ORGANOSILICON BIOTECHNOLOGY: A BIO-INSPIRED APPROACH TO THE HYDROLYSIS OF ALKOXYSILANES and THE LIPASE-CATALYZED SYNTHESIS OF SILOXANE-CONTAINING POLYESTERS AND POLYAMIDES

The first part of this thesis studied the capacity of amino acids and enzymes to catalyze the hydrolysis and condensation of tetraethoxysilane and phenyltrimethoxysilane. Selected amino acids were shown to accelerate the hydrolysis and condensation of tetraethoxysilane under ambient temperature, pressure and at neutral pH (pH 7±0.02). The nature of the side chain of the amino acid was important in promoting hydrolysis and condensation. Several proteases were shown to have a capacity to hydrolyze tri- and tet-ra- alkoxysilanes under the same mild reaction conditions. The second part of this thesis employed an immobilized Candida antarctica lipase B (Novozym-435, N435) to produce siloxane-containing polyesters, polyamides, and polyester amides under solvent-free conditions. Enzymatic activity was shown to be temperature dependent, increasing until enzyme denaturation became the dominant pro-cess, which typically occurred between 120-130ᵒC. The residual activity of N435 was, on average, greater than 90%, when used in the synthesis of disiloxane-containing polyesters, regardless of the polymerization temperature except at the very highest temperatures, 140-150ᵒC. A study of the thermal tolerance of N435 determined that, over ten reaction cycles, there was a decrease in the initial rate of polymerization with each consecutive use of the catalyst. No change in the degree of monomer conversion after a 24 hour reaction cycle was found.

Enzyme glycation influences product yields during oligosaccharide synthesis by reverse hydrolysis

Possible evidence is presented for Maillard glycation of enzymes during oligosaccharide synthesis by reverse hydrolysis. In 70% (w/v) mannose solutions, 1,2-alpha-mannosidase from Penicillium citrinum lost 40% and alpha-mannosidase from almonds lost 60% activity at 55 degreesC over 2 weeks. Oligosaccharide yields were 15 and 45% respectively. Higher molecular weight glycation adducts were formed in a time-dependent manner as seen by MALDI-TOF. Inhibitors of the Maillard. reaction were able to partially alleviate these effects resulting in reduced loss of enzyme activity and oligosaccharide yield increases of 27-53% relative to the control. (C) 2004 Elsevier B.V. All rights reserved.

Expression of four beta-galactosidases from Bifidobacterium bifidum NCIMB41171 and their contribution on the hydrolysis and synthesis of galactooligosaccharides

This paper deals with two aspects tightly related to the enzymatic characteristics and expression of four beta-galactosidases (BbgI, BbgII, BbgIII and BbgIV) from Bifidobacterium bifidum NCIMB41171. The growth patterns of this strain indicated a preference towards complex (i.e. lactose, galactooligosaccharides (GOSs)) rather than simple carbohydrates (i.e. glucose and galactose) and a collaborative action and synergistic relation of more than one beta-galactosidase isoenzyme for either lactose or GOS hydrolysis and subsequent assimilation. Native polyacrylamide gel electrophoresis analysis of protein extracts from cells growing on different carbohydrates (i.e. glucose, lactose or GOS) indicated that two lactose hydrolysing enzymes (BbgI and BbgIII) and one GOS hydrolysing enzyme (BbgII) were constitutively expressed, whereas a fourth lactose hydrolysing enzyme (BbgIV) was induced in the presence of lactose or different GOS fractions. Furthermore, the beta-galactosidase expression profiles of B. bifidum cells and the transgalactosylating properties of each individual isoenzyme, with lactose as substrate, clearly indicated that mainly three isoenzymes (BbgI, BbgIII and BbgIV) are implicated in GOS synthesis when whole B. bifidum cells are utilised. Two of the isoenzymes (BbgI and BbgIV) proved to have better transgalactosylating properties giving yields ranging from 42% to 47% whereas the rest (BbgI and BbgIII) showed lower yields (15% and 29%, respectively).

Regioselective synthesis of mannobiose and mannotriose by reverse hydrolysis using a novel 1,6-alpha-D-mannosidase from Aspergillus phoenicis

A novel 1,6-alpha-D-mannosidase was produced by Aspergillus phoenicis grown on a commercial manno-oligosaccharide preparation in liquid culture. The enzyme hydrolysed only alpha-D-Manp-(1 --> 6)-D-Manp and did not act on alpha-D-Manp-(1 --> 2)-D-Manp, or alpha-D-Manp-(1 --> 3)-D-Manp. The 1,6-alpha-D-mannosidase was used for synthesis of manno-oligosaccharides by reverse hydrolysis reaction. The highest yields, expressed as percentages (w/w) of total sugar, were similar to21% mannobiose and similar to5% mannotriose, and they were obtained with 45% (w/w) initial mannose concentration at pH 4.5 after 12 days incubation at 55 degreesC. The disaccharide and trisaccharide products were separated and their structures determined by methylation analysis. Only 1-6 linkages were found in both of them. (C) 2003 Elsevier B.V. All rights reserved.

Effect of enzymatic pretreatment and increasing the organic loading rate of lipid-rich wastewater treated in a hybrid UASB reactor

This study aimed at evaluating the effect of increasing organic loading rates and of enzyme pretreatment on the stability and efficiency of a hybrid upflow anaerobic sludge blanket reactor (UASBh) treating dairy effluent. The UASBh was submitted to the following average organic loading rates (OLR) 0.98 Kg.m(-3).d(-1), 4.58 Kg.m(-3).d(-1), 8.89 Kg.m(-3).d(-1) and 15.73 Kg.m(-3).d(-1), and with the higher value, the reactor was fed with effluent with and without an enzymatic pretreatment to hydrolyze fats. The hydraulic detention time was 24 h, and the temperature was 30 +/- 2 degrees C. The reactor was equipped with a superior foam bed and showed good efficiency and stability until an OLR of 8.89 Kg.m(-3).d(-1). The foam bed was efficient for solid retention and residual volatile acid concentration consumption. The enzymatic pretreatment did not contribute to the process stability, propitiating loss in both biomass and system efficiency. Specific methanogenic activity tests indicated the presence of inhibition after the sludge had been submitted to the pretreated effluent It was concluded that continuous exposure to the hydrolysis products or to the enzyme caused a dramatic drop in the efficiency and stability of the process, and the single exposure of the biomass to this condition did not inhibit methane formation. (C) 2011 Elsevier B.V. All rights reserved.

AMP hydrolysis in soluble and microsomal rat cardiac cell fractions: kinetic characterization and molecular identification of 5 `-nucleotidase

The present study describes the enzymatic properties and molecular identification of 5`-nucleotidase in soluble and microsomal fractions from rat cardiac ventricles. Using AMP as a substrate, the results showed that the cation and the concentration required for maximal activity in the two fractions was magnesium at a final concentration of 1 mM. The pH optimum for both fractions was 9.5. The apparent K-m (Michaelis constant) values calculated from the Eadie-Hofstee plot were 59.7 +/- 10.4 mu M and 134.8 +/- 32.1 mu M, with V-max values of 6.7 +/- 0.4 and 143.8 +/- 23.8 nmol P-i/min/mg of protein (means +/- S.D., n = 4) from soluble and microsomal fractions respectively. Western blotting analysis of ecto-5`-nucleotidase revealed a 70 kDa protein in both fractions, with the major proportion present in the microsomal fraction. The presence of these enzymes in the heart probably has a physiological function in adenosine signalling. Furthermore, the presence of ecto-5`-nucleotidase in the microsomal fraction could have a role in the modulation of the excitation-contraction-coupling process through involvement of the Ca2+ influx into the sarcoplasmic reticulum. The measurement of maximal enzyme activities in the two fractions highlights the potential capacity of the different pathways of purine metabolism in the heart.

Optimization of fibrolytic enzyme production by Aspergillus japonicus C03 with potential application in ruminant feed and their effects on tropical forages hydrolysis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

FACTORS THAT AFFECT THE ENZYMATIC DEGRADATION OF NATURAL STARCH GRANULES - EFFECT OF THE SIZE OF THE GRANULES

In this study the relationship between the enzymatic susceptibility and the size of the com and cassava starch granules was studied. The starch granules were separated by size and classified according to their average diameter in: a) larger than 16 mum; b) between 15 and 10 mum and c) smaller than 10 mum. The starch granules of various sizes were hydrolyzed by bacterial alpha-amylase and fungal amyloglucosidase. The results showed a relationship between the enzymatic susceptibility and the size of the starch granules; smaller size of the starch granules resulted in a higher percentage of hydrolysis. A basic difference in the mode of action of enzymes on small and large granules was observed. Enzymatic attack on the large granules was characterized by considerable surface corrosion, mainly at the radial axis. For small granules, the enzymatic action occurred on the surface of the granules and was characterized by an erosion with solubilization of the granules. Chemical and physical analysis of the starches suggested that hydrolysis should occur mainly at the amorphous areas of the granules.

Electrogravimetric Real-Time and in Situ Michaelis-Menten Enzimatic Kinetics: Progress Curve of Acetylcholinesterase Hydrolysis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Purification and Characterization of an Ethanol-Tolerant β-Glucosidase from Sporidiobolus pararoseus and Its Potential for Hydrolysis of Wine Aroma Precursors

An extracellular ethanol-tolerant β-glucosidase from Sporidiobolus pararoseus was purified to homogeneity and characterized, and its potential use for the enhancement of wine aroma was investigated. The crude enzymatic extract was purified in four steps (concentration, dialysis, ultrafiltration, and chromatography) with a yield of around 40 % for total activity. The purified enzyme (designated Sp-βgl-P) showed a specific activity of approximately 20.0 U/mg, an estimated molecular mass of 63 kDa after sodium dodecyl sulfate polyacrylamide gel electrophoresis, and isoelectric point of 5.0 by isoelectric focusing. Sp-βgl-P has optimal activity at pH 4.0 and at 55 °C. It was stable in a broad pH range at low temperatures and it was tolerant to ethanol and glucose, indicating suitable properties for winemaking. The hydrolysis of glycosidic terpenes was analyzed by adding Sp-βgl-P directly to the wines. The released terpene compounds were evaluated by gas chromatography/mass spectrometry. The enzymatic treatment significantly increased the amount of free terpenes, suggesting that this enzyme could potentially be applicable in wine aroma improvement. © 2013 Springer Science+Business Media New York.

Improved pretreatments applied to the sugarcane bagasse and release of lignin and hemicellulose from the cellulose-enriched fractions by sulfuric acid hydrolysis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Identification and enzymatic characterization of acid phosphatase from Burkholderia gladioli

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Preliminary Characterization of Novel Extra-cellular Lipase from Penicillium crustosum Under Solid-State Fermentation and its Potential Application for Triglycerides Hydrolysis

Current studies about lipase production involve the use of agro-industrial residues and newly isolated microorganisms aimed at increasing economic attractiveness of the process. Based on these aspects, the main objective of this work is to perform the partial characterization of enzymatic extracts produced by a newly isolated Penicillium crustosum in solid-state fermentation. Lipase extract presented optimal temperature and pH of 37 A degrees C and 9-10, respectively. The concentrated enzymatic extract showed more stability at 25 A degrees C and pH 7. The enzymes kept 100% of their enzymatic activity until 60 days of storage at 4 and -10 A degrees C. The stability under calcium salts indicated that the hydrolytic activity presented decay with the increase of calcium concentration. The specificity under several substrates indicated good enzyme activities in triglycerides from C4 to C18.

Carbon isotope fractionation during calcium carbonate precipitation induced by urease-catalysed hydrolysis of urea

Stable carbon isotopic fractionation during calcium carbonate precipitation induced by urease-catalysed hydrolysis of urea was experimentally investigated in artificial water at a constant temperature of 30 degrees C. Carbon isotope fractionation during urea hydrolysis follows a Rayleigh distillation trend characterized by a C-13-enrichment factor of -20 to -22 parts per thousand. CaCO3 precipitate is up to 17.9 parts per thousand C-13-depleted relative to the urea substrate (-48.9 +/- 0.07 parts per thousand). Initial CaCO3 precipitate forms close to isotopic equilibrium with dissolved inorganic carbon. Subsequent precipitation occurs at -2 to -3 parts per thousand offset from isotopic equilibrium, suggesting that the initial delta C-13 value of CaCO3 is reset through dissolution followed by reprecipitation with urease molecules playing a role in offsetting the delta C-13 value of CaCO3 from isotopic equilibrium. Potentially, this isotopic systematics may provide a tool for the diagnosis of ureolytically-formed carbonate cements used as sealing agent. Moreover, it may serve as a basis to develop a carbon isotope tool for the quantification of ureolytically-induced CO2 sequestration. Finally, it suggests carbon isotope disequilibrium as a hallmark of past enzymatic activity in ancient microbial carbonate formation. (C) 2012 Elsevier B.V. All rights reserved.

Effects of pretreatment on morphology, chemical composition and enzymatic digestibility of eucalyptus bark: a potentially valuable source of fermentable sugars for biofuel production – part 1

Abstract Background In recent years, the growing demand for biofuels has encouraged the search for different sources of underutilized lignocellulosic feedstocks that are available in sufficient abundance to be used for sustainable biofuel production. Much attention has been focused on biomass from grass. However, large amounts of timber residues such as eucalyptus bark are available and represent a potential source for conversion to bioethanol. In the present paper, we investigate the effects of a delignification process with increasing sodium hydroxide concentrations, preceded or not by diluted acid, on the bark of two eucalyptus clones: Eucalyptus grandis (EG) and the hybrid, E. grandis x urophylla (HGU). The enzymatic digestibility and total cellulose conversion were measured, along with the effect on the composition of the solid and the liquor fractions. Barks were also assessed using Fourier-transform infrared spectroscopy (FTIR), solid-state nuclear magnetic resonance (NMR), X-Ray diffraction, and scanning electron microscopy (SEM). Results Compositional analysis revealed an increase in the cellulose content, reaching around 81% and 76% of glucose for HGU and EG, respectively, using a two-step treatment with HCl 1%, followed by 4% NaOH. Lignin removal was 84% (HGU) and 79% (EG), while the hemicellulose removal was 95% and 97% for HGU and EG, respectively. However, when we applied a one-step treatment, with 4% NaOH, higher hydrolysis efficiencies were found after 48 h for both clones, reaching almost 100% for HGU and 80% for EG, in spite of the lower lignin and hemicellulose removal. Total cellulose conversion increased from 5% and 7% to around 65% for HGU and 59% for EG. NMR and FTIR provided important insight into the lignin and hemicellulose removal and SEM studies shed light on the cell-wall unstructuring after pretreatment and lignin migration and precipitation on the fibers surface, which explain the different hydrolysis rates found for the clones. Conclusion Our results show that the single step alkaline pretreatment improves the enzymatic digestibility of Eucalyptus bark. Furthermore, the chemical and physical methods combined in this study provide a better comprehension of the pretreatment effects on cell-wall and the factors that influence enzymatic digestibility of this forest residue.