Cloning, expression and characterization of the endoglucanase gene from Bacillus subtilis UMC7 isolated from the gut of the indigenous termite Macrotermes malaccensis in Escherichia coli

Background Bacillus subtilis UMC7 isolated from the gut of termite Macrotermes malaccensis has the ability to secrete a significant amount of extracellular endoglucanase, with an enzyme activity of 0.12 ± 0.01 μmol/min/mL. However, for economically viable industrial applications, the enzyme...

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Autores principales:	Wei,Kelvin Swee Chuan, Teoh,Teow Chong, Koshy,Philip, Salmah,Ismail, Zainudin,Arifin
Lenguaje:	English
Publicado:	Pontificia Universidad Católica de Valparaíso 2015
Materias:	β-1,4-Endoglucanase Carboxyl-methylcellulose Extracellular enzyme activity Metal ions effect
Acceso en línea:	http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582015000200007
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spelling	oai:scielo:S0717-345820150002000072015-05-08Cloning, expression and characterization of the endoglucanase gene from Bacillus subtilis UMC7 isolated from the gut of the indigenous termite Macrotermes malaccensis in Escherichia coliWei,Kelvin Swee ChuanTeoh,Teow ChongKoshy,PhilipSalmah,IsmailZainudin,Arifin β-1,4-Endoglucanase Carboxyl-methylcellulose Extracellular enzyme activity Metal ions effect Background Bacillus subtilis UMC7 isolated from the gut of termite Macrotermes malaccensis has the ability to secrete a significant amount of extracellular endoglucanase, with an enzyme activity of 0.12 ± 0.01 μmol/min/mL. However, for economically viable industrial applications, the enzyme needs to be expressed in a heterologous host to overcome the low enzyme production from the wild-type strain. Results The endoglucanase gene from B. subtilis UMC7 was successfully cloned and expressed. A higher enzyme activity was observed in the intracellular fraction of the recombinant clone (0.51 ± 0.02 μmol/min/mL) compared with the cell-bound fraction (0.37 ± 0.02 μmol/min/mL) and the extracellular fraction (0.33 ± 0.01 μmol/min/mL). The recombinant endoglucanase was approximately 56 kDa, with optimal enzyme activity at 60°C and pH 6.0. The activity of the enzyme was enhanced by the addition of Ca2 +. However, the enzyme was inhibited by other metal ions in the following order: Fe3 + > Ni2 + > Cu2 + > Mn2 + = Zn2 + > Mg2 + > Cd2 + > Cr2 +. The enzyme was able to hydrolyze both low- and high-viscosity carboxymethyl-cellulose (CMC), avicel, cotton linter, filter paper and avicel but not starch, xylan, chitin, pectin and p-nitrophenyl α-d-glucopyranoside. Conclusions The recombinant endoglucanase showed a threefold increase in extracellular enzyme activity compared with the wild-type strain. This result revealed the potential of endoglucanase expression in E. coli, which can be induced for the overexpression of the enzyme. The enzyme has a broad range of activity with high specificity toward cellulose.info:eu-repo/semantics/openAccessPontificia Universidad Católica de ValparaísoElectronic Journal of Biotechnology v.18 n.2 20152015-03-01text/htmlhttp://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582015000200007en10.1016/j.ejbt.2014.12.007
institution	Scielo Chile
collection	Scielo Chile
language	English
topic	β-1,4-Endoglucanase Carboxyl-methylcellulose Extracellular enzyme activity Metal ions effect
spellingShingle	β-1,4-Endoglucanase Carboxyl-methylcellulose Extracellular enzyme activity Metal ions effect Wei,Kelvin Swee Chuan Teoh,Teow Chong Koshy,Philip Salmah,Ismail Zainudin,Arifin Cloning, expression and characterization of the endoglucanase gene from Bacillus subtilis UMC7 isolated from the gut of the indigenous termite Macrotermes malaccensis in Escherichia coli
description	Background Bacillus subtilis UMC7 isolated from the gut of termite Macrotermes malaccensis has the ability to secrete a significant amount of extracellular endoglucanase, with an enzyme activity of 0.12 ± 0.01 μmol/min/mL. However, for economically viable industrial applications, the enzyme needs to be expressed in a heterologous host to overcome the low enzyme production from the wild-type strain. Results The endoglucanase gene from B. subtilis UMC7 was successfully cloned and expressed. A higher enzyme activity was observed in the intracellular fraction of the recombinant clone (0.51 ± 0.02 μmol/min/mL) compared with the cell-bound fraction (0.37 ± 0.02 μmol/min/mL) and the extracellular fraction (0.33 ± 0.01 μmol/min/mL). The recombinant endoglucanase was approximately 56 kDa, with optimal enzyme activity at 60°C and pH 6.0. The activity of the enzyme was enhanced by the addition of Ca2 +. However, the enzyme was inhibited by other metal ions in the following order: Fe3 + > Ni2 + > Cu2 + > Mn2 + = Zn2 + > Mg2 + > Cd2 + > Cr2 +. The enzyme was able to hydrolyze both low- and high-viscosity carboxymethyl-cellulose (CMC), avicel, cotton linter, filter paper and avicel but not starch, xylan, chitin, pectin and p-nitrophenyl α-d-glucopyranoside. Conclusions The recombinant endoglucanase showed a threefold increase in extracellular enzyme activity compared with the wild-type strain. This result revealed the potential of endoglucanase expression in E. coli, which can be induced for the overexpression of the enzyme. The enzyme has a broad range of activity with high specificity toward cellulose.
author	Wei,Kelvin Swee Chuan Teoh,Teow Chong Koshy,Philip Salmah,Ismail Zainudin,Arifin
author_facet	Wei,Kelvin Swee Chuan Teoh,Teow Chong Koshy,Philip Salmah,Ismail Zainudin,Arifin
author_sort	Wei,Kelvin Swee Chuan
title	Cloning, expression and characterization of the endoglucanase gene from Bacillus subtilis UMC7 isolated from the gut of the indigenous termite Macrotermes malaccensis in Escherichia coli
title_short	Cloning, expression and characterization of the endoglucanase gene from Bacillus subtilis UMC7 isolated from the gut of the indigenous termite Macrotermes malaccensis in Escherichia coli
title_full	Cloning, expression and characterization of the endoglucanase gene from Bacillus subtilis UMC7 isolated from the gut of the indigenous termite Macrotermes malaccensis in Escherichia coli
title_fullStr	Cloning, expression and characterization of the endoglucanase gene from Bacillus subtilis UMC7 isolated from the gut of the indigenous termite Macrotermes malaccensis in Escherichia coli
title_full_unstemmed	Cloning, expression and characterization of the endoglucanase gene from Bacillus subtilis UMC7 isolated from the gut of the indigenous termite Macrotermes malaccensis in Escherichia coli
title_sort	cloning, expression and characterization of the endoglucanase gene from bacillus subtilis umc7 isolated from the gut of the indigenous termite macrotermes malaccensis in escherichia coli
publisher	Pontificia Universidad Católica de Valparaíso
publishDate	2015
url	http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582015000200007
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Cloning, expression and characterization of the endoglucanase gene from Bacillus subtilis UMC7 isolated from the gut of the indigenous termite Macrotermes malaccensis in Escherichia coli

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