title: Caldolase: an alkaline serine protease from Thermus strain TOK₃

authors: Saravani, G.A.

abstract: The object of this investigation was the isolation of extreme thermophiles producing extracellular proteases, and the biochemical characterisation of a stable, chelator-insensitive protease. 
Two plate assay systems were developed for the initial screening of proteases. The first involved the incorporation of various protease inhibitors (particularly chelating agents) in casein agar plates, the second the inclusion of a variety of native and chromogenic proteins in the agar plates. In conjunction, these methods provided a basis for screening extreme thermophiles for particular proteases, and enabled the identification of fourteen new proteases. Included amongst them was an extracellular serine protease from a Thermus strain designated Tok₃, which was selected for further study.
The protease was purified to homogeneity by ammonium-sulphate precipitation followed by ion exchange on DEAE-cellulose and QAE-Sephadex, affinity chromatography on CBZ-phe-TETA-Sepharose-4B, gel filtration chromatography on either Sephadex G-75 or TSK G3000 SW using HPLC system and finally Polyacrylamide gel electrophoresis.
For convenience the protease was assigned a trivial name, and the term Caldolase was chosen. The prefix Caldo- is derived from Latin Caldo (hot) and the suffix ‘ase’ is a general term for enzymes. 
The specific activity of the pure enzyme was estimated to be 38,000 proteolytic units per mg (PU mg ml⁻¹) at 75°C using casein as the substrate. The purified enzyme had a pH optimum of 9.5 with an isoelectric point of 8.9. Caldolase demonstrated greatest stability between pH 7 and 10. The molecular weight of the protease was estimated by exclusion chromatography on Sephadex G-75 and TSK G3000 SW to be 25,000 daltons.
The enzyme was inhibited by serine inhibitors (DFP, PMSF and di-phenyl carbamyl chloride), partially inhibited by heavy metal (CUCl₂), but not inhibited by metal chelators (EDTA, EGTA, 0-phenanthroline, and NEPIS), Cysteine inhibitors (PCMB, iodo-acetamide, and N-ethylmaleimide) or trypsin inhibitors. These results indicate that Caldolase is an alkaline serine protease. 
Neither Ca²⁺ nor Zn²⁺ ions were detected in the highly purified protease. The presence of four disulphide bonds per molecule of the enzyme was indicated with dithionitro-benzoate. No free sulphydryl groups were found. The purified protease contained approximately 10% carbohydrate. The amino acid composition of Caldolase was determined. 
The enzyme exhibited strong substrate inhibition when using casein, azo-casein, and azo-albumin as substrates. No substrate inhibition was observed when low-molecular weight synthetic substrates were used, indicating that substrate inhibition using casein and azo-albumin substrates may be due to steric hindrance rather than binding to the active site of the enzyme.
The Arrhenius plots for both casein and peptide substrates were curved, but without any clearly marked discontinuity. It is concluded that the effect of temperature on the enzyme conformation is continuous rather than occurring at a particular temperature. No significant differences were observed in Kₘ values at various temperatures between 45° and 85°C.
Caldolase hydrolysed several protein substrates, including casein, albumin, ovalbumin, haemoglobin, collagen, fibrin and elastin, and a number of synthetic chromogenic peptides. It also possessed esterase activity. The enzyme was not able to hydrolyse peptides possessing fewer than four groups (amino acid residues and terminal blocking group). Caldolase did not hydrolyse Bradykinin (Arg-Pro-Pro-Gly-Phe-Ser-Pro-phe-Arg). In contrast enzyme hydrolysis of insulin B chain resulted in a very complex pattern, suggesting a low degree of specificity. 
The enzyme was very thermostable. Half-life values were: 100°C, 5 min; 90°C, 45 min; and 80°C, 840 min. Caldolase was stable in denaturing agents (GuHCl, urea) at 22°C, but not at 85°C. Exposure of the enzyme to various organic solvents caused no significant loss of catalytic activity.
Ionic strength had a marked effect on enzyme stability. The combination of low salt concentration (below 0.3M NaCl) and low temperatures (under 75°C) results in reversible enzyme denaturation. However, at high temperatures (above 80°C) this phenomenon is rapidly followed by autolysis by the remaining active enzyme.

text: Caldolase: an alkaline serine protease from Thermus strain TOK₃ The object of this investigation was the isolation of extreme thermophiles producing extracellular proteases, and the biochemical characterisation of a stable, chelator-insensitive protease. 
Two plate assay systems were developed for the initial screening of proteases. The first involved the incorporation of various protease inhibitors (particularly chelating agents) in casein agar plates, the second the inclusion of a variety of native and chromogenic proteins in the agar plates. In conjunction, these methods provided a basis for screening extreme thermophiles for particular proteases, and enabled the identification of fourteen new proteases. Included amongst them was an extracellular serine protease from a Thermus strain designated Tok₃, which was selected for further study.
The protease was purified to homogeneity by ammonium-sulphate precipitation followed by ion exchange on DEAE-cellulose and QAE-Sephadex, affinity chromatography on CBZ-phe-TETA-Sepharose-4B, gel filtration chromatography on either Sephadex G-75 or TSK G3000 SW using HPLC system and finally Polyacrylamide gel electrophoresis.
For convenience the protease was assigned a trivial name, and the term Caldolase was chosen. The prefix Caldo- is derived from Latin Caldo (hot) and the suffix ‘ase’ is a general term for enzymes. 
The specific activity of the pure enzyme was estimated to be 38,000 proteolytic units per mg (PU mg ml⁻¹) at 75°C using casein as the substrate. The purified enzyme had a pH optimum of 9.5 with an isoelectric point of 8.9. Caldolase demonstrated greatest stability between pH 7 and 10. The molecular weight of the protease was estimated by exclusion chromatography on Sephadex G-75 and TSK G3000 SW to be 25,000 daltons.
The enzyme was inhibited by serine inhibitors (DFP, PMSF and di-phenyl carbamyl chloride), partially inhibited by heavy metal (CUCl₂), but not inhibited by metal chelators (EDTA, EGTA, 0-phenanthroline, and NEPIS), Cysteine inhibitors (PCMB, iodo-acetamide, and N-ethylmaleimide) or trypsin inhibitors. These results indicate that Caldolase is an alkaline serine protease. 
Neither Ca²⁺ nor Zn²⁺ ions were detected in the highly purified protease. The presence of four disulphide bonds per molecule of the enzyme was indicated with dithionitro-benzoate. No free sulphydryl groups were found. The purified protease contained approximately 10% carbohydrate. The amino acid composition of Caldolase was determined. 
The enzyme exhibited strong substrate inhibition when using casein, azo-casein, and azo-albumin as substrates. No substrate inhibition was observed when low-molecular weight synthetic substrates were used, indicating that substrate inhibition using casein and azo-albumin substrates may be due to steric hindrance rather than binding to the active site of the enzyme.
The Arrhenius plots for both casein and peptide substrates were curved, but without any clearly marked discontinuity. It is concluded that the effect of temperature on the enzyme conformation is continuous rather than occurring at a particular temperature. No significant differences were observed in Kₘ values at various temperatures between 45° and 85°C.
Caldolase hydrolysed several protein substrates, including casein, albumin, ovalbumin, haemoglobin, collagen, fibrin and elastin, and a number of synthetic chromogenic peptides. It also possessed esterase activity. The enzyme was not able to hydrolyse peptides possessing fewer than four groups (amino acid residues and terminal blocking group). Caldolase did not hydrolyse Bradykinin (Arg-Pro-Pro-Gly-Phe-Ser-Pro-phe-Arg). In contrast enzyme hydrolysis of insulin B chain resulted in a very complex pattern, suggesting a low degree of specificity. 
The enzyme was very thermostable. Half-life values were: 100°C, 5 min; 90°C, 45 min; and 80°C, 840 min. Caldolase was stable in denaturing agents (GuHCl, urea) at 22°C, but not at 85°C. Exposure of the enzyme to various organic solvents caused no significant loss of catalytic activity.
Ionic strength had a marked effect on enzyme stability. The combination of low salt concentration (below 0.3M NaCl) and low temperatures (under 75°C) results in reversible enzyme denaturation. However, at high temperatures (above 80°C) this phenomenon is rapidly followed by autolysis by the remaining active enzyme.

year: 1985

title: Assessing the internal structure of the COPINE scale

authors: Merdian, Hannah Lena

subjects: child pornography

abstract: The COPINE scale allows standardised classification of a person's child pornography collection while taking into account contextual considerations. However, despite its frequent usage in research studies, the scale has never been empirically validated with regards to its psychometric properties. This study describes a validation design to assess reliability and construct validity of the scale while considering ethical concerns. An online survey was conducted amongst psychological staff members of Correctional Services NZ as well as postgraduate students from a New Zealand University. Participants were asked to rate verbal descriptions of each item level of the original scale according to its perceived seriousness of the offence, suggesting a higher penalty for more serious image offending. Results revealed that students and correctional staff showed high inter-rater agreement in their rankings, and that rankings increased with higher-level images as defined in the original scale. Two areas of difference to the original scale ranks were identified, concerning levels ‘Posing’ and ‘Sexual Explicit Activities’. The implications of these findings are discussed.

text: Assessing the internal structure of the COPINE scale The COPINE scale allows standardised classification of a person's child pornography collection while taking into account contextual considerations. However, despite its frequent usage in research studies, the scale has never been empirically validated with regards to its psychometric properties. This study describes a validation design to assess reliability and construct validity of the scale while considering ethical concerns. An online survey was conducted amongst psychological staff members of Correctional Services NZ as well as postgraduate students from a New Zealand University. Participants were asked to rate verbal descriptions of each item level of the original scale according to its perceived seriousness of the offence, suggesting a higher penalty for more serious image offending. Results revealed that students and correctional staff showed high inter-rater agreement in their rankings, and that rankings increased with higher-level images as defined in the original scale. Two areas of difference to the original scale ranks were identified, concerning levels ‘Posing’ and ‘Sexual Explicit Activities’. The implications of these findings are discussed.

year: 2013

title: Sustainability and Indigeneity

authors: Daya-Winterbottom, Trevor

abstract: In the lead up to the Rio Conference on Environment and Development 1992, New Zealand enshrined the concept of the sustainable management of natural and physical resources in statute law by enacting the Resource Management Act 1991. Implementing the concept of sustainable management in domestic law gave rise to a tension between anthropocentric and ecological approaches for implementing the statute that were not resolved by the Courts until 2014. However, the adoption of an ecological approach to implementing the statute did not assuage the desire for statutory reform unlocked by the previous debate. Most recently, the reform trajectory has resulted in Parliamentary scrutiny of an exposure draft Bill that replaces the concept of sustainable management with a new concept of the welfare and wellbeing of the environment rooted firmly in indigenous perspectives on earth governance, guardianship, and intergenerational equity. My paper will critically interrogate the implications of adopting the concept of the welfare and wellbeing of the environment for the future of sustainability, and what implications this approach could have for environmental law generally in the Anthropocene both comparatively and internationally.

text: Sustainability and Indigeneity In the lead up to the Rio Conference on Environment and Development 1992, New Zealand enshrined the concept of the sustainable management of natural and physical resources in statute law by enacting the Resource Management Act 1991. Implementing the concept of sustainable management in domestic law gave rise to a tension between anthropocentric and ecological approaches for implementing the statute that were not resolved by the Courts until 2014. However, the adoption of an ecological approach to implementing the statute did not assuage the desire for statutory reform unlocked by the previous debate. Most recently, the reform trajectory has resulted in Parliamentary scrutiny of an exposure draft Bill that replaces the concept of sustainable management with a new concept of the welfare and wellbeing of the environment rooted firmly in indigenous perspectives on earth governance, guardianship, and intergenerational equity. My paper will critically interrogate the implications of adopting the concept of the welfare and wellbeing of the environment for the future of sustainability, and what implications this approach could have for environmental law generally in the Anthropocene both comparatively and internationally.

year: 2022