Purification and biochemical characterization of an alkaline pectin lyase from Fusarium decemcellulare MTCC 2079 suitable for Crotolaria juncea fiber retting

An extracellular pectin lyase secreted by Fusarium decemcellulare MTCC 2079 under solid state fermentation condition has been purified to electrophoretic homogeniety by using ammonium sulfate fractionation, carboxymethyl cellulose and gel filtration (Sephadex G-100) column chromatographies. The purified enzyme showed single protein band corresponding to molecular mass 45 +/- 01 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme had maximum activity at pH 9.0 and showed maximum stability in the pH range of 9.0-12.0. The optimum temperature of the purified enzyme was 50 degrees C and it showed maximum stability upto 40 degrees C. The energy of activation for the thermal denaturation (Ea) was 59.06 kJ mol(-1) K-1. The K-m and k(cat) values using citrus pectin as the substrate were 0.125mgml(-1) and 72.9 s(-1) in 100mM sodium carbonate buffer pH 9.0 at 50 degrees C. The biophysical studies on pectin lyase showed that its secondary structure belongs to alpha+beta class of protein with comparatively less of beta-sheets. Purified pectin lyase showed efficient retting of Crotolaria juncea fibers.

Biochemical characterisation and application of lipases produced by Aspergillus sp on solid-state fermentation using three substrates

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

Purification and properties of polygalacturonase produced by thermophilic fungus Thermoascus aurantiacus CBMAI-756 on solid-state fermentation

Polygalacturonases are enzymes involved in the degradation of pectic substances, being extensively used in food industries, textile processing, degumming of plant rough fibres, and treatment of pectic wastewaters. Polygalacturonase (PG) production by thermophilic fungus Thermoascus aurantiacus on solid-state fermentation was carried out in culture media containing sugar cane bagasse and orange bagasse in proportions of 30% and 70% (w/w) at 45°C for 4 days. PG obtained was purified by gel filtration and ion-exchange chromatography. The highest activity was found between pH 4.5 and 5.5, and the enzyme preserved more than 80% of its activity at pH values between 5.0 and 6.5. At pH values between 3.0 and 4.5, PG retained about 73% of the original activity, whereas at pH 10.0 it remained around 44%. The optimum temperature was 60–65°C. The enzyme was completely stable when incubated for 1 hour at 50°C. At 55°C and 60°C, the activity decreased 55% and 90%, respectively. The apparent molecular weight was 29.3 kDa, Km of 1.58 mg/mL and Vmax of 1553.1μmol/min/mg. The presence of Zn+2, Mn+2, and Hg+2 inhibited 59%, 77%, and 100% of enzyme activity, respectively. The hydrolysis product suggests that polygalacturonase was shown to be an endo/exoenzyme.

Immobilization of a Plant Lipase from Pachira aquatica in Alginate and Alginate/PVA Beads

This study reports the immobilization of a new lipase isolated from oleaginous seeds of Pachira aquatica, using beads of calcium alginate (Alg) and poly(vinyl alcohol) (PVA). We evaluated the morphology, number of cycles of reuse, optimum temperature, and temperature stability of both immobilization methods compared to the free enzyme. The immobilized enzymes were more stable than the free enzyme, keeping 60% of the original activity after 4 h at 50°C. The immobilized lipase was reused several times, with activity decreasing to approximately 50% after 5 cycles. Both the free and immobilized enzymes were found to be optimally active between 30 and 40°C.

Reuse of Cyclodextrin Glycosyltransferase through Immobilization on Magnetic Carriers

National Council for Scientific and Technological Development (CNPq)

Produção de fitase por Rhizopus microsporus var. microsporus: purificação, caracterização bioquímica e aplicação

Pós-graduação em Biotecnologia - IQ

Improvement of gamete quality and its short-term storage: an approach for biotechnology in laboratory fish

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

Desenvolvimento de um bioprocesso utilizando-se resíduos para produção de amilases por Rhizopus oligosporus e etanol por Saccharomyces cerevisiae

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Produção, imobilização e aplicação de lipase de Fusarium verticillioides

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Nanopartículas magnéticas como suporte para imobilização de lipases

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Improvement of fungal arabinofuranosidase thermal stability by reversible immobilization

A gene encoding a-L-arabinofuranosidase (abfA) from Aspergillus niveus was identified, cloned, and successfully expressed in Aspergillus nidulans. Based on amino acid sequence comparison, the 88.6 kDa enzyme could be assigned to the GH family 51. The characterization of the purified recombinant AbfA revealed that the enzyme was active at a limited pH range (pH 4.0-5.0) and an optimum temperature of 70 degrees C. The AbfA was able to hydrolyze arabinoxylan, xylan from birchwood, debranched arabinan, and 4-nitrophenyl arabinofuranoside. Synergistic reactions using both AbfA and endoxylanase were also assessed. The highest degree of synergy was obtained after the sequential treatment of the substrate with endoxylanase, followed by AbfA, which was observed to release noticeably more reducing sugars than that of either enzyme acting individually. The immobilization of AbfA was performed via ionic adsorption onto various supports: agarose activated by polyethyleneimine polymers, cyanogen bromide activated Sepharose, DEAE-Sepharose, and Sepharose-Q The Sepharose-Q derivative remained fully active at pH 5 after 360 min at 60 degrees C, whereas the free AbfA was inactivated after 60 min. A synergistic effect of arabinoxylan hydrolysis by AbfA immobilized in Sepharose-Q and endoxylanase immobilized in glyoxyl agarose was also observed. The stabilization of arabinofuranosidases using immobilization tools is a novel and interesting topic. (C) 2012 Elsevier Ltd. All rights reserved.

Immobilization of a recombinant endo-1,5-arabinanase secreted by Aspergillus nidulans strain A773

An endo-1,5-arabinanase (abnA) encoding gene from Aspergillus niveus was identified, cloned and successfully expressed in Aspergillus nidulans strain A773. Based on amino acid sequence comparison, the 34-kDa enzyme could be assigned to CAZy GH family 43. Characterization of purified recombinant endo-1,5-arabinanase (AbnA) revealed that it is active at a wide pH range (pH 4.0-7.0) and an optimum temperature at 70 degrees C. The immobilization of the AbnA was performed via covalent binding onto agarose-modified supports: glyoxyl iminodiacetic acid-Ni2+, glyoxyl amine, glyoxyl (4% and 10%) and cyanogen bromide activated sepharose. The yield of immobilization was similar on glyoxyl amine and glyoxyl (96%), and higher than glyoxyl iminodiacetic acid-Ni2+ (43%) support. The thermal inactivation of these immobilized preparations showed that the stability of the AbnA immobilized on glyoxyl 4 and 10% was improved by 4.0 and 10.3-fold factor at 70 degrees C. The half-life of glyoxyl 4% derivative at 60 degrees C was >48 h (pH 5), 9 h (pH 7) and 88 min (pH 9). The major hydrolysis product of debranched arabinan or arabinopentaose by glyoxyl agarose-immobilized AbnA was arabinobiose. (C) 2012 Elsevier B.V. All rights reserved.

Purification, characterization and structural determination of chitinases produced by Moniliophthora perniciosa

The enzyme chitinase from Moniliophthora perniciosa the causative agent of the witches' broom disease in Theobroma cacao, was partially purified with ammonium sulfate and filtration by Sephacryl S-200 using sodium phosphate as an extraction buffer. Response surface methodology (RSM) was used to determine the optimum pH and temperature conditions. Four different isoenzymes were obtained: ChitMp I, ChitMp II, ChitMp III and ChitMp IV. ChitMp I had an optimum temperature at 44-73ºC and an optimum pH at 7.0-8.4. ChitMp II had an optimum temperature at 45-73ºC and an optimum pH at 7.0-8.4. ChitMp III had an optimum temperature at 54-67ºC and an optimum pH at 7.3-8.8. ChitMp IV had an optimum temperature at 60ºC and an optimum pH at 7.0. For the computational biology, the primary sequence was determined in silico from the database of the Genome/Proteome Project of M. perniciosa, yielding a sequence with 564 bp and 188 amino acids that was used for the three-dimensional design in a comparative modeling methodology. The generated models were submitted to validation using Procheck 3.0 and ANOLEA. The model proposed for the chitinase was subjected to a dynamic analysis over a 1 ns interval, resulting in a model with 91.7% of the residues occupying favorable places on the Ramachandran plot and an RMS of 2.68.

Global planktonic foraminifera seasonality

Shell fluxes of planktonic Foraminifera species vary intra-annually in a pattern that appears to follow the seasonal cycle. However, the variation in the timing and prominence of seasonal flux maxima in space and among species remains poorly constrained. Thus, although changing seasonality may result in a flux-weighted temperature offset of more than 5° C within a species, this effect is often ignored in the interpretation of Foraminifera-based paleoceanographic records. To address this issue we present an analysis of the intra-annual pattern of shell flux variability in 37 globally distributed time series. The existence of a seasonal component in flux variability was objectively characterised using periodic regression. This analysis yielded estimates of the number, timing and prominence of seasonal flux maxima. Over 80% of the flux series across all species showed a statistically significant periodic component, indicating that a considerable part of the intra-annual flux variability is predictable. Temperature appears to be a powerful predictor of flux seasonality, but its effect differs among species. Three different modes of seasonality are distinguishable. Tropical and subtropical species (Globigerinoides ruber (white and pink varieties), Neogloboquadrina dutertrei, Globigerinoides sacculifer, Orbulina universa, Globigerinella siphonifera, Pulleniatina obliquiloculata, Globorotalia menardii, Globoturborotalita rubescens, Globoturborotalita tenella and Globigerinoides conglobatus) appear to have a less predictable flux pattern, with random peak timing in warm waters. In colder waters, seasonality is more prevalent: peak fluxes occur shortly after summer temperature maxima and peak prominence increases. This tendency is stronger in species with a narrower temperature range, implying that warm-adapted species find it increasingly difficult to reproduce outside their optimum temperature range and that, with decreasing mean temperature, their flux is progressively more focussed in the warm season. The second group includes the temperate to cold-water species Globigerina bulloides, Globigerinita glutinata, Turborotalita quinqueloba, Neogloboquadrina incompta, Neogloboquadrina pachyderma, Globorotalia scitula, Globigerinella calida, Globigerina falconensis, Globorotalia theyeri and Globigerinita uvula. These species show a highly predictable seasonal pattern, with one to two peaks a year, which occur earlier in warmer waters. Peak prominence in this group is independent of temperature. The earlier-when-warmer pattern in this group is related to the timing of productivity maxima. Finally, the deep-dwelling Globorotalia truncatulinoides and Globorotalia inflata show a regular and pronounced peak in winter and spring. The remarkably low flux outside the main pulse may indicate a long reproductive cycle of these species. Overall, our analysis indicates that the seasonality of planktonic Foraminifera shell flux is predictable and reveals the existence of distinct modes of phenology among species. We evaluate the effect of changing seasonality on paleoceanographic reconstructions and find that, irrespective of the seasonality mode, the actual magnitude of environmental change will be underestimated. The observed constraints on flux seasonality can serve as the basis for predictive modelling of flux pattern. As long as the diversity of species seasonality is accounted for in such models, the results can be used to improve reconstructions of the magnitude of environmental change in paleoceanographic records.

Efficient gene activation in cultured mammalian cells mediated by FLP recombinase-expressing recombinant adenovirus

A recombinant adenovirus (rAd) expressing Cre recombinase derived from bacteriophage P1 has already been extensively used for the conditional gene activation and inactivation strategies in mammalian systems. In this study, we generated AxCAFLP, a rAd expressing FLP recombinase derived from Saccharomyces cerevisiae and carried out quantitative comparisons with Cre-expressing rAd in both in vitro and in cultured cells to provide another efficient gene regulation system in mammalian cells. In the in vitro experiments, the relative recombination efficiency of FLP expressed in 293 cells infected with FLP-expressing rAd was approximately one-thirtieth that of Cre even at 30°C, the optimum temperature for FLP activity, and was approximately one-ninetieth at 37°C. Co-infection experiments in HeLa cells using a target rAd conditionally expressing LacZ under the control of FLP showed that an FLP-expressing rAd, infected at a multiplicity of infection (MOI) of 5, was able to activate the transgene in almost 100% of HeLa cells whereas the Cre-expressing rAd was sufficient at an MOI of 0.2. Since an MOI of 5 is ordinarily used in rAd experiments, these results showed that the FLP-expressing rAd is useful for gene activation strategies and is probably applicable to a sequential gene regulation system in combination with Cre-expressing rAd in mammalian cells.