title: An extracellular protease from an extreme thermophile

authors: Cowan, Don A.

abstract: The major extracellular protease of the Thermus-like organism Thermus T-351, has been isolated by Sephadex SP-C25 ion exchange chromatography, affinity chromatography on CBZ-D-phe-TETA-Sepharose and Sephadex G75 gel filtration chromatography. The purified enzyme, assigned the title “Caldolysin”, was shown to be homogeneous by a variety of electrophoretic techniques. Two other minor extracellular proteolytic enzymes (totally 12% to 25% of initial enzyme activity) were separated during ion exchange chromatography, but have not been studied. The molecular weight and isoelectric point of purified Caldolysin were estimated to be approximately 20,000 Daltons and 8.5, respectively. The presence of 6 disulphide bonds was indicated by the reaction with dithionitro-benzoate. No free sulphydryl groups were detected. 
When tested against a wide range of inhibitors, significant responses to EDTA, EGTA, and iodoacetic acid, but not o-phenanthroline or p-chloromercuri benzoate, were noted. Together with the presence of lytic activity (Caldolysin lysed a broad range of Gram-negative bacteria but only few Gram-positive organisms), this permitted the classification of Caldolysin as a metal-chelator-sensitive lytic protease. A number of similarities with Myxobacter and Sorangium lytic proteases have been noted. 
It was shown that calcium was essential for the stability of Caldolysin, but not for its activity. No evidence was found for the presence of an active site-bound metal ion. This is a significant dissimilarity to Thermolysin, which contains a single catalytic zinc atom.
Casein, albumin, haemoglobin, collagen, elastin, and fibrin were all hydrolysed to varying degrees by Caldolysin. An apparent minimum size requirement for activity (a minimum of 3 or 4 amino acid residues) precluded the use of amino acid and dipeptide substrate analogues for the measurement of proteolytic activity. A preliminary determination of specificity indicated a preference for small neutral aliphatic amino acids on either side of the point of hydrolysis. No esterase activity was detected. Optimal hydrolysis of both casein and albumin occurred at approximately pH 8.
Temperature-activity relationships indicated that Caldolysin underwent conformational changes at 50°C and 92°C. Loss of enzyme activity below the latter temperature was thought to be primarily the result of autolysis while above this temperature denaturation occurred. Arrhenius plots showed a break at about 50°C, above which a reduction in activation energy occurred. The Kₘ at 40°C was considerably higher than that at either 65°C or 85°C.
Caldolysin is one of the most stable enzymes known. Thermostability was greater than that of most other documented proteases (t ¹/₂ (80° C) = 30 hours; t ¹/₂ (85 °C) = 5-6 hours; t ¹/₂ (90°C) = 1 hour; t ¹/₂ (95°C) = 30 minutes; t ¹/₂ (100°C) = 4 minutes) . In the absence of calcium, thermostability was reduced considerably (t ¹/₂ 80°C) = 8 minutes). Other metal ions provided only a slight thermostabilising effect compared with Ca [Ca» Zn>Sr>Mg>Co>Ba>Cu]. Neither the presence of high ionic concentrations nor the presence of non-ionic solutes had any major effect on thermostability. However, high and low pH values significantly reduced thermostability at 75°C and 90°C. At room temperature in solution, little loss of enzyme activity was noted at pH’s from 7-11 during a 95 day period. Activity half-lives at pH 3.6, 5, and 11.8 were 28 days, more than 60 days, and 4 days, respectively. Little activity loss resulted from storage of the enzyme in the frozen or lyophilised state for a period of six months. Caldolysin was shown to be very stable in the presence of denaturing agents (8M urea, 6M guanidine.HC1, 1% sodium dodecylsulphate) at ambient temperatures. At 75°C, a 50% loss of activity occurred within 59 minutes, 53 minutes, and 320 minutes, respectively.
A feature of the Michaelis-Menten kinetics of Caldolysin is marked substrate inhibition (Kₘ (azoalbumin) = 0.028; Kₛ´ (azoalbumin) = 3) which, however, does not occur with small substrates or when the enzyme is immobilised. The enzyme also exhibits an initial activation during incubation at temperatures above 65°C. This effect has been correlated with the ionic strength, the incubation temperature, and the concentration of non-enzyme protein present. The latter causes initial inhibition which is reversed on heating. It is postulated that activation/inhibition and substrate inhibition are related, and are caused by the presence of non-catalytic protein-binding sites in the molecule which are discrete from the active site.
Caldolysin was successfully immobilised to glass beads, CM-cellulose, and Sepharose 4B, with enzyme activity retentions of 1%, 31% and 73%, respectively. The Kₘ of Caldolysin was unchanged in the immobilised state, but the pH optimum of Sepharose-caldolysin was reduced by approximately one pH unit. Thermal stability of both apo-and holo-caldolysin-Sepharose was increased by 3-4-fold.

text: An extracellular protease from an extreme thermophile The major extracellular protease of the Thermus-like organism Thermus T-351, has been isolated by Sephadex SP-C25 ion exchange chromatography, affinity chromatography on CBZ-D-phe-TETA-Sepharose and Sephadex G75 gel filtration chromatography. The purified enzyme, assigned the title “Caldolysin”, was shown to be homogeneous by a variety of electrophoretic techniques. Two other minor extracellular proteolytic enzymes (totally 12% to 25% of initial enzyme activity) were separated during ion exchange chromatography, but have not been studied. The molecular weight and isoelectric point of purified Caldolysin were estimated to be approximately 20,000 Daltons and 8.5, respectively. The presence of 6 disulphide bonds was indicated by the reaction with dithionitro-benzoate. No free sulphydryl groups were detected. 
When tested against a wide range of inhibitors, significant responses to EDTA, EGTA, and iodoacetic acid, but not o-phenanthroline or p-chloromercuri benzoate, were noted. Together with the presence of lytic activity (Caldolysin lysed a broad range of Gram-negative bacteria but only few Gram-positive organisms), this permitted the classification of Caldolysin as a metal-chelator-sensitive lytic protease. A number of similarities with Myxobacter and Sorangium lytic proteases have been noted. 
It was shown that calcium was essential for the stability of Caldolysin, but not for its activity. No evidence was found for the presence of an active site-bound metal ion. This is a significant dissimilarity to Thermolysin, which contains a single catalytic zinc atom.
Casein, albumin, haemoglobin, collagen, elastin, and fibrin were all hydrolysed to varying degrees by Caldolysin. An apparent minimum size requirement for activity (a minimum of 3 or 4 amino acid residues) precluded the use of amino acid and dipeptide substrate analogues for the measurement of proteolytic activity. A preliminary determination of specificity indicated a preference for small neutral aliphatic amino acids on either side of the point of hydrolysis. No esterase activity was detected. Optimal hydrolysis of both casein and albumin occurred at approximately pH 8.
Temperature-activity relationships indicated that Caldolysin underwent conformational changes at 50°C and 92°C. Loss of enzyme activity below the latter temperature was thought to be primarily the result of autolysis while above this temperature denaturation occurred. Arrhenius plots showed a break at about 50°C, above which a reduction in activation energy occurred. The Kₘ at 40°C was considerably higher than that at either 65°C or 85°C.
Caldolysin is one of the most stable enzymes known. Thermostability was greater than that of most other documented proteases (t ¹/₂ (80° C) = 30 hours; t ¹/₂ (85 °C) = 5-6 hours; t ¹/₂ (90°C) = 1 hour; t ¹/₂ (95°C) = 30 minutes; t ¹/₂ (100°C) = 4 minutes) . In the absence of calcium, thermostability was reduced considerably (t ¹/₂ 80°C) = 8 minutes). Other metal ions provided only a slight thermostabilising effect compared with Ca [Ca» Zn>Sr>Mg>Co>Ba>Cu]. Neither the presence of high ionic concentrations nor the presence of non-ionic solutes had any major effect on thermostability. However, high and low pH values significantly reduced thermostability at 75°C and 90°C. At room temperature in solution, little loss of enzyme activity was noted at pH’s from 7-11 during a 95 day period. Activity half-lives at pH 3.6, 5, and 11.8 were 28 days, more than 60 days, and 4 days, respectively. Little activity loss resulted from storage of the enzyme in the frozen or lyophilised state for a period of six months. Caldolysin was shown to be very stable in the presence of denaturing agents (8M urea, 6M guanidine.HC1, 1% sodium dodecylsulphate) at ambient temperatures. At 75°C, a 50% loss of activity occurred within 59 minutes, 53 minutes, and 320 minutes, respectively.
A feature of the Michaelis-Menten kinetics of Caldolysin is marked substrate inhibition (Kₘ (azoalbumin) = 0.028; Kₛ´ (azoalbumin) = 3) which, however, does not occur with small substrates or when the enzyme is immobilised. The enzyme also exhibits an initial activation during incubation at temperatures above 65°C. This effect has been correlated with the ionic strength, the incubation temperature, and the concentration of non-enzyme protein present. The latter causes initial inhibition which is reversed on heating. It is postulated that activation/inhibition and substrate inhibition are related, and are caused by the presence of non-catalytic protein-binding sites in the molecule which are discrete from the active site.
Caldolysin was successfully immobilised to glass beads, CM-cellulose, and Sepharose 4B, with enzyme activity retentions of 1%, 31% and 73%, respectively. The Kₘ of Caldolysin was unchanged in the immobilised state, but the pH optimum of Sepharose-caldolysin was reduced by approximately one pH unit. Thermal stability of both apo-and holo-caldolysin-Sepharose was increased by 3-4-fold.

year: 1980

title: The Taupo eruption occurred in 232 ± 10 CE, and not later

authors: Hogg, Alan G.

abstract: The Taupo eruption1 deposit is an isochronous marker bed that spans much of New Zealand’s North Island and pre-dates human arrival² . Holdaway et al. (2018; HDK18 hereafter)³ propose that the current Taupo eruption date is inaccurate and that the eruption occurred “…decades to two centuries…” after the published wiggle-match estimate of 232 ± 10 CE (2 s.d.)⁴ derived from a tanekaha (Phyllocladus trichomanoides) tree at the Pureora buried forest Site⁵,⁶. HDK18 propose that trees growing at Pureora (and other near-source areas) that were killed and buried by the climactic ignimbrite event were affected by ¹⁴C-depleted (magmatic) CO₂. HDK18’s proposal utilises a wide range of published ¹⁴C data, but their work results in assertions that are implausible. Four parts to their hypothesis are considered here

text: The Taupo eruption occurred in 232 ± 10 CE, and not later The Taupo eruption1 deposit is an isochronous marker bed that spans much of New Zealand’s North Island and pre-dates human arrival² . Holdaway et al. (2018; HDK18 hereafter)³ propose that the current Taupo eruption date is inaccurate and that the eruption occurred “…decades to two centuries…” after the published wiggle-match estimate of 232 ± 10 CE (2 s.d.)⁴ derived from a tanekaha (Phyllocladus trichomanoides) tree at the Pureora buried forest Site⁵,⁶. HDK18 propose that trees growing at Pureora (and other near-source areas) that were killed and buried by the climactic ignimbrite event were affected by ¹⁴C-depleted (magmatic) CO₂. HDK18’s proposal utilises a wide range of published ¹⁴C data, but their work results in assertions that are implausible. Four parts to their hypothesis are considered here

year: 2018

title: Exploring female art-making through reflective practice: A multi-dimensional cultural, spiritual and embodied experience

authors: Bright, Deborah Ann

subjects: female

abstract: This thesis explores some of the many elements and influences of lived experience that are present in acts of art-making. Reflective practice in art-making adds the intentional and purposive act of reflective learning to the experience of art-making. In an academic context the challenge arises for a researcher to adequately represent the breadth and richness of the lived experiences of reflective practice in artmaking. This breadth and richness may embrace elements of art production and influences including gender, culture, spirituality, creativity, creative processes, embodied knowing, the place of conversations and learning as a result of facilitated reflective practice. Adequate representation is particularly vital because art-makers may communicate best through non-verbal means, including embodied knowing and their artmaking, rather than through words. Flexibility of approach and representation is even more important when the art-maker participants engage in various art-making areas and identify themselves as part of indigenous, dominant and/or non-dominant cultures within Aotearoa New Zealand. In this thesis, by applying a feminist participatory approach, informed by kaupapa Maori, I affirmed that the lived experiences of reflective practice in art-making can be adequately represented in a thesis. By such means, I sought to empower the participants, including myself, by providing a process through which they might increase confidence in their own art-making practices and professional artistry. This approach was necessary because no single or even double blending of worldviews can embrace such a range of variables. However, an interweaving of feminist and participatory, informed by indigenous peoples’ worldviews, provides the scope for such a study in terms of fundamental beliefs, ways of knowing, values, issues of power, methodologies and methods. The ways of knowing include collaborative, constructed, cultural, embodied, experiential, indigenous, presentational, propositional, spiritual and subjective and writing as a way of knowing; such a range was vital to this thesis. As artmaker co-participant, I was able to engage in conversation with the other art-makers as facilitator of their reflective practice in art-making and to represent their lived experiences of reflective practice in art-making through verbal and non-verbal means and art-making, in ways that were comfortable culturally to all participants. ii I represent and discuss my findings through formal academic writing, personal narrative and a DVD. The DVD includes a video montage and an image narrative that contribute to the discussion on embodied knowing (Chapter 7), a video of my own dance work, which contributes to the discussion on creativity and visual images (Chapter 6), and a copy of a poster referred to at various times in the findings and discussion chapters. Finally, I conclude that a feminist participatory approach, informed by indigenous peoples’ worldviews and including facilitated reflective practice, may have application to other fields of research and practice including other areas of the arts, teaching, sport and leisure and in the wider field of social sciences.

text: Exploring female art-making through reflective practice: A multi-dimensional cultural, spiritual and embodied experience This thesis explores some of the many elements and influences of lived experience that are present in acts of art-making. Reflective practice in art-making adds the intentional and purposive act of reflective learning to the experience of art-making. In an academic context the challenge arises for a researcher to adequately represent the breadth and richness of the lived experiences of reflective practice in artmaking. This breadth and richness may embrace elements of art production and influences including gender, culture, spirituality, creativity, creative processes, embodied knowing, the place of conversations and learning as a result of facilitated reflective practice. Adequate representation is particularly vital because art-makers may communicate best through non-verbal means, including embodied knowing and their artmaking, rather than through words. Flexibility of approach and representation is even more important when the art-maker participants engage in various art-making areas and identify themselves as part of indigenous, dominant and/or non-dominant cultures within Aotearoa New Zealand. In this thesis, by applying a feminist participatory approach, informed by kaupapa Maori, I affirmed that the lived experiences of reflective practice in art-making can be adequately represented in a thesis. By such means, I sought to empower the participants, including myself, by providing a process through which they might increase confidence in their own art-making practices and professional artistry. This approach was necessary because no single or even double blending of worldviews can embrace such a range of variables. However, an interweaving of feminist and participatory, informed by indigenous peoples’ worldviews, provides the scope for such a study in terms of fundamental beliefs, ways of knowing, values, issues of power, methodologies and methods. The ways of knowing include collaborative, constructed, cultural, embodied, experiential, indigenous, presentational, propositional, spiritual and subjective and writing as a way of knowing; such a range was vital to this thesis. As artmaker co-participant, I was able to engage in conversation with the other art-makers as facilitator of their reflective practice in art-making and to represent their lived experiences of reflective practice in art-making through verbal and non-verbal means and art-making, in ways that were comfortable culturally to all participants. ii I represent and discuss my findings through formal academic writing, personal narrative and a DVD. The DVD includes a video montage and an image narrative that contribute to the discussion on embodied knowing (Chapter 7), a video of my own dance work, which contributes to the discussion on creativity and visual images (Chapter 6), and a copy of a poster referred to at various times in the findings and discussion chapters. Finally, I conclude that a feminist participatory approach, informed by indigenous peoples’ worldviews and including facilitated reflective practice, may have application to other fields of research and practice including other areas of the arts, teaching, sport and leisure and in the wider field of social sciences.

year: 2010