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.

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.

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.

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.

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.

Demonstration of an endo-beta-galactosidase and an endo-beta-xylosidase that degrade the proteoglycan linkage region.

Purified proteochondroitin sulfate was incubated at pH 4.0 with a rabbit liver lysosomal enzyme fraction. The formed degradation products were isolated by gel filtration followed by reduction with NaB3H4. After acid hydrolysis of the reduced digest, the hydrolysate was passed through Dowex 50W-X8 and 1-X2 columns. The separated neutral sugars were then subjected to paper chromatography, which demonstrated that galactose and xylose had been at the reducing termini of chondroitin sulfate after incubation with the liver lysosomal preparation. These results suggest the presence in the liver lysosomal preparation of an endo-beta-galactosidase and an endo-beta-xylosidase which act at the linkage region of proteochondroitin sulfate.

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.

Purification and properties of thermostable beta-xylosidase from immature stalks of Saccharum officinarum L. (sugar cane).

Thermostable beta-xylosidase was purified from immature sugar cane stalks to an electrophoretically homogeneous form by ammonium sulfate fractionation, ion-exchange chromatography on DEAE-cellulose and P-cellulose columns, heat treatment (70 degrees C, 20 min) and gel filtration on a Sephadex G-100 column. The purification was about 165-fold in specific activity with a high recovery of 43%. The apparent molecular weight of the enzyme, as determined by gel filtration, was 62,000. In SDS-polyacrylamide gel electrophoresis, the purified enzyme was homogeneous and consisted of only one polypeptide, having a molecular weight of approximately 62,000. The optimum temperature and pH were found to be 75 degrees C and 4.85, respectively. The enzyme was thermostable and especially stable in the presence of D-xylose. The enzyme retained full activity after incubation at 70 degrees C for 60 min in the presence of 0.1% D-xylose and when heated at 75 degrees C in the presence of 1% D-xylose, the enzyme was stable up to 30 min. Among the various sugars tested, D-xylose was found to be most effective stabilizer. The Km and Vmax values were 2.05 mM and 20.4 mumol/mg/min, respectively. The substrate specificity of purified sugar cane beta-xylosidase was investigated with 16 substrates. It was not able to hydrolyze any p-nitrophenyl glycopyranosides, larch wood xylan, or sugar cane except for p- and o-nitrophenyl-beta-D-xylopyranosides. The enzyme hydrolyzed p-nitrophenyl-beta-D-xylopyranoside more rapidly than o-nitrophenyl-beta-D-xylopyranoside. The hydrolysis of p-nitrophenyl-beta-D-xylopyranoside was markedly inhibited by AgNO3, HgCl2, and D-xylose. Competitive inhibition was shown to occur with both HgCl2 and D-xylose. AgNO3 was found to be a non-competitive inhibitor. The enzyme lost 20% of its activity by photo-oxidation in the presence of methylen blue for 8 h. By polyacrylamide disc gel electrophoresis, the enzyme was found to contain carbohydrate. The enzyme was then hydrolyzed and the carbohydrate content found to be 13.5%, the constituent sugars being arabinose and galactose.

Significance of the detection of beta-galactosidase and of beta-xylosidase in the taxonomic study of the genus Haemophilus.

A total of 314 strains of Haemophilus, isolated from clinical samples, were studied for the production of beta-galactosidase and beta-xylosidase. None of the H. influenzae strains studied (9 beta-lactamase positive strains and 129 beta-lactamase negative strains) possessed these enzymes. Both enzymes were almost constantly observed among strains of H. paraphrophilus (10 strains studied) and of H. paraphrohaemolyticus (9 strains studied). Among the other species (H. parainfluenzae, 55 strains; H. haemolyticus, 5 strains; H. parahaemolyticus, 97 strains), beta-galactosidase was present in about 30% of the strains studied whereas beta-xylosidase was detected occasionally (3% of the strains studied). Detection of these two enzymes could be a valuable test for the taxonomic study of the genus Haemophilus. However, the type of substrate used for the detection of beta-xylosidase is important: use of the para-nitro-phenyl-beta-xylopyranoside yielded more positive results than the use of its ortho-isomer.

Taxonomy of Acinetobacter: the usefulness of beta-D-xyloside xylohydrolase for strain differentiation.

One hundred and twenty-two clinical isolates of Acinetobacter were studied for the presence of beta-galactosidase and of beta-xylosidase, for biochemical characteristics, and for genetic interspecies transformation tests. All strains lacked beta-galactosidase; in contrast, beta-xylosidase was always present in the oxidative strains. This test proved to be of value for separating strains able to form acid from carbohydrates (A. anitratum and A. haemolyticus spp haemolyticus) from the non-oxidative strains (A. lwoffi and A. haemolyticus spp alcaligenes). However, the genetic relationship of all strains tested warrants further study before Acinetobacters are grouped into clearly defined species.

Homologues of catalytic domains of Cellulomonas glucanases found in fungal and Bacillus glycosidases.

We demonstrate homology between the catalytic domains of exoglucanase (1,4-beta-D-glucan cellobiohydrolase, EC 3.2.1.91) from Cellulomonas fimi and those of endoxylanases (1,4-beta-D-xylan xylanohydrolases, EC 3.2.1.8) from Bacillus sp. strain C-125 and the fungus Cryptococcus albidus; and between the catalytic domains of endoglucanase (1,4-(1,3:1,4)-beta-D-glucan 4-glucanohydrolase, EC 3.2.1.4) from Cellulomonas fimi and exoglucanase II from Trichoderma reesei. These five enzymes apparently evolved by reshuffling of two catalytic domains and several substrate-binding domains.

Multiplicity and expression of xylanases in the rumen fungus Neocallimastix frontalis.

The time course production of xylanolytic enzymes by the rumen anaerobic fungus Neocallimastix frontalis was studied during growth on different carbon sources and revealed using isoelectric focusing and immunoblotting. A constant low level of endoxylanase expression was observed in glucose medium. A high level of xylanase activity was detected in methyl glucoside medium corresponding to the induction of new isoforms which were repressed by the presence of glucose. beta-Xylosidases were constitutively produced at a high level and remained mainly associated to the fungal cells. Polyclonal antibodies raised against the endoxylanases XYLI and XYLII revealed that XYLI was secreted to the different culture media showing a characteristic pattern of constitutive expression, while anti-XYLII recognized several polypeptides larger than XYLII indicating the production of multiple antigenically related enzymes during growth on the inducing substrate.

Screening of non-Saccharomyces wine yeasts for the production of beta-D-xylosidase activity.

Fifty-four yeast strains belonging to the genera Candida, Dekkera, Hanseniaspora, Metschnikowia, Pichia, Rhodotorula, Schizosaccharomyces and Zygosaccharomyces, mainly isolated from grapes and wines, were screened for the production of beta-D-xylosidase activity. Beta-D-xylosidase activity was only detected in eight yeast strains belonging to the genera Hanseniaspora (H. osmophila and H. uvarum) and Pichia (P. anomala). Beta-D-xylosidase preparations active against p-nitrophenyl-beta-D-xyloside were characterised with respect to their optimal pH and temperature conditions. H. uvarum 11105 and 11107 and P. anomala 10320 beta-D-xylosidase preparations were active at pH and temperature ranges and at concentrations of glucose and ethanol usually found during winemaking processes.

Aroma improving in microvinification processes by the use of a recombinant wine yeast strain expressing the Aspergillus nidulans xlnA gene.

A recombinant wine yeast strain has been constructed expressing the gene coding for beta-(1,4)-endoxylanase from Aspergillus nidulans under the control of the yeast actin gene promoter. The resulting recombinant strain is able to secrete active xylanase enzyme into the culture medium. Wines obtained by microvinification with the control and the recombinant wine yeast strain did not differ in their physicochemical characteristics although an increase in fruity aroma was organoleptically detected in the wine produced by the recombinant yeast. Also, an increase in the concentration of some esters, higher alcohols and terpenes was observed in the case of the recombinant strain.

Preparation and application of a fluorogenic substrate for endo-beta-xylosidase.

The present paper describes a fluorometric assay for galactosaminoglycan-degrading endo-beta-xylosidase, utilizing glycosaminoglycan chains bearing a 4-methylumbelliferyl group at the reducing terminus as a substrate. This fluorogenic substrate is synthesized by human skin fibroblasts cultured in the presence of a fluorogenic xyloside, 4-methylumbelliferyl-beta-D-xyloside. The assay is based on measurement of the fluorescence of 4-methylumbelliferone, enzymatically liberated from the synthetic substrate by endo-beta-xylosidase. We examined the applicability of the assay for analysis of endo-beta-xylosidase activity.

Syntheses and testing of substrates and mechanism-based inactivators for xylanases.

The syntheses of the 2,5- and 3,4-dinitrophenyl beta-xylobiosides by two separate routes are described, as well as the syntheses of the 2,4-dinitrophenyl beta-glycosides of 2-chloro-2-deoxy-xylobiose and 2-deoxy-2-fluoro-xylobiose. Both the 3,4- and 2,5-dinitrophenyl beta-xylobiosides proved to be good substrates for the Bacillus subtilis xylanase, with kcat/Km values of 1.0 and 34.4 mM-1 s-1, respectively. Excellent time-dependent inactivation of the exoxylanase/glucanase from Cellulomonas fimi was provided by 2,4-dinitrophenyl 2-deoxy-2-fluoro-beta-xylobioside, according to inactivation parameters of ki = 0.057 min-1 and Ki = 0.0035 mM.

Development of a 96-well enzyme-linked solid-phase assay for beta-glucanase and xylanase.

An enzyme-linked sorbent assay (ELSA) for estimating beta-glucanase activity was developed on the basis of the use of biotinylated beta-glucan as a solid-phase substrate. The assay involves the coating of titer plate wells with biotinylated beta-glucan, the partial hydrolysis of this substrate with beta-glucanase, the reaction of the biotin from the unhydrolyzed substrate with an alkaline phosphatase-streptavidin complex, and quantitation of the remaining beta-glucan using alkaline phosphatase. The activity of the bound indicator enzyme, alkaline phosphatase, is proportionally related to the beta-glucanase activity in the sample. The ELSA is simple, can be readily adapted to the routine assay of a large number of samples (as many as 200 per person/day), and has good precision (CV = 4.0-6.4%) and high sensitivity (detects as low as 0. 001 mU of beta-glucanase/assay). A similar assay was developed for xylanase using biotinylated arabinoxylan. The ELSA provides a simple and sensitive estimate of beta-glucanase and xylanase activity.

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.