Partial purification and characterisation of a wheat leaf N-acetyl-℗-D-hexosaminidase and its role in defensive lignin deposition
Partial purification and characterisation of a wheat leaf N-acetyl-℗-D-hexosaminidase and its role in defensive lignin deposition
A wheat leaf N-acetyl-β-D-hexosaminidase (N-HEX) (EC. 3. 2. 1. 52), possessing considerable chitobiase activity, has been partially purified and characterised. This enzyme, termed N-HEX B, readily hydrolyses lignification-eliciting chitin oligosaccharides to their inactive forms. The evidence presented in this thesis supports the view that this enzyme is distinct from a previously purified wheat leaf enzyme, N-HEX A, which was found to possess virtually no activity to these substrates.
Some basic properties of N-HEX B have been established. Maximum yields of N-HEX B were extracted in McIlvaine's buffer at pH 7.0, although the highest specific activity was extracted at pH 4.0. The enzyme exhibited optimum activity at pH 4.0 with 50% of maximal activity between pH 3.0 and 5.8. The enzyme activity of crude extracts showed greatest stability when stored at pH 5.0 and 4^oC. N-HEX B was present in wheat seeds and mature leaves, with maximum specific activity observed in 15 day old leaves. Similar levels of N-HEX B activity were extracted from 5 different cultivars of wheat. The enzyme was located predominantly in the cytosol. Initial purification steps resulted in relatively stable preparations of N-HEX B. A 2497-fold purification of the enzyme was achieved following ammonium sulphate cutting, ultrafiltration and affinity chromatography on Concanavalin A-Sepharose, giving rise to a maximum specific activity of 1023 pkats mg^-1. The native molecular weights of both enzymes, estimated by gel filtration, were between 145 and 190 kDa. Separation of the N-HEX-A and B activities was achieved by anion exchange chromatography and preparatory isoelectric focusing (IEF) (approximate pI's of 4.74 and 5.25 respectively). Gel filtration, anion exchange chromatography and preparatory IEF gave rise to initial increases in specific activity but resulted in rapid inactivation of the enzyme. Addition of Triton X-100 partially stabilised enzyme activity following IEF. The evidence from anion exchange and IEF is consistent with the N-HEX A and B activities residing on separate molecules. However, both enzymes cross reacted with polyclonal antibody raised to N-HEX A, suggesting that the two activities are serologically related.
University of Southampton
1994
Jordan, Nicholas David
(1994)
Partial purification and characterisation of a wheat leaf N-acetyl-℗-D-hexosaminidase and its role in defensive lignin deposition.
University of Southampton, Doctoral Thesis.
Record type:
Thesis
(Doctoral)
Abstract
A wheat leaf N-acetyl-β-D-hexosaminidase (N-HEX) (EC. 3. 2. 1. 52), possessing considerable chitobiase activity, has been partially purified and characterised. This enzyme, termed N-HEX B, readily hydrolyses lignification-eliciting chitin oligosaccharides to their inactive forms. The evidence presented in this thesis supports the view that this enzyme is distinct from a previously purified wheat leaf enzyme, N-HEX A, which was found to possess virtually no activity to these substrates.
Some basic properties of N-HEX B have been established. Maximum yields of N-HEX B were extracted in McIlvaine's buffer at pH 7.0, although the highest specific activity was extracted at pH 4.0. The enzyme exhibited optimum activity at pH 4.0 with 50% of maximal activity between pH 3.0 and 5.8. The enzyme activity of crude extracts showed greatest stability when stored at pH 5.0 and 4^oC. N-HEX B was present in wheat seeds and mature leaves, with maximum specific activity observed in 15 day old leaves. Similar levels of N-HEX B activity were extracted from 5 different cultivars of wheat. The enzyme was located predominantly in the cytosol. Initial purification steps resulted in relatively stable preparations of N-HEX B. A 2497-fold purification of the enzyme was achieved following ammonium sulphate cutting, ultrafiltration and affinity chromatography on Concanavalin A-Sepharose, giving rise to a maximum specific activity of 1023 pkats mg^-1. The native molecular weights of both enzymes, estimated by gel filtration, were between 145 and 190 kDa. Separation of the N-HEX-A and B activities was achieved by anion exchange chromatography and preparatory isoelectric focusing (IEF) (approximate pI's of 4.74 and 5.25 respectively). Gel filtration, anion exchange chromatography and preparatory IEF gave rise to initial increases in specific activity but resulted in rapid inactivation of the enzyme. Addition of Triton X-100 partially stabilised enzyme activity following IEF. The evidence from anion exchange and IEF is consistent with the N-HEX A and B activities residing on separate molecules. However, both enzymes cross reacted with polyclonal antibody raised to N-HEX A, suggesting that the two activities are serologically related.
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Published date: 1994
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Local EPrints ID: 458355
URI: http://eprints.soton.ac.uk/id/eprint/458355
PURE UUID: ffb2d268-f7e6-4615-8a34-b25a06a21c07
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Date deposited: 04 Jul 2022 16:47
Last modified: 04 Jul 2022 16:47
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Author:
Nicholas David Jordan
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