Orientador: Pedro de Oliva Neto / Banca: Sandra Regina Ceccato Antonini / Banca: Eutimio Gustavo Fernandez Nuñez / Resumo: As amilases pertencem à classe das hidrolases e são enzimas de grande importância para aplicações industriais representando de 25 a 33% do mercado mundial de enzimas. Há uma gama extensiva de aplicações e um enorme interesse na descoberta de enzimas com melhores propriedades na degradação do amido. O presente trabalho visou otimizar a tecnologia de produção de amilases por Rhizopus microsporus var. oligosporus em fermentação em estado sólido com farelo de trigo. Em uma segunda etapa o extrato enzimático foi parcialmente caracterizado e utilizado para produção de xarope de glicose. Foi determinada a concentração ideal de sais para suplementação do meio de cultivo, a influência da umidade e inóculo na produção amilolítica utilizando-se um delineamento central composto rotacional (DCCR). Estabeleceu-se o protocolo para extração enzimática e verificou-se a produção ao longo do tempo em diferentes temperaturas de fermentação. A atividade enzimática foi determinada a partir da quantificação dos açúcares redutores liberados durante reação de hidrólise de amido. A menor concentração otimizada foi de 1,25% (m/m) de sulfato de amônio, 0,25% de fosfato de potássio e 1,25% de ureia. O melhor teor de umidade para a produção amilolítica foi de 50% e a extração mais eficiente ocorreu com 10 mL de água destilada por grama de substrato. A máxima produção enzimática ocorreu nos cultivos a 30 °C durante 120 h. A melhor temperatura para atividade amilolítica foi de 60 a 65 °C. A melhor estabilidade térmica (80%) foi obtida até 50 °C por 1 hora de incubação. A enzima produzida apresentou maior atividade em uma faixa de pH entre 4,0 e 5,0. A estabilidade foi superior a 80% após 1 hora de contato em pH de 3,0 a 7,0. O extrato enzimático bruto foi capaz de realizar a conversão total de amido em açúcar redutor após 6 horas de reação a 55 °C. Para a farinha de trigo tipo II foi obtido um rendimento... / Abstract: The amylases belong to the class of the hydrolases and they are enzymes of great importance to industrial applications, representing from 25% to 33% of the worldwide enzymes business. There is a wide range of applications and a huge interest in the discovery of enzymes with better properties in the starch degradation. The present work aims to optimize the technology of production of amylases by Rhizopus microsporus var. oligosporus in solid state fermentation with wheat bran. In second stage the enzymatic extract was partially characterized and used in glucose syrup production. The ideal concentration of salts to supplement the culture medium was determined, as well as the influence of moisture and inoculum on amylase production using a central composite rotational design (DCCR). The best protocol for enzymatic extraction and profiles of amylase production at different temperatures of fermentation were determined. The enzymatic activity was measured by the quantification of reducing sugars released during hydrolysis reaction of starch. The lowest optimal concentration was 1.25% (w/w) ammonium sulfate, 0.25% potassium phosphate and 1.25% urea. The best moisture level for amylolytic production was 50%, and the most efficient extraction was with 10 ml of distilled water per gram of substrate. The maximum enzyme production in the cultures was at 30 °C for 120 h. The best temperature for amylolytic activity was 60 to 65 °C, and better stability (80%) was obtained up to 50 °C for 1 hour of incubation. The enzyme produced showed greater activity in a pH range 4.0-5.0, and more than 80% of stability after 1 hour of contact in a pH range 3.0-7.0. The crude enzymatic extract was able to perform the total conversion of starch to reducing sugar in 6 hours of reaction at 55 °C. For type II wheat flour was obtained a yield of 83% in 6.5 hours, and for wheat bran, 62% in 7 hours. The product of the catalytic reaction was concentrated to obtain 20% ... / Mestre

No description available.

The proteasome is renowned for its high molecular weight, multisubunit and mulicatalytic nature. One of its many suggested roles is the degradation of myofibrillar proteins, and therefore it has been proposed to play a role in the meat tenderisation process. The aim of this study was therefore to isolate, purify and characterise the 20S proteasome from ostrich skeletal muscle, with a view to ultimately investigating its role in the tenderisation process of ostrich meat. The 20S proteasome was successfully isolated and purified from ostrich skeletal muscle using Toyopearl Super Q-650S, Sephacryl S-300, hydroxylapatite and Mono Q chromatographies. The intact molecule showed a molecular weight of 725 K and a pI of 6.67. The subunits showed a molecular weight range of 22.2-33.5 K and a pI range of 3-9. 2D-PAGE revealed at least 14 polypeptides. The amino acid composition of the intact enzyme and of each of the eight subunits separating on SDSPAGE, as well as the N-terminal sequences of five of the eight subunits, were determined. The trypsinlike (Tr-L), chymotrypsin-like (ChT-L), peptidylglutamyl peptide hydrolase (PGPH) and caseinolytic activities showed pH optima of 11, 9, 7-8 and 10.3, and temperature optima of 40, 60, 70 and 60oC, respectively. The pH stability range for all four activities was 5-12. The ChT-L and PGPH activities showed thermostabilities up to 60oC, whereas the Tr-L and caseinolytic activities were stable up to 40o C. The enzyme showed complex kinetics. It was inhibited by the peptide aldehyde Z-LLL-CHO and cysteine protease inhibitors. Cations had negligible effects on the enzyme, excepting for Ca2+ and Mg2+. Of the detergents tested, SDS had the most potent stimulatory effect, particularly on the PGPH and caseinolytic activities. The fatty acid studies showed that unsaturation enhanced the ChT-L and the caseinolytic activities, while it completely suppressed the Tr-L activity. Heating at 60oC for 1-2 min stimulated the caseinolytic and PGPH activities. The studies on the role of ostrich skeletal muscle 20S proteasome in ostrich meat tenderisation suggested a definite but minor role of this enzyme, based on the fact that it remained active throughout the 12 days of storage of ostrich M. iliofibularis meat at 4oC and that it participated in myofibril degradation of post-mortem muscle, but to a small degree. These results support the proposal that the proteasome comes into play after the calpains have initiated degradation. However, there was a lack of improvement in tenderness values and minimal myofibrillar degradation over the 12-day storage period of the ostrich M. iliofibularis meat, leading to the conclusion that the tenderisation of this meat was incomplete after 12 days.

It has been well-established that Pseudomonas species possess extremely versatile metabolic systems allowing them to utilise a wide range of nutrient sources and, furthermore, that the regulation of these enzyme systems involves highly evolved and sophisticated regulatory machinery. This study examined the complexity of metabolic regulation in Pseudomonas using the hydantoin-hydrolysing system of the environmental isolate, Pseudomonas putida RU-KM3s. In this system, the genes encoding dihydropyrimidinase and β-ureidopropionase (dhp and bup) are arranged divergently on the chromosome, separated by a 616 bp intergenic region involved in the transcriptional regulation of these genes. The focus was on the transcriptional regulation of dhp expression. DHP activity was found to be sensitive to several environmental signals including growth phase, carbon catabolite repression (CCR), substrate induction and quorum sensing (QS). Bioinformatic analysis of the intergenic region upstream of dhp revealed a number of putative binding sites for transcriptional regulators, including recognition sequences for the alternate sigma factors σ54 and σ38, as well as for the global regulators Anr (for anaerobic regulator) and Vfr (for virulence factor regulator). The targeted disruption of the genes encoding the transcriptional regulators, Vfr and the major CCR protein, Crc, resulted in a partial relief from repression for the vfr- mutant under quorum sensing conditions and a general decrease in activity in the crc- mutant. This data suggested that both Vfr and Crc were involved in regulating DHP activity. Mutational analysis of the dhp promoter revealed that at least two sites were involved in regulating transcriptional activity, one which mediated activation and the other repression. These sites were designated as a putative Anr box, situated 232 bp from the start codon of dhp, and a CRP-like binding site, at a position 213 bp upstream of dhp. Taken together, this data shows the involvement of several global regulatory factors in controlling the expression of dhp. A complex synergistic model was proposed for the transcriptional regulation of dhp, involving alternate sigma factors in addition to both global and specific regulators and responding to a number of environmental signals associated with growth phase, including nutrient availability, cell density and oxygen status.

Cathepsin D, a muscle proteinase, participates in lysosomally mediated protein degradation in vivo. This enzyme has been proposed to play a significant role in the postmortem proteolysis process apparently associated with tenderisation. The lack of data on the postmortem characteristics of ostrich meat, especially on the ageing process and its influence on meat tenderness, called for an investigation into this process. There is no data available for purified ostrich cathepsin D, and the aim of this study was, therefore, to isolate, purify and characterise cathepsin D from ostrich skeletal muscle and subsequently investigate the possible role that it may have in the tenderisation process of meat. Cathepsin D was successfully isolated and purified from ostrich skeletal muscle using pepstatin A-agarose chromatography. The purified enzyme was composed of two subunits (14 and 29kDa). The amino acid composition as well as the N-terminal amino acid sequence of both subunits were determined. Kinetic parameters (Km and Vm), thermodynamic parameters (Ea, ∆H, ∆S and ∆G) and functional characteristics (effect of pH, temperature and various inhibitors on cathepsin D activity) were determined and are reported in this study. Ostrich muscle cathepsin D showed a pH optimum of 4 and a temperature optimum of 45°C. The activity of cathepsin D was strongly inhibited by pepstatin A and DTT. Purified ostrich cathepsin D displayed kinetic and functional properties similar to previously reported values from various species. The effect of storage on the activity of cathepsin D was investigated over a 30 day period. It was established that substantial postmortem cathepsin D activity remained throughout the storage period, to implicate cathepsin D, fulfilling a possible role in meat maturation.

The ostrich is becoming an important source of meat for humans in developed and developing countries. This study was conducted to purify and characterize myofibrilbound serine protease (MBSP) and its endogenous inhibitor (MBSPI) from skeletal muscle of the ostrich. It is well documented that MBSP is tightly bound to myofibrils and its endogenous inhibitor has been purified from the same tissue of other studied mammalian species. Literature supports an association of MBSP and its endogenous inhibitor with the degradation of myofribrillar proteins, resulting in the softening of muscle that lead to the conversion of muscle into meat with the control of the inhibitor. MBSP was successfully dissociated from washed myofibrils by 40 percent ethylene glycol at pH 8.5. Following centrifugation, MBSP was partially purified in two chromatographic steps, namely Toyopearl Super Q 650S and p-aminobenzamidine-Agarose. On the other hand, MBSPI was fractionated from the sarcoplasmic fraction using 75 percent ammonium sulfate saturation, followed by centrifugation and partially purified by three chromatographic steps, namely Toyopearl Super Q 650S, Superdex 200 and HiTrap SP HR. Ostrich MBSP was physicochemically and kinetically characterized, while MBSPI was only physicochemically characterized. Ostrich MBSP revealed an Mr of 21 kDa, cleaving synthetic fluorogenic substrates specifically at the carboxyl side of arginine residues. Optimum pH and temperature of ostrich MBSP were 8.0 and 40˚C, respectively. Kinetic parameters (Km and Vmax values) were calculated from Lineweaver-Burk plots. The characteristics of ostrich MBSP were compared to the values obtained for commercial bovine trypsin in this study, as well as that obtained for MBSP from various fish species and mouse. The results suggest that ostrich MBSP is a trypsin-like serine protease, thereby confirming the existence of MBSP in ostrich skeletal muscle. Partially purified ostrich MBSPI (Mr 17 kDa) (one form) shares 100 percent identity to myoglobin from the same species, while 2 other forms of MBSPI (Mr values of 35 and 36 kDa) exhibited high sequence identity to glyceraldehyde 3- phosphate dehydrogenase (GAPDH) (76 percent) from human and rat.

CHAPTER ONE reviews the literature, discussing aspects of transition metal mediated asymmetric catalysis in the presence of enantiomerically pure ligands. CHAPTER TWO discusses the asymmetric addition of dialkyl-zinc reagents to aromatic aldehydes. The work presented is particularly concerned with the design and construction of enantiomerically pure oxazoline ligands tethered to alcohols These ligands have proved effective in the acceleration of the alkylation reaction and are able to influence good levels of asymmetric induction in the resultant secondary alcohol products CHAPTER THREE examines the electronic (and steric) effects of enantiomerically pure oxazoline ligands for the palladium catalysed allylic substitution reaction. Using ligands possessing two electronically different donor atoms, it is possible to create electronic distortion upon the intermediate allyl complex. In doing so it is possible to direct nucleophilic addition to one carbon centre preferentially to the other, resulting in asymmetric induction. Manipulation of these ligands enables control in the extent of electron distortion inflicted upon the allyl complex and consequently influences the levels of enantioselectivity observed. CHAPTER FOUR investigates the ability of hydrolytic enzymes to kinetically resolve a series of allylic acetates, under varying conditions. Lipases appeared superior to esterases for the substrates employed. In particular cis-3-acetoxy-5-carbomethoxycyclohexene was smoothly resolved m high yield and enantioselectivity. CHAPTER FIVE reports on the potentiality of a dynamic resolution of allylic acetates, using hydrolytic enzymes in the presence of a palladium catalyst. A proposed mechanism is discussed. Initial results are promising, however, the sensitivity of the reaction is realised and optimisation of conditions still needs to be addressed.

In recent years, two mechanisms have been proposed for the enzymatic formation of acetoacetate by liver extracts. One of these, the "HMG-CoA cycle", involves the condensation of acetyl-CoA and acetoacetyl-CoA to form β-hydroxy- β-methylglutaryl-CoA (HMG-CoA) via the action of the HMG-CoA condensing enzyme, with the release of free coenzyme A (CoASH) (reaction 1). Acetyl-CoA + acetoacetyI-CoA + H₂O⇆ HMG-CoA + CoASH (1) followed by cleavage of the HMG-CoA to acetyl-CoA and free acetoacetate, via the action of the HMG-CoA cleavage enzyme (reaction 2). HMG-CoA⇆ acetoacetate + acetyl-CoA (2) The second mechanism which has been proposed involves a direct deacylation of acetoacetyl CoA through the action of a specific acetoacetyl-CoA thioesterase (reaction). Acetoacetyl-CoA + H₂O→ acetoacetate + CoASH (3) Evidence is presented which indicates acetoacetate formation by a soluble enzyme system from bicarbonate extracts of whole beef liver proceeds largely, if not exclusively, via HMG-CoA (reactions 1 and 2). Both the HMG-CoA condensing and cleavage enzymes have been partially purified from beef liver bicarbonate extracts, each free of contamination by the other, in good yields. The level of activity of these two enzymes is sufficiently high to account for all the acetoacetate formed by liver tissue. The possibility that the specific acetoacetyl-CoA thioesterase may play a minor role in the enzymatic synthesis of acetoacetate is also discussed. The intracellular and tissue localization of the enzymes of acetoacetate formation is also discussed. In liver homogenates, most, if not all, of the acetoacetate-synthesizing activity appears to be associated with the mitochondrion. Evidence is also presented that the primary reason for the inability of extrahepatic tissue preparations to catalyze the accumulation of acetoacetate may be the lack of one of the enzymes involved, i.e., the HMG-CoA condensing enzyme, and not merely further metabolic degradation of acetoacetate, as has generally been assumed. An enzyme fraction in chicken liver extracts which inhibits the in vitro formation of acetoacetate by chicken liver homogenates has also been studied. Evidence is presented that this enzyme fraction exerts its effect through the inactivation of coenzyme A. Preliminary observations indicate that this enzyme may be a 3’-nucleotidase, removing the 3’- phosphate of coenzyme A, forming dephosphocoenzyme A. The occurrence of a highly active β-hydroxybutyryl dehydrogenase in extracts of dry culture of C. kluyveri has been noted. This enzyme differs from the similar enzyme reported in mammalian tissues, in that it is very specific for triphosphopyridine nucleotide, and is virtually inactive with diphosphopyridine nucleotide (DPN) (reaction). Acetoacetyl-CoA + TPNH + H⁺⇆β - hydroxybutyryl-CoA (4) + TPN⁺ / Medicine, Faculty of / Anesthesiology, Pharmacology and Therapeutics, Department of / Graduate

Proteolytic enzymes are common in plants but are usually specifi to endogenous protein. Plant proteases with specificities applicable to the food industry include papain, ficin and bromelain. Other plants have been used in traditional methods of food preparation for their proteolytic action on food components. The following species were investigated for propagation in tissue culture: Carica papaya, Ficus carica, Cynara cardunculus, Galium verum, Circium arvense, Dieffenbachia amoena, D. picta and Ananas comosus. Tissues of the first five of these demonstrated proteolytic activity by clearing of milk turbidity in agar medium. Commercial papain and ficin preparations are currently obtained from latex of immature papaya and fig fruit, respectively. This investigation was conducted, in part, to determine the feasibility of producing these two enzymes by the in vitro cell culture technique. Standard method of aseptic seed germination and leaf tissue excision were employed for callus initiation. Cell suspension cultures derived from callus were maintained in B5 medium at 28 °C in darkness. Proteolytic activity was determined by a modification of the Food Chemicals Codex method for papain and protein content was determined by Bradford's dye-binding, method. Production of protein and protease varied among cell cultures, but could be influenced by changes to some nutritional factors. Fig cells were grown in medium supplemented with single amino acids in the presence of either nitrate or ammonia as a source of inorganic nitrogen. All nitrate-based media produced higher yields of cell dry weight than ammonia-based media. Glutamic and aspartic acids were most stimulatory growth, protein accumulation and protease activity of fig cells. Skimmed milk, added at 3% (v/v), was a highly effective growth stimulant, and also resulted in higher protein and protease levels than the amino acids. Fresh casein and whey, added individually, produced similar results to skimmed milk. Citric acid, added at the level found in the 3% milk supplement, also caused stimulation of fig cell growth, protein synthesis and protease activity not significantly different from skimmed milk. It appears that nitrogen accumulation and reduction in fig cells may have been limited by an energy requirement which could be satisfied with the addition of citric acid or milk whey to the basal medium. / Land and Food Systems, Faculty of / Graduate

No description available.