一株丝状真菌对半纤维素利用的初步研究

本文筛选出一株能利用木糖产乙醇的丝状真菌Z7，对其利用木糖和半纤维素水解产物产乙醇的发酵条件进行了研究，并对Z7 利用玉米芯产木聚糖酶的条件进行了优化。全文分为三部分： 第一部分：目标微生物筛选、纯化及系统发育分析。以木糖为唯一碳源，采用梯度稀释和平板化线法从高温、中温酒曲中分离到16 株能利用木糖良好生长的丝状真菌；通过发酵试验复筛，获得一株能产乙醇的丝状真菌Z7；综合形态学和ITS 序列分析，初步鉴定为Aspergillus flavus。 第二部分：Z7 的乙醇发酵条件研究。以木糖为碳源，通过单因素试验确定最佳氮源和发酵温度；通过正交试验及SPSS 软件分析得到了不同N、P、K 成分对乙醇、残糖和菌体干重的影响。获得最佳的发酵条件为：（g/L）木糖50，尿素1, NH4NO3 1, K2HPO4 2 , KCl 0.5 , MgSO4.7H2O 0.5 , NaNO3 1 , pH 自然，培养温度33 ℃。以玉米芯半纤维素稀酸水解液为底物进行乙醇发酵，根据稀酸水解的单糖释放量和乙醇产量，确定115 ℃，1 h 为最佳玉米芯预处理条件；结合最佳发酵条件，添加1 g/L 的吐温20 能获得最大的乙醇浓度8.31 g/L。因此，Aspergillus flavus Z7 能利用半纤维素水解产物产乙醇，其中木糖的利用率80%以上。 第三部分：Z7 利用玉米芯产木聚糖酶条件优化。Aspergillus flavus Z7 在具有产乙醇能力的同时还具有产木聚糖酶的能力。本文通过单因素和正交试验得到最佳产酶培养基组分为：(g/L)玉米芯20，尿素2, 酵母膏2.5, K2HPO4 5，NaNO31, MgSO4.7H2O 1。单因素试验表明，用纱布代替塑料布密封摇瓶封口能显著提高产酶量；Z7 在碱性条件下具有更强的产酶性能。在最优条件下发酵，能产生最大木聚糖酶活122.23IU/mL。通过薄层分析，验证了Z7 产生的木聚糖酶具有水解木聚糖生成木糖及木寡糖的能力。 A strain of filamentous fungus which can produce ethanol by using the xylose was isolated in this research. The ethanol fermention conditions from xylose and dilute-acid hydrolyzate of the corn core were studied. The conditions of xylanase production by Z7 were also optimized. The paper involved three parts. Part1: Isolation, purification and phylogenetic analysis of the microbe. By using xylose as the single carbon source and the pla te streaking method, several filamentous fungi were isolated from the wine starter; through the fermentation test, a filamentous fungus Z7 which can produce ethanol was further recognized; furthermore, according to the morphologic observation and ITS seque nces analysis, Z7 was identified as Aspergillus flavus at the first step. Part2: Research on the condition of ethanol fermentation by Z7. By single factor experiment, the optional nitrogen resource and temperature of the fermentation were fixed; meanwhile, through the orthogonal array tests and the analysis of statistic software SPSS, the optional component of the culture medium and the fermentation condition were organized as follows: (g/L) xylose 50, urea 1, NH4NO3 1, K2HPO4 2, KCl 0.5 , MgSO4.7H2O 0.5, NaNO31, pH nature, temperature 33℃. Based on these optimal parameters, the fermentation of dilute-acid hydrolyzate of the corn core was carried on by Z7. According to the quantities of released sugar monomers and content of the ethanol, 115℃ in 1h is the best pretreatment condition; the maximal ethanol content can be obtained when 1g/L Tween 20 was added to. Therefore, the filamentous fungus Aspergillus flavus can use the hydrolysate of hemicellulose to produce ethanol, and the rate of xylose utilization was over 80%. Part3: Optimization of Z7’s xylanase producing condition from corn core. Aspergillus flavus Z7, which can utilize xylose or the hydrolysate of hemicellulose to produce ethanol, also had the ability of xylanase production. The optional component of the culture medium were fixed by the single factor experiment and the orthogonal array tests, and they were organized as follows: (g/L) corn core 20, Urea 2, Yeast extract 2.5, K2HPO4 5, NaNO31, MgSO4.7H2O 1; it was testified by the single factor experiment that sealing the shaking flasks with pledget other than plastic paper can obviously increase the xylanase activity; moreover, Z7 showed better xylanase production ability when in the alkali environment. Under the optional fermentation condition, the maximal xylanase activity 122.23IU/mL was proved. Through the analysis of thin- layer chromatography (TLC), the ability of xylanase from Z7, which can hydrolyze xylan to xylose monomer and oligomer, was vividly displayed.

Structural and Functional Characterization of a Novel Heterodimeric Kinesin in Candida albicans

Kinesins are molecular motors that transport intracellular cargos along microtubules (MTs) and influence the organization and dynamics of the MT cytoskeleton. Their force-generating functions arise from conformational changes in their motor domain as ATP is bound and hydrolyzed, and products are released. In the budding yeast Saccharomyces cerevisiae, the Kar3 kinesin forms heterodimers with one of two non-catalytic kinesin-like proteins, Cik1 and Vik1, which lack the ability to bind ATP, and yet they retain the capacity to bind MTs. Cik1 and Vik1 also influence and respond to the MT-binding and nucleotide states of Kar3, and differentially regulate the functions of Kar3 during yeast mating and mitosis. The mechanism by which Kar3/Cik1 and Kar3/Vik1 dimers operate remains unknown, but has important implications for understanding mechanical coordination between subunits of motor complexes that traverse cytoskeletal tracks. In this study, we show that the opportunistic human fungal pathogen Candida albicans (Ca) harbors a single version of this unique form of heterodimeric kinesin and we present the first in vitro characterization of this motor. Like its budding yeast counterpart, the Vik1-like subunit binds directly to MTs and strengthens the MT-binding affinity of the heterodimer. However, in contrast to ScKar3/Cik1 and ScKar3/Vik1, CaKar3/Vik1 exhibits weaker overall MT-binding affinity and lower ATPase activity. Preliminary investigations using a multiple motor motility assay indicate CaKar3/Vik1 may not be motile. Using a maltose binding protein tagging system, we determined the X-ray crystal structure of the CaKar3 motor domain and observed notable differences in its nucleotide-binding pocket relative to ScKar3 that appear to represent a previously unobserved state of the active site. Together, these studies broaden our knowledge of novel kinesin motor assemblies and shed new light on structurally dynamic regions of Kar3/Vik1-like motor complexes that help mediate mechanical coordination of its subunits.

Isolation of Alternaria alternata from an emollient cream:implications for public health

A specimen of emollient cream, which was observed to be contaminated peripherally with a filamentous fungus was examined for the presence of fungi and the resulting fungal colonies were examined phenotypically and genotypically. Subsequent DNA extraction and PCR amplification of the large internal transcribed spacer region [ITS1-5.8S-ITS2] yielded an amplicon of 512 bp. Sequence analysis identified this as Alternaria alternata at the 100% homology level with all 512/512 bases called. This organism has been previously reported as a cause of opportunistic infections involving skin and immunocompromised patients. This is the first report of an emollient cream as a source of this organism. It highlights the need for proper management of such preparations in order to minimize the potential spread of fungi to susceptible patient populations.

Bioethanol production from a sub-product of pulping industry

A vida da sociedade atual é dependente dos recursos fósseis, tanto a nível de energia como de materiais. No entanto, tem-se verificado uma redução das reservas destes recursos, ao mesmo tempo que as necessidades da sociedade continuam a aumentar, tornando cada vez mais necessárias, a produção de biocombustíveis e produtos químicos. Atualmente o etanol é produzido industrialmente a partir da cana-de-açúcar e milho, matérias-primas usadas na alimentação humana e animal. Este fato desencadeou o aumento de preços dos alimentos em todo o mundo e, como consequência, provocou uma série de distúrbios sociais. Os subprodutos industriais, recursos independentes das cadeias alimentares, têm-se posicionado como fonte de matérias-primas potenciais para bioprocessamento. Neste sentido, surgem os subprodutos gerados em grande quantidade pela indústria papeleira. Os licores de cozimento da madeira ao sulfito ácido (SSLs) são uma matériaprima promissora, uma vez que durante este processo os polissacarídeos da madeira são hidrolisados originando açúcares fermentáveis. A composição dos SSLs varia consoante o tipo de madeira usada no processo de cozimento (de árvores resinosas, folhosas ou a mistura de ambas). O bioprocessamento do SSL proveniente de folhosas (HSSL) é uma metodologia ainda pouco explorada. O HSSL contém elevadas concentrações de açúcares (35-45 g.L-1), na sua maioria pentoses. A fermentação destes açúcares a bioetanol é ainda um desafio, uma vez que nem todos os microrganismos são capazes de fermentar as pentoses a etanol. De entre as leveduras capazes de fermentar naturalmente as pentoses, destaca-se a Scheffersomyces stipitis, que apresenta uma elevada eficiência de fermentação. No entanto, o HSSL contém também compostos conhecidos por inibirem o crescimento de microrganismos, dificultando assim o seu bioprocessamento. Neste sentido, o principal objetivo deste trabalho foi a produção de bioetanol pela levedura S. stipitis a partir de HSSL, resultante do cozimento ao sulfito ácido da madeira de Eucalyptus globulus. Para alcançar este objetivo, estudaram-se duas estratégias de operação diferentes. Em primeiro lugar estudou-se a bio-desintoxicação do HSSL com o fungo filamentoso Paecilomyces variotii, conhecido por crescer em resíduos industriais. Estudaram-se duas tecnologias fermentativas diferentes para a biodesintoxicação do HSSL: um reator descontínuo e um reator descontínuo sequencial (SBR). A remoção biológica de inibidores do HSSL foi mais eficaz quando se usou o SBR. P. variotii assimilou alguns inibidores microbianos como o ácido acético, o ácido gálico e o pirogalol, entre outros. Após esta desintoxicação, o HSSL foi submetido à fermentação com S. stipitis, na qual foi atingida a concentração máxima de etanol de 2.36 g.L-1 com um rendimento de 0.17 g.g-1. P. variotti, além de desintoxicar o HSSL, também é útil na produção de proteína microbiana (SCP) para a alimentação animal pois, a sua biomassa é rica em proteína. O estudo da produção de SCP por P. variotii foi efetuado num SBR com HSSL sem suplementos e suplementado com sais. A melhor produção de biomassa foi obtida no HSSL sem adição de sais, tendo-se obtido um teor de proteína elevado (82,8%), com uma baixa concentração de DNA (1,1%). A proteína continha 6 aminoácidos essenciais, mostrando potencial para o uso desta SCP na alimentação animal e, eventualmente, em nutrição humana. Assim, a indústria papeleira poderá integrar a produção de bioetanol após a produção SCP e melhorar a sustentabilidade da indústria de pastas. A segunda estratégia consistiu em adaptar a levedura S. stipitis ao HSSL de modo a que esta levedura conseguisse crescer e fermentar o HSSL sem remoção de inibidores. Operou-se um reator contínuo (CSTR) com concentrações crescentes de HSSL, entre 20 % e 60 % (v/v) durante 382 gerações em HSSL, com uma taxa de diluição de 0.20 h-1. A população adaptada, recolhida no final do CSTR (POP), apresentou uma melhoria na fermentação do HSSL (60 %), quando comparada com a estirpe original (PAR). Após esta adaptação, a concentração máxima de etanol obtida foi de 6.93 g.L-1, com um rendimento de 0.26 g.g-1. POP possuía também a capacidade de metabolizar, possivelmente por ativação de vias oxidativas, compostos derivados da lenhina e taninos dissolvidos no HSSL, conhecidos inibidores microbianos. Por fim, verificou-se também que a pré-cultura da levedura em 60 % de HSSL fez com que a estirpe PAR melhorasse o processo fermentativo em HSSL, em comparação com o ensaio sem pré-cultura em HSSL. No entanto, no caso da estirpe POP, o seu metabolismo foi redirecionado para a metabolização dos inibidores sendo que a produção de etanol decresceu.

β-glucosidases from Aspergillus unguis NII 08123: Molecular characterization, properties and applications

Lignocellulosic biomass is probably the best alternative resource for biofuel production and it is composed mainly of cellulose, hemicelluloses and lignin. Cellulose is the most abundant among the three and conversion of cellulose to glucose is catalyzed by the enzyme cellulase. Cellulases are groups of enzymes act synergistically upon cellulose to produce glucose and comprise of endoglucanase, cellobiohydrolase and β-glucosidase. β -glucosidase assumes great importance due to the fact that it is the rate limiting enzyme. Endoglucanases (EG) produces nicks in the cellulose polymer exposing reducing and non reducing ends, cellobiohydrolases (CBH) acts upon the reducing or non reducing ends to liberate cellobiose units, and β - glucosidases (BGL) cleaves the cellobiose to liberate glucose completing the hydrolysis. . β -glucosidases undergo feedback inhibition by their own product- β glucose, and cellobiose which is their substrate. Few filamentous fungi produce glucose tolerant β - glucosidases which can overcome this inhibition by tolerating the product concentration to a particular threshold. The present study had targeted a filamentous fungus producing glucose tolerant β - glucosidase which was identified by morphological as well as molecular method. The fungus showed 99% similarity to Aspergillus unguis strain which comes under the Aspergillus nidulans group where most of the glucose tolerant β -glucosidase belongs. The culture was designated the strain number NII 08123 and was deposited in the NII culture collection at CSIR-NIIST. β -glucosidase multiplicity is a common occurrence in fungal world and in A.unguis this was demonstrated using zymogram analysis. A total 5 extracellular isoforms were detected in fungus and the expression levels of these five isoforms varied based on the carbon source available in the medium. Three of these 5 isoforms were expressed in higher levels as identified by the increased fluorescence (due to larger amounts of MUG breakdown by enzyme action) and was speculated to contribute significantly to the total _- β glucosidase activity. These isoforms were named as BGL 1, BGL3 and BGL 5. Among the three, BGL5 was demonstrated to be the glucose tolerant β -glucosidase and this was a low molecular weight protein. Major fraction was a high molecular weight protein but with lesser tolerance to glucose. BGL 3 was between the two in both activity and glucose tolerance.121 Glucose tolerant .β -glucosidase was purified and characterized and kinetic analysis showed that the glucose inhibition constant (Ki) of the protein is 800mM and Km and Vmax of the enzyme was found to be 4.854 mM and 2.946 mol min-1mg protein-1respectively. The optimumtemperature was 60°C and pH 6.0. The molecular weight of the purified protein was ~10kDa in both SDS as well as Native PAGE indicating that the glucose tolerant BGL is a monomeric protein.The major β -glucosidase, BGL1 had a pH and temperature optima of 5.0 and 60 °C respectively. The apparent molecular weight of the Native protein is 240kDa. The Vmax and Km was 78.8 mol min-1mg protein-1 and 0.326mM respectively. Degenerate primers were designed for glycosyl hydrolase families 1, 3 and 5 and the BGL genes were amplified from genomic DNA of Aspergillus unguis. The sequence analyses performed on the amplicons results confirmed the presence of all the three genes. Amplicon with a size of ~500bp was sequenced and which matched to a GH1 –BGL from Aspergillus oryzae. GH3 degenerate primers producing amplicons were sequenced and the sequences matched to β - glucosidase of GH3 family from Aspergillus nidulans and Aspergillus acculateus. GH5 degenerate primers also gave amplification and sequencing results indicated the presence of GH5 family BGL gene in the Aspergillus unguis genomic DNA.From the partial gene sequencing results, specific as well as degenerate primers were designed for TAIL PCR. Sequencing results of the 1.0 Kb amplicon matched Aspergillus nidulans β -glucosidase gene which belongs to the GH1 family. The sequence mainly covered the N-Terminal region of the matching peptide. All the three BGL proteins ie. BGL1, BGL3 and BGL5 were purified by chromatography an electro elution from Native PAGE gels and were subjected to MALDI-TOF mass spectrometric analysis. The results showed that BGL1 peptide mass matched to . β -glucosidase-I of Aspergillus flavus which is a 92kDa protein with 69% protein coverage. The glucose tolerant β -glucosidase BGL5 mass matched to the catalytic C-terminal domain of β -glucosidase-F from Emericella nidulans, but the protein coverage was very low compared to the size of the Emericella nidulans protein. While comparing the size of BGL5 from Aspergillus unguis, the protein sequence coverage is more than 80%. BGL F is a glycosyl hydrolase family 3 protein.The properties of BGL5 seem to be very unique, in that it is a GH3 β -glucosidase with a very low molecular weight of ~10kDa and at the same time having catalytic activity and glucose 122 tolerance which is as yet un-described in GH β -glucosidases. The occurrence of a fully functional 10kDA protein with glucose tolerant BGL activity has tremendous implications both from the points of understanding the structure function relationships as well as for applications of BGL enzymes. BGL-3 showed similarity to BGL1 of Aspergillus aculateus which was another GH3 β -glucosidase. It may be noted that though PCR could detect GH1, GH3 and GH5 β-glucosidases in the fungus, the major isoforms BGL1 BGL3 and BGL5 were all GH3 family enzymes. This would imply that β-glucosidases belonging to other families may also co-exist in the fungus and the other minor isoforms detected in zymograms may account for them. In biomass hydrolysis, GT-BGL containing BGL enzyme was supplemented to cellulase and the performances of blends were compared with a cocktail where commercial β- glucosidase was supplemented to the biomass hydrolyzing enzyme preparation. The cocktail supplemented with A unguis BGL preparation yielded 555mg/g sugar in 12h compared to the commercial enzyme preparation which gave only 333mg/g in the same period and the maximum sugar yield of 858 mg/g was attained in 36h by the cocktail containing A. unguis BGL. While the commercial enzyme achieved almost similar sugar yield in 24h, there was rapid drop in sugar concentration after that, indicating probably the conversion of glucose back to di-or oligosaccharides by the transglycosylation activity of the BGl in that preparation. Compared this, the A.unguis enzyme containing preparation supported peak yields for longer duration (upto 48h) which is important for biomass conversion to other products since the hydrolysate has to undergo certain unit operations before it goes into the next stage ie – fermentation in any bioprocesses for production of either fuels or chemicals.. Most importantly the Aspergillus unguis BGL preparation yields approximately 1.6 fold increase in the sugar release compared to the commercial BGL within 12h of time interval and 2.25 fold increase in the sugar release compared to the control ie. Cellulase without BGL supplementation. The current study therefore leads to the identification of a potent new isolate producing glucose tolerant β - glucosidase. The organism identified as Aspergillus unguis comes under the Aspergillus nidulans group where most of the GT-BGL producers belong and the detailed studies showed that the glucose tolerant β -glucosidase was a very low molecular weight protein which probably belongs to the glycosyl hydrolase family 3. Inhibition kinetic studies helped to understand the Ki and it is the second highest among the nidulans group of Aspergilli. This has promoted us for a detailed study regarding the mechanism of glucose tolerance. The proteomic 123 analyses clearly indicate the presence of GH3 catalytic domain in the protein. Since the size of the protein is very low and still its active and showed glucose tolerance it is speculated that this could be an entirely new protein or the modification of the existing β -glucosidase with only the catalytic domain present in it. Hydrolysis experiments also qualify this BGL, a suitable candidate for the enzyme cocktail development for biomass hydrolysis

Fungal community associated with marine macroalgae from Antarctica

Filamentous fungi and yeasts associated with the marine algae Adenocystis utricularis, Desmarestia anceps, and Palmaria decipiens from Antarctica were studied. A total of 75 fungal isolates, represented by 27 filamentous fungi and 48 yeasts, were isolated from the three algal species and identified by morphological, physiological, and sequence analyses of the internal transcribed spacer region and D1/D2 variable domains of the large-subunit rRNA gene. The filamentous fungi and yeasts obtained were identified as belonging to the genera Geomyces, Antarctomyces, Oidiodendron, Penicillium, Phaeosphaeria, Aureobasidium, Cryptococcus, Leucosporidium, Metschnikowia, and Rhodotorula. The prevalent species were the filamentous fungus Geomyces pannorum and the yeast Metschnikowia australis. Two fungal species isolated in our study, Antarctomyces psychrotrophicus and M. australis, are endemic to Antarctica. This work is the first study of fungi associated with Antarctic marine macroalgae, and contributes to the taxonomy and ecology of the marine fungi living in polar environments. These fungal species may have an important role in the ecosystem and in organic matter recycling.

Biochemical characterization of potential virulence markers in the human fungal pathogen Pseudallescheria boydii

The ubiquitous Pseudallescheria boydii (anamorph Scedosporium apiospermum) is a saprophytic filamentous fungus recognized as a potent etiologic agent of a wide variety of infections in immunocompromised as well as in immunocompetent patients. Very little is known about the virulence factors expressed by this fungal pathogen. The present review provides an overview of recent discoveries related to the identification and biochemical characterization of potential virulence attributes produced by P. boydii, with special emphasis on surface and released molecules. These structures include polysaccharides (glucans), glycopeptides (peptidorhamnomannans), glycolipids (glucosylceramides) and hydrolytic enzymes (proteases, phosphatases and superoxide dismutase), which have been implicated in some fundamental cellular processes in P. boydii including growth, differentiation and interaction with host molecules. Elucidation of the structure of cell surface components as well as the secreted molecules, especially those that function as virulence determinants, is of great relevance to understand the pathogenic mechanisms of P. boydii.

Purification, and Biochemical and Biophysical Characterization of Cellobiohydrolase I from Trichoderma harzianum IOC 3844

Because of its elevated cellulolytic activity, the filamentous fungus Trichoderma harzianum has a considerable potential in biomass hydrolysis applications. Trichoderma harzianum cellobiohydrolase I (ThCBHI), an exoglucanase, is an important enzyme in the process of cellulose degradation. Here, we report an easy single-step ion-exchange chromatographic method for purification of ThCBHI and its initial biophysical and biochemical characterization. The ThCBHI produced by induction with microcrystalline cellulose under submerged fermentation was purified on DEAE-Sephadex A-50 media and its identity was confirmed by mass spectrometry. The ThCBHI biochemical characterization showed that the protein has a molecular mass of 66 kDa and pi of 5.23. As confirmed by small-angle X-ray scattering (SAXS), both full-length ThCBHI and its catalytic core domain (CCD) obtained by digestion with papain are monomeric in solution. Secondary structure analysis of ThCBHI by circular dichroism revealed alpha-helices and beta-strands contents in the 28% and 38% range, respectively. The intrinsic fluorescence emission maximum of 337 nm was accounted for as different degrees of exposure of ThCBHI tryptophan residues to water. Moreover, ThCBHI displayed maximum activity at pH 5.0 and temperature of 50 degrees C with specific activities against Avicel and p-nitrophenyl-beta-D-cellobioside of 1.25 U/mg and 1.53 U/mg, respectively.

Palma forrageira (Opuntia ficus indica e Nopalea cochenillifera) como matéria-prima para produção de etanol celulósico e enzimas celulolíticas

The need for new sources of energy and the concern about the environment have pushed the search for renewable energy sources such as ethanol. The use of lignocellulosic biomass as substrate appears as an important alternative because of the abundance of this raw material and for it does not compete with food production. However, the process still meets difficulties of implementation, including the cost for production of enzymes that degrade cellulose to fermentable sugars. The aim of this study was to evaluate the behavior of the species of cactus pear Opuntia ficus indica and Nopalea cochenillifera, commonly found in northeastern Brazil, as raw materials for the production of: 1) cellulosic ethanol by simultaneous saccharification and fermentation (SSF) process, using two different strains of Saccharomyces cerevisiae (PE-2 and LNF CA-11), and 2) cellulolytic enzymes by semi-solid state fermentation (SSSF) using the filamentous fungus Penicillium chrysogenum. Before alcoholic fermentation process, the material was conditioned and pretreated by three different strategies: alkaline hydrogen peroxide, alkaline using NaOH and acid using H2SO4 followed by alkaline delignification with NaOH. Analysis of composition, crystallinity and enzymatic digestibility were carried out with the material before and after pretreatment. In addition, scanning electron microscopy images were used to compare qualitatively the material and observe the effects of pretreatments. An experimental design 2² with triplicate at the central point was used to evaluate the influence of temperature (30, 40 and 45 °C) and the initial charge of substrate (3, 4 and 5% cellulose) in the SSF process using the material obtained through the best condition and testing both strains of S. cerevisiae, one of them flocculent (LNF CA-11). For cellulase production, the filamentous fungus P. chrysogenum was tested with N. cochenillifera in the raw condition (without pretreatment) and pretrated hydrothermically, varying the pH of the fermentative medium (3, 5 and 7). The characterization of cactus pear resulted in 31.55% cellulose, 17.12% hemicellulose and 10.25% lignin for N. cochenillifera and 34.86% cellulose, 19.97% hemicellulose and 15.72% lignin for O. ficus indica. It has also been determined, to N. cochenillifera and O. ficus indica, the content of pectin (5.44% and 5.55% of calcium pectate, respectively), extractives (26.90% and 9.69%, respectively) and ashes (5.40% and 5.95%). Pretreatment using alkaline hydrogen peroxide resulted in the best cellulose recovery results (86.16% for N. cochenillifera and 93.59% for O. ficus indica) and delignification (48.79% and 23.84% for N. cochenillifera and O. ficus indica, respectively). This pretreatment was also the only one which did not increase the crystallinity index of the samples, in the case of O. ficus indica. However, when analyzing the enzymatic digestibility of cellulose, alkali pretreatment was the one which showed the best yields and therefore it was chosen for the tests in SSF. The experiments showed higher yield of conversion of cellulose to ethanol by PE-2 strain using the pretreated N. cochenillifera (93.81%) at 40 °C using 4% initial charge of cellulose. N. cochenillifera gave better yields than O. ficus indica and PE-2 strain showed better performance than CA-11. N. cochenillifera proved to be a substrate that can be used in the SSSF for enzymes production, reaching values of 1.00 U/g of CMCase and 0.85 FPU/g. The pretreatment was not effective to increase the enzymatic activity values

Production of pectinase by solid-state fermentation with Penicillium viridicatum RFC3

Endo-polygalacturonase (endo-PG), exo-polygalacturonase (exo-PG) and pectin liase (PL) were produced by solid-state fermentation of a mixture of orange bagasse and wheat bran (1:1) with the filamentous fungus Penicillium viridicatum RFC3. This substrate was prepared with two moisture contents, 70% and 80%, and each was fermented in two types of container, Erlenmeyer flask and polypropylene pack. When Erlenmeyer flasks were used, the medium containing 80% of initial moisture afforded higher PL production while neither exo- nor endo-PG production was influenced by substrate moisture. The highest enzyme activities obtained were 0.70 U mL(-1) for endo-PG, 8.90 U mL(-1) for exo-PG, and 41.30 U mL(-1) for PL. However, when the fermentation was done in polypropylene packs, higher production of all three enzymes was obtained at 70% moisture (0.7 and 8.33 U mL(-1) for endo- and exo-PG and 100 U mL(-1) for PL). An increase in the pH and decrease in the reducing sugar content of the medium was observed. The fungus was able to produce pectin esterase and other depolymerizing enzymes such as xylanase, CMCase, protease and amylase. (c) 2005 Elsevier Ltd. All rights reserved.

Effect of the addition of invert sugar on the production of cephalosporin C in a fed-batch bioreactor

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

Thermostable invertases from Paecylomyces variotii produced under submerged and solid-state fermentation using agroindustrial residues

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

Extracellular tannase from Emericella nidulans showing hypertolerance to temperature and organic solvents

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

Production of pectate lyase by penicillium viridicatum RFC3 in solid-state and submerged fermentation

Pectate lyase (PL) was produced by the filamentous fungus Penicillium viridicatum RFC3 in solid-state cultures of a mixture of orange bagasse and wheat bran (1 : 1 w/w), or orange bagasse, wheat bran and sugarcane bagasse (1 : 1 : 0.5 w/w), and in a submerged liquid culture with orange bagasse and wheat bran (3%) as the carbon source. PL production was highest (1,500U mL -1 or 300Ug -1 of substrate) in solid-state fermentation (SSF) on wheat bran and orange bagasse at 96 hours. PL production in submerged fermentation (SmF) was influenced by the initial pH of the medium. With the initial pH adjusted to 4.5, 5.0, and 5.5, the peak activity was observed after 72, 48, and 24 hours of fermentation, respectively, when the pH of the medium reached the value 5.0. PL from SSF and SmF were loaded on Sephadex-G75 columns and six activity peaks were obtained from crude enzyme fromSSF and designated PL I, II, III, IV, V, andVI, while five peaks were obtained fromcrude enzyme fromSmF and labeled PL I', II', III', IV', and VII'. Crude enzyme and fraction III from each fermentative process were tested further. The optimum pH for crude PL from either process was 5.5, while that for PL III was 8.0. The maximum activity of enzymes from SSF was observed at 35°C, but crude enzyme was more thermotolerant than PL III, maintaining its maximum activity up to 45°C. Crude enzyme from SmF and PL III' showed thermophilic profiles of activity, with maximum activity at 60 and 55°C, respectively. In the absence of substrate, the crude enzyme from SSF was stable over the pH range 3.0-10.0 and PL III was most stable in the pH range 4.0-7.0. Crude enzyme from SmF retained 70%-80% of its maximum activity in the acid-neutral pH range (4.0-7.0), but PIII showed high stability at alkaline pH (7.5-9.5). PL from SSF was more thermolabile than that from SmF. The latter maintained 60% of its initial activity after 1 h at 55°C. The differing behavior of the enzymes with respect to pH and temperature suggests that they are different isozymes. Copyright © 2010 Viviani Ferreira et al.

Purificação e caracterização bioquímica da invertase extracelular produzida pelo fungo filamentoso Aspergillus terreus

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