an apparent molecular weight of 40 kDa. It displayed the maximum activity at pH 6.0 and 50°C, and was stable at a pH range of 4.0-6.5 and at below 45°C. Its activity was not significantly affected by K+, Ca2 +, Mg2 +, Cu2 +, Zn2 +, Mn2 +, Na+ and EDTA, but activated by Fe2 +. The Km and Vmax toward 4-nitrophenyl ferulate were 5.5 mM and 69.0 U/mg, respectively. Conclusions The two-copy strain GSKZaFA20 showed a 4.4-fold increase in extracellular enzyme activity compared with the one-copy strain GSKFA3. Construction of two-copy strain improved secretion of recombinant AnFaeA in P. pastoris.info:eu-repo/semantics/openAccessPontificia Universidad Católica de ValparaísoElectronic Journal of Biotechnology v.18 n.5 20152015-09-01text/htmlhttp://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582015000500002en10.1016/j.ejbt.2015.06.004
institution Scielo Chile
collection Scielo Chile
language English
topic Aspergillus niger
Feruloyl esterase
Pichia pastoris
spellingShingle Aspergillus niger
Feruloyl esterase
Pichia pastoris
Zhou,Min
Huang,Zhiqing
Zhou,Bo
Luo,Yanliu
Jia,Gang
Liu,Guangmang
Zhao,Hua
Chen,Xiaoling
Construction and expression of two-copy engineered yeast of feruloyl esterase
description Background Aspergillus niger has the ability to secrete feruloyl esterase. However, for economically viable industrial applications, it is necessary to increase their catalytic activities and/or protein yields to satisfy the increasing needs for feruloyl esterases. Results The gene AnFaeA that encodes a type A feruloyl esterase was successfully expressed in Pichia pastoris by a two-copy engineered yeast. After a screen in shaker flask, a one-copy strain GSKFA3 having the highest feruloyl esterase activity of 2.4 U/mL was obtained. Then, the pPICZaA-AnFaeA plasmid was transformed into GSKFA3 and the transformants were grown on YPDS plates with antibiotic Zeocin. After cultivation, a two-copy strain GSKZaFA20 with the highest feruloyl esterase activity of 15.49 U/mL was obtained. The expressed protein (recombinant AnFaeA) may be a glycoprotein with an apparent molecular weight of 40 kDa. It displayed the maximum activity at pH 6.0 and 50°C, and was stable at a pH range of 4.0-6.5 and at below 45°C. Its activity was not significantly affected by K+, Ca2 +, Mg2 +, Cu2 +, Zn2 +, Mn2 +, Na+ and EDTA, but activated by Fe2 +. The Km and Vmax toward 4-nitrophenyl ferulate were 5.5 mM and 69.0 U/mg, respectively. Conclusions The two-copy strain GSKZaFA20 showed a 4.4-fold increase in extracellular enzyme activity compared with the one-copy strain GSKFA3. Construction of two-copy strain improved secretion of recombinant AnFaeA in P. pastoris.
author Zhou,Min
Huang,Zhiqing
Zhou,Bo
Luo,Yanliu
Jia,Gang
Liu,Guangmang
Zhao,Hua
Chen,Xiaoling
author_facet Zhou,Min
Huang,Zhiqing
Zhou,Bo
Luo,Yanliu
Jia,Gang
Liu,Guangmang
Zhao,Hua
Chen,Xiaoling
author_sort Zhou,Min
title Construction and expression of two-copy engineered yeast of feruloyl esterase
title_short Construction and expression of two-copy engineered yeast of feruloyl esterase
title_full Construction and expression of two-copy engineered yeast of feruloyl esterase
title_fullStr Construction and expression of two-copy engineered yeast of feruloyl esterase
title_full_unstemmed Construction and expression of two-copy engineered yeast of feruloyl esterase
title_sort construction and expression of two-copy engineered yeast of feruloyl esterase
publisher Pontificia Universidad Católica de Valparaíso
publishDate 2015
url http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582015000500002
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AT huangzhiqing constructionandexpressionoftwocopyengineeredyeastofferuloylesterase
AT zhoubo constructionandexpressionoftwocopyengineeredyeastofferuloylesterase
AT luoyanliu constructionandexpressionoftwocopyengineeredyeastofferuloylesterase
AT jiagang constructionandexpressionoftwocopyengineeredyeastofferuloylesterase
AT liuguangmang constructionandexpressionoftwocopyengineeredyeastofferuloylesterase
AT zhaohua constructionandexpressionoftwocopyengineeredyeastofferuloylesterase
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