Manoel F. Guimaraes

Bioproduction of carotenoids: The comparative use of raw sugarcane juice and depolymerized bagasse by Phaffia rhodozyma

Brown sugar, raw sugarcane juice, and enzymatic hydrolyzates from amilaceous or ligno(hemi)cellulosic sources like cane bagasse turned out to be a minimal but satisfactory culture medium for the production of astaxanthin in Phaffia rhodozyma. The condition for this was a small supplementation (1 g/l) of a nitrogen source. Urea resulted in a preferential yeast biomass increase as compared to soya meal and tannery shavings, the major beneficial effect of which was in the pigment content of the yeast cells.

Acetobacter Cellulosic Biofilms Search for New Modulators of Cellulogenesis and Native Membrane Treatments

Since natural substances like pseudoxanthins exert a positive effect on the cellulogenic ability ofAcetobacter xylinum when producing cellulosic pellicles suitable for skin burn therapy, new defined and complex modulators were sought. Ca2+ and Mg2+ (4 mM) were strongly stimulatory. Na+ had no effect and K+ was inhibitory. Ammonium dihydrogen phosphate (0.12 g/L) ensured the same nitrogen supply as the same concentration of yeast extract as measured by cellomembrane dry wt./yield albeit higher yeast extract supplies produced thicker membranes. Corn steep liquor (CSL) was also progressively beneficial from 0.125 to 0.5 mL/L, and this yield could be further improved by the combination of CSL with a tea infusion (source of caffeine). Uridine (precursor for UDP-Glc, sugar donor in cellulose biosynthesis), guanine, guanosine, and its butirylated derivatives (precursors for the positive modulator of cellulose synthetase, di-cGMP) resulted in only moderate stimulation. Sodium phytate and betaine were also slightly stimulatory. The fibrilar product from a newAcetobacter isolate (Ax-M) was characterized as cellulose by comparison with the solid-state13C-NMR of algal cellulose. Its X-ray diffractogram was a confirmatory analysis. After incorporation of tamarind xyloglucan to previously air-dried cellulosic pellicles, diffractometry displayed only slight differences. Mercerized (5M NaOH) fresh cellulosic biofilms underwent drastic size reduction (3.5-fold), turning compact nut still flexible if maintained wet.

Culture of the Astaxanthinogenic Yeast Phaffia rhodozyma in Low-Cost Media

Growth of the yeastPhaffia rhodozyma was carried out in a simplified medium based on less expensive nutrient sources, such as diluted sugar cane juice, urea, and sodium phosphate. The usual content of the astaxanthin, an oxygenated pink carotenoid useful for fish flesh staining, was improved along with with good cell yields (respective values of >1300 μg/g cells and >5 g cells/L were observed). Yeast invertase and urease must therefore play an important role in the implementation of low-cost culture media.

Screening of Bothrops snake venoms for L-amino acid oxidase activity

Toxins, enzymes, and biologically active peptides are the main components of snake venoms from the genus Bothrops. Following the venom inoculation, the local effects are hemorrhage, edema, and myonecrosis. Nineteen different species of Brazilian Bothrops were screened for protein content and L-amino acid oxidase activity. B. cotiara, formerly found in the South of Brazil, is now threatened with extinction. Its venom contains a highly hemorrhagic fraction and, as expected from the deep yellow color of the corresponding lyophilized powder, a high L-amino acid oxidase CLAO) activity was also characterized. Flavin adenine dinucleotide (FAD) is its associate coenzyme. B. cotiara venom LAO catalyzed the oxidative deamination of several L-amino acids, and the best substrates were methionine, leucine, tryptophan, and phenylalanine, hence, its potential application for the use in biosensors for aspartame .determination and for the removal of amino acids from plasma. High levels for LAO were also found in other species than B. cotiara. In addition, the technique of isoelectric focusing (IEF) was employed as a powerful tool to study the isoor multienzyme distribution for LAO activity in the B. cotiara snake venom. Index Entries: Bothrops cotiara venom; LAO/L-amino acid oxidase; hemorrhagic activity; LAO isoelectrofocusing; LAO zymogram. *Author to whom all correspondence and reprint requests should be addressed. Applied Biochemistry and Biotechnology ] 9 7 WoL 51/52, 1995

Astaxanthinogenesis in the yeast Phaffia rhodozyma - optimization of low-cost culture media and yeast cell-wall lysis

Astaxanthin is a diketo-dihydroxy-carotenoid produced byPhaffia rhodozyma, a basidiomicetous yeast. A low-cost fermentation medium consisting of raw sugarcane juice and urea was developed to exploit the active sucrolytic/urelolytic enzyme apparatus inherent to the yeast. As compared to the beneficial effect of 0.1 g% urea, a ready nitrogen source, mild phosphoric pre inversion of juice sucrose to glucose and fructose, promptly fermentable carbon sources, resulted in smaller benefits. Corn steep liquor (CSL) was found to be a valuable supplement for both yeast biomass yield (9.2 g dry cells/L) and astaxanthin production (1.3 mg/g cells). Distillery effluent (vinace), despite only a slightly positive effect on yeast growth, allowed for the highest pigment productivity (1.9 mg/g cells). Trace amounts of Ni2 (1 mg/L, as a cofactor for urease) resulted in controversial effects, namely, biomass decrease and astaxanthin increase, with no effect on the release (and uptake) of ammonium ion from urea. Since the synthesized astaxanthin is associated with the yeast cell and the pigment requires facilitated release for aquaculture uses (farmed fish meat staining), an investigation of the yeast cell wall was undertaken using detergent-treated cells. The composition of the rigid yeast envelope was found to be heterogeneous. Its partial acid or enzymatic depolymerization revealed glucose and xylose as common monomeric units of the cell-wall glycopolymers. Yeast cell-wall partial depolymerization with appropriate hydrolases may improve the pigment bioavailability for captive aquatic species and poultry.Astaxanthin is a diketo-dihydroxy-carotenoid produced by Phaffia rhodozyma, a basidiomicetous yeast. A low-cost fermentation medium consisting of raw sugarcane juice and urea was developed to exploit the active sucrolytic/urelolytic enzyme apparatus inherent to the yeast. As compared to the beneficial effect of 0.1 g% urea, a ready nitrogen source, mild phosphoric pre inversion of juice sucrose to glucose and fructose, promptly fermentable carbon sources, resulted in smaller benefits. Corn steep liquor (CSL) was found to be a valuable supplement for both yeast biomass yield (9.2 g dry cells/L) and astaxanthin production (1.3 mg/g cells). Distillery effluent (vinace), despite only a slightly positive effect on yeast growth, allowed for the highest pigment productivity (1.9 mg/g cells). Trace amounts of Ni2 (1 mg/L, as a cofactor for urease) resulted in controversial effects, namely, biomass decrease and astaxanthin increase, with no effect on the release (and uptake) of ammonium ion from urea. Since the synthesized astaxanthin is associated with the yeast cell and the pigment requires facilitated release for aquaculture uses (farmed fish meat staining), an investigation of the yeast cell wall was undertaken using detergent-treated cells. The composition of the rigid yeast envelope was found to be heterogeneous. Its partial acid or enzymatic depolymerization revealed glucose and xylose as common monomeric units of the cell-wall glycopolymers. Yeast cell-wall partial depolymerization with appropriate hydrolases may improve the pigment bioavailability for captive aquatic species and poultry.

Production and Purification of CGTase of Alkalophylic Bacillus Isolated from Brazilian Soil

Alkalophylic bacilli that produce cyclodextringlycosyltransferase (CGTase) were isolated from Brazilian soil, with a scheme of two plating steps. In the first step, the bacterial isolate forms a halo in the cultivation medium that contains γ-cyclodextrin (CD) complexing dyes. The CGTase of an isolate was purified 157-fold by biospecific affinity chromatography, with β-CD showing a mol wt of 77,580 Daltons. It produces a γ- to β-CD ratio of 0.156 and a small amount of α-CD, using maltodextrin 10% as substrate, at 50°C, pH 8.0 and 22 h reaction time, reaching 21.4% conversion of the substrate to cyclodextrins. In the second screening step, the isolates chosen give larger halos with β-CD complexing dyes, and smaller halos with β-CD complexing dyes, leading to a 30% improvement in γ-CD selectivity, although at lower total yield for cyclodextrins (11.5%).

Screening ofBothrops snake venoms forl-amino acid oxidase activity

Toxins, enzymes, and biologically active peptides are the main components of snake venoms from the genusBothrops. Following the venom inoculation, the local effects are hemorrhage, edema, and myonecrosis.Nineteen different species of BrazilianBothrops were screened for protein content andl-amino acid oxidase activity.B. cotiara, formerly found in the South of Brazil, is now threatened with extinction. Its venom contains a highly hemorrhagic fraction and, as expected from the deep yellow color of the corresponding lyophilized powder, a highl-amino acid oxidase (LAO) activity was also characterized. Flavin adenine dinucleotide (FAD) is its associate coenzyme.B. cotiara venom LAO catalyzed the oxidative deamination of severall-amino acids, and the best substrates were methionine, leucine, tryptophan, and phenylalanine, hence, its potential application for the use in biosensors for aspartame determination and for the removal of amino acids from plasma. High levels for LAO were also found in other species thanB. cotiara. In addition, the technique of isoelectric focusing (IEF) was employed as a powerful tool to study the iso-or multienzyme distribution for LAO activity in theB. cotiara snake venom.

Astaxanthinogenesis in the Yeast Phaffia rhodozyma

Astaxanthin is a diketo-dihydroxy-carotenoid produced by Phaffia rhodozyma, a basidiomicetous yeast. A low-cost fermentation medium consisting of raw sugarcane juice and urea was developed to exploit the active sucrolytic/urelolytic enzyme apparatus inherent to the yeast. As compared to the beneficial effect of 0.1 g% urea, a ready nitrogen source, mild phosphoric pre inversion of juice sucrose to glucose and fructose, promptly fermentable carbon sources, resulted in smaller benefits. Corn steep liquor (CSL) was found to be a valuable supplement for both yeast biomass yield (9.2 g dry cells/L) and astaxanthin production (1.3 mg/g cells). Distillery effluent (vinace), despite only a slightly positive effect on yeast growth, allowed for the highest pigment productivity (1.9 mg/g cells). Trace amounts of Ni2 (1 mg/L, as a cofactor for urease) resulted in controversial effects, namely, biomass decrease and astaxanthin increase, with no effect on the release (and uptake) of ammonium ion from urea. Since the synthesized astaxanthin is associated with the yeast cell and the pigment requires facilitated release for aquaculture uses (farmed fish meat staining), an investigation of the yeast cell wall was undertaken using detergent-treated cells. The composition of the rigid yeast envelope was found to be heterogeneous. Its partial acid or enzymatic depolymerization revealed glucose and xylose as common monomeric units of the cell-wall glycopolymers. Yeast cell-wall partial depolymerization with appropriate hydrolases may improve the pigment bioavailability for captive aquatic species and poultry.

Screening of acetogenin-producing plants in Brazilian flora

The acetogenins are strongly bioactive natural compounds present in the bark, roots, leaves, and seeds of manyAnnonaceae plants. They are modified fatty acids and their cytotoxicities have been determined for different biological models including the in vitro growth inhibition of several human cancer cell lines.Very low acetogenin yield (< 0.1 g%) has been found previously in native phytobiomass, and we have now investigated the nonpredatory exploitation of the seeds as acetogenin sources characterizing the seed triacylglycerols (dominant fraction; > 90% of the whole lipid extracts) as potential valuable by-products.Supercritical fluid extraction (SFE) was utilized as an alternative for both kinds of lipid biomass, acetogenins and fats, and manipulation ofAnnona expiants for obtaining in vitro callus and/or new plants.

Stabilization and reutilization of Bacillus megaterium glucose dehydrogenase by immobilization

Glucose dehydrogenase (GDH) fromBacillus megaterium was immobilized using aminopropyl controlled-pore silica (CPS, average pore sizes of 170 and 500 Å) as a support and glutaraldehyde as a bifunctional crosslinking agent. The CPS-immobilized enzyme could be reused 12 times and the best results were obtained using aminopropyl CPS-500 and bovine serum albumin as a feeder for stabilizing the protein layer on the support. DEAE-Sephadex (A-25 and A-50) was also used as a support for immobilizing GDH, with yields of around 42% for A-25 and 25–30% for A-50. The effect of pH on the immobilization procedure showed pH 6.5 to be better than pH 7.5 with respect to the recovery of enzyme activity. Both preparations of DEAE-Sephadex immobilized GDH could be reused several times and were thermostable at 40°C for 7 h. The kinetic parameters as Michaelis constant and maximum rate were determined for the immobilized enzyme and compared with those for the freeform.