by Yuen Wai Keung. / Thesis submitted in: December 1999. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 99-112). / Abstracts in English and Chinese. / Acknowledgement --- p.i / Abstract --- p.ii / Abstract (Chinese Version) --- p.iv / Abbreviations --- p.vi / Table of Contents --- p.viii / Chapter chapter one --- introduction --- p.1 / Chapter 1.1 --- Glutathione S-transferase --- p.2 / Chapter 1.1.1 --- Introduction --- p.2 / Chapter 1.1.2 --- Classification of mammalian GST --- p.2 / Chapter 1.1.3 --- Classification of insect GST --- p.7 / Chapter 1.1.4 --- Substrate specificity --- p.11 / Chapter 1.2 --- The chironomidae --- p.13 / Chapter 1.2.1 --- Biology and life history of chironomidae --- p.13 / Chapter 1.3 --- Chironomidae larvae --- p.16 / Chapter 1.3.1 --- Bloodworm t --- p.6 / Chapter 1.3.2 --- Sources of chironomidae larvae --- p.17 / Chapter 1.4 --- Aim of research --- p.18 / Chapter chapter two --- materials and methods --- p.20 / Chapter 2.1 --- Screening of GST in different subcellular fractions --- p.21 / Chapter 2.1.1 --- Preparation of mitochondria --- p.21 / Chapter 2.1.2 --- Preparation of microsomes --- p.22 / Chapter 2.1.3 --- Preparation of cytosol --- p.22 / Chapter 2.2 --- Assay for GST activity --- p.23 / Chapter 2.2.1 --- Activity Units --- p.23 / Chapter 2.3 --- Protein assay --- p.23 / Chapter 2.4 --- Preparation of glutathione-affinity column --- p.25 / Chapter 2.5 --- Purification of cytosolic GSTs --- p.26 / Chapter 2.5.1 --- Preparation of cytosol --- p.26 / Chapter 2.5.2 --- Chromatography on Sephadex G25 --- p.26 / Chapter 2.5.3 --- Affinity Chromatography --- p.26 / Chapter 2.5.3.1 --- Specific elution of GSTs --- p.26 / Chapter 2.5.3.2 --- Non-specific elution of GSTs --- p.27 / Chapter 2.5.4 --- Fast Protein Liquid Chromatography with Mono Q --- p.27 / Chapter 2.6 --- Determination of molecular mass --- p.29 / Chapter 2.6.1 --- Subunit molecular mass --- p.29 / Chapter 2.6.2 --- Native molecular mass --- p.31 / Chapter 2.7 --- Isoelectric focusing PAGE --- p.31 / Chapter 2.8 --- Enzyme activities and kinetic studies --- p.34 / Chapter 2.8.1 --- Optimum pH --- p.34 / Chapter 2.8.2 --- Heat inactivation assay --- p.34 / Chapter 2.8.3 --- Km and Vmax --- p.34 / Chapter 2.8.4 --- Substrate specificity --- p.35 / Chapter 2.8.5 --- Glutathione peroxidase activity --- p.38 / Chapter 2.9 --- N-terminal amino acid sequence analysis --- p.39 / Chapter 2.9.1 --- Semidry electroblotting --- p.39 / Chapter 2.9.2 --- Staining of proteins on PVDF membrane --- p.40 / Chapter 2.9.3 --- N-terminal amino acid sequence analysis --- p.40 / Chapter 2.9.4 --- On-membrane deblocking of protein --- p.40 / Chapter 2.9.5 --- BLAST search --- p.41 / Chapter chapter three --- results --- p.42 / Chapter 3.1 --- Screening of GST in different subcellular fractions --- p.43 / Chapter 3.2 --- Purification of cytosolic GSTs by chromatography --- p.45 / Chapter 3.2.1 --- Sephadex G25 column --- p.45 / Chapter 3.2.2 --- GSH affinity column --- p.45 / Chapter 3.2.3 --- Mono-Q column --- p.45 / Chapter 3.3 --- Determination of molecular mass --- p.53 / Chapter 3.3.1 --- Subunit molecular mass --- p.53 / Chapter 3.3.2 --- Native molecular mass --- p.53 / Chapter 3.4 --- Isoelectric point determination --- p.53 / Chapter 3.5 --- Enzymes activities and kinetic studies --- p.57 / Chapter 3.5.1 --- Optimum pH --- p.57 / Chapter 3.5.2 --- Thermostability --- p.57 / Chapter 3.5.3 --- Km and Vmax --- p.57 / Chapter 3.5.4 --- Substrate specificity --- p.76 / Chapter 3.5.5 --- Glutathione peroxidase Activity --- p.76 / Chapter 3.6 --- N-terminal amino acid sequence analysis --- p.83 / Chapter chapter four --- discussion --- p.89 / Chapter 4.1 --- GST in different subcellular fractions --- p.90 / Chapter 4.2 --- Purification of cytosolic GST --- p.91 / Chapter 4.3 --- Physical properties --- p.93 / Chapter 4.3.1 --- Subunit molecular mass --- p.93 / Chapter 4.3.2 --- Native molecular mass --- p.93 / Chapter 4.3.3 --- Isoelectric point --- p.95 / Chapter 4.4 --- Kinetic properties --- p.94 / Chapter 4.4.1 --- Optimum pH --- p.94 / Chapter 4.4.2 --- Thermostability --- p.95 / Chapter 4.4.3 --- Km and Vmax --- p.95 / Chapter 4.4.4 --- Substrate specificity --- p.96 / Chapter 4.4.5 --- Glutathione peroxidase activity --- p.96 / Chapter 4.5 --- N-terminal amino acid sequence data --- p.97 / Chapter 4.6 --- Conclusion --- p.98 / references --- p.99

Being the most abundant carbon-containing terrestrial biopolymer, lignocellulose serves as one of the best candidate feedstocks for biofuel production. The current cost-ineffective method for lignocellulose pretreatment is one of the major barriers that hinder the development of biofuel production. This leads to an exploration in the potential application of lignocellulolytic enzymes in this biorefinery process. Taking advantage of the strong activity of ligninolytic enzymes in L. edodes, we aimed at cloning and heterologously expressing these enzymes. The present project applied a yeast expression system, Pichia pastoris, as a laboratory-scale platform for heterologous expression of one of our target ligninolytic enzymes, manganese peroxidase (MnP). We successfully cloned and expressed recombinant MnP. Its enzymatic activity was the highest when grown in the presence of hemoglobin. Our long-term goal is to establish a platform for the large-scale production of recombinant lignocellulotyic enzymes at low-cost, which would strengthen their application in biofuel production. / The shitake mushroom, Lentinula edodes, is one of the most commonly consumed edible mushrooms in Asian countries. It is a saprophyte that naturally colonizes dead wood. As a member of wood-decaying white rot basidiomycete, L. edodes is able to depolymerize lignin and hydrolyze wood polysaccharides. However, the enzymatic mechanism for its lignocellulolytic system is poorly understood. Examination on the L. edodes genome and transcriptome revealed a unique lignocellulolytic system. L. edodes has a diverse enzymatic arsenal for lignin degradation. The enzymes include laccase, manganese peroxidase, cellobiose dehydrogenase and various lignin degrading auxiliary enzymes. When compared to another white rot fungus Phanerochaete chrysosporium, L. edodes possesses more hemicellulase- and pectinase-coding genes, and fewer genes encoding cellulases, suggesting that it preferentially attacks non-cellulosic polysaccharides. The transcription analysis on genes related to antioxidative mechanisms also offers insights to the oxidative stress encountered by mycelium during the free radical-mediated lignin degradation. / Kwok, Sze Wai. / Adviser: Hoi-Shan Kwan. / Source: Dissertation Abstracts International, Volume: 73-01, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 141-160). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Lai Chi-wai. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 93-100). / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.I / ABSTRACT --- p.II / 摘要 --- p.IV / ABBREVIATION --- p.VI / TABLE OF CONTENTS --- p.VII / LIST OF FIGURES --- p.XII / LIST OF TABLES --- p.XIV / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- Catalases --- p.2 / Chapter 1.2 --- Classification of catalases --- p.3 / Chapter 1.2.1 --- Catalase peroxidase (HPI) --- p.3 / Chapter 1.2.2 --- Monofunctional catalases (HPII) --- p.6 / Chapter 1.2.2.1 --- NADPH in catalases --- p.9 / Chapter 1.2.3 --- Mn-catalases --- p.11 / Chapter 1.3 --- Sources and cytotoxic effects of hydrogen peroxide --- p.13 / Chapter 1.4 --- The Chironomidae --- p.14 / Chapter 1.4.1 --- Life cycle of Chironomidae --- p.14 / Chapter 1.4.2 --- Bloodworms --- p.18 / Chapter 1.4.3 --- Sources of bloodworms --- p.19 / Chapter 1.5 --- Aim of the project --- p.22 / Chapter 1.6 --- Application of the project --- p.22 / Chapter CHAPTER 2 --- MATERIALS AND METHODS --- p.24 / Chapter 2.1 --- Protein determination --- p.25 / Chapter 2.2 --- In vitro activity assays --- p.27 / Chapter 2.2.1 --- Catalase activity assay --- p.27 / Chapter 2.2.2 --- Peroxidase activity assay --- p.27 / Chapter 2.3 --- Screening of catalase in different subcellular fractions --- p.28 / Chapter 2.3.1 --- Preparation of mitochondrial fractions --- p.28 / Chapter 2.3.2 --- Preparation of microsomal fractions --- p.29 / Chapter 2.3.3 --- Preparation of cytosolic fractions --- p.29 / Chapter 2.3.4 --- Preparation of post-mitochondrial fractions --- p.29 / Chapter 2.4 --- Purification of post-mitochondrial catalase --- p.29 / Chapter 2.4.1 --- Preparation of post-mitochondrial fractions --- p.30 / Chapter 2.4.2 --- Ethanol-chloroform precipitation --- p.30 / Chapter 2.4.3 --- Affinity chromatography --- p.30 / Chapter 2.4.4 --- Cation exchange chromatography --- p.31 / Chapter 2.5 --- Molecular mass determination --- p.34 / Chapter 2.6 --- Isoelectric focusing --- p.39 / Chapter 2.7 --- Kinetic studies of the purified enzyme --- p.42 / Chapter 2.7.1 --- Optimal pH --- p.42 / Chapter 2.7.2 --- Thermal stability --- p.42 / Chapter 2.7.3 --- Km and Vmax --- p.42 / Chapter 2.7.4 --- Inhibition studies --- p.43 / Chapter 2.7.4.1 --- "3-amino-1,2,4-triazole" --- p.43 / Chapter 2.7.4.2 --- Potassium cyanide and sodium azide --- p.43 / Chapter 2.8 --- Spectroscopic analysis --- p.44 / Chapter 2.8.1 --- Native enzyme --- p.44 / Chapter 2.8.2 --- Denatured enzyme --- p.44 / Chapter 2.8.3 --- Determination of pyridine hemochrome --- p.44 / Chapter 2.9 --- N-terminal amino acid sequence analysis for blotted protein --- p.45 / Chapter 2.9.1 --- Semi-dry electroblotting --- p.45 / Chapter 2.9.2 --- Protein staining on PVDF membrane --- p.46 / Chapter 2.9.3 --- N-terminal amino acid sequence analysis --- p.46 / Chapter 2.9.4 --- N-terminal deblocking of protein bound on PVDF membrane… --- p.47 / Chapter 2.9.5 --- BLAST® search --- p.48 / Chapter CHAPTER 3 --- RESULTS --- p.49 / Chapter 3.1 --- Catalase in different sub-cellular fractions --- p.50 / Chapter 3.2 --- Purification of post-mitochondrial catalase --- p.51 / Chapter 3.2.1 --- Ethanol-chloroform precipitation --- p.51 / Chapter 3.2.2 --- Affinity chromatography --- p.51 / Chapter 3.2.3 --- Cation exchange chromatography --- p.52 / Chapter 3.3 --- Determination of molecular mass --- p.57 / Chapter 3.4 --- Determination of isoelectric point --- p.57 / Chapter 3.5 --- Kinetic studies of the catalase --- p.62 / Chapter 3.5.1 --- Optimal pH --- p.62 / Chapter 3.5.2 --- Thermal stability --- p.62 / Chapter 3.5.3 --- Km and Vmax --- p.65 / Chapter 3.5.4 --- Inhibition studies --- p.65 / Chapter 3.5.4.1 --- "3-amino-1,2,4-triazole" --- p.65 / Chapter 3.5.4.2 --- Potassium cyanide and sodium azide --- p.65 / Chapter 3.5.5 --- Catalase peroxidase activity --- p.66 / Chapter 3.6 --- Spectroscopic analysis --- p.73 / Chapter 3.6.1 --- Native enzyme --- p.73 / Chapter 3.6.2 --- Denatured enzyme --- p.73 / Chapter 3.6.2.1 --- Potassium cyanide --- p.73 / Chapter 3.6.2.2 --- Sodium azide --- p.73 / Chapter 3.6.3 --- Pyridine hemochrome characterization --- p.73 / Chapter 3.7 --- N-terminal amino acid sequence analysis --- p.79 / Chapter CHAPTER 4 --- DISCUSSION --- p.81 / Chapter 4.1 --- Subcellular locations of catalase in bloodworms --- p.82 / Chapter 4.2 --- Purification of post-mitochondrial catalase --- p.82 / Chapter 4.3 --- Physical properties of the purified enzyme --- p.84 / Chapter 4.3.1 --- Native and subunit molecular mass --- p.84 / Chapter 4.3.2 --- Isoelectric point --- p.85 / Chapter 4.4 --- Kinetic properties of the purified enzyme --- p.85 / Chapter 4.4.1 --- Optimal pH --- p.85 / Chapter 4.4.2 --- Thermal stability --- p.85 / Chapter 4.4.3 --- Km and Vmax --- p.87 / Chapter 4.4.4 --- Inhibition studies --- p.87 / Chapter 4.4.5 --- Catalase peroxidase activity --- p.87 / Chapter 4.5 --- Spectroscopic analysis --- p.88 / Chapter 4.5.1 --- Native and denatured enzyme --- p.88 / Chapter 4.5.2 --- Pyridine hemochrome characterization --- p.88 / Chapter 4.6 --- N-terminal amino acid analysis --- p.89 / Chapter 4.7 --- Conclusions --- p.89 / REFERENCES --- p.93

In humans and most eukaryotes, thymidylate synthase (TSase) serves as a key enzyme that catalyzes the reductive methylation of deoxyuridine monophosphate (dUMP) to synthesize deoxythymidine monophosphate (dTMP), a key component of DNA. The N5, N10- methylene-5,6,7,8-tetrahydrofolate (MTHF) serves as both the methylene donor and the hydride donor while generating dihydrofolate (H2folate) as the byproduct. However, in 2002, Myllykallio reported the discovery of flavin-dependent thymidylate synthase (FDTS) that also functions to maintain the dTMP pool, although the mechanism is different. Since then, considerable progress was made in characterizing this enzyme. It was found that structurally FDTS is substantially different from TSase both with respect to structure and with respect to the mechanistic pathway of catalysis. In the FDTS-catalyzed methylation of dUMP, MTHF serves only as the methylene donor, generating tetrahydrofolate (H4folate), unlike TSase, and FDTS utilizes NADPH as a reductant. Activity of the enzyme depends on the presence of the noncovalently bound prosthetic group, flavin adenine dinucleotide (FAD). Interestingly, the enzyme FDTS is present in several human pathogens that cause diseases including syphilis, tuberculosis, anthrax poisoning, typhus, botulism, peptic ulcers and more, but is absent in humans; thus, it poses an attractive target for antibiotics. In the modern world, antibiotic resistance is a menace; consequently, new targets for new antibiotics are being sought. Hence, elucidating the chemical mechanism of FDTS is of paramount interest, as we and others believe this could allow for rational design of drugs that selectively target these pathogens with minimal human toxicity. Although several chemical mechanisms for FDTS catalysis have been put forward, complete understanding has still not been achieved. One of the primary concerns was the role of FAD in catalysis, and we found – as described in Chapter II and III – that FAD is a methylene carrier rather than just a hydride donor, as previously postulated. Secondly, all mechanisms proposed so far predict the presence of a noncovalently bound putative exocyclic methylene intermediate (an isomer of dTMP) occurring in the catalytic pathway of FDTS. However, direct evidence to prove its existence was lacking. Recently, we have been able to synthesize this intermediate, as described in Chapter IV. As shown in Chapter V and VI, we used steady-state kinetics, isotopic substitution and NMR studies to test this intermediate with FDTS. We believe our findings will greatly improve the understanding of this enzyme and will impact drug design by government agencies, pharmaceutical companies, and academic laboratories.

Graduation date: 1997

The principal methylglyoxal (MG)-detoxifying system in most living organisms is the two metalloenzyme Glyoxalase system. Glyoxalase I (GlxI) initially converts the non-enzymatically formed MG-GSH hemithioacetal to the thioester S,D-lactoylglutathione. The hydrolase, Glyoxalase II(GlxII) regenerates GSH and liberates the product D-lactate. Ni2+/Co2+- and Zn2+-activated GlxI enzymes exist in nature. The Ni2+/Co2+-activated GlxI are not active as Zn2+-holoenzymes in spite of the structural similarities to the Zn2+-dependent enzymes. The Zn2+-GlxI enzymes have been investigated heavily relative to the Ni2+/Co2+-activated enzymes, which have been isolated more recently. As part of this study the three GlxI homologs isolated from Pseudomonas aeruginosa were characterized. The homologous genes encode GlxI enzymes of both metal activation type. The Zn2+-activated P. aeruginosa GlxI is difficult to de-metallate compared to the Ni2+/Co2+-activated enzymesreflecting a difference in metal-binding/insertion between the two types of GlxI. The E. coli GlxII was isolated and characterized to determine whether Ni2+/Co2+-activation is a characteristic of the Glx system as a whole in this organism. Inductively coupled plasma mass spectrometry on purified E. coli GlxII confirms that the active protein is a binuclear Zn2+-metalloenzyme. The results to date indicate a detectable isotope effect for the Cd2+-holoenzyme but not the Ni2+-reconstituted enzyme. Chemical crosslinking experiments indicate that the SlyD Ni2+ metallochaperone does not form a complex with E.coli GlxI. This indicates that the E. coli active site is not metallated in vivo by this accessory protein. The principal biophysical experiment in this project was determining of Ni2+-binding stoichiometry for E. coli GlxI by 1H-15N heteronuclear single quantum coherence (HSQC) NMR. The GlxI dimer reorganization ceases when the metal:dimer stoichiometry reaches 0.5 during apoenzyme titration. This finding supports previous studies that indicate half-of-the-sites metal binding in this enzyme.

The principal methylglyoxal (MG)-detoxifying system in most living organisms is the two metalloenzyme Glyoxalase system. Glyoxalase I (GlxI) initially converts the non-enzymatically formed MG-GSH hemithioacetal to the thioester S,D-lactoylglutathione. The hydrolase, Glyoxalase II(GlxII) regenerates GSH and liberates the product D-lactate. Ni2+/Co2+- and Zn2+-activated GlxI enzymes exist in nature. The Ni2+/Co2+-activated GlxI are not active as Zn2+-holoenzymes in spite of the structural similarities to the Zn2+-dependent enzymes. The Zn2+-GlxI enzymes have been investigated heavily relative to the Ni2+/Co2+-activated enzymes, which have been isolated more recently. As part of this study the three GlxI homologs isolated from Pseudomonas aeruginosa were characterized. The homologous genes encode GlxI enzymes of both metal activation type. The Zn2+-activated P. aeruginosa GlxI is difficult to de-metallate compared to the Ni2+/Co2+-activated enzymesreflecting a difference in metal-binding/insertion between the two types of GlxI. The E. coli GlxII was isolated and characterized to determine whether Ni2+/Co2+-activation is a characteristic of the Glx system as a whole in this organism. Inductively coupled plasma mass spectrometry on purified E. coli GlxII confirms that the active protein is a binuclear Zn2+-metalloenzyme. The results to date indicate a detectable isotope effect for the Cd2+-holoenzyme but not the Ni2+-reconstituted enzyme. Chemical crosslinking experiments indicate that the SlyD Ni2+ metallochaperone does not form a complex with E.coli GlxI. This indicates that the E. coli active site is not metallated in vivo by this accessory protein. The principal biophysical experiment in this project was determining of Ni2+-binding stoichiometry for E. coli GlxI by 1H-15N heteronuclear single quantum coherence (HSQC) NMR. The GlxI dimer reorganization ceases when the metal:dimer stoichiometry reaches 0.5 during apoenzyme titration. This finding supports previous studies that indicate half-of-the-sites metal binding in this enzyme.

The evolutionary biology of 14 species of Maniolini (Nymphalidae: Satyrinae) was studied. Electrophoretic analysis of 35 enzyme loci identified a larger number of alleles than an1 levels of polymorphism similar to those found in other Lepidoptera. In Maniola jurtina, some populations exhibited a massive heterozygote deficit and sex associated allele frequency differentiation at the GOT-l locus. Allele frequencies in pre- and post-aestivation jurtina from southern Europe were significantly different. At some loci, significant annual differences in allele frequencies were noted. A significant correlation between geographic and genetic distance in allele frequencies was observed, but no correlation was detected between heterozygosity and land area in insular populations. Cluster analysis and nonmetric multidimensional scaling per~ormed on electrophoretic data from populations of Maniola jurtina revealed a dichotomy between 'Eastern' and 'Western' subspecies groups. The analysis of Manioline species fitted existing taxonomies. Genetic differences between Maniola species were much smaller than those between Pyronia and Hyponephele species. Ultrastructural studies of the Maniola Jullien organs revealed a species-specific tooth pattern on the inner surfaces. It is suggested that these structures may be sound production mechanisms of great evolutionary significance to the species. Maniolini ova were studied and it is suggested that their form and chorionic sculpturing owe much to selection induced by oviposition strategy. Chaetotaxy of first instar larvae was undertaken and morphometric analysis of setal lengths was found to be useful, but not unambiguous. Multivariate analysis of chaetotaxy data showed a significant correlation with electrophoretic data. viii The evolution and zoogeography of Maniola is discussed. It is suggested that disjunction, founder effect, rapid post-glacial colonisation and bottlenecking have played a major roles in effecting rapid speciation. It is further suggested that all Maniola species are very recent, perhaps having evolved within the last 50,000 years, and some species almost certainly have evolved in postglacial times •

Duas variações de resíduo agroindustrial foram analisadas como meio de cultura para o bioprocesso de fermentação semissólida pelo fungo Fusarium oxysporum, com o objetivo de obter a melhor produção de peptidases e lipases. Essas enzimas foram microencapsuladas por spray drying, visando garantir sua estabilidade e investigar outros prováveis benefícios obtidos pela técnica. A utilização de planejamento experimental permitiu analisar os efeitos e interações entre as variáveis operacionais do processo (temperatura de secagem, proporção de adjuvantes e relação entre adjuvantes). A caracterização bioquímica e físico-química do extrato enzimático e das micropartículas também foram estudadas. O emprego de farelo de trigo como substrato demonstrou maior produção enzimática que o uso de farelo de algodão. A fermentação produziu uma serinopeptidase e uma lipase, ambas com característica alcalina, com alta estabilidade em ampla faixa de pH e certa estabilidade em diferentes temperaturas. Para ambas as enzimas, observou-se modulação positiva da atividade frente à maioria dos íons estudados e forte inibição pelo surfactante SDS, enquanto a lipase demonstrou superatividade frente a CTAB. A caracterização enzimática permite sugerir a aplicação dessas enzimas na formulação de detergentes enzimáticos, indústria de couro, indústria de papel, agroquímicos, síntese de biopolímeros e biodísel. No processo de microencapsulação, a temperatura foi a variável operacional mais importante para a estabilidade, enquanto a quantidade de adjuvantes em relação à quantidade de extrato enzimático influenciou nas condições de manipulação. O estudo demonstrou que a técnica de microencapsulação por spray drying resultou em grande benefício no armazenamento das enzimas, por aumentar consideravelmente sua estabilidade e melhorar as propriedades físicas do extrato. / Two variations of agroindustrial residue were analyzed as culture medium for the bioprocess of solid-state fermentation by the fungus Fusarium oxysporum, in order to achieve the best production of peptidases and lipases. These enzymes were microencapsulated by spray drying in order to ensure its stability and investigate other potential benefits obtained by the technique. The use of experimental design allowed us to analyze the effects and interactions between the operating variables of the process (drying temperature, proportion of adjuvants and relation among adjuvants). Biochemical and physico-chemical characterization of the enzymatic extract and of the microparticles were also studied. The use of wheat bran as substrate demonstrated a greater enzyme production then the use of cottonseed meal. The fermentation produced serinepeptidases and lipases, both alkaline, with high stability over a wide pH range and some stability at different temperatures. For both enzymes, there was up regulation of activity with most of the ions analyzed and strong inhibition against the surfactant SDS, whereas lipase demonstrated superactivity against CTAB. The enzymatic characterization suggests the application of these enzymes in the formulation of enzymatic detergents, leather industry, paper industry, agrochemicals, synthesis of biopolymers and biodiesel. In the microencapsulation process, the temperature was the most important operating variable for stability, while the amount of adjuvants in relation to the amount of enzymatic extract influenced in terms of handling. The study demonstrated that the technique of microencapsulation by spray drying resulted in benefits for the storage of enzymes, by increasing considerably its stability and improve the physical properties of the extract.

The effects of temperature upon acetylcholinesterase (AChE) from the nervous system of fish were studied to determine if such compensatory phenomena as thermal accommodation, thermal acclimation and evolutionary adaptation to temperature as displayed by this physiological system could be observed and interpreted at the level of enzyme function. At probable physiological substrate concentrations the rate of acetylcholine (ACh) hydrolysis by AChE from rainbow trout (Salmo gairdnerii) and electric eel remains relatively unaffected by assay temperature over the temperature ranges normally experienced by these animals. Plots of Km versus temperature for these enzymes yield U shaped curves with minimum Km values occurring at temperatures close to the minimum habitat temperature. It is proposed that thermal accommodation of reaction rate is achieved throughout the habitat temperature range by temperature directed changes in enzyme-substrate affinity. Thermal acclimation in rainbow trout, and probably in speckled trout (Salvelinus fontinalis) and lake trout (Salvelinus namaychus) is accompanied by alterations in the relative proportions of two electrophoretically distinct AChE variants displaying different and adaptive Km-temperature relationships. Since the minimum Km values and energies of activation of the two rainbow trout enzymes are similar, and the specific activities of the enzymes are essentially identical following acclimation of fish to 2° and 17°C, it is suggested that rate compensation of AChE activity may not occur at different acclimation temperatures. However, the possibility remains that changes in such factors as pH, ionic environment and membrane lipids which accompany the acclimation process may act to stabilize reaction rates. Comparisons of AChE enzymes from rainbow trout, electric eel and the Antarctic fish Trematomus borchgrevinki indicate that the evolutionary adaptation of AChE function in species inhabiting different thermal environments is based upon selection for a Km-temperature relationship that will allow thermal accommodation of reaction rate over the temperature range normally encountered. Shifts in the Km-temperature relationship during speciation are interpreted in terms of changes in enzyme conformation following the accumulation of amino acid substitutions. Possible mechanisms by which two AChE enzymes could be incorporated into the trout central nervous system were considered and a hypothesis involving hybridization between fish populations was tested with trout inter-species crosses. It was observed that hybrids formed between speckled and lake trout contained a greater number of electrophoretically distinct AChE variants than did either parent and further, the presence of similar thermally controlled AChE complexes in rainbow, speckled and lake trout indicated that the original incorporation of multiple AChE enzymes into the rainbow trout probably occurred prior to the evolutionary divergence of these three species. It is concluded from this study that changes in enzyme-substrate affinity with temperature, and the temperature directed production of enzyme variants displaying adaptive Km-temperature relationships, are both important mechanisms for controlling catalytic activity in an enzyme system which functions over a wide range of temperatures. / Science, Faculty of / Zoology, Department of / Graduate