title: Characterisation of a thermostable pepstatin-insensitive acid proteinase from a Bacillus sp.

authors: Prescott, Mark

subjects: acid proteinase

abstract: An acid proteinase, Wai 21a, produced by a thermophilic Bacillus species (strain Wai 21a) has been purified to homogeneity by cation-exchange chromatography, phenyl-Sepharose chromatography and anion-exchange chromatography. A pI of 3.8 was determined by isoelectric focussing. The protein contained some associated carbohydrate (20 mol hexose equiv/mol proteinase). Optimal proteolytic activity was observed at pH 3.0 (at 60°C). The Leu¹⁵-Tyr¹⁶ bond was the major site of hydrolysis for the oxidized B chain of insulin. Enzyme activity was not affected by inhibitors of the cysteine, metallo or serine class of proteinases. The aspartate proteinase inhibitor, pepstatin, did not inhibit enzyme activity. Inhibition of enzyme activity by 1,2-epoxy-3-(p-nitrophenoxy)-propane indicated the presence of at least one carboxyl group essential to the catalytic mechanism of the enzyme. Proteinase activity was inhibited by diazoacetyl-  -norleucine methyl ester in a slow and non-specific manner atypical of pepstatin-sensitive aspartate proteinases. Wai 21a proteinase may be classified as member of the pepstatin-insensitive group of aspartate proteinases. The thermal stability at pH 3.0 and 60°C increased 2.1-fold (t1/2, 4.5–9.7 hr) in the presence of 5 mM Ca⁺⁺. An increase in both pH (3.0–4.5) and Ca⁺⁺ concentration (0–30 mM) resulted in a 15-fold increase (t1/2, 15–230 min) in thermal stability at 75°C. The amino acid composition of Wai 21a proteinase was found to be similar to other pepstatin-insensitive proteinases from bacterial sources and in particular similar to the thermostable enzyme, kumamolysin.

text: Characterisation of a thermostable pepstatin-insensitive acid proteinase from a Bacillus sp. An acid proteinase, Wai 21a, produced by a thermophilic Bacillus species (strain Wai 21a) has been purified to homogeneity by cation-exchange chromatography, phenyl-Sepharose chromatography and anion-exchange chromatography. A pI of 3.8 was determined by isoelectric focussing. The protein contained some associated carbohydrate (20 mol hexose equiv/mol proteinase). Optimal proteolytic activity was observed at pH 3.0 (at 60°C). The Leu¹⁵-Tyr¹⁶ bond was the major site of hydrolysis for the oxidized B chain of insulin. Enzyme activity was not affected by inhibitors of the cysteine, metallo or serine class of proteinases. The aspartate proteinase inhibitor, pepstatin, did not inhibit enzyme activity. Inhibition of enzyme activity by 1,2-epoxy-3-(p-nitrophenoxy)-propane indicated the presence of at least one carboxyl group essential to the catalytic mechanism of the enzyme. Proteinase activity was inhibited by diazoacetyl-  -norleucine methyl ester in a slow and non-specific manner atypical of pepstatin-sensitive aspartate proteinases. Wai 21a proteinase may be classified as member of the pepstatin-insensitive group of aspartate proteinases. The thermal stability at pH 3.0 and 60°C increased 2.1-fold (t1/2, 4.5–9.7 hr) in the presence of 5 mM Ca⁺⁺. An increase in both pH (3.0–4.5) and Ca⁺⁺ concentration (0–30 mM) resulted in a 15-fold increase (t1/2, 15–230 min) in thermal stability at 75°C. The amino acid composition of Wai 21a proteinase was found to be similar to other pepstatin-insensitive proteinases from bacterial sources and in particular similar to the thermostable enzyme, kumamolysin.

year: 1995

title: Effect of abomasal prebiotic supplementation on sheep faecal microbiota

authors: Li, Y.

subjects: prebiotic

abstract: The effect of abomasal fructo-oligosaccharides supplementation on sheep faecal microbiota was investigated in a balanced, two Latin square, cross-over design experiment. Ten fistulated sheep were managed in five consecutive periods, with each of five treatments (an 'acidified saline' control or one of four prebiotic candidates chosen to represent different types of oligosaccharides: Arabinogalactan, Fibruline, Raftilose, or Yacon) administered to two sheep in each period. Seven grams of fructo-oligosaccharides were used daily for each animal. In each period, fresh faecal samples were collected before, during and after 'supplementation' for the analysis of bifidobacteria, lactobacilli, Escherichia coli, sulphite-reducing clostridia, total anaerobes, pH and dry matter. The treatments with Fibruline, Raftilose and Yacon increased the sheep faecal bifidobacteria after 9 days of daily dosing. Compared with the control, the Raftilose treatment caused the greatest increase by 2.128 log10 CFU/g of faeces (P<0.01), followed by the Yacon and Fibruline treatments, by 0.990 (P<0.05) and 0.925 log10 CFU/g of faeces (P=0.05), respectively. Both Raftilose and Yacon treatments increased the number of lactobacilli by 1.286 (P<0.05) and 1.138 log10 CFU/g of faeces (P<0.05) after 9 days of daily dosing. All tested fructo-oligosaccharides had no effect on sulphite-reducing clostridia and E. coli. There were no changes in sheep faecal pH or dry matter between the treatments. These data provide the first evidence that the bifidogenic effects of fructo-oligosaccharides, previously shown to be gained in the large intestine of monogastric animals, are also efficacious in the ruminant.

text: Effect of abomasal prebiotic supplementation on sheep faecal microbiota The effect of abomasal fructo-oligosaccharides supplementation on sheep faecal microbiota was investigated in a balanced, two Latin square, cross-over design experiment. Ten fistulated sheep were managed in five consecutive periods, with each of five treatments (an 'acidified saline' control or one of four prebiotic candidates chosen to represent different types of oligosaccharides: Arabinogalactan, Fibruline, Raftilose, or Yacon) administered to two sheep in each period. Seven grams of fructo-oligosaccharides were used daily for each animal. In each period, fresh faecal samples were collected before, during and after 'supplementation' for the analysis of bifidobacteria, lactobacilli, Escherichia coli, sulphite-reducing clostridia, total anaerobes, pH and dry matter. The treatments with Fibruline, Raftilose and Yacon increased the sheep faecal bifidobacteria after 9 days of daily dosing. Compared with the control, the Raftilose treatment caused the greatest increase by 2.128 log10 CFU/g of faeces (P<0.01), followed by the Yacon and Fibruline treatments, by 0.990 (P<0.05) and 0.925 log10 CFU/g of faeces (P=0.05), respectively. Both Raftilose and Yacon treatments increased the number of lactobacilli by 1.286 (P<0.05) and 1.138 log10 CFU/g of faeces (P<0.05) after 9 days of daily dosing. All tested fructo-oligosaccharides had no effect on sulphite-reducing clostridia and E. coli. There were no changes in sheep faecal pH or dry matter between the treatments. These data provide the first evidence that the bifidogenic effects of fructo-oligosaccharides, previously shown to be gained in the large intestine of monogastric animals, are also efficacious in the ruminant.

year: 2010

title: A Portfolio of Compositions

authors: Dobson, Elizabeth Jane

subjects: music

abstract: The pieces in this portfolio cover a wide range of methods and styles - from work for a single vocalising percussionist, to singing bowl and electronics and a work for full orchestra. The aim of this portfolio is to demonstrate my technical and musical proficiency as well as to give an overview of my personal composing philosophy and style.
The portfolio opens with a work for orchestra, A Study in Scarlet. It differs from my previous orchestral works and is based on a musical code of Sir Arthur Conan Doyle's book, titled A Study in Scarlet. This was read in the 2009 Todd/ NSZO Young Composer Readings. The second work is .manatu.. It features tinkly piano, wind chimes and ethnic instrumentalists in a chamber ensemble to create a musical sound world. This was written for and premiered at Bang on a Can's summer music festival in Massachusetts 2009. The piano work .ātanga. is an adaptation of .manatu. for solo pianist and was written for the Dame Malvina Major Foundations Showcase Concert in New Plymouth. This adaptation proved to be a worthwhile exercise in efficient use of music material. Wrong Number is a Janet Frame poem set for soprano and real-time effect processing of audio. Confessions - Part I and II, for solo violin and string orchestra is based on the Fibonacci series and explores my love for string sonorities. Sand Song was written for a friend who asked me to set a poem she wrote. Here I demonstrate my love for setting text and voice to say something beautiful and precise. This song now stands as a eulogy for her Father. Parihaka, for vocalising percussionist, is a setting of Apirana Taylor's poem Parihaka and was written for Australian performer Louise Devenish. I explore further in the realm of live acoustic and electroacoustic music with Orison a piece I wrote for myself to perform live with singing bowl and Ableton Live. Finally The Headlines Today for five improvising musicians explores the music of shouting and the vernacular of place.

text: A Portfolio of Compositions The pieces in this portfolio cover a wide range of methods and styles - from work for a single vocalising percussionist, to singing bowl and electronics and a work for full orchestra. The aim of this portfolio is to demonstrate my technical and musical proficiency as well as to give an overview of my personal composing philosophy and style.
The portfolio opens with a work for orchestra, A Study in Scarlet. It differs from my previous orchestral works and is based on a musical code of Sir Arthur Conan Doyle's book, titled A Study in Scarlet. This was read in the 2009 Todd/ NSZO Young Composer Readings. The second work is .manatu.. It features tinkly piano, wind chimes and ethnic instrumentalists in a chamber ensemble to create a musical sound world. This was written for and premiered at Bang on a Can's summer music festival in Massachusetts 2009. The piano work .ātanga. is an adaptation of .manatu. for solo pianist and was written for the Dame Malvina Major Foundations Showcase Concert in New Plymouth. This adaptation proved to be a worthwhile exercise in efficient use of music material. Wrong Number is a Janet Frame poem set for soprano and real-time effect processing of audio. Confessions - Part I and II, for solo violin and string orchestra is based on the Fibonacci series and explores my love for string sonorities. Sand Song was written for a friend who asked me to set a poem she wrote. Here I demonstrate my love for setting text and voice to say something beautiful and precise. This song now stands as a eulogy for her Father. Parihaka, for vocalising percussionist, is a setting of Apirana Taylor's poem Parihaka and was written for Australian performer Louise Devenish. I explore further in the realm of live acoustic and electroacoustic music with Orison a piece I wrote for myself to perform live with singing bowl and Ableton Live. Finally The Headlines Today for five improvising musicians explores the music of shouting and the vernacular of place.

year: 2010