Aqueous two-phase partitioning and characterization of xylanase produced by Streptomyces geysiriensis from low cost lignocellulosic substrates.

Microbial production of xylanase is gaining the commercial importance, due to its wide range of applications from paper and pulp to food and feed industries. Streptomyces geysiriensis was used for the production of extracellular xylanase from lignocellulosic substrates such as rice bran and saw dust, under solid-state fermentation. The influence of pH, temperature and incubation period for the maximum production of xylanase was investigated with 1:2 (w/v) of substrate to moisture ratio at 100 rpm shaking conditions. The maximum production was recorded after 5 days of fermentation with pH 8.0 at 40 °C. The scale-up was done based on the results of optimized parameters using 3 L Applikon autoclavable bioreactor with maximum yield of 186 U/ml after 4 days of fermentation. Extracellular xylanase was separated by partitioning in aqueous two-phase system consisting of 20% polyethylene glycol 6000 and 12% K2HPO4 with maximum yield of 93.97%. The investigation of the effect of pH and temperature and its incubation time showed that xylanase was retained its activity in a pH range of 6.5-8.5, with thermal stability from 20 °C to 60 °C up to 180 min. The presence of metal ions was found to inhibit the activity of xylanase especially Cu2+ and Zn2+. Xylanase was stable both at 4 °C and room temperature (35 °C) for 30 and 9 days respectively. The kinetic parameters Km (0.48 mg/ml) and Vmax (8.33 U/mg) were determined using birchwood xylan as substrate.

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.

Xyloglucan in the primary cell wall: assessment by FESEM, selective enzyme digestions and nanogold affinity tags.

Xyloglucan has been hypothesized to bind extensively to cellulose microfibril surfaces and to tether microfibrils into a load-bearing network, thereby playing a central role in wall mechanics and growth, but this view is challenged by newer results. Here we combined high-resolution imaging by field emission scanning electron microscopy (FESEM) with nanogold affinity tags and selective endoglucanase treatments to assess the spatial location and conformation of xyloglucan in onion cell walls. FESEM imaging of xyloglucanase-digested cell walls revealed an altered microfibril organization but did not yield clear evidence of xyloglucan conformations. Backscattered electron detection provided excellent detection of nanogold affinity tags in the context of wall fibrillar organization. Labelling with xyloglucan-specific CBM76 conjugated with nanogold showed that xyloglucans were associated with fibril surfaces in both extended and coiled conformations, but tethered configurations were not observed. Labelling with nanogold-conjugated CBM3, which binds the hydrophobic surface of crystalline cellulose, was infrequent until the wall was predigested with xyloglucanase, whereupon microfibril labelling was extensive. When tamarind xyloglucan was allowed to bind to xyloglucan-depleted onion walls, CBM76 labelling gave positive evidence for xyloglucans in both extended and coiled conformations, yet xyloglucan chains were not directly visible by FESEM. These results indicate that an appreciable, but still small, surface of cellulose microfibrils in the onion wall is tightly bound with extended xyloglucan chains and that some of the xyloglucan has a coiled conformation.

Fast automated online xylanase activity assay using HPAEC-PAD.

In contrast to biochemical reactions, which are often carried out under automatic control and maintained overnight, the automation of chemical analysis is usually neglected. Samples are either analyzed in a rudimentary fashion using in situ techniques, or aliquots are withdrawn and stored to facilitate more precise offline measurements, which can result in sampling and storage errors. Therefore, in this study, we implemented automated reaction control, sampling, and analysis. As an example, the activities of xylanases on xylotetraose and soluble xylan were examined using high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The reaction was performed in HPLC vials inside a temperature-controlled Dionex™ AS-AP autosampler. It was started automatically when the autosampler pipetted substrate and enzyme solution into the reaction vial. Afterwards, samples from the reaction vial were injected repeatedly for 60 min onto a CarboPac™ PA100 column for analysis. Due to the rapidity of the reaction, the analytical method and the gradient elution of 200 mM sodium hydroxide solution and 100 mM sodium hydroxide with 500 mM sodium acetate were adapted to allow for an overall separation time of 13 min and a detection limit of 0.35-1.83 mg/L (depending on the xylooligomer). This analytical method was applied to measure the soluble short-chain products (xylose, xylobiose, xylotriose, xylotetraose, xylopentaose, and longer xylooligomers) that arise during enzymatic hydrolysis. Based on that, the activities of three endoxylanases (EX) were determined as 294 U/mg for EX from Aspergillus niger, 1.69 U/mg for EX from Bacillus stearothermophilus, and 0.36 U/mg for EX from Bacillus subtilis. Graphical abstract Xylanase activity assay automation.

Cost-effective and concurrent production of industrially valuable xylano-pectinolytic enzymes by a bacterial isolate Bacillus pumilus AJK.

Simultaneous production of xylanase and pectinase by Bacillus pumilus AJK under submerged fermentation was investigated in this study. Under optimized conditions, it produced 315 ± 16 IU/mL acidic xylanase, 290 ± 20 IU/mL alkaline xylanase, and 88 ± 9 IU/mL pectinase. The production of xylano-pectinolytic enzymes was the highest after inoculating media (containing 2% each of wheat bran and Citrus limetta peel, 0.5% peptone, 10 mM MgSO4, pH 7.0) with 2% of 21-hr-old culture and incubated at 37°C for 60 hr at 200 rpm. Xylanase retained 100% activity from pH 6.0 to10.0 after 3 hr of incubation, while pectinase showed 100% stability from pH 6.0 to 9.0 even after 6 hr of incubation. Cost-effective and concurrent production of xylanase and pectinase by a bacterial isolate in the same production media suggests its potential for various biotechnological applications. This is the first report of simultaneous production of industrially important extracellular xylano-pectinolytic enzymes by B. pumilus.

Custom fabrication of biomass containment devices using 3-D printing enables bacterial growth analyses with complex insoluble substrates.

Physiological studies of recalcitrant polysaccharide degradation are challenging for several reasons, one of which is the difficulty in obtaining a reproducibly accurate real-time measurement of bacterial growth using insoluble substrates. Current methods suffer from several problems including (i) high background noise due to the insoluble material interspersed with cells, (ii) high consumable and reagent cost and (iii) significant time delay between sampling and data acquisition. A customizable substrate and cell separation device would provide an option to study bacterial growth using optical density measurements. To test this hypothesis we used 3-D printing to create biomass containment devices that allow interaction between insoluble substrates and microbial cells but do not interfere with spectrophotometer measurements. Evaluation of materials available for 3-D printing indicated that UV-cured acrylic plastic was the best material, being superior to nylon or stainless steel when examined for heat tolerance, reactivity, and ability to be sterilized. Cost analysis of the 3-D printed devices indicated they are a competitive way to quantitate bacterial growth compared to viable cell counting or protein measurements, and experimental conditions were scalable over a 100-fold range. The presence of the devices did not alter growth phenotypes when using either soluble substrates or insoluble substrates. We applied biomass containment to characterize growth of Cellvibrio japonicus on authentic lignocellulose (non-pretreated corn stover), and found physiological evidence that xylan is a significant nutritional source despite an abundance of cellulose present.

Influence of twin-screw extrusion on soluble arabinoxylans and corn fiber gum from corn fiber.

BACKGROUND: The effect of feed moisture content and screw speed in the extrusion process with and without chemical pretreatment of corn fiber was investigated. Different chemical pretreatment methods (NaOH and H2 SO4 solution) were compared. The improvement of reducing sugar, soluble arabinoxylans (SAX) content and the yield of corn fiber gum was measured. RESULTS: A high reducing sugar content was obtained in the filtrate fraction from the extruded destarched corn fiber (EDCF) with H2SO4 pretreatment. Feed moisture content most effectively improved both reducing sugar and SAX content of filtrate. Increasing feed moisture content and screw speed resulted in a higher SAX content in the filtrate of the EDCF with NaOH pretreatment. The SAX content of the residual solid from the EDCF with NaOH pretreatment was higher compared to H2SO4 pretreated and unpretreated samples and significantly increased with decreasing feed moisture content. The screw speed did not have a major impact after enzyme hydrolysis. The yield of corn fiber gum was increased by 12% using NaOH pretreatment combined with extrusion process as compared to the destarched corn fiber. CONCLUSION: The results show the great potential of the extrusion process as an effective pretreatment for disruption the lignocelluloses of corn fiber, leading to conversion of cellulose to glucose and hemicelluloses to SAX and isolation of corn fiber gum.

Assessment of the biomass hydrolysis potential in bacterial isolates from a volcanic environment: biosynthesis of the corresponding activities.

The biomass degrading enzymatic potential of 101 thermophilic bacterial strains isolated from a volcanic environment (Santorini, Aegean Sea, Greece) was assessed. 80 % of the strains showed xylanolytic activity in Congo Red plates, while only eight could simultaneously hydrolyze cellulose. Fifteen isolates were selected on the basis of their increased enzyme production, the majority of which was identified as Geobacilli through 16S rDNA analysis. In addition, the enzymatic profile was evaluated in liquid cultures using various carbon sources, a procedure that revealed lack of correlation on xylanase levels between the two cultivation modes and the inability of solid CMC cultures to fully unravel the cellulose degrading potential of the isolates. Strain SP24, showing more than 99 % 16S DNA similarity with Geobacillus sp. was further studied for its unique ability to simultaneously exhibit cellulase, xylanase, ß-glucosidase and ß-xylosidase activities. The first two enzymes were produced mainly extracellularly, while the ß-glycosidic activities were primarily detected in the cytosol. Maximum enzyme production by this strain was attained using a combination of wheat bran and xylan in the growth medium. Bioreactor cultures showed that aeration was necessary for both enhanced growth and enzyme production. Aeration had a strong positive effect on cellulase production while it negatively affected expression of ß-glucosidase. Xylanase and ß-xylosidase production was practically unaffected by aeration levels.

Investigation of enzyme formulation on pretreated switchgrass.

This work studied the benefits of adding different enzyme cocktails (cellulase, xylanase, ß-glucosidase) to pretreated switchgrass. Pretreatment methods included ammonia fiber expansion (AFEX), dilute-acid (DA), liquid hot water (LHW), lime, lime+ball-milling, soaking in aqueous ammonia (SAA), and sulfur dioxide (SO(2)). The compositions of the pretreated materials were analyzed and showed a strong correlation between initial xylan composition and the benefits of xylanase addition. Adding xylanase dramatically improved xylan yields for SAA (+8.4%) and AFEX (+6.3%), and showed negligible improvement (0-2%) for the pretreatments with low xylan content (dilute-acid, SO(2)). Xylanase addition also improved overall yields with lime+ball-milling and SO(2) achieving the highest overall yields from pretreated biomass (98.3% and 93.2%, respectively). Lime+ball-milling obtained an enzymatic yield of 92.3kg of sugar digested/kg of protein loaded.

Assessment on the fermentability of xylooligosaccharides from rice husks by probiotic bacteria.

Liquors from rice husk autohydrolysis, containing xylooligosaccharides (XOS), other saccharides, and nonsaccharide compounds, were refined by membrane processing to increase the proportion of substituted XOS in refined liquors. XOS were assayed for composition and degree of polymerization (DP) distribution and hydrolyzed with commercial enzymes for obtaining XOS with DP in the range of 2-6. Nanofiltered, hydrolyzed liquors were subjected to ion exchange processing to yield a final product containing monosaccharides, XOS (accounting for 55.6% of the nonvolatile solutes), and other nonvolatile compounds. The solution obtained after enzymatic hydrolysis with commercial xylanases (in which 82.8% of XOS were in the DP range of 2-6) was examined as a medium for promoting the growth of Bifidobacterium adolescentis CECT 5781, B. longum CECT 4503, B. infantis CECT 4551, and B. breve CECT 4839. The growth rate of B. adolescentis (0.58 h(-1)) was higher than the ones determined for B. longum, B. infantis, and B. breve (0.37, 0.30, and 0.40 h(-1), respectively). The percentage of total XOS consumption by B. adolescentis was 77% after 24 h, the highest percentage of utilization corresponding to xylotriose (90%), followed by xylobiose (84%), xylotetraose (83%), and xylopentaose (71%).

Use of xylan-rich cost effective agro-residues in the production of xylanase by Streptomyces cyaneus SN32.

AIM: The present study aimed at optimization of cultural and nutritional parameters for enhanced production of xylanase from Streptomyces cyaneus SN32. METHODS AND RESULTS: The xylanase production by S. cyaneus SN32 on most of the agro-residues tested in this study was more, as compared with the xylanase yield in the medium supplemented with commercial xylan. The presence of wheat bran as carbon source in the medium induced the highest production of xylanase followed by corn cob. Utilization of maize stalk, gram husk and black gram husk for microbial xylanase production has been reported first time in the present study. Among all the organic and inorganic sources of nitrogen tested in the study, peptone was found to be the best in stimulating xylanase production by S. cyaneus SN32. CONCLUSION: The production of xylanase from this thermoalkalophilic actinomycete has been enhanced 1.44-fold. To the best of our knowledge, the magnitude of enzyme yield i.e. 720 IU ml(-1) by S. cyaneus SN32 has not been reported for any other actinomycete so far. SIGNIFICANCE AND IMPACT OF STUDY: Present studies revealed that thermoalkalophilic S. cyaneus SN32, because of its simple nutritional requirements and its ability to exhibit considerably good enzyme yield, is a potent xylanase producer for its economical application in various industries.

Rational affinity purification of native Streptomyces family 10 xylanase.

Xylanase SoXyn10A from Streptomyces olivaceoviridis E-86 comprises a family 10 catalytic module linked to a family 13 carbohydrate-binding module (SoCBM13). The SoCBM13 has a beta-trefoil structure, with binding sites in each subdomain (alpha, beta and gamma). Subdomain alpha, but not subdomains beta and gamma, binds tightly to lactose. It was, therefore, thought that immobilized lactose could be used for the affinity purification of SoXyn10A. Lactosyl-Sepharose was prepared and tested as an affinity matrix. SoXyn10A produced from the cloned xyn10A gene by Escherichia coli, and native SoXyn10A in culture supernatants from S. olivaceoviridis, were purified to homogeneity in a single step by affinity chromatography using this matrix. This simple purification of SoXyn10A makes the enzyme an attractive candidate for applications requiring xylanase. The CBM also has the potential for use as an affinity tag for the purification of other proteins.