title: Comparative characterisation of esterases and lipases for biosensors

authors: Lind, Penelope A.

abstract: Twenty four carboxylesterases and lipases from various microbial sources were compared with commercial pig liver esterase in order to determine which would be most appropriate for use in a biosensor application and in the elucidation of the relationship between protein hydration and gas phase activity. This involved comparison of appropriate kinetic parameters against the esters ethyl acetate, methyl butyrate and ethyl butyrate and their equivalent p-nitrophenylated synthetic model substrates.

Comparison of functional pH ranges of the various esterases revealed Lipase B from Candida rugosa to be amongst the least inhibited when pH fell to between 4 and 5. C. rugosa Lipase B also exhibited the highest specific activities towards p-nitrophenyl acetate and butyrate, hydrolysed all three of the above esters and did not show protease activity against azocasein. For these reasons C. rugosa Lipase B was judged to be the best suited to the ester biosensor application, and so was partially purified and further characterised.

Investigation of the effects of acyl chain length of the ester substrates on C. rugosa Lipase B activity revealed two peaks of preferential activity across the range C₂ − C₁₈. The first of these peaks, at C₄ indicative of true carboxylesterase activity, was more marked than that of pig liver esterase. The specificity of Lipase B (Yₘₐₓ/Kₘ) for ethyl acetate was 58 and 23 times lower than that for methyl butyrate and ethyl butyrate, respectively (compared to 4.7 and 2.5 times lower, respectively for pig liver esterase). The second, larger peak in the substrate size preference profile, for a C₁₂ acyl chain length, indicated a predominance of lipase activity.
Concurrent analysis of thermal- and pH-stability showed C. rugosa Lipase B to be stable below 40°C and between pH 5.5 and 7.0, with optimal stability at pH 5.5. In these respects, it is superior to pig liver esterase, which is highly susceptible to denaturation by storage above 30°C or below pH 5.0.

The influence of enzyme hydration on the hydrolysis of ethyl butyrate by C. rugosa Lipase B and pig liver esterase was investigated through the vapor-solid mode approach where each enzyme was studied as a hydrated solid phase in equilibrium with a substrate vapor phase. The effect of enzyme hydration was evaluated through measurements made in controlled atmospheres of known relative humidity. Although it was previously concluded that enzyme activity required a minimal water content of 0.2 gH₂O.g⁻¹  protein (0.2 h), we have shown that activity is possible at much lower (<0.03 h) hydration. C. rugosa Lipase B and pig liver esterase were hydrolytically active when exposed to mixtures of ethyl butyrate vapor over a range of relative humidities from 7 to 90%, equivalent to C. rugosa Lipase B and pig liver esterase water contents of between 0.054-0.47 h and 0.029-0.60 h, respectively. C.rugosa Lipase B hydrolytic activity was highly dependent on the degree of protein hydration and exhibited a linear relationship with respect to protein water content. For pig liver esterase activity, the effect of increasing hydration on activity was less marked where the increase in PLE activity began to stabilize at protein water contents over 0.17 h. Although no activity was seen at 0 h, since the reaction catalyzed (the hydrolysis of ethyl butyrate) requires water as a second substrate, this work does not discount the possibility of activity at lower hydration.

text: Comparative characterisation of esterases and lipases for biosensors Twenty four carboxylesterases and lipases from various microbial sources were compared with commercial pig liver esterase in order to determine which would be most appropriate for use in a biosensor application and in the elucidation of the relationship between protein hydration and gas phase activity. This involved comparison of appropriate kinetic parameters against the esters ethyl acetate, methyl butyrate and ethyl butyrate and their equivalent p-nitrophenylated synthetic model substrates.

Comparison of functional pH ranges of the various esterases revealed Lipase B from Candida rugosa to be amongst the least inhibited when pH fell to between 4 and 5. C. rugosa Lipase B also exhibited the highest specific activities towards p-nitrophenyl acetate and butyrate, hydrolysed all three of the above esters and did not show protease activity against azocasein. For these reasons C. rugosa Lipase B was judged to be the best suited to the ester biosensor application, and so was partially purified and further characterised.

Investigation of the effects of acyl chain length of the ester substrates on C. rugosa Lipase B activity revealed two peaks of preferential activity across the range C₂ − C₁₈. The first of these peaks, at C₄ indicative of true carboxylesterase activity, was more marked than that of pig liver esterase. The specificity of Lipase B (Yₘₐₓ/Kₘ) for ethyl acetate was 58 and 23 times lower than that for methyl butyrate and ethyl butyrate, respectively (compared to 4.7 and 2.5 times lower, respectively for pig liver esterase). The second, larger peak in the substrate size preference profile, for a C₁₂ acyl chain length, indicated a predominance of lipase activity.
Concurrent analysis of thermal- and pH-stability showed C. rugosa Lipase B to be stable below 40°C and between pH 5.5 and 7.0, with optimal stability at pH 5.5. In these respects, it is superior to pig liver esterase, which is highly susceptible to denaturation by storage above 30°C or below pH 5.0.

The influence of enzyme hydration on the hydrolysis of ethyl butyrate by C. rugosa Lipase B and pig liver esterase was investigated through the vapor-solid mode approach where each enzyme was studied as a hydrated solid phase in equilibrium with a substrate vapor phase. The effect of enzyme hydration was evaluated through measurements made in controlled atmospheres of known relative humidity. Although it was previously concluded that enzyme activity required a minimal water content of 0.2 gH₂O.g⁻¹  protein (0.2 h), we have shown that activity is possible at much lower (<0.03 h) hydration. C. rugosa Lipase B and pig liver esterase were hydrolytically active when exposed to mixtures of ethyl butyrate vapor over a range of relative humidities from 7 to 90%, equivalent to C. rugosa Lipase B and pig liver esterase water contents of between 0.054-0.47 h and 0.029-0.60 h, respectively. C.rugosa Lipase B hydrolytic activity was highly dependent on the degree of protein hydration and exhibited a linear relationship with respect to protein water content. For pig liver esterase activity, the effect of increasing hydration on activity was less marked where the increase in PLE activity began to stabilize at protein water contents over 0.17 h. Although no activity was seen at 0 h, since the reaction catalyzed (the hydrolysis of ethyl butyrate) requires water as a second substrate, this work does not discount the possibility of activity at lower hydration.

year: 2002

title: Effects of anthropogenic land use on temperate mangrove forests

authors: Thomson, Timothy

subjects: Temperate mangrove forests

abstract: Coastal ecosystems worldwide are facing unprecedented challenges due to ever increasing human activities, resulting in increased land-derived stressors that can threaten the health and integrity of these systems. Mangrove forests are unique ecosystems that play a crucial role in filtering nutrients, retaining pollutants, and sequestering carbon. However, to what extent they are impacted by an intensification of human pressures is unclear and empirical evidence of landscape scale effects of a holistic view of the ecosystem is scarce. This thesis investigates the effects of human land use on several aspects of temperate mangrove ecosystems and aims to identify potential vulnerabilities of ecosystem processes to provide a holistic view on the impacts that humas are having on these important ecosystems.
By working across a broad gradient of human land use, the first chapter establishes a connection between human land use intensity in catchments and regionally generalizable trends in mangrove forest structure and physicochemical parameters in sediments. Highlighting the integrated nature of anthropogenic impacts, this chapter emphasizes the need for holistic evaluations in assessing responses of complex natural systems to change.
The second chapter investigates environmental controls on organic matter degradation in mangrove forests. Results from this study emphasize the importance of considering multiple factors influencing degradation processes, that vary with the quality of the decaying organic matter. Furthermore, using the human land use gradient from the previous chapter, this study identifies an impact of human land use through eutrophication on the rate of labile organic matter loss from mangrove sediments.
The third chapter provides insights into bacterial communities in temperate mangrove sediments, revealing the persistent effects of nutrient enrichment even after the biochemical signal is lost. The study highlights the spatial resolution needed to detect impacts on microbial communities, warning that functional changes may persist beyond observable eutrophication effects.

The synthesis of these chapters provides a holistic view of the effects of anthropogenic impacts on temperate mangrove ecosystems across various scales. While some components of the system may benefit from moderate amounts of nutrient enrichment, others can deteriorate, ultimately diminishing overall service provisioning of the system. To understand and quantify these effects, this thesis calls for a nuanced understanding of disturbance effects on mangrove ecosystems, emphasizing their complexity and the interconnectedness of their components. It adds a valuable information about the functional resilience of mangrove forests in the face of human pressures and highlights the importance of an ecosystem-based perspective when assessing the effects of external stressors on these important ecosystems.

text: Effects of anthropogenic land use on temperate mangrove forests Coastal ecosystems worldwide are facing unprecedented challenges due to ever increasing human activities, resulting in increased land-derived stressors that can threaten the health and integrity of these systems. Mangrove forests are unique ecosystems that play a crucial role in filtering nutrients, retaining pollutants, and sequestering carbon. However, to what extent they are impacted by an intensification of human pressures is unclear and empirical evidence of landscape scale effects of a holistic view of the ecosystem is scarce. This thesis investigates the effects of human land use on several aspects of temperate mangrove ecosystems and aims to identify potential vulnerabilities of ecosystem processes to provide a holistic view on the impacts that humas are having on these important ecosystems.
By working across a broad gradient of human land use, the first chapter establishes a connection between human land use intensity in catchments and regionally generalizable trends in mangrove forest structure and physicochemical parameters in sediments. Highlighting the integrated nature of anthropogenic impacts, this chapter emphasizes the need for holistic evaluations in assessing responses of complex natural systems to change.
The second chapter investigates environmental controls on organic matter degradation in mangrove forests. Results from this study emphasize the importance of considering multiple factors influencing degradation processes, that vary with the quality of the decaying organic matter. Furthermore, using the human land use gradient from the previous chapter, this study identifies an impact of human land use through eutrophication on the rate of labile organic matter loss from mangrove sediments.
The third chapter provides insights into bacterial communities in temperate mangrove sediments, revealing the persistent effects of nutrient enrichment even after the biochemical signal is lost. The study highlights the spatial resolution needed to detect impacts on microbial communities, warning that functional changes may persist beyond observable eutrophication effects.

The synthesis of these chapters provides a holistic view of the effects of anthropogenic impacts on temperate mangrove ecosystems across various scales. While some components of the system may benefit from moderate amounts of nutrient enrichment, others can deteriorate, ultimately diminishing overall service provisioning of the system. To understand and quantify these effects, this thesis calls for a nuanced understanding of disturbance effects on mangrove ecosystems, emphasizing their complexity and the interconnectedness of their components. It adds a valuable information about the functional resilience of mangrove forests in the face of human pressures and highlights the importance of an ecosystem-based perspective when assessing the effects of external stressors on these important ecosystems.

year: 2024

title: E pā tō reo e Te Matatini, Herewini Parata

authors: Maxwell, Te Kahautu

abstract: Waita ā-ringa (action song) Lyrics composed by Te Kahautu Maxwell, Musical score composed by Te Whānau Mitai, Eric Kurei. Performed by Ōpōtiki Mai Tawhiti (Kapa Haka group) (3mins)

Waiata ā Tira: Aroha
Whakaeke: Pinepine Te Kura, Hau Te Kura
Mōteatea: Te Ao o Te Pārerā
Waiata ā Ringa: E pā to reo, Te Matatini, Herewini Parata
Poi: E ia e ia
Haka: Te Kōtiritiri, Te Kōtaratara
Whakawātea: Ōranga Tōiti, tōnui kōrero

text: E pā tō reo e Te Matatini, Herewini Parata Waita ā-ringa (action song) Lyrics composed by Te Kahautu Maxwell, Musical score composed by Te Whānau Mitai, Eric Kurei. Performed by Ōpōtiki Mai Tawhiti (Kapa Haka group) (3mins)

Waiata ā Tira: Aroha
Whakaeke: Pinepine Te Kura, Hau Te Kura
Mōteatea: Te Ao o Te Pārerā
Waiata ā Ringa: E pā to reo, Te Matatini, Herewini Parata
Poi: E ia e ia
Haka: Te Kōtiritiri, Te Kōtaratara
Whakawātea: Ōranga Tōiti, tōnui kōrero

year: 2023-02-22