title: Purification and characterization of Ak.1 protease, a thermostable subtilisin with a disulphide bond in the substrate-binding cleft

authors: Toogood, Helen S.

subjects: calcium binding

abstract: Ak.1 protease, a thermostable subtilisin isolated originally from Bacillus st. Ak.1, was purified to homogeneity from the Escherichia coli clone PB5517. It is active against substrates containing neutral or hydrophobic branched-chain amino acids at the P₁ site, such as valine, alanine or phenylalanine. The Km and kcat of the enzyme decrease with decreasing temperature, though not to the same degree with all substrates, suggesting that specificity changes with temperature. The protease is markedly stabilized by Ca²⁺ ions. At 70 °C, a 10-fold increase in Ca²⁺ concentration increases the half-life by three orders of magnitude. Ak.1 protease is stabilized by Ca²⁺ to a greater extent than is thermitase. This may be due, in part, to the presence of an extra Ca²⁺-binding site in Ak.1 protease. Other metal ions, such as Sr²⁺, increase the thermostability of the enzyme, but to a significantly lower degree than does Ca²⁺. The structure of the protease showed the presence of a disulphide bond located within the active-site cleft. This bond influences both enzyme activity and thermostability. The disulphide bond appears to have a dual role: maintaining the integrity of the substrate-binding cleft and increasing the thermostability of the protease. The protease was originally investigated to determine its usefulness in the clean-up of DNA at high temperatures. However, it was found that this protease has a limited substrate specificity, so this application was not explored further.

text: Purification and characterization of Ak.1 protease, a thermostable subtilisin with a disulphide bond in the substrate-binding cleft Ak.1 protease, a thermostable subtilisin isolated originally from Bacillus st. Ak.1, was purified to homogeneity from the Escherichia coli clone PB5517. It is active against substrates containing neutral or hydrophobic branched-chain amino acids at the P₁ site, such as valine, alanine or phenylalanine. The Km and kcat of the enzyme decrease with decreasing temperature, though not to the same degree with all substrates, suggesting that specificity changes with temperature. The protease is markedly stabilized by Ca²⁺ ions. At 70 °C, a 10-fold increase in Ca²⁺ concentration increases the half-life by three orders of magnitude. Ak.1 protease is stabilized by Ca²⁺ to a greater extent than is thermitase. This may be due, in part, to the presence of an extra Ca²⁺-binding site in Ak.1 protease. Other metal ions, such as Sr²⁺, increase the thermostability of the enzyme, but to a significantly lower degree than does Ca²⁺. The structure of the protease showed the presence of a disulphide bond located within the active-site cleft. This bond influences both enzyme activity and thermostability. The disulphide bond appears to have a dual role: maintaining the integrity of the substrate-binding cleft and increasing the thermostability of the protease. The protease was originally investigated to determine its usefulness in the clean-up of DNA at high temperatures. However, it was found that this protease has a limited substrate specificity, so this application was not explored further.

year: 2000

title: Indigenous education: addressing current issues and developments

authors: May, Stephen

subjects: indigenous education

abstract: This introduction to a special issue of Comparative Education focuses on current issues and developments in
both the theory and practice of indigenous education from around the world.

text: Indigenous education: addressing current issues and developments This introduction to a special issue of Comparative Education focuses on current issues and developments in
both the theory and practice of indigenous education from around the world.

year: 2003-04-01

title: Community-university collaborations: creating hybrid research and collective identities

authors: Cave, Jennifer Barbara Jane

subjects: collaborative

abstract: In this article we explore the politics of community-university collaborative research and activism. We are scholars based in universities as well as members of various 'communities'-queer, Pasifika and Māori-hence we regularly venture beyond the formal research spaces of the academy and enter into critical collaborative research with others working in 'the community'. In what follows we first outline collaborative community-based research literature that advocates hybrid research collectivities. Second, we give some context and background to our methodologies that have enabled us to re-think collaborative research. Third, examples from our research are offered to illustrate the ways in which collaborative research constructs new collective identities. Finally, we conclude by arguing that social science scholars working across university-community boundaries may expand and multiply hybrid research collectives, and thereby effect positive social change at many levels

text: Community-university collaborations: creating hybrid research and collective identities In this article we explore the politics of community-university collaborative research and activism. We are scholars based in universities as well as members of various 'communities'-queer, Pasifika and Māori-hence we regularly venture beyond the formal research spaces of the academy and enter into critical collaborative research with others working in 'the community'. In what follows we first outline collaborative community-based research literature that advocates hybrid research collectivities. Second, we give some context and background to our methodologies that have enabled us to re-think collaborative research. Third, examples from our research are offered to illustrate the ways in which collaborative research constructs new collective identities. Finally, we conclude by arguing that social science scholars working across university-community boundaries may expand and multiply hybrid research collectives, and thereby effect positive social change at many levels

year: 2012