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: Workaholism and Employee Well-Being

authors: Horton, Tanya Elizabeth

subjects: Workaholism

abstract: The term workaholism, patterned after the word alcoholism, first appeared in a book by Oates (1971) in which he described workaholism as a compulsive or uncontrollable need to work incessantly, resulting in negative consequences. Research has yielded mixed results in relation to the impact workaholism can have on people’s lives. Some authors view workaholism in positive terms (Machlowitz, 1980), while others view it in negative terms (Robinson, 1998). This study focused on the relationship between workaholism and health and well-being. An online, self report questionnaire, which included the Workaholism Battery (Spence & Robbins, 1992), was completed by 136 employees throughout New Zealand. Additional measures included work→family conflict, family→work conflict, family satisfaction, anxiety/depression, social dysfunction, positive psychological well-being, negative psychological well-being and physical health symptoms. Participants were classified into one of six groups, consisting of the enthusiastic workaholics, unenthusiastic workaholics, unengaged workers, disenchanted workers, work enthusiasts and relaxed workers. The unenthusiastic workaholics and the enthusiastic workaholics made up the “workaholic” group, and the unengaged workers, disenchanted workers, work enthusiasts and relaxed workers made up the “non-workaholic” group. The main finding of this study was that there were few differences between workaholics and non-workaholics in relation to family→work conflict, family satisfaction, positive psychological well-being, negative psychological well-being, anxiety/depression, social dysfunction and physical health symptoms. The only difference between the workaholics and non-workaholics was that enthusiastic workaholics reported significantly higher levels of work→family conflict compared to relaxed workers. Another important finding of this study was that different types of workaholics reported significantly different levels of psychological well-being. Unenthusiastic workaholics reported significantly lower levels of positive psychological well-being, and significantly higher levels of negative psychological well-being compared to the enthusiastic workaholics. These results suggest that, with the exception of the comparatively low levels of psychological well-being the unenthusiastic workaholics reported in relation to the enthusiastic workaholics, workaholism may not be as harmful as previously thought. They also provide support for the continued differentiation of multiple types of workaholics, as the unenthusiastic workaholics and the enthusiastic workaholics differed significantly on their reported levels of psychological well-being. Having an excessive drive to work was significantly associated with poor health and well-being, whereas enjoyment of work was associated significantly with high positive levels of health and well-being. Work involvement was much more inconsistently related to health and well-being. On this basis, it may be inferred that excessive drive to work may be the harmful element in workaholism as it produces negative health and lifestyle outcomes, while enjoyment may be a productive factor. Finally, a number of significant relationships were found between the health and well-being variables, suggesting that an individual’s physical, mental and emotional health might be related to one another. The present data suggests that differentiation between different types of workaholics is important. The present data also challenges the negative stereotype of workaholism, and emphasises the importance of developing strategies to better manage workaholism within the workplace.

text: Workaholism and Employee Well-Being The term workaholism, patterned after the word alcoholism, first appeared in a book by Oates (1971) in which he described workaholism as a compulsive or uncontrollable need to work incessantly, resulting in negative consequences. Research has yielded mixed results in relation to the impact workaholism can have on people’s lives. Some authors view workaholism in positive terms (Machlowitz, 1980), while others view it in negative terms (Robinson, 1998). This study focused on the relationship between workaholism and health and well-being. An online, self report questionnaire, which included the Workaholism Battery (Spence & Robbins, 1992), was completed by 136 employees throughout New Zealand. Additional measures included work→family conflict, family→work conflict, family satisfaction, anxiety/depression, social dysfunction, positive psychological well-being, negative psychological well-being and physical health symptoms. Participants were classified into one of six groups, consisting of the enthusiastic workaholics, unenthusiastic workaholics, unengaged workers, disenchanted workers, work enthusiasts and relaxed workers. The unenthusiastic workaholics and the enthusiastic workaholics made up the “workaholic” group, and the unengaged workers, disenchanted workers, work enthusiasts and relaxed workers made up the “non-workaholic” group. The main finding of this study was that there were few differences between workaholics and non-workaholics in relation to family→work conflict, family satisfaction, positive psychological well-being, negative psychological well-being, anxiety/depression, social dysfunction and physical health symptoms. The only difference between the workaholics and non-workaholics was that enthusiastic workaholics reported significantly higher levels of work→family conflict compared to relaxed workers. Another important finding of this study was that different types of workaholics reported significantly different levels of psychological well-being. Unenthusiastic workaholics reported significantly lower levels of positive psychological well-being, and significantly higher levels of negative psychological well-being compared to the enthusiastic workaholics. These results suggest that, with the exception of the comparatively low levels of psychological well-being the unenthusiastic workaholics reported in relation to the enthusiastic workaholics, workaholism may not be as harmful as previously thought. They also provide support for the continued differentiation of multiple types of workaholics, as the unenthusiastic workaholics and the enthusiastic workaholics differed significantly on their reported levels of psychological well-being. Having an excessive drive to work was significantly associated with poor health and well-being, whereas enjoyment of work was associated significantly with high positive levels of health and well-being. Work involvement was much more inconsistently related to health and well-being. On this basis, it may be inferred that excessive drive to work may be the harmful element in workaholism as it produces negative health and lifestyle outcomes, while enjoyment may be a productive factor. Finally, a number of significant relationships were found between the health and well-being variables, suggesting that an individual’s physical, mental and emotional health might be related to one another. The present data suggests that differentiation between different types of workaholics is important. The present data also challenges the negative stereotype of workaholism, and emphasises the importance of developing strategies to better manage workaholism within the workplace.

year: 2011

title: Final report of the 2018 Census External Data Quality Panel

authors: Bedford, Richard

abstract: At the time this report was submitted for publication in January 2020, Stats NZ was finalizing its release schedule for census products that allow users to produce their own tabulations and statistical analyses using census data (see Appendix 2 for a summary of release dates for 2018 Census products).
In early 2020, Stats NZ will release 2018 Census data for use in the Data Lab, a distributed set of secure computer sites where users can access, with approval, microdata for research purposes. By March 2020, 2018 Census data will be in the Integrated Data Infrastructure (IDI) and available for use by approved researchers and policy analysts who wish to negotiate access to the IDI.

text: Final report of the 2018 Census External Data Quality Panel At the time this report was submitted for publication in January 2020, Stats NZ was finalizing its release schedule for census products that allow users to produce their own tabulations and statistical analyses using census data (see Appendix 2 for a summary of release dates for 2018 Census products).
In early 2020, Stats NZ will release 2018 Census data for use in the Data Lab, a distributed set of secure computer sites where users can access, with approval, microdata for research purposes. By March 2020, 2018 Census data will be in the Integrated Data Infrastructure (IDI) and available for use by approved researchers and policy analysts who wish to negotiate access to the IDI.

year: 2020