title: Purification and characterization of a thermostable proteinase isolated from Thermus sp. strain Rt41A

authors: Peek, Keith

subjects: enzyme

abstract: Thermus sp. strain Rt41A produces an extracellular thermostable alkaline proteinase. The enzyme has a high isoelectric point (10.25–10.5) which can be exploited in purification by using cation-exchange chromatography. The proteinase was purified to homogeneity and has a molecular mass of 32.5 kDa by SDS/PAGE. It is a glycoprotein, containing 0.7% carbohydrate as glucose equivalents, and has four half-cystine residues present as two disulphide bonds. Maximum proteolytic activity was observed at pH 8.0 against azocasein and greater than 75% of this activity was retained in the pH range 7.0–10.0. Substrate inhibition was observed with casein and azocasein. The enzyme was stable in the pH range 5.0–10.0 and maximum activity, in a 10-min assay, was observed at 90°C with 5 mM CaCl2 present. No loss of activity was observed after 24 h at 70°C and the half-lives at 80°C and 90°C were 13.5 h and 20 min, respectively. Removal of Ca2+ reduced the temperature for maximum proteolytic activity against azocasein to 60°C and the half-life at 70°C was 2.85 min. The enzyme was stable at low and high ionic strength and in the presence of denaturing reagents and organic solvents.
Rt41A proteinase cleaved a number of synthetic amino acid p-nitrophenol esters, the kinetic data indicating that small aliphatic or aromatic amino acids were the preferred residue at the P1 position. The kinetic data for the hydrolysis of a number of peptide p-nitroanilide substrates are also reported. Primary cleavage of the oxidized insulin B chain occurred at sites where the P1′ amino acid was aromatic. Minor cleavage sites (24 h incubation) were for amino acids with aliphatic side chains at the P1′ position. The esterase and insulin cleavage data indicate the specificity is similar for both the P1 and P1′ sites.

text: Purification and characterization of a thermostable proteinase isolated from Thermus sp. strain Rt41A Thermus sp. strain Rt41A produces an extracellular thermostable alkaline proteinase. The enzyme has a high isoelectric point (10.25–10.5) which can be exploited in purification by using cation-exchange chromatography. The proteinase was purified to homogeneity and has a molecular mass of 32.5 kDa by SDS/PAGE. It is a glycoprotein, containing 0.7% carbohydrate as glucose equivalents, and has four half-cystine residues present as two disulphide bonds. Maximum proteolytic activity was observed at pH 8.0 against azocasein and greater than 75% of this activity was retained in the pH range 7.0–10.0. Substrate inhibition was observed with casein and azocasein. The enzyme was stable in the pH range 5.0–10.0 and maximum activity, in a 10-min assay, was observed at 90°C with 5 mM CaCl2 present. No loss of activity was observed after 24 h at 70°C and the half-lives at 80°C and 90°C were 13.5 h and 20 min, respectively. Removal of Ca2+ reduced the temperature for maximum proteolytic activity against azocasein to 60°C and the half-life at 70°C was 2.85 min. The enzyme was stable at low and high ionic strength and in the presence of denaturing reagents and organic solvents.
Rt41A proteinase cleaved a number of synthetic amino acid p-nitrophenol esters, the kinetic data indicating that small aliphatic or aromatic amino acids were the preferred residue at the P1 position. The kinetic data for the hydrolysis of a number of peptide p-nitroanilide substrates are also reported. Primary cleavage of the oxidized insulin B chain occurred at sites where the P1′ amino acid was aromatic. Minor cleavage sites (24 h incubation) were for amino acids with aliphatic side chains at the P1′ position. The esterase and insulin cleavage data indicate the specificity is similar for both the P1 and P1′ sites.

year: 1992

title: The educational philosophy of Sosaku Kobayashi

authors: Edwards, Alexander

abstract: This thesis discusses the educational philosophy of Sosaku Kobayashi, the principal and founder of Tomoe Gakuen, the innovative school featured in the novel Totto-chan: The Little Girl at the Window. It begins with an introduction to the novel and its author, situating Tomoe Gakuen within the broader context of early-20th-century Japanese education. After discussing the features of the progressive education movement, I then explore the key principles, values, and teaching style that Sosaku Kobayashi employs, as illustrated through the anecdotes in the novel, and the goals they are intended to achieve. Following this, I discuss how learner-centered education has influenced modern curricula worldwide since the Second World War and examine the ongoing dialogue between its detractors and advocates. This segues into a comparison between the learning philosophy of Sosaku Kobayashi and other prominent styles of progressive education, namely Montessori, Steiner, and outdoor-focused education. Finally, I explore what Kobayashi’s philosophy might have looked like had it developed into a broader educational movement and continued to flourish into the 21st century beyond Tomoe Gakuen.

text: The educational philosophy of Sosaku Kobayashi This thesis discusses the educational philosophy of Sosaku Kobayashi, the principal and founder of Tomoe Gakuen, the innovative school featured in the novel Totto-chan: The Little Girl at the Window. It begins with an introduction to the novel and its author, situating Tomoe Gakuen within the broader context of early-20th-century Japanese education. After discussing the features of the progressive education movement, I then explore the key principles, values, and teaching style that Sosaku Kobayashi employs, as illustrated through the anecdotes in the novel, and the goals they are intended to achieve. Following this, I discuss how learner-centered education has influenced modern curricula worldwide since the Second World War and examine the ongoing dialogue between its detractors and advocates. This segues into a comparison between the learning philosophy of Sosaku Kobayashi and other prominent styles of progressive education, namely Montessori, Steiner, and outdoor-focused education. Finally, I explore what Kobayashi’s philosophy might have looked like had it developed into a broader educational movement and continued to flourish into the 21st century beyond Tomoe Gakuen.

year: 2025

title: Environmental ethics in a New Zealand context

authors: Gunn, Alastair S.

subjects: environmental ethics

abstract: Environmental ethics has many strands and roots but there is increasing for a holistic approach. Such an approach, which is consistent with te ao Maori and tikanga Maori, is especially appropriate for New Zealand.

text: Environmental ethics in a New Zealand context Environmental ethics has many strands and roots but there is increasing for a holistic approach. Such an approach, which is consistent with te ao Maori and tikanga Maori, is especially appropriate for New Zealand.

year: 2007