Soil enzyme responses to land use change in the tropical rainforest of the Colombian Amazon region.

Soil enzymes mediate key processes and functions of the soils, such as organic matter decomposition and nutrient cycling in both natural and agricultural ecosystems. Here, we studied the activity of five extracellular soil enzymes involved in the C, N, and P-mineralizing process in both litter and surface soil layer of rainforest in the northwest region of the Colombian Amazon and the response of those soil enzymes to land use change. The experimental study design included six study sites for comparing long-term pasture systems to native forest and regeneration practices after pasture, within the main landscapes of the region, mountain and hill landscapes separately. Results showed considerable enzymatic activity in the litter layer of the forest, highlighting the vital role of this compartment in the nutrient cycling of low fertility soils from tropical regions. With the land use transition to pastures, changes in soil enzymatic activities were driven by the management of pastures, with SOC and N losses and reduced absolute activity of soil enzymes in long-term pastures under continuous grazing (25 years). However, the enzyme activities expressed per unit of SOC did not show changes in C and N-acquiring enzymes, suggesting a higher mineralization potential in pastures. Enzymatic stoichiometry analysis indicated a microbial P limitation that could lead to a high catabolic activity with a potential increase in the use of SOC by microbial communities in the search for P, thus affecting soil C sequestration, soil quality and the provision of soil-related ecosystem services.

Development of novel economical methodologies for zymographic analysis of purified xylano-pectinolytic enzymes using agrowaste-based substrates.

In this study, zymographic analysis for xylanase and pectinase enzymes has been carried out using agrowaste residues, wheat bran and citrus peel as well as their extracts. Isozymic forms of xylanase as well as pectinase enzyme displayed comparable zymographic bands onto agar petriplates containing either commercial substrates (xylan and pectin), agrowaste-based substrates (wheat bran and citrus peel), or polysaccharides extracted from these agrowastes (crude xylan and pectin extracted from wheat bran and citrus peel, respectively), indicating the fact that agro residues and their extracts can be utilized as a substitute of cost-intensive commercial substrates, xylan and pectin for zymographic analysis. This is the first report revealing the zymographic analysis of xylano-pectinolytic enzymes using agro-based solid residues particles or polysaccharides extracted from agro-based residues.

Inhibition of extracellular proteases improves the production of a xylanase in Parageobacillus thermoglucosidasius.

BACKGROUND: Parageobacillus thermoglucosidasius is a thermophilic and ethanol-producing bacterium capable of utilising both hexose and pentose sugars for fermentation. The organism has been proposed to be a suitable organism for the production of bioethanol from lignocellulosic feedstocks. These feedstocks may be difficult to degrade, and a potential strategy to optimise this process is to engineer strains that secrete hydrolases that liberate increased amounts of sugars from those feedstocks. However, very little is known about protein transport in P. thermoglucosidasius and the limitations of that process, and as a first step we investigated whether there were bottlenecks in the secretion of a model protein. RESULTS: A secretory enzyme, xylanase (XynA1), was produced with and without its signal peptide. Cell cultures were fractionated into cytoplasm, membrane, cell wall, and extracellular milieu protein extracts, which were analysed using immunoblotting and enzyme activity assays. The main bottleneck identified was proteolytic degradation of XynA1 during or after its translocation. A combination of mass spectrometry and bioinformatics indicated the presence of several proteases that might be involved in this process. CONCLUSION: The creation of protease-deficient strains may be beneficial towards the development of P. thermoglucosidasius as a platform organism for industrial processes.

A novel sucrose-based expression system for heterologous proteins expression in thermotolerant methylotrophic yeast Ogataea thermomethanolica.

The thermotolerant methylotrophic yeast Ogataea thermomethanolica TBRC656 is a potential host for heterologous protein expression. In this study, a novel expression system was developed for O. thermomethanolica based on the maltase (mal) gene promoter from this organism. The OtMal promoter function was tested for expression of fungal enzymes as reporter genes. Measurement of xylanase reporter enzyme activity showed that the OtMal promoter was repressed during growth on glucose and was activated by sucrose. When sucrose was used as a carbon source, the OtMal promoter was approximately twice as strong as the constitutive OtGAP promoter. Comparison of the OtMal promoter with the methanol-inducible OtAOX promoter showed that OtMal promoter drove 1.2 and 1.7-fold higher expression of xylanase and phytase reporter, respectively, than OtAOX promoter under inducing conditions at 24 h. Our results indicated that this novel expression system could be useful for the production of heterologous proteins from sucrose in yeast O. thermomethanolica.

Untapped bacterial diversity and metabolic potential within Unkeshwar hot springs, India.

Hot springs support diverse and interesting groups of microorganisms adapted to extreme conditions and gaining attention in biotechnological applications. However, due to limitations of cultivation methods, a majority of such extremophiles remain uncultivated and unexplored. The advent of multiple cultivation conditions and specialized culture media could possibly aid to access the unexplored microbial portion of hot springs. In the present study, different media and isolation strategies were applied to isolate hitherto unexplored bacterial taxa in the water samples collected from Unkeshwar hot springs, India. Molecular, phylogenetic and predictive functional characterization of the isolated bacterial population was done using 16S rRNA sequencing coupled with Tax4Fun tools. Furthermore, representative isolates were screened for important enzymes (cellulase, xylanase, amylase, and protease) and heavy metal tolerance (chromium, arsenic) properties. A total of 454 bacterial isolates obtained were mapped into 57 unique bacterial genera and 4 different bacterial phyla. Interestingly, 37 genera not previously isolated from Indian hot springs, were isolated for the first time in the present study. However, most of these genera (23 out of 37) were reported only in metagenomics studies from Indian and global hot springs. Furthermore, around 14 genera not previously cultivated and not detected in metagenomics studies of hot springs are documented here. The metabolic potential was ascertained by determining the abundance of specific genes using in silico based Tax4Fun tool, which identified around 315 metabolic pathways for metabolism of carbohydrates, synthesis of secondary metabolites and degradation of xenobiotic compounds. Bioprospection study revealed that 33 and 25 bacterial genera were positive for enzyme production and resistance to the heavy metals, respectively. The present study revealed the advantages of cultivation methods using a comprehensive multiple isolation approach for exploring untapped and unique bacterial diversity, and also utilities for various biotechnological and environmental applications.

Determination and optimization of Vitamin B complex in xylanase enzyme treated polished rice by response surface methodology.

The present study provides information about the concentrations of Vitamin B (thiamine, riboflavin, pyridoxine and niacin) in polished brown rice treated with xylanase. Xylanase enzyme was produced from Aspergillus awamori MTCC 9166. Brown rice was treated with 60-100% enzyme (40 ml of buffer -undiluted) for 30 to 150 min (with variation of 30 min) at 30 degrees C to 50 degrees C (with variation of 5 degrees C) to attain a saturated moisture level of 35.5 g100(-1)g .The enzyme acted upon selective degradation (polishing time 10-50 sec) of bran layer facilitating retention of more vital nutrients along with the vitamins. Vitamin B content, detected through HPLC and optimized by response surface methodology (RSM) with central composite design (CCRD), demonstrated that selective degradation of bran layers for polished rice facilitated increase of thiamine (57%), riboflavin (48%), pyridoxine (90%) and niacin (55%) concentration in bio polished rice over normally milled rice.Enzyme treated bio-polished rice was considered to be better source of vitamin B complex than mechanically milled rice, hence more nutritionally efficacious.

Functional diversity and properties of multiple xylanases from Penicillium oxalicum GZ-2.

A multiple xylanase system with high levels of xylanase activity produced from Penicillium oxalicum GZ-2 using agricultural waste as a substrate has been previously reported. However, the eco-physiological properties and origin of the multiplicity of xylanases remain unclear. In the present study, eight active bands were detected using zymography, and all bands were identified as putative xylanases using MALDI-TOF-MS/MS. These putative xylanases are encoded by six different xylanase genes. To evaluate the functions and eco-physiological properties of xylanase genes, xyn10A, xyn11A, xyn10B and xyn11B were expressed in Pichia pastoris. The recombinant enzymes xyn10A and xyn10B belong to the glycoside hydrolase (GH) family 10 xylanases, while xyn11A and xyn11B belong to GH11 xylanases. Biochemical analysis of the recombinant proteins revealed that all enzymes exhibited xylanase activity against xylans but with different substrate specificities, properties and kinetic parameters. These results demonstrated that the production of multiple xylanases in P. oxalicum GZ-2 was attributed to the genetic redundancy of xylanases and the post-translational modifications, providing insight into a more diverse xylanase system for the efficient degradation of complex hemicelluloses.

Molecular characterization of a Xylanase-producing fungus isolated from fouled soil

Xylanase (EC 3. 2. 1. 8), hydrolyzes xylo-oligosaccharides into D-xylose and required for complete hydrolysis of native cellulose and biomass conversion. It has broad range of applications in the pulp and paper, pharmaceutical and Agri-food industries. Fifty fungal species were isolated from the fouled soil around an oil refinery and screened for the production of xylanase enzyme by enrichment culture techniques. The isolated fungal strain was identified as Hypocrea lixii SS1 based on the results of biochemical tests and 18s rRNA sequencing. The phylogenetic tree was constructed using the MEGA 5 software. Further, Hypocrea lixii SS1 was tested for the ability to utilize the sunflower oil sludge (waste from the oil industry) as the sole carbon source for xylanase production. The growth characteristics of Hypocrea lixii SS1 were also studied and maximum growth was found on the 7th day of incubation. The fungus showed a remarkable xylanase production of 38.9 U/mL. Xylanase was purified using a combination of 0-50% NH4SO2 precipitation, DEAE-sepharose and Sephacryl S-200 chromatography. Single peak obtained in RP-HPLC confirms the purity of xylanase. Further the enzyme produced was affirmed as xylanase with its molecular weight (29 kDa) using SDS-PAGE.

Screening of thermotolerant and thermophilic fungi aiming β-xylosidase and arabinanase production

Plant cell wall is mainly composed by cellulose, hemicellulose and lignin. The heterogeneous structure and composition of the hemicellulose are key impediments to its depolymerization and subsequent use in fermentation processes. Thus, this study aimed to perform a screening of thermophilic and thermotolerant filamentous fungi collected from different regions of the São Paulo state, and analyze the production of β-xylosidase and arabinanase at different temperatures. These enzymes are important to cell wall degradation and synthesis of end products as xylose and arabinose, respectively, which are significant sugars to fermentation and ethanol production. A total of 12 fungal species were analyzed and 9 of them grew at 45 ºC, suggesting a thermophilic or thermotolerant character. Additionally Aspergillus thermomutatus anamorph of Neosartorya and A. parasiticus grew at 50 ºC. Aspergillus niger and Aspergillus thermomutatus were the filamentous fungi with the most expressive production of β-xylosidase and arabinanase, respectively. In general for most of the tested microorganisms, β-xylosidase and arabinanase activities from mycelial extract (intracellular form) were higher in cultures grown at high temperatures (35-40 ºC), while the correspondent extracellular activities were favorably secreted from cultures at 30 ºC. This study contributes to catalogue isolated fungi of the state of São Paulo, and these findings could be promising sources for thermophilic and thermotolerant microorganisms, which are industrially important due to their enzymes.

Molecular characterization of a Xylanase-producing fungus isolated from fouled soil.

Xylanase (EC 3. 2. 1. 8), hydrolyzes xylo-oligosaccharides into D-xylose and required for complete hydrolysis of native cellulose and biomass conversion. It has broad range of applications in the pulp and paper, pharmaceutical and Agri-food industries. Fifty fungal species were isolated from the fouled soil around an oil refinery and screened for the production of xylanase enzyme by enrichment culture techniques. The isolated fungal strain was identified as Hypocrea lixii SS1 based on the results of biochemical tests and 18s rRNA sequencing. The phylogenetic tree was constructed using the MEGA 5 software. Further, Hypocrea lixii SS1 was tested for the ability to utilize the sunflower oil sludge (waste from the oil industry) as the sole carbon source for xylanase production. The growth characteristics of Hypocrea lixii SS1 were also studied and maximum growth was found on the 7(th) day of incubation. The fungus showed a remarkable xylanase production of 38.9 U/mL. Xylanase was purified using a combination of 0-50% NH4SO2 precipitation, DEAE-sepharose and Sephacryl S-200 chromatography. Single peak obtained in RP-HPLC confirms the purity of xylanase. Further the enzyme produced was affirmed as xylanase with its molecular weight (29 kDa) using SDS-PAGE.

Screening of thermotolerant and thermophilic fungi aiming ß-xylosidase and arabinanase production.

Plant cell wall is mainly composed by cellulose, hemicellulose and lignin. The heterogeneous structure and composition of the hemicellulose are key impediments to its depolymerization and subsequent use in fermentation processes. Thus, this study aimed to perform a screening of thermophilic and thermotolerant filamentous fungi collected from different regions of the São Paulo state, and analyze the production of ß-xylosidase and arabinanase at different temperatures. These enzymes are important to cell wall degradation and synthesis of end products as xylose and arabinose, respectively, which are significant sugars to fermentation and ethanol production. A total of 12 fungal species were analyzed and 9 of them grew at 45 °C, suggesting a thermophilic or thermotolerant character. Additionally Aspergillus thermomutatus anamorph of Neosartorya and A. parasiticus grew at 50 °C. Aspergillus niger and Aspergillus thermomutatus were the filamentous fungi with the most expressive production of ß-xylosidase and arabinanase, respectively. In general for most of the tested microorganisms, ß-xylosidase and arabinanase activities from mycelial extract (intracellular form) were higher in cultures grown at high temperatures (35-40 °C), while the correspondent extracellular activities were favorably secreted from cultures at 30 °C. This study contributes to catalogue isolated fungi of the state of São Paulo, and these findings could be promising sources for thermophilic and thermotolerant microorganisms, which are industrially important due to their enzymes.

Richness of endophytic fungi isolated from Opuntia ficus-indica Mill. (Cactaceae) and preliminary screening for enzyme production.

Opuntia ficus-indica Mill. (forage cactus) is farmed with relative success in the semi-arid region of the Brazilian northeast for commercial purposes, particularly as forage and food. Endophytic microorganisms are those that can be isolated inside plant tissues and can be a new source to production of enzymes with different potentialities. The objective of this study was to describe the richness of endophytic fungi from O. ficus-indica and to detect the capacity of these species to produce extracellular hydrolytic enzymes. Forty-four endophytic fungi species were isolated. Among them, the most commonly found were Cladosporium cladosporioides (20.43%) and C. sphaerospermum (15.99%). Acremonium terricola, Monodictys castaneae, Penicillium glandicola, Phoma tropica and Tetraploa aristata are being reported for the first time as endophytic fungi for Brazil. The majority of isolated fungi exhibited enzymatic potential. Aspergillus japonicus and P. glandicola presented pectinolytic activity. Xylaria sp. was the most important among the other 14 species with positive cellulase activity. All 24 isolates analysed were xylanase-positive. Protease was best produced by isolate PF103. The results indicate that there is a significant richness of endophytic fungi in O. ficus-indica, and that these isolates indicate promising potential for deployment in biotechnological processes involving production of pectinases, cellulases, xylanases and proteases.

Miniaturized enzyme production and development of micro-assays for cellulolytic and xylanolytic enzymes.

Miniaturized fungal cultivation and enzyme assays were developed. Cultivation for enzyme production was performed in 50mL conical tubes. In addition, the miniaturized enzyme assays reduced the amount of enzymes and reagents necessary. These procedures can be adopted in screening fungi to determine if they produce cellulolytic and xylanolytic enzymes.

Saprotrophic basidiomycete mycelia and their interspecific interactions affect the spatial distribution of extracellular enzymes in soil.

Saprotrophic cord-forming basidiomycetes are important decomposers of lignocellulosic substrates in soil. The production of extracellular hydrolytic enzymes was studied during the growth of two saprotrophic basidiomycetes, Hypholoma fasciculare and Phanerochaete velutina, across the surface of nonsterile soil microcosms, along with the effects of these basidiomycetes on fungi and bacteria within the soil. Higher activities of α-glucosidase, ß-glucosidase, cellobiohydrolase, ß-xylosidase, phosphomonoesterase and phosphodiesterase, but not of arylsulphatase, were recorded beneath the mycelia. Despite the fact that H. fasciculare, with exploitative hyphal growth, produced much denser hyphal cover on the soil surface than P. velutina, with explorative growth, both fungi produced similar amounts of extracellular enzymes. In the areas where the mycelia of H. fasciculare and P. velutina interacted, the activities of N-acetylglucosaminidase, α-glucosidase and phosphomonoesterase, the enzymes potentially involved in hyphal cell wall damage, and the utilization of compounds released from damaged hyphae of interacting fungi, were particularly increased. No significant differences in fungal biomass were observed between basidiomycete-colonized and noncolonized soil, but bacterial biomass was reduced in soil with H. fasciculare. The increases in the activities of ß-xylosidase, ß-glucosidase, phosphomonoesterase and cellobiohydrolase with increasing fungal:bacterial biomass ratio indicate the positive effects of fungal enzymes on nutrient release and bacterial abundance, which is reflected in the positive correlation of bacterial and fungal biomass content.

The effect of conditioning temperature and exogenous xylanase addition on the viscosity of wheat-based diets and the performance of broiler chickens.

1. A total of 1920 Ross broiler chicks were used in a 6 treatment by 8 replicate growth study to investigate the effect of conditioning temperature and exogenous xylanase addition on diet characteristics and bird performance. 2. Increasing conditioning temperature from 80 to 90 degrees C increased the viscosity of the diets by approximately 6 mPa.s and reduced body weight gain of the broiler chickens by 7%. 3. Furthermore, there was a significant interaction between temperature and the addition of exogenous xylanase, with a proportionately greater positive response to added xylanase in the diets that were pelleted at higher temperatures. 4. Addition of exogenous xylanase also increased the proportion of soluble arabinoxylan in the feed and reduced dietary viscosity, which is likely to be related to the degree of polymerisation of the soluble carbohydrate. 5. These results suggest that conditioning temperature should be considered as a factor when attempting to predict the response of broilers to added xylanase, especially when high-viscosity or 'low-quality' wheat is being used. 6. It can be concluded that pelleting wheat-based diets above 80 degrees C can compromise bird performance and that, if higher temperatures are to be employed, the use of exogenous xylanase is critical to maintain productivity.

Growth and pectinase production by Aspergillus Mexican strain protoplast regenerated under acidic stress.

Protoplasts from Aspergillus sp. FP-180 and Aspergillus awamori NRRL- 3112 were released and regenerated at extreme acidic conditions. The best conditions for protoplast release were 0.8 M KCI, pH 5.8, and 3 h of digestion using mycelia from 12- to 16-h cultures from either Aspergillus sp. FP-180 or A. awamori NRRL-3112. The addition of fresh mycelia to an ongoing digestion after 1 h increased protoplast 4.5-5 times. A regeneration efficiency of 90% was attained at pH 6.0, and it was possible to regenerate protoplasts at pH 1.7 with a regeneration efficiency of 0.5% for Aspergillus sp. FP-180. The LpH-10 strain, derived from protoplast from Aspergillus sp FP-180, was able to regenerate at pH 1.7 and grow at pH values as low as 1.5, values at which the original strain is unable to grow. Regeneration at extreme pH improved the performance of LpH-10 strain. It showed a twofold increase in cell growth at pH 2.0 in liquid culture and a higher pectinolytic activity in relation to that produced by the original strain.

Xylan-hydrolyzing enzyme system from Bacillus pumilus CBMAI 0008 and its effects on Eucalyptus grandis kraft pulp for pulp bleaching improvement.

The extracellular productions of beta-xylanase, beta-xylosidase, beta-glucosidase, beta-mannanase, arabinosidase, alpha-glucuronidase, alpha-galactosidase and Fpase from Bacillus pumilus CBMAI 0008 were investigated with three different xylan sources as substrate. The enzymatic profiles on birchwood, Eucalyptus grandis and oat were studied at alkaline and acidic pH conditions. B. pumilus CBMAI 0008 grown on the three carbon sources produced mainly beta-xylanase. At pH 10, the levels of xylanase were 328, 160 and 136 U/ml, for birch, oat and E. grandis, respectively. beta-Mannanase production was induced on E. grandis (5 U/ml) and arabinofuranosidase on oat (5 U/ml). Although small quantities of alpha-glucuronidase had been produced at pH 10, activity at pH 4.8 was 1.5 U/ml, higher than observed for Aspergillus sp. in literature reports. Preliminary assays carried out on E. grandis kraft pulp from an industrial paper mill (RIPASA S.A. Celulose e Papel, Limeira, SP, Brazil) showed a reduction of 0.3% of chlorine use in the pulp treated with the enzymes, resulting in increased brightness, compared to conventional bleaching. The enzymes were more efficient if applied before the initial bleaching sequence, in a non-pre-oxygenated pulp.

Bifunctional family 3 glycoside hydrolases from barley with alpha -L-arabinofuranosidase and beta -D-xylosidase activity. Characterization, primary structures, and COOH-terminal processing.

An alpha-l-arabinofuranosidase and a beta-d-xylosidase, designated ARA-I and XYL, respectively, have been purified about 1,000-fold from extracts of 5-day-old barley (Hordeum vulgare L.) seedlings using ammonium sulfate fractional precipitation, ion exchange chromatography, chromatofocusing, and size-exclusion chromatography. The ARA-I has an apparent molecular mass of 67 kDa and an isoelectric point of 5.5, and its catalytic efficiency during hydrolysis of 4'-nitrophenyl alpha-l-arabinofuranoside is only slightly higher than during hydrolysis of 4'-nitrophenyl beta-d-xyloside. Thus, the enzyme is actually a bifunctional alpha-l-arabinofuranosidase/beta-d-xylosidase. In contrast, the XYL enzyme, which also has an apparent molecular mass of 67 kDa and an isoelectric point of 6.7, preferentially hydrolyzes 4'-nitrophenyl beta-d-xyloside, with a catalytic efficiency approximately 30-fold higher than with 4'-nitrophenyl alpha-l-arabinofuranoside. The enzymes hydrolyze wheat flour arabinoxylan slowly but rapidly hydrolyze oligosaccharide products released from this polysaccharide by (1 --> 4)-beta-d-xylan endohydrolase. Both enzymes hydrolyze (1 --> 4)-beta-d-xylopentaose, and ARA-I can also degrade (1 --> 5)-alpha-l-arabinofuranohexaose. ARA-I and XYL cDNAs encode mature proteins of 748 amino acid residues which have calculated molecular masses of 79.2 and 80.5 kDa, respectively. Both are family 3 glycoside hydrolases. The discrepancies between the apparent molecular masses obtained for the purified enzymes and those predicted from the cDNAs are attributable to COOH-terminal processing, through which about 130 amino acid residues are removed from the primary translation product. The genes encoding the ARA-I and XYL have been mapped to chromosomes 2H and 6H, respectively. ARA-I transcripts are most abundant in young roots, young leaves, and developing grain, whereas XYL mRNA is detected in most barley tissues.

Determination of xylanase, beta-glucanase, and cellulase activity.

A simple, robust and highly reproducible method for the determination of xylanase, beta-glucanase, and cellulase in commercial feed enzyme preparations is described. The method is based on measurement of reducing moieties released by the enzymes from arabinoxylan, beta-glucan, or carboxymethylcellulose (CMC) and is independent of enzyme standards.

Enzymatic activity of endophytic fungi of six native seedling species from Doi Suthep-Pui National Park, Thailand.

Endophytic fungi were isolated from the stems, petioles, midribs, and leaves of seedlings of six native tree species collected from Doi Suthep-Pui National Park, Thailand. Endophytes were isolated from all tissue samples investigated, and taxa included five ascomycetes, eight anamorphic taxa, and numerous sterile mycelia. Twenty-six strains were tested for their ability to produce cellulase, mannanase, proteinase, and xylanase. The ability to produce these enzymes was distributed amongst the strains tested. Rainforest seedlings supported a diverse array of endophytes that have a wide range of enzymatic activities. The implication of enzyme production in relation to lifestyle abilities of the endophytes is discussed.