Daniel, Roy M.Schofield, Linley Rose2026-06-162026-06-161990https://hdl.handle.net/10289/18354Heat treatment was a successful purification step for five xylanolytic and cellulolytic enzymes cloned from the extreme thermophile ‘Caldocellum saccharolyticum’ strain Tp8T6.3.3.1. Temperature and length of heat treatment can be altered to give high recovery, or a high degree of purification. Higher purification could be gained by heat treatment at an acidic pH. Various studies showed that further factors, such as protein concentration, were also important. The substrate specificities of the five enzymes were investigated. The β-xylosidase had a very narrow substrate specificity, with high activity only on o- or p-nitrophenyl β-D-xylopyranoside and no activity on xylobiose. It showed some transferase activity. The xylanase had high activity on xylan, releasing xylobiose and some xylotriose. It also hydrolysed carboxymethylcellulose, 4-methylumbelliferyl β-D-cellobioside (MUC) and p-nitrophenyl β-D-cellobioside but could not hydrolyse xylobiose. It showed transferase activity on p-nitrophenyl β-D-xylopyranoside. The β-glucosidase also had broad substrate specificity, with activity on cellobiose and aryl glycosides, but not xylobiose. The carboxymethylcellulase acted as an endoglucanase, with high activity only on carboxymethylcellulose. The MUCase appeared to have two catalytic sites. The N-terminal domain acted as an exoglycobiohydrolase, with high activity on xylan, MUC and p-nitrophenyl β-D-cellobioside, while the C-terminal domain acted as an endoglucanase, with high activity on carboxymethylcellulose. The xylanase was purified and found to have a molecular weight of 42 000 and an isoelectric point of approximately 5.0. It showed optimum activity at pH 5.0 - 7.7 and had an activation energy of 44kJmol⁻¹. The half life at 75°C was 20 minutes in the presence of 0.5mgml⁻¹ bovine serum albumin. The xylanase was stable at room temperature at pH 4.5 - 11.5 in the presence of 0.5mgml⁻¹ bovine serum albumin. Xylose did not appear to inhibit the xylanase, but xylobiose may be inhibitory. The Kₘ for oat spelts xylan was 0.02% (w/v) at pH 6.0. The xylanase had higher activity on oat spelts and sugar cane bagasse xylan than larchwood xylan. The two catalytic residues were predicted to be positioned at the N-terminus of the xylanase. A truncated MUCase containing only one catalytic domain (the exoglycobiohydrolase) was studied. It retained activity on the substrates that the entire protein (containing all domains) hydrolysed. The truncated MUCase had a molecular weight of 84 000. Activities on MUC and Avicel were stable for at least 24 hours at 75°C. The activation energy of the truncated MUCase on MUC was lower (39kJmol⁻¹) than that of the entire protein (55kJmol⁻¹). The two catalytic residues of the exoglycobiohydrolase domain were predicted to be positioned at the N-terminus of the MUCase. The two catalytic residues of the endoglucanase domain, present in the entire protein, were predicted to be in the middle of the C-terminal endoglucanase domain.enAll items in Research Commons are provided for private study and research purposes and are protected by copyright with all rights reserved unless otherwise indicated.Thesis