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Recombinant Expression in Pichia pastoris and Characterization of GH36 α-Galactosidases

Dadson, Baaba LU (2024) KEMR30 20241
Department of Chemistry
Abstract
α-Galactosidases from Aspergillus nidulans (AglC) and Penicillium subrubescens (PsGal36-2527) are exo-acting enzymes that can be found in the Glycoside Hydrolase (GH) 36 phylogenetic cluster with eukaryotic α-galactosidases. In utilizing enzymes for industrial applications, finding enzymes with a broad range of substrate specificities and high catalytic efficiencies is quite challenging. It has become important to discover and produce novel enzymes such as the ones from GH36 family. This study seeks to recombinantly produce and characterize fungal GH36 α-galactosidase in terms of its stability and catalytic potential on different substrates. The expression of the enzyme was induced by growing Pichia pastoris transformants expressing AglC... (More)
α-Galactosidases from Aspergillus nidulans (AglC) and Penicillium subrubescens (PsGal36-2527) are exo-acting enzymes that can be found in the Glycoside Hydrolase (GH) 36 phylogenetic cluster with eukaryotic α-galactosidases. In utilizing enzymes for industrial applications, finding enzymes with a broad range of substrate specificities and high catalytic efficiencies is quite challenging. It has become important to discover and produce novel enzymes such as the ones from GH36 family. This study seeks to recombinantly produce and characterize fungal GH36 α-galactosidase in terms of its stability and catalytic potential on different substrates. The expression of the enzyme was induced by growing Pichia pastoris transformants expressing AglC and PsGal36-2527 in Buffered Methanol-Complex Medium (BMMY). His-tag Affinity Chromatography was used to purify the expressed AglC followed by a purity analysis on SDS-PAGE and MALDI-TOF. Enzyme activity assays were done to obtain specific activity and other kinetic parameters using the artificial substrate para-Nitrophenyl-α-D-Galactopyranoside (pNPαGal). Enzyme activity assay and SDS-PAGE revealed success in the production of AglC with high purity. AglC showed optimum activity at pH 5.5 and temperature 40°C but had relatively low affinity to pNPαGal compared to previous studies. However, no enzyme activity was observed for PsGal36-2527 nor was it detected on SDS-PAGE. The expressed AglC was secreted with very little contamination. However, its relatively lower affinity to pNPαGal could be due to loss of enzyme activity over a long period of storage. Null activity of PsGal36-2527 in the supernatant and cell lysate suggests that the PsGal36-2527 was misfolded or degraded. (Less)
Popular Abstract
Achieving Sustainability with Plant-Degrading Enzymes
According to the World Economic Forum, plants contribute to a significant amount of the total biomass generated on earth. Plant biomass may include wood, straw and energy plants such as sugarcane. They can be used to generate renewable materials and energy such as transport fuel, biochemicals and heat to generate power.
It is important to understand the structure of plants to extract useful parts of the plant. This can be achieved with the aid of carbohydrate-degrading enzymes such as glycoside hydrolases. Enzymes are biomolecules that speed up chemical reactions. Glycoside hydrolases catalyze the breakdown of carbohydrate structures into simpler sugar structures and units. Some... (More)
Achieving Sustainability with Plant-Degrading Enzymes
According to the World Economic Forum, plants contribute to a significant amount of the total biomass generated on earth. Plant biomass may include wood, straw and energy plants such as sugarcane. They can be used to generate renewable materials and energy such as transport fuel, biochemicals and heat to generate power.
It is important to understand the structure of plants to extract useful parts of the plant. This can be achieved with the aid of carbohydrate-degrading enzymes such as glycoside hydrolases. Enzymes are biomolecules that speed up chemical reactions. Glycoside hydrolases catalyze the breakdown of carbohydrate structures into simpler sugar structures and units. Some industrial applications of such enzymes include the production of food stabilizers, prebiotics and detergents.
In this study, we are interested in producing a type of glycoside hydrolase known as α-galactosidases. This enzyme catalyses the release of terminally linked galactose units from carbohydrate structures. We are specifically interested in α-galactosidases from fungal sources. Using genetic engineering techniques, the enzyme was expressed in a different fungal host. The activity of the expressed enzyme was determined using a synthetic substrate which includes the structure of galactose linked to another compound that allows the quantification of the substrate. This activity assay was used to determine the stability and kinetic characteristics of the enzyme. The results showed a success in the production of the enzyme with high purity. It also catalysed the breakdown of the substrate and showed good stability at temperatures below 60°C. However, its kinetic parameters showed a relatively lower affinity to the substrate compared to other recombinantly expressed α-galactosidases in previous studies. (Less)
Please use this url to cite or link to this publication:
author
Dadson, Baaba LU
supervisor
organization
course
KEMR30 20241
year
type
H2 - Master's Degree (Two Years)
subject
keywords
Biochemistry, α-Galactosidase, Galactomannan, Glycoside Hydrolase, Recombinant expression, Pichia pastoris
language
English
id
9163676
date added to LUP
2024-06-18 11:27:59
date last changed
2024-06-18 11:27:59
@misc{9163676,
  abstract     = {{α-Galactosidases from Aspergillus nidulans (AglC) and Penicillium subrubescens (PsGal36-2527) are exo-acting enzymes that can be found in the Glycoside Hydrolase (GH) 36 phylogenetic cluster with eukaryotic α-galactosidases. In utilizing enzymes for industrial applications, finding enzymes with a broad range of substrate specificities and high catalytic efficiencies is quite challenging. It has become important to discover and produce novel enzymes such as the ones from GH36 family. This study seeks to recombinantly produce and characterize fungal GH36 α-galactosidase in terms of its stability and catalytic potential on different substrates. The expression of the enzyme was induced by growing Pichia pastoris transformants expressing AglC and PsGal36-2527 in Buffered Methanol-Complex Medium (BMMY). His-tag Affinity Chromatography was used to purify the expressed AglC followed by a purity analysis on SDS-PAGE and MALDI-TOF. Enzyme activity assays were done to obtain specific activity and other kinetic parameters using the artificial substrate para-Nitrophenyl-α-D-Galactopyranoside (pNPαGal). Enzyme activity assay and SDS-PAGE revealed success in the production of AglC with high purity. AglC showed optimum activity at pH 5.5 and temperature 40°C but had relatively low affinity to pNPαGal compared to previous studies. However, no enzyme activity was observed for PsGal36-2527 nor was it detected on SDS-PAGE. The expressed AglC was secreted with very little contamination. However, its relatively lower affinity to pNPαGal could be due to loss of enzyme activity over a long period of storage. Null activity of PsGal36-2527 in the supernatant and cell lysate suggests that the PsGal36-2527 was misfolded or degraded.}},
  author       = {{Dadson, Baaba}},
  language     = {{eng}},
  note         = {{Student Paper}},
  title        = {{Recombinant Expression in Pichia pastoris and Characterization of GH36 α-Galactosidases}},
  year         = {{2024}},
}