Subcellular extraction and enzyme characterisation of polyphenol oxidase and peroxidase in Cinnamon myrtle

© 2016, Franciszek Górski Institute of Plant Physiology, Polish Academy of Sciences, Kraków. Polyphenol oxidase (PPO) and peroxidase (POD) from Backhousia myrtifolia leaf and floral tissues were characterised. PPO from both tissues showed maximum activity at pH 6.0 and 10.0 at 25 °C, whereas POD act...

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Main Authors: Sarana Sommano, Wilawan Kumpoun, Nor Azma Yusuf
Format: Journal
Published: 2018
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http://cmuir.cmu.ac.th/jspui/handle/6653943832/46295
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spelling th-cmuir.6653943832-462952018-04-25T07:33:20Z Subcellular extraction and enzyme characterisation of polyphenol oxidase and peroxidase in Cinnamon myrtle Sarana Sommano Wilawan Kumpoun Nor Azma Yusuf Biochemistry, Genetics and Molecular Biology Agricultural and Biological Sciences © 2016, Franciszek Górski Institute of Plant Physiology, Polish Academy of Sciences, Kraków. Polyphenol oxidase (PPO) and peroxidase (POD) from Backhousia myrtifolia leaf and floral tissues were characterised. PPO from both tissues showed maximum activity at pH 6.0 and 10.0 at 25 °C, whereas POD activity optima were at pH 5.0 and 9.0 at 25 °C for leaf tissues. The same pH optima for POD activity were evident at 20 °C for floral tissues. With regard to substrate specificity, B. myrtifolia PPOs were of both monophenolase (tyrosine) and diphenolase (l-DOPA) types. POD activity was highest when catechol was used as a substrate for oxidisation. K m ranged from 0.6 to 1.0 mM with l-DOPA as the substrate for PPO, and from 0.1 to 0.4 mM with H 2 O 2 and constant catechol (10 mM) as substrates for POD. In both tissues types, glutathione was a non-competitive inhibitor to PPO at the lower concentrations of 0.1–1 mM, but was uncompe titive at the higher concentrations of 10.0 mM. Sodium azide at concentrations ranging from 0.005 to 0.5 mM was a competitive inhibitor to POD. Subcellular extraction methods showed that PPO and POD were localised in the membrane fraction. Cationic native PAGE performed for both enzymes was only able to detect PPO activity. Using Western blot analyses, low molecular weight PPO isozymes from leaf tissue were identified ( < 10 kDa). Five POD isozymes (20–80 kDa) were detected in both tissue types. These ‘isoform’ patterns were investigated by two-dimensional gel electrophoresis (2DGE). PPOs were mainly neutral (pI 6–7), while POD isoforms had acidic, neutral and alkaline forms. 2018-04-25T06:52:25Z 2018-04-25T06:52:25Z 2017-01-01 Journal 01375881 2-s2.0-85007481401 10.1007/s11738-016-2339-8 https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=85007481401&origin=inward http://cmuir.cmu.ac.th/jspui/handle/6653943832/46295
institution Chiang Mai University
building Chiang Mai University Library
country Thailand
collection CMU Intellectual Repository
topic Biochemistry, Genetics and Molecular Biology
Agricultural and Biological Sciences
spellingShingle Biochemistry, Genetics and Molecular Biology
Agricultural and Biological Sciences
Sarana Sommano
Wilawan Kumpoun
Nor Azma Yusuf
Subcellular extraction and enzyme characterisation of polyphenol oxidase and peroxidase in Cinnamon myrtle
description © 2016, Franciszek Górski Institute of Plant Physiology, Polish Academy of Sciences, Kraków. Polyphenol oxidase (PPO) and peroxidase (POD) from Backhousia myrtifolia leaf and floral tissues were characterised. PPO from both tissues showed maximum activity at pH 6.0 and 10.0 at 25 °C, whereas POD activity optima were at pH 5.0 and 9.0 at 25 °C for leaf tissues. The same pH optima for POD activity were evident at 20 °C for floral tissues. With regard to substrate specificity, B. myrtifolia PPOs were of both monophenolase (tyrosine) and diphenolase (l-DOPA) types. POD activity was highest when catechol was used as a substrate for oxidisation. K m ranged from 0.6 to 1.0 mM with l-DOPA as the substrate for PPO, and from 0.1 to 0.4 mM with H 2 O 2 and constant catechol (10 mM) as substrates for POD. In both tissues types, glutathione was a non-competitive inhibitor to PPO at the lower concentrations of 0.1–1 mM, but was uncompe titive at the higher concentrations of 10.0 mM. Sodium azide at concentrations ranging from 0.005 to 0.5 mM was a competitive inhibitor to POD. Subcellular extraction methods showed that PPO and POD were localised in the membrane fraction. Cationic native PAGE performed for both enzymes was only able to detect PPO activity. Using Western blot analyses, low molecular weight PPO isozymes from leaf tissue were identified ( < 10 kDa). Five POD isozymes (20–80 kDa) were detected in both tissue types. These ‘isoform’ patterns were investigated by two-dimensional gel electrophoresis (2DGE). PPOs were mainly neutral (pI 6–7), while POD isoforms had acidic, neutral and alkaline forms.
format Journal
author Sarana Sommano
Wilawan Kumpoun
Nor Azma Yusuf
author_facet Sarana Sommano
Wilawan Kumpoun
Nor Azma Yusuf
author_sort Sarana Sommano
title Subcellular extraction and enzyme characterisation of polyphenol oxidase and peroxidase in Cinnamon myrtle
title_short Subcellular extraction and enzyme characterisation of polyphenol oxidase and peroxidase in Cinnamon myrtle
title_full Subcellular extraction and enzyme characterisation of polyphenol oxidase and peroxidase in Cinnamon myrtle
title_fullStr Subcellular extraction and enzyme characterisation of polyphenol oxidase and peroxidase in Cinnamon myrtle
title_full_unstemmed Subcellular extraction and enzyme characterisation of polyphenol oxidase and peroxidase in Cinnamon myrtle
title_sort subcellular extraction and enzyme characterisation of polyphenol oxidase and peroxidase in cinnamon myrtle
publishDate 2018
url https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=85007481401&origin=inward
http://cmuir.cmu.ac.th/jspui/handle/6653943832/46295
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