Metabolic engineering of Saccharomyces cerevisiae for second-generation ethanol production from xylo-oligosaccharides and acetate.

Simultaneous intracellular depolymerization of xylo-oligosaccharides (XOS) and acetate fermentation by engineered Saccharomyces cerevisiae offers significant potential for more cost-effective second-generation (2G) ethanol production. In the present work, the previously engineered S. cerevisiae strain, SR8A6S3, expressing enzymes for xylose assimilation along with an optimized route for acetate reduction, was used as the host for expressing two ß-xylosidases, GH43-2 and GH43-7, and a xylodextrin transporter, CDT-2, from Neurospora crassa, yielding the engineered SR8A6S3-CDT-2-GH34-2/7 strain. Both ß-xylosidases and the transporter were introduced by replacing two endogenous genes, GRE3 and SOR1, that encode aldose reductase and sorbitol (xylitol) dehydrogenase, respectively, and catalyse steps in xylitol production. The engineered strain, SR8A6S3-CDT-2-GH34-2/7 (sor1Δ gre3Δ), produced ethanol through simultaneous XOS, xylose, and acetate co-utilization. The mutant strain produced 60% more ethanol and 12% less xylitol than the control strain when a hemicellulosic hydrolysate was used as a mono- and oligosaccharide source. Similarly, the ethanol yield was 84% higher for the engineered strain using hydrolysed xylan, compared with the parental strain. Xylan, a common polysaccharide in lignocellulosic residues, enables recombinant strains to outcompete contaminants in fermentation tanks, as XOS transport and breakdown occur intracellularly. Furthermore, acetic acid is a ubiquitous toxic component in lignocellulosic hydrolysates, deriving from hemicellulose and lignin breakdown. Therefore, the consumption of XOS, xylose, and acetate expands the capabilities of S. cerevisiae for utilization of all of the carbohydrate in lignocellulose, potentially increasing the efficiency of 2G biofuel production.

Estratégias para recuperação de xilooligossacarídeos do licor de resíduos de eucalipto para avaliação do seu efeito estimulante em Staphylococcus xylosus / Strategies for xylooligosaccharides recovery from eucalyptus waste liquor to evaluate their stimulatory effect on Staphylococcus xylosus

Xilooligossacarídeos (XOS) são reconhecidos pelo seu potencial prebiótico relevante para diversos setores industriais e foram obtidos após o pré-tratamento hidrotérmico da biomassa lignocelulósica residual de galhos de eucalipto. Subprodutos inibitórios são gerados durante o processo de solubilização dos oligossacarídeos e acabam comprometendo a utilização do licor em microrganismos. Neste trabalho, o processo de destoxificação, hidrólise enzimática e atividade estimulantes de crescimento da bactéria Staphylococcus xylosus foram estabelecidos. Os resultados mostraram que a adsorção com carvão ativado em pó removeu cerca de 55% do ácido acético e mais de 90% do ácido fórmico, compostos fenólicos, lignina solúvel, furfural e 5-hidroximetilfurfural, e que a soma dos oligossacarídeos xilobiose (X2) e xilotriose (X3) foram maximizadas de 0,57 g/L para 1,21 g/L com 110 U/gXOS da enzima endoxilanase e 6,3% do licor destoxificado na hidrólise enzimática. O consumo de cerca de 63% de X2 e de 46% de X3 pela bactéria em meio basal deficiente em fontes de carbono, mas acrescido com os oligômeros, proporcionou maior crescimento celular em relação aos meios basais com alta composição de carbono, com e sem XOS, revelando seu potencial prebiótico pelo efeito estimulante de crescimento. (AU)

Xylooligosaccharides (XOS) are recognized for their prebiotic potential relevant to several industrial sectors and were obtained after hydrothermal pretreatment of residual lignocellulosic biomass from eucalyptus branches. Inhibitory by-products are generated during the solubilization process of oligosaccharides and end up compromising the utilization of the liquor in microorganisms. In this work, the detoxification process, enzymatic hydrolysis and growth stimulating activity of Staphylococcus xylosus bacteria were established. The results showed that adsorption with powdered activated carbon removed about 55% of acetic acid and more than 90% of formic acid, phenolic compounds, soluble lignin, furfural, and 5-hydroxymethyl furfural and the sum of the oligosaccharides xylobiose (X2) and xylotriose (X3) were maximized from 0.57 g/L to 1.21 g/L with 110 U/gXOS of the enzyme endoxylanase and 6.3% of the detoxified liquor in the enzymatic hydrolysis. The consumption of X2 and X3 were about 63% and 46%, respectively, by the bacteria in basal medium deficient in carbon sources, but in medium added with the oligomers, provided higher cell growth compared to basal medium with high carbon composition, with and without XOS, revealing its prebiotic potential by its growth-stimulating effect. (AU)

Deletion of AA9 Lytic Polysaccharide Monooxygenases Impacts A. nidulans Secretome and Growth on Lignocellulose.

Lytic polysaccharide monooxygenases (LPMOs) are oxidative enzymes found in viruses, archaea, and bacteria as well as eukaryotes, such as fungi, algae and insects, actively contributing to the degradation of different polysaccharides. In Aspergillus nidulans, LPMOs from family AA9 (AnLPMO9s), along with an AA3 cellobiose dehydrogenase (AnCDH1), are cosecreted upon growth on crystalline cellulose and lignocellulosic substrates, indicating their role in the degradation of plant cell wall components. Functional analysis revealed that three target LPMO9s (AnLPMO9C, AnLPMO9F and AnLPMO9G) correspond to cellulose-active enzymes with distinct regioselectivity and activity on cellulose with different proportions of crystalline and amorphous regions. AnLPMO9s deletion and overexpression studies corroborate functional data. The abundantly secreted AnLPMO9F is a major component of the extracellular cellulolytic system, while AnLPMO9G was less abundant and constantly secreted, and acts preferentially on crystalline regions of cellulose, uniquely displaying activity on highly crystalline algae cellulose. Single or double deletion of AnLPMO9s resulted in about 25% reduction in fungal growth on sugarcane straw but not on Avicel, demonstrating the contribution of LPMO9s for the saprophytic fungal lifestyle relies on the degradation of complex lignocellulosic substrates. Although the deletion of AnCDH1 slightly reduced the cellulolytic activity, it did not affect fungal growth indicating the existence of alternative electron donors to LPMOs. Additionally, double or triple knockouts of these enzymes had no accumulative deleterious effect on the cellulolytic activity nor on fungal growth, regardless of the deleted gene. Overexpression of AnLPMO9s in a cellulose-induced secretome background confirmed the importance and applicability of AnLPMO9G to improve lignocellulose saccharification. IMPORTANCE Fungal lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that boost plant biomass degradation in combination with glycoside hydrolases. Secretion of LPMO9s arsenal by Aspergillus nidulans is influenced by the substrate and time of induction. These findings along with the biochemical characterization of novel fungal LPMO9s have implications on our understanding of their concerted action, allowing rational engineering of fungal strains for biotechnological applications such as plant biomass degradation. Additionally, the role of oxidative players in fungal growth on plant biomass was evaluated by deletion and overexpression experiments using a model fungal system.

Xylo-Oligosaccharide Utilization by Engineered Saccharomyces cerevisiae to Produce Ethanol.

The engineering of xylo-oligosaccharide-consuming Saccharomyces cerevisiae strains is a promising approach for more effective utilization of lignocellulosic biomass and the development of economic industrial fermentation processes. Extending the sugar consumption range without catabolite repression by including the metabolism of oligomers instead of only monomers would significantly improve second-generation ethanol production This review focuses on different aspects of the action mechanisms of xylan-degrading enzymes from bacteria and fungi, and their insertion in S. cerevisiae strains to obtain microbial cell factories able of consume these complex sugars and convert them to ethanol. Emphasis is given to different strategies for ethanol production from both extracellular and intracellular xylo-oligosaccharide utilization by S. cerevisiae strains. The suitability of S. cerevisiae for ethanol production combined with its genetic tractability indicates that it can play an important role in xylan bioconversion through the heterologous expression of xylanases from other microorganisms.

Multi-omics analysis provides insights into lignocellulosic biomass degradation by Laetiporus sulphureus ATCC 52600.

BACKGROUND: Wood-decay basidiomycetes are effective for the degradation of highly lignified and recalcitrant plant substrates. The degradation of lignocellulosic materials by brown-rot strains is carried out by carbohydrate-active enzymes and non-enzymatic Fenton mechanism. Differences in the lignocellulose catabolism among closely related brown rots are not completely understood. Here, a multi-omics approach provided a global understanding of the strategies employed by L. sulphureus ATCC 52600 for lignocellulose degradation. RESULTS: The genome of Laetiporus sulphureus ATCC 52600 was sequenced and phylogenomic analysis supported monophyletic clades for the Order Polyporales and classification of this species within the family Laetiporaceae. Additionally, the plasticity of its metabolism was revealed in growth analysis on mono- and disaccharides, and polysaccharides such as cellulose, hemicelluloses, and polygalacturonic acid. The response of this fungus to the presence of lignocellulosic substrates was analyzed by transcriptomics and proteomics and evidenced the occurrence of an integrated oxidative-hydrolytic metabolism. The transcriptomic profile in response to a short cultivation period on sugarcane bagasse revealed 125 upregulated transcripts, which included CAZymes (redox enzymes and hemicellulases) as well as non-CAZy redox enzymes and genes related to the synthesis of low-molecular-weight compounds. The exoproteome produced in response to extended cultivation time on Avicel, and steam-exploded sugarcane bagasse, sugarcane straw, and Eucalyptus revealed 112 proteins. Contrasting with the mainly oxidative profile observed in the transcriptome, the secretomes showed a diverse hydrolytic repertoire including constitutive cellulases and hemicellulases, in addition to 19 upregulated CAZymes. The secretome induced for 7 days on sugarcane bagasse, representative of the late response, was applied in the saccharification of hydrothermally pretreated grass (sugarcane straw) and softwood (pine) by supplementing a commercial cocktail. CONCLUSION: This study shows the singularity of L. sulphureus ATCC 52600 compared to other Polyporales brown rots, regarding the presence of cellobiohydrolase and peroxidase class II. The multi-omics analysis reinforces the oxidative-hydrolytic metabolism involved in lignocellulose deconstruction, providing insights into the overall mechanisms as well as specific proteins of each step.

Screening of potential endoglucanases, hydrolysis conditions and different sugarcane straws pretreatments for cello-oligosaccharides production.

Cello-oligosaccharides (COS) are oligomers with 2 to 6 ß-1,4-linked glucose units, with potential applications in the food/feed and bioenergy industrial sectors. In this study, the combination of five heterologous expressed endoglucanases varying the temperature and pH conditions were evaluated by design of experiments for COS production. Afterwards, the best combination was tested to produce COS from different pretreated sugarcane straws: ionic liquid, diluted acid, hydrothermal and steam-explosion. The results showed that steam explosion pretreated sugarcane straw treated with CtCel9R enzyme at 50 °C and pH 5.0 yielded 13.4 mg COS g biomass-1, 5-18-fold higher compared to the other pretreated straws. Under the conditions evaluated, the removal of hemicellulose and decrease in the cellulose crystallinity can benefits the enzymatic hydrolysis. This is the first study that combined the evaluation of different enzymes, conditions, and sugarcane straw pretreatments to optimize COS production in a single step without glucose formation.

An alkaline active feruloyl-CoA synthetase from soil metagenome as a potential key enzyme for lignin valorization strategies.

Ferulic acid (FA), a low-molecular weight aromatic compound derived from lignin, represents a high-value molecule, used for applications in the cosmetic and pharmaceutical industries. FA can be further enzymatically converted in other commercially interesting molecules, such as vanillin and bioplastics. In several organisms, these transformations often start with a common step of FA activation via CoA-thioesterification, catalyzed by feruloyl-CoA synthetases (Fcs). In this context, these enzymes are of biotechnological interest for conversion of lignin-derived FA into high value chemicals. In this study, we describe the first structural characterization of a prokaryotic Fcs, named FCS1, isolated from a lignin-degrading microbial consortium. The FCS1 optimum pH and temperature were 9 and 37°C, respectively, with Km of 0.12 mM and Vmax of 36.82 U/mg. The circular dichroism spectra indicated a notable secondary structure stability at alkaline pH values and high temperatures. This secondary structure stability corroborates the activity data, which remains high until pH 9. The Small Angle X-Ray Scattering analyses resulted on the tertiary/quaternary structure and the low-resolution envelope in solution of FCS1, which was modeled as a homodimer using the hyperthermophilic nucleoside diphosphate-forming acetyl-CoA synthetase from Candidatus Korachaeum cryptofilum. This study contributes to the field of research by establishing the first biophysical and structural characterization for Fcs, and our data may be used for comparison against novel enzymes of this class that to be studied in the future.

Assessment of agricultural biomass residues to replace fossil fuel and hydroelectric power energy: A spatial approach.

Despite the recent discoveries of considerable fossil fuel reserves, Brazil is one of the only great economic and industrial powers with very high amounts of renewable energy in its electricity matrix. Approximately 79.3% of the electric energy supply comes from renewable resources, of which hydroelectric power represents 70.6%. The two primary concerns regarding hydroelectricity are the damage caused to the environment by the construction of dams and the uncertainty of the supply in cases of long drought seasons. This article presents an analysis on the availability and energy exploitation of sugarcane straw and forest residues derived from eucalyptus for decentralized generation using a Geographic Information System-based model. The potential bioelectricity and bioethanol production from sugarcane and eucalyptus biomass in the Administrative Region of Campinas (ARC) is higher than the demand in this region. The results provide guidelines for designing alternatives to the intended Nationally Determined Contributions in Brazil within the scope of the ARC, and they can be used to provide energy empowerment, electric matrix diversification, and new policies that address the residue availability and demand.

Chitosan/pectin polyelectrolyte complex as a pH indicator.

A polyelectrolyte complex (PEC) matrix formed between chitosan and pectin was developed to entrap a bioactive compound (anthocyanin), obtaining an useful pH indicator device. Polysaccharides of opposite charges such as chitosan and pectin can have a very strong intermolecular interaction. The innovation lies in obtaining a new system based on natural and biodegradable compounds, which is simple to manufacture, to indicate variation in pH by visual changes in colour. This device has potential applications in food packaging. The PEC was studied using chitosan and pectin solutions at different pHs values (3.0, 4.0, 5.0 and 5.5) and pectin/chitosan molar ratios (1.0 to 10/1.0 to 5.0). PEC films were homogeneous and showed the highest yield (60.0%) at pH 5.5. Diffusion tests indicated efficient bioactive compound entrapment in the PEC matrix. Thermogravimetric analysis (TGA), scanning electron microscopy (SEM) and Fourier transform infrared (FTIR) spectroscopy indicate the compatibility between the polymers and bioactive compound.

Cell mass energetic yields of fed-batch culture by Lipomyces starkeyi.

Estimation of the energy capacity of a microbial cell mass on the basis of its lipid content and elemental composition can be used for the comparative evaluation of different microbial sources of biodiesel. Lipomyces starkeyi cell mass concentration reached 94.6 g/L with 37.4% of lipids in a fed-batch process using xylose and urea as substrates. The fatty acid composition of the yeast oil was quite similar to that of palm oil. L. starkeyi converted more than 80% of the energy contained in xylose into cell mass energy yield. The approach used in this study makes it possible to determine the energy of a cell mass by its elemental composition. A heat of combustion (Q c) of 25.7 (kJ/g) was obtained for the cell mass after 142 h of fed-batch cultivation, which represents approximately 56% of the energy content of diesel oil (45.4 kJ/g). The Q c of the triacylglycerols produced was 48.9 (kJ/g), indicating the potential of this oleaginous yeast for biodiesel production. Our work developed here provides a simple and efficient tool for characterization of this cell mass to further our understanding of its use as a feedstock for bioenergy production.

Produção de biodiesel de terceira geração a partir de microalgas / Third generation biodiesel production from microalgae

O objetivo do estudo foi avaliar o biodiesel de 3 a geração produzido a partir do cultivo heterotrófico da microalga Phormidium sp., empregando amido de mandioca como fonte de carbono orgânico. Um planejamento experimental foi realizado para determinar as condições ótimas de temperatura e razão C/N. A partir da obtenção das melhores condições de cultivo desenvolveram-se cultivos em batelada e batelada alimentada em biorreator e avaliaram-se as características do biodiesel produzido. Os resultados indicaram que a temperatura de 30ºC e a razão C/N de 68 são as condições ideais do processo. As maiores produtividades em biomassa (50,41mgL-1h-1) e lipídica (7,49mgL-1h-1) foram obtidas no cultivo em batelada. Os ácidos graxos com maior representatividade foram os ácidos caproico (65,29%) e oleico (23,88%). As propriedades de combustão do biodiesel: conteúdo de ésteres (99,9%), número de cetano (54,88), índice de iodo (21,47gI2100g-1), grau de instauração (23,88%) e ponto de entupimento de filtro a frio (39,21ºC) se mostraram adequadas às principais normativas nacionais e internacionais.

The aim of the study was to evaluate the third generation biodiesel produced from heterotrophic cultivation of the microalgae Phormidium sp. employing cassava starch as source of organic carbon. An experimental design was performed to determine the optimal conditions of temperature and C/N ratio. From the best growing conditions, was developed cultivations in batch and fed-batch in a bioreactor and evaluated the biodiesel quality. The results indicate that the temperature of 30ºC and the C/N ratio of 68 are the ideal conditions of the process. The highest biomass productivity (50.41mgL-1h-1) and lipid productivity (7.49mgL-1h-1) were obtained in batch cultivations. The fatty acids most representative were caproic acid (65.29%) and oleic acid (23.88%). The fuel properties of biodiesel: ester content of 99.9%, cetane number of 54.88, iodine value of 21.47gI100g-1, unsaturation degree of 23.88% and a cold filter plugging point of 39.21ºC, comply with the main international and national standards.

Assessment of different carbohydrates as exogenous carbon source in cultivation of cyanobacteria.

Glucose is the substrate most widely used as exogenous carbon source for heterotrophic cultivation of cyanobacteria. Due to limited information about the use of different carbohydrates as carbon sources to support cyanobacterial heterotrophic metabolism, the objective of this work was to evaluate different monosaccharides (arabinose, fructose, galactose, glucose, mannose and xylose), disaccharides (lactose, maltose, sucrose and trehalose) and polysaccharides (carboxymethylcellulose, cassava starch, Hi-maize(®), maltodextrin Corn Globe 1805(®) and xylan) as exogenous carbon source for heterotrophic culture of cyanobacterium Phormidium sp. The batch cultivation using fructose as organic carbon source resulted in the highest (p < 0.05) cell biomass (5,540 mg/L) in parallel with the highest (p < 0.05) substrate yield coefficient (0.67 mg(biomass)/mg(fructose)). Mannose was the carbon source with the highest (p < 0.05) substrate consumption rate (3,185.7 mg/L/day) and maltodextrin was the carbohydrate with major potential to produce biomass (1,072.8 mg(biomass)/L/day) and lipids (160.8 mg(lipids)/L/day). Qualitatively, the fatty acid profiles of the lipid extract from Phormidium sp. showed predominance of saturated chains for the cultures grown with most of the carbon sources, with the exception of the ones grown with xylose and maltodextrin.

Changes in hyphal morphology due to chitosan treatment in some fungal species

In this work, changes in the hyphal morphology due to chitosan treatment in some fungal species were studied. Scanning electron microscope (SEM) observations revealed that chitosans with molar fraction of acetyl groups (F A 0.16 and 0.18) and degree of polymerization (DP 1,089 and 1,242) had a direct effect on the morphology of the chitosan-treated fungi, reflecting its potential for causing a delay in the growth of Alternaria alternata (500 µg × mL-1), Botrytis cinerea (1,000 µg × mL-1), Penicillium expansum (1,000 µg × mL-1) and Rhizopus stolonifer (500 µg × mL-1). Mycelial aggregation and structural changes such as excessive branching, swelling of the cell wall and hyphae size reduction were observed in the micrographs.

Production and characterization of amylases from Zea mays malt

In this work the α and β-amylase enzymes were obtained from maize (Zea mays) malt and were biochemistry characterized. A germination study to obtain the maize malt with good amylase activity was made. The maize seeds were germinated in laboratory and the enzymatic activity was measured daily. Activity dependence to germination time were fitted to an exponential model (A=A0eµt), which showed that the behaviour of enzymatic activity in the germination process was similar to the growth of the microorganism. Its model could be applied to describe the mechanism of α-amylase production for each maize varieties and others cereals. Maize malt characterization showed that α and β-amylase had optimal pH between 4-6.5, optimal temperature 50 and 90ºC, and molecular weight of 67.4 and 47.5kDa, respectively. This work contributed with the advances in biotechnology generating of conditions for application of a new and of low price amylases source.

Neste trabalho as enzimas α e β-amilases foram obtidas de malte de milho e depois foram caracterizadas bioquimicamente. Um estudo da germinação foi feito para obtenção do malte com boa atividade amilásica. A germinação ocorreu em escala laboratorial e a atividade enzimática foi medida diariamente. Um modelo exponencial do tipo A=A0eµt foi ajustado a dependência do tempo de germinação com a atividade, mostrando que o comportamento da atividade enzimática no processo de germinação é semelhante ao crescimento de microorganismos. Este modelo pode ser aplicado para descrever o mecanismo de produção da α-amilase para cada variedade de milho e de outros cereais. A caracterização do malte de milho mostrou que as α e β-amilase têm pH ótimo entre 4,0-6,5, temperatura ótima de 50 e 90ºC, e massa molar de 67,4 e 47,5 kDa, respectivamente. Este trabalho contribuiu com os avanços da biotecnologia gerando condições de emprego de uma nova e barata fonte de amilases.

Toluene gas phase biofiltration by Paecilomyces lilacinus and isolation and identification of a hydrophobin protein produced thereof.

Paecilomyces lilacinus consumed toluene as the sole carbon source in a gas-phase biofilter packed with perlite obtaining an average elimination capacity of 50 g m(-3) h(-1), a removal efficiency of 53%, and a final biomass of 31.6 mg biomass g dry support(-1). Hydrophobin proteins from the mycelium produced in the biofilter were purified by formic acid extraction and precipitated by electrobubbling, and the molecular weight was found to be 10.6 +/- 0.3 kDa. The peptide mass fingerprinting analysis of the purified hydrophobin by matrix-assisted laser desorption/ionization time-of-flight resulted in the identification of two peptides that presented high homology with sequences of class I hydrophobin proteins from other ascomycetous fungi when compared against the National Center for Biotechnology Information database. The yield of hydrophobin (PLHYD) from P. lilacinus was 1.1 mg PLHYD g biomass(-1). These proteins modified the hydrophobicity of Teflon by lowering the contact angle from 130.1 (+/-2) degrees to 57.0 (+/-5) degrees supporting hot sodium dodecyl sulfate washing. This work is the first report about biodegradation of toluene by the nematophagous fungus P. lilacinus in a gas-phase biofilter and the identification of its hydrophobin protein.

Caffeine degradation by Rhizopus delemar in packed bed column bioreactor using coffee husk as substrate

Diversos microrganismos incluindo bactérias, fungos e leveduras são capazes de assimilar a cafeína de meios sintéticos ou de resíduos de café. Existem poucos trabalhos sobre a via de degradação da cafeína em fungos filamentosos, principalmente por fermentação no estado sólido (FES). Estudos de degradação da cafeína por fungos filamentosos em FES usando casca de café como substrato vêm sendo realizados. O objetivo deste trabalho foi investigar a via de degradação da cafeína por Rhizopus delemar em biorreator de colunas aeradas e comparar este metabolismo de degradação com o da fermentação em frascos de vidro. As metilxantinas foram quantificadas por análises em HPLC. Os experimentos foram realizados com as condições otimizadas previamente: pH 6,5, 28ºC, 106 espores/g substrato, vazão de ar 60 mL/min e 73% de umidade inicial. Após 90 horas de fermentação, 65% da cafeína foi reduzida, resultando 0,19% de cafeína e 0,014% de teofilina na casca de café. Esta cepa provou ter habilidade para degradar cafeína e teofilina por FES em biorreator de colunas.

Caffeine degradation by Rhizopus delemar in packed bed column bioreactor using coffee husk as substrate

Various microorganisms including bacteria, yeast and fungi can degrade caffeine. There are few publications about caffeine degradation pathway in filamentous fungi, mainly by solid-state fermentation (SSF). Studies were carried out on degradation of caffeine and their metabolites by filamentous fungi in SSF using coffee husk as substrate. The purpose of this work was to investigate the caffeine degradation pathway by Rhizopus delemar in packed bed column fermenter and to compare this degradation metabolism with glass flasks fermentation. The methylxanthines were quantified by HPLC analysis. The experiments were realized with the optimized conditions in previous experiments: pH 6.5, 28ºC, inoculation rate 10(6) spores/g substrate, aeration rate 60 mL/min and initial moisture 73%. Under these conditions, after 72 hous of fermentation was achieved only 0.19% of caffeine and 0.014% of theophylline in the coffee husk. The strain proved to be able for caffeine and theophylline degradation by SSF in packed bed column bioreactor.

Diversos microrganismos incluindo bactérias, fungos e leveduras são capazes de assimilar a cafeína de meios sintéticos ou de resíduos de café. Existem poucos trabalhos sobre a via de degradação da cafeína em fungos filamentosos, principalmente por fermentação no estado sólido (FES). Estudos de degradação da cafeína por fungos filamentosos em FES usando casca de café como substrato vêm sendo realizados. O objetivo deste trabalho foi investigar a via de degradação da cafeína por Rhizopus delemar em biorreator de colunas aeradas e comparar este metabolismo de degradação com o da fermentação em frascos de vidro. As metilxantinas foram quantificadas por análises em HPLC. Os experimentos foram realizados com as condições otimizadas previamente: pH 6,5, 28ºC, 10(6) espores/g substrato, vazão de ar 60 mL/min e 73% de umidade inicial. Após 90 horas de fermentação, 65% da cafeína foi reduzida, resultando 0,19% de cafeína e 0,014% de teofilina na casca de café. Esta cepa provou ter habilidade para degradar cafeína e teofilina por FES em biorreator de colunas.

Characterization of alkaline xylanases from "Bacillus pumilus"

Alkaline xylanases produced by four different strains of "Bacillus pumilus" were characterized. The optimal pH and temperature were pH 9.0 and 60ºC for strain 13(a), and pH 8.0 and 55ºC for strains 5(2), 5(14), and 4(a). Under these conditions the following activities were found after 10 min in the presence of 1(per cent) xylan (birchwood): 328 U.ml(-1), 131 U.ml(-1), 90 U.ml(-1), and 167 U.ml(-1), respectively, for the four strains. The enzymes were stable at 40ºC, with 40(per cent) of the xylanase activity remaining after 2 hours for the enzymes of strain 5(2) and 60(per cent) for the other three strains. Stability at 50ºC was improved by addition of glycerol. Taking into account the conditions under which kraft pulps are bleached during the manufacture of paper xylanases from "B. pumilus" exhibit favorable potential for application to bleaching in the paper making process.

Purification of microbial (beta)-galactosidase from Kluyveromyces fragilis by bioaffinity partitioning

This work investigated the partitioning of (beta)-galactosidase from "Kluyveromyces fragilis" in aqueous two-phase systems (ATPS) by bioaffinity. PEG 4000 was chemically activated with thresyl chloride, and the biospecific ligand p-aminophenyl 1-thio-(beta)-D-galactopyranoside (APGP) was attached to the activated PEG 4000. A new two-step method for extraction and purification of the enzyme (beta)-galactosidase from "Kluyveromyces fragilis" was developed. In the first step, a system composed of 6(per cent) PEG 4000-APGP and 8(per cent) dextran 505 was used, where (beta)-galactosidase was strongly partitioned to the top phase (K = 2.330). In the second step, a system formed of 13(per cent) Peg-APGP and 9(per cent) phosphate salt was used to revert the value of the partition coefficient of (beta)-galactosidase (K = 2.0E-5) in order to provide the purification and recovery of 39(per cent) of the enzyme in the bottom salt-rich phase

Isolamento de princípios ativos da própolis por cromatografia em papel bidimensional e doseamento espectrofotométrico / Isolation of active principles of propolis through bidimensional paper chromatography and spectrophotometric determination

Através de cromatografia em papel bidimensional e doseamento espectrofotométrico de flavonóides totais, foi proposto um método de controle de qualidade para produtos contendo própolis. Este método foi aplicado a 21 amostras comerciais cujos rótulos declaravam a presença de própolis, mostrou ser acessível a laboratórios comuns e apresentou alta sensibilidade (AU).