No description available.

Using fresh papaya latex as the starting material, this thesis describes methods for the isolation and purification of three major proteinases by column adsorption chromatography; a comparative study of certain salient chemical and physical properties of these enzymes is presented. Reference is made to some conflicting results in the literature concerning papain studies, and an effort has been made in this work, by further experimentation, to account for some of the discrepancies. A supplementary study of biochemical interest has been the collecting of latex from the same growing fruit at fixed time intervals : analysis of the samples shows the changes in the yields of latex and enzyme content during the six months that the fruit takes to ripen.

Yeung, Ching Yee. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 112-122). / Abstracts in English and Chinese. / Acknowledgments --- p.I / Abstract --- p.II / 摘要 --- p.III / Content --- p.IV / Abbreviations and symbols --- p.XI / List of tables and figures --- p.XV / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Acylphosphatase --- p.1 / Chapter 1.2 --- Human acylphosphatase --- p.4 / Chapter 1.3 --- Hyperthermophilic Pyrococcus horikoshii acylphosphatase --- p.5 / Chapter 1.4 --- Human common-type acylphosphatase as a mesophilic homologue of Pyrococcus horikoshii acylphosphatase --- p.8 / Chapter 1.5 --- Enzyme-substrate interaction of acylphosphatase --- p.9 / Chapter Chapter 2 --- Materials and methods --- p.10 / Chapter 2.1 --- Preparation of Escherichia coli competent cells --- p.10 / Chapter 2.2 --- SDS-polyacrylamide gel electrophoresis --- p.11 / Chapter 2.2.1 --- Preparation of polyacrylamide gel --- p.11 / Chapter 2.2.2 --- SDS-polyacrylamide gel electrophoresis (SDS-PAGE) --- p.12 / Chapter 2.2.3 --- Staining of protein in polyacrylamide gel by Coommassie Brillant Blue R250 --- p.12 / Chapter 2.3 --- Expression and purification of Protein --- p.13 / Chapter 2.3.1 --- "General bacterial culture, harvesting and lysis" --- p.13 / Chapter 2.3.2 --- Purification of acylphosphatase --- p.14 / Chapter 2.3.2.1 --- Ion-exchange chromatography --- p.14 / Chapter 2.3.2.2 --- Size excision chromatography --- p.15 / Chapter 2.3.3 --- Protein concentration determination --- p.16 / Chapter 2.4 --- X-ray crystallography --- p.17 / Chapter 2.4.1 --- Crystallization of Hu CT AcP --- p.17 / Chapter 2.4.2 --- Model building and structural refinement --- p.18 / Chapter 2.4.3 --- Crystallization of Hu CT AcP -substate analogue complex --- p.19 / Chapter 2.5 --- Enzymatic Assay --- p.21 / Chapter 2.5.1 --- Preparation of benzoyl phosphate --- p.21 / Chapter 2.5.2 --- Purity check of the BP synthesized --- p.22 / Chapter 2.5.3 --- Determination of kinetic parameters of Hu CT AcP --- p.25 / Chapter 2.5.4 --- Determination of Ki value of substrate analogue --- p.27 / Chapter 2.6 --- Isothermal titration calorimetry --- p.28 / Chapter 2.7 --- Reagents and Buffers --- p.30 / Chapter 2.7.1 --- Reagent for competent cell preparation --- p.30 / Chapter 2.7.2 --- Media for bacterial culture --- p.31 / Chapter 2.7.3 --- Reagent for SDS-PAGE --- p.32 / Chapter 2.7.4 --- Buffer for AcP purification --- p.33 / Chapter 2.7.5 --- Buffer for enzymatic assay and ITC --- p.33 / Chapter Chapter 3 --- Structural determination of human common-type acylphosphatase --- p.34 / Chapter 3.1 --- Introduction --- p.34 / Chapter 3.2 --- Expression and purification of Hu CT AcP --- p.35 / Chapter 3.3 --- Structure of Hu CT AcP was determined by X-ray crystallography --- p.37 / Chapter 3.3.1 --- Crystallization of Hu CT AcP --- p.37 / Chapter 3.3.2 --- Model building and structural refinement --- p.41 / Chapter 3.3.3 --- Hu CT AcP shares a same α/β sandwich fold structure as other AcP --- p.43 / Chapter 3.4 --- Discussion --- p.46 / Chapter 3.4.1 --- Active site structure of Hu CT AcP is the same as those of bovine CT AcP and Ph AcP --- p.46 / Chapter 3.4.2 --- Absence of salt bridge between the active site residue and the C-terminal may contribute to the higher catalytic efficiency of Hu CT AcP --- p.52 / Chapter Chapter 4 --- Characterization of interaction between acylphosphatase and substrate analogues --- p.56 / Chapter 4.1 --- Introduction --- p.56 / Chapter 4.2 --- Selected substrate analogues --- p.57 / Chapter 4.3 --- Characterization of AcP-substrate analogue interaction by enzymatic assay --- p.59 / Chapter 4.3.1 --- Enzyme kinetics of Hu CT AcP was determined by the continuous optical assay of BP hydrolysis --- p.59 / Chapter 4.3.2 --- Substrate analogues were found to be competitive inhibitor to the AcP-catalyzed BP hydrolysis --- p.61 / Chapter 4.3.3 --- S-BA was the best competitive inhibitor against AcP-catalyzed BP hydrolysis --- p.64 / Chapter 4.3.4 --- S-BA was shown to be a competitive inhibitor for both Hu CT and Ph AcP --- p.66 / Chapter 4.4 --- Characterization of AcP-substrate analogue interaction by thermodynamic study --- p.68 / Chapter 4.4.1 --- Enthalpy change was observed for the association between substrate analogue and AcP --- p.68 / Chapter 4.4.2 --- S-BA was shown to bind Hu CT AcP with high affinity in ITC study --- p.68 / Chapter 4.5 --- S-BA was found to be the best substrate analogue for AcP --- p.72 / Chapter 4.6 --- Discussion --- p.73 / Chapter 4.6.1 --- Structure-affinity study of substrate analogue reveals chemical structures essential to interaction with AcP --- p.73 / Chapter 4.6.2 --- Structure-affinity study of substrate analogues is consistent with docking model of AcP with acetyl phosphate --- p.75 / Chapter 4.6.3 --- Validation of docking model by crystal complex structure --- p.78 / Chapter 4.6.4 --- Structural basis of substrate inhibition in Hu CT AcP --- p.80 / Chapter 4.6.4.1 --- Substrate inhibition is observed in Hu CT AcP --- p.80 / Chapter 4.6.4.2 --- Non-productive binding and substrate inhibition in AcP --- p.80 / Chapter Chapter 5 --- Investigation on the effect of salt bridge on acylphosphatase- substrate analogue interaction --- p.84 / Chapter 5.1 --- Introduction --- p.84 / Chapter 5.2 --- Thermodynamic study on the binding of S-BA with AcPs --- p.87 / Chapter 5.2.1 --- Determination of thermodynamic parameters of interaction between AcP and substrate analogue --- p.87 / Chapter 5.2.2 --- Determination of thermodynamic parameters as a function of temperature --- p.90 / Chapter 5.3 --- Discussion --- p.93 / Chapter 5.3.1 --- The presence of salt bridge leads to a reduced flexibility at the substrate binding active site --- p.93 / Chapter 5.3.2 --- The single salt bridge reduces the flexibility of active site in both study on thermodynamics of binding and thermodynamics of activation --- p.94 / Chapter 5.3.3 --- Temperature dependence of the thermodynamic parameters and heat capacity change ΔCp --- p.97 / Chapter 5.3.3.1 --- Change in heat capacity reveals the nature of the complex interface --- p.97 / Chapter 5.3.3.2 --- Determination of heat capacity change ΔCp --- p.98 / Chapter Chapter 6 --- Structural determination of acylphosphatase-substrate analogue complex --- p.102 / Chapter 6.1 --- Introduction --- p.102 / Chapter 6.2 --- Soaking and cocrystallization failed to give cocrystal structure of Hu CT AcP and S-BA --- p.103 / Chapter 6.4 --- Discussion --- p.106 / Chapter 6.4.1 --- Hu CT AcP and S-BA is not compatible with cocrystal formation --- p.106 / Chapter 6.5 --- Future prospect --- p.107 / Chapter 6.5.1 --- Structure determination by NMR spectroscopy --- p.107 / Chapter 6.5.2 --- Structure determination of AcP with aluminofluoride complexes --- p.108 / Chapter Chapter 7 --- Conclusion --- p.109 / Reference --- p.112

Thesis (Ph. D.)--University of Adelaide, Dept. of Plant Science, 1998. / Includes bibliographical references (leaves 114-141).

No description available.

An enrichment procedure led to the isolation, by the cellulose roll tube method, of a number of actively cellulolytic anaerobic thermophilic bacteria. Two isolates were terminally sporing rods and were tentatively identified as Clostridium thermocellulaseum (Enebo, 1951). Strain M-7 (0.6 μm x 4.0 μm) from manure grew optimally at 58°C to 63°C, pH 6.0 to 6.5 and did not require organic nitrogen. Strain C-19 (0.3 μm x 4.5 μm) from compost was similar but grew optimally at 50°C to 68°C, pH 7.5. Both utilized cellobiose and a wide range of other sugars. Strain C-19 did not utilize glucose, raffinose and inositol but did use inulin. The mean generation times in a rich nutrient medium containing cellobiose were 35 min for strain M-7 and 25 min for strain C-19. Strain M-7 had a mean generation time of 2 hr when grown on cellulose. Yeast extract (0.5%) stimulated growth and cellulase production by strain M-7 but was inhibitory at higher concentrations. Other organic nitrogen sources acted similarly. Cellulose at 1.0% gave maximum cellulase production after 72 hr incubation of strain M-7. Higher concentrations of cellulose were not completely degraded in 72 hr. Strain M-7 did not produce cellulase when grown on any carbon source other than cellulose substrates. The addition of cellobiose (0.3%) and glucose (0.4%) prevented cellulose hydrolysis in cellulose medium. This may have been repression of synthesis but cellulase was inhibited by both sugars. Both C₁, cellulase (degrades native cellulose) and Cx cellulase (β-1,4-glucanase) activities in strain M-7 cultures were assayed by measuring the liberation of reducing sugars, using dinitrosalicylic acid. Both activities had optima at pH 6.5 and 67°C. Cx cellulase could conveniently be assayed by a new automated procedure. Strain M-7 was very actively cellulolytic when compared to previously microbial species. The 48 hr culture contained Cx activity (56 μg glucose/min/ml from carboxymethyl cellulose) and C₁ activity (8 μg glucose/min/ml from cotton fibres); the ratio of C₁,:Cx was 1:7. The cellulase(s) from strain M-7 were extra-cellular, produced during exponential growth but were not free in the growth medium until 50% of the cellulose was hydrolyzed. Glucose and cellobiose were the only soluble products liberated by the cellulase from cellulose. ZnCl₂ precipitation appeared to be a good method for the concentration of cellulase activity but subsequent purification was not successful. Isoelectric focusing indicated the presence of four Cx cellulases (pI 4.5, 6.3, 6.8, and 8.7). DEAE-Sephadex chromatography indicated three Cx components. It is concluded that C. thermocellulaseum M-7 produces cellulase(s) capable of rapidly hydrolyzing native cellulose. The rapid production and high activity of cellulases from this organism strongly support the basic premise that increased hydrolysis of cellulose is possible at elevated temperature. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

No description available.

Reproduction de : Thèse de doctorat : Biologie et Biochimie Appliquée : Reims : 2004. / Titre provenant de l'écran titre. Bibliogr. f. 141-172.

Ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), a protein of 223 amino acids, is a member of deubiquitinating enzymes and it is one of the most abundant proteins in the brain. Although the in vivo functions of UCH-L1 are still unclear, its abundance and specificity for neurons indicate that it may serve an important role in neuronal cell function or dysfunction. Indeed, an isoleucine 93 to methionine amino acid mutation (I93M) in UCH-L1 was identified to be linked to an autosomal dominant form of Parkinson’s disease, while the serine 18 to tyrosine amino acid mutation (S18Y) in UCH-L1 is linked to a decreased susceptibility to Parkinson’s disease. To investigate the effects of these mutations on the structure of human UCH-L1, the mutant proteins have been successfully over-expressed, biophysically characterized and compared with the wild-type UCH-L1 using circular dichroism and NMR spectroscopy. While the data from circular dichroism and NMR chemical shift perturbation analysis suggested that the S18Y point mutation only slightly perturbs the global structure, the effect of the I93M point mutation was found to be more profound. In particular, the structural perturbations caused by I93M substitution are not only observed near the site of mutation, but are also found at more distant sites. These structural perturbations may be significant for the function of UCH-L1 and explain the reduced hydrolase activity (~55 % of wild-type) observed in UCH-L1-I93M, as the geometry of the catalytic triad (C90, H161 and D176) is likely to be distorted by this substitution. To provide further insights into the effect of serine 18 to tyrosine (S18Y) mutation on the structure and function of UCH-L1, the three-dimensional solution structure of UCH-L1-S18Y was determined by NMR spectroscopy. The solution structure of UCH-L1-S18Y reveals a monomer with a typical fold of papain-like cysteine proteases and consists of a six-membered antiparallel β-sheet surrounded by eight α-helices. Although the global structure is very similar to the crystal structure of wild-type UCH-L1, both the altered hydrogen bond network and the surface charge distributions have demonstrated that the S18Y substitution could lead to profound structural changes. In particular, the analysis of the difference in the dimeric interfaces of the wild-type and the S18Y mutant showed that the serine to tyrosine mutation can significantly affect the distribution of the surface-exposed residues involved in the dimeric interface. It is thought that such observed differences might weaken the stability of the UCH-L1 dimer and hence may explain the reduced dimerization-dependent ligase activity of UCH-L1-S18Y in comparison to the wild-type UCH-L1. / published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2005. / Härtill 4 uppsatser.