Production and potential applications of a xylanase from a new strain of Streptomyces cuspidosporus

Enzyme production by a new mesophilic Streptomyces isolate was investigated which grew optimally on 1% (w/v) xylan and 10% (w/v) wheat bran at pH 7 and 37 °C. Xylan induced only CMCase (0.29 U/ml) besides xylanase (22–35 U/ml, 40–49 U/mg protein). Wheat bran induced xylanase (105 U/ml, 17.5 U/mg protein), CMCase (0.74 U/ml), -xylosidase (0.009 U/ml), -glucosidase (0.026 U/ml), -L-arabinofuranosidase (0.049 U/ml), amylase (1.6 U/ml) and phytase (0.432 U/ml). The isolate was amenable to solid state cultivation and produced increased levels of xylanase (146 U/ml, 28 U/mg protein). The pH and temperature optima of the crude xylanase activity were 5.5 and 65 °C respectively. The pI was 6.0 as determined by PEG precipitation. The crude enzyme was applied in treatment of paper pulp and predigestion of poultry feed and was found to be effective in releasing sugars from both and soluble phosphorus from the latter.     

Statistical screening and optimization of process variables for xylanase production utilizing alkali-pretreated rice husk

With the objective of the production of xylanase, local raw material (rice husk) and the indigenous isolate, Aspergillus niger ITCC 7678, were studied. Optimization of the cultivation system for enhancing xylanase production was studied via submerged fermentation. Statistical procedures were employed to study the effect of process variables, such as alkali-pretreated rice husk (as carbon source), NaNO₃ (as nitrogen source), KH₂PO₄, KCl, Tween 80 (as surfactant), MgSO₄, FeSO₄·7H₂O, pH, particle size, agitation, and temperature, on xylanase production by A. niger. The effect and significance of the variables was studied using Plackett–Burman (PBD) and central composite statistical design (CCD). It was found that alkali pretreated rice husk (weight/volume), pH, temperature, and NaNO₃ significantly influence xylanase production. So, these four factors were further optimized by CCD, and it was found that maximum xylanase activity of 10.9 IU/ml was observed at (6.5 % w/v) rice husk, pH (5.5), temperature (32.5 °C), and NaNO₃ (0.35 % w/v) concentration. Under optimum conditions, xylanase production was also studied at the bioreactor level and showed 12.8 % enhanced xylanase activity.     

Utilization of agro-industrial waste for xylanase production by Aspergillus foetidus MTCC 4898 under solid state fermentation and its application in saccharification

Xylanase production by Aspergillus foetidus MTCC 4898 was carried out under solid state fermentation using wheat bran and anaerobically treated distillery spent wash. Response surface methodology involving Box–Behnken design was employed for optimizing xylanase production. The interactions among various fermentation parameters viz. moisture to substrate ratio, inoculum size, initial pH, effluent concentration and incubation time were investigated and modeled. The predicted xylanase activity under optimized parameters was 8200–8400 U/g and validated xylanase activity was 8450 U/g with very poor cellulase activity. Crude xylanase was used for enzymatic saccharification of agroresidues like wheat straw, rice straw and corncobs. Dilute NaOH and ammonia pretreatments were found to be beneficial for the efficient enzymatic hydrolysis of all the three substrates. Dilute NaOH pretreated wheat straw, rice straw and corncobs yielded 4, 4.2, 4.6 g/l reducing sugars, respectively whereas ammonia treated wheat straw, rice straw and corncobs yielded 4.9, 4.7, 4.6 g/l reducing sugars, respectively. The hydrolyzates were analysed by HPTLC. Xylose was found to be the major end product with traces of glucose in the enzymatic hydrolyzates of all the substrates.     

Spelt and emmer flours: characterization of the lactic acid bacteria microbiota and selection of mixed starters for bread making

Aims: This study aimed at characterizing the lactic acid bacteria microbiota and selecting mixed endogenous starters to be used for sourdough fermentation of spelt or emmer flours. Methods and Results: Identification of lactic acid bacteria was carried out by partial sequencing of the 16S rRNA, recA, 16S/23S rRNA spacer region and pheS genes. Spelt flour showed the largest biodiversity, while Lactobacillus plantarum dominated in emmer flour. Isolates were subjected to RAPD‐PCR analysis and screened based on the kinetics of growth and acidification, quotient of fermentation and liberation of free amino acids (FAA) during sourdough fermentation. After selection, mixed starters were used according to a two‐step fermentation process. Wheat flour was fermented by the same starters. Spelt and emmer sourdoughs had slightly higher pH than wheat sourdoughs but titratable acidity, concentration of FAA and phytase activity were higher. Specific volume and crumb grain of emmer and, especially, spelt breads approached those of wheat breads. Sensory analysis confirmed the suitability of spelt and emmer for bread making. Conclusions: The sourdough biotechnology was indispensable to completely exploit the potential of spelt and emmer flours. Significance and Impact of the Study: Results filled up the lack of knowledge on the lactic acid bacteria microbiota and technological performances of spelt and emmer flours.     

Use of agricultural surpluses for production of biomass by marine microalgae

The marine microalga Phaeodactylum tricornutum was cultivated in semi-continuous culture under mixotrophic conditions with the soluble fractions of potato, rye and wheat flours that had been naturally fermented, at 2% or 4% (w/v). The rye flour produced the highest microalgal cellular density of 90×10⁶ cells.ml^-1 when supplemented with NaNO₃ and NaH₂PO₄. The autotrophic control only gave 57×10⁶ cells.ml^-1. The value of agricultural surpluses, such as rye flour, can therefore be increased by its use in the production of valuable, microalgal biomass which is rich in protein, pigments and fatty acids.     

Production and characterization of a xylanase from Cyathus stercoreus

Cyathus stercoreus grown on wheat straw had a higher xylanase activity than when it was grown on rice husk or extracted hemicellulose. Inclusion of casein hydrolysate, Tween 80 and Mn²⁺ (at 0.02%, 0.2% and 0.075%, respectively) increased the production of extracellular xylanase. Optimal yield of xylanase (0.73 U/ml) was at pH 5.6 after 9 to 12 days at 30°C. The xylanase was stable at pH 4.5 to 7.5 for 2h but above 50°C its stability fell sharply.The authors are with the Department of Microbiology, University of Delhi South Campus, Benito Juarez Road, New Delhi-110021, India;     

Secretome analysis of thermophilic mould Myceliophthora thermophila cultivated on rice straw and hydrolysis of lignocellulosic biomass for bioethanol production

Abstract

Myceliophthora thermophila encodes for large number of carbohydrate-active enzymes (CAZymes) involved in lignocellulosic biomass degradation. The mould was grown on rice straw in solid state fermentation at pH 5.0 and 45?°C that produced high levels of cellulolytic and xylanolytic enzymes i.e. 2218.12, 515.23, 478.23, 13.34?U/g DMR for xylanase, CMCase, FPase and β-glucosidase, respectively. The secretome analysis of M. thermophila BJAMDU5 by mass spectroscopy, described 124 different proteins with majority of CAZymes consisting of glycosyl hydrolases (GH), lytic polysaccharide mono-oxygenases (LPMO), carbohydrate esterases (CE) and polysaccharide lyases (PL). Furthermore, the enzyme cocktail of the mould was evaluated for hydrolysis of steam treated rice straw that produced 184.59?mg/g substrate reducing sugars after 24?h, which was used for production of bioethanol by using fast fermenting yeast Saccharomyces cerevisiae resulting in high production of bioethanol.     

Organic Cultivation of Triticum turgidum subsp. durum Is Reflected in the Flour-Sourdough Fermentation-Bread Axis

Triticum turgidum subsp. durum was grown according to four farming systems: conventional (C_ONV), organic with cow manure (O_MAN) or green manure (O_LEG), and without inputs (NO_INPUT). Some chemical and technological characteristics differed between C_ONV and organic flours. As shown by two-dimensional electrophoresis (2-DE) analysis, O_MAN and O_LEG flours showed the highest number of gliadins, and O_MAN flour also had the highest number of high-molecular-mass glutenins. Type I sourdoughs were prepared at the laboratory level through a back-slopping procedure, and the bacterial ecology during sourdough preparation was described by 16S rRNA gene pyrosequencing. Before fermentation, the dough made with C_ONV flour showed the highest bacterial diversity. Flours were variously contaminated by genera belonging to the Proteobacteria, Firmicutes, and Actinobacteria. Mature sourdoughs were completely and stably dominated by lactic acid bacteria. The diversity of Firmicutes was the highest for mature sourdoughs made with organic and, especially, NO_INPUT flours. Beta diversity analysis based on the weighted UniFrac distance showed differences between doughs and sourdoughs. Those made with C_ONV flour were separated from the other with organic flours. Lactic acid bacterium microbiota structure was qualitatively confirmed through the culturing method. As shown by PCR-denaturing gradient gel electrophoresis (DGGE) analysis, yeasts belonging to the genera Saccharomyces, Candida, Kazachstania, and Rhodotorula occurred in all sourdoughs. Levels of bound phenolic acids and phytase and antioxidant activities differed depending on the farming system. Mature sourdoughs were used for bread making. Technological characteristics were superior in the breads made with organic sourdoughs. The farming system is another determinant affecting the sourdough microbiota. The organic cultivation of durum wheat was reflected along the flour-sourdough fermentation-bread axis.     

Concomitant production of cellulase and xylanase by thermophilic mould <Emphasis Type="Italic">Sporotrichum thermophile</Emphasis> in solid state fermentation and their applicability in bread making

Sporotrichum thermophile BJAMDU5 secreted high titres of xylanolytic and cellulolytic enzymes in solid state fermentation using mixture of wheat straw and cotton oil cake (ratio 1:1) at 45?°C, pH 5.0 after 72 h inoculated with 2.9?×?10⁷ CFU/mL conidiospores. Supplementation of solid medium with lactose and ammonium sulphate further enhanced the production of hydrolytic enzymes. Among different surfactants studied, Tween 80 enhanced the production of all enzymes [3455 U/g DMR (dry mouldy residue), 879.26 U/g DMR, 976.28 U/g DMR and 35.10 U/g DMR for xylanase, CMCase (Carboxymethylcellulase), FPase (Filter paper activity) and β-glucosidase, respectively] as compared to other surfactants. Recycling of solid substrate reduced the production of all these enzymes after second cycle. End products analysis by TLC showed the ability of hydrolytic enzymes of S. thermophile to liberate monomeric (xylose and glucose) as well as oligomeric (xylobiose, cellobiose and higher ones) sugars. Supplementation of enzyme resulted in improved nutritional properties of the bread. Formation of oligomeric sugars by xylanase enzyme of S. thermophile BJAMDU5 make it a good candidate in food industry.     

Phytase from Citrobacter koseri PM-7: Enhanced production using statistical method and application in ameliorating mineral bioaccessibility and protein digestibility of high-phytate food

The present study was aimed at enhancing phytase (Phy-Ck) production from Citrobacter koseri PM-7 using response surface methodology (RSM) and improving the bioaccessibility of minerals (Fe and Zn) and protein digestibility in high-phytate food using Phy-Ck. A five-variable and three-level central composite design of RSM using wheat bran (6.681%, w/v), inoculum level (2.5%, v/v), and triton X-100 (0.2%, v/v) resulted in up to 5.57-fold (1.047 U/ml) improvement in Phy-Ck yield from C. koseri PM-7 when compared with fermentation media I and II. The model was successfully validated in the design space by taking a random set of variable combinations. Treatment of high-phytate food with partially purified Phy-Ck showed improvement in mineral bioaccessibility maximally for defatted sesame flour (DSF) (Fe 45.5%; Zn 50.7%) followed by wheat flour (WF) (Fe 13.5%; Zn 14.4%), green gram flour (GGF) (Fe 0.7%; Zn 3.8%) and defatted groundnut flour (DGF) (Zn 5.6%). The in vitro protein digestibility (IVPD) of WF increased from 48.83 to 65.04%, GGF from 45.04 to 57.12%, and DSF from 47.34 to 55.7% after Phy-Ck treatment.     

Medium optimization of a hydrophilic solvent‐stable protease from Serratia sp. SYBC H

Two statistical methods were used for medium optimization for a hydrophilic solvent‐stable protease production by Serratia sp. SYBC H with duckweed as the nitrogen source. Orthogonal design was applied to find the significant variables, then response surface methodology (RSM), including Box–Behnken central composite experiments, was used to determine the optimal concentrations and interaction of the significant variables. Results demonstrated that duckweed powder, wheat flour, Tween 80, sodium chloride had significant effects on the solvent‐stable protease production. The interaction between duckweed and wheat flour was significant. The optimal level of the variables for the maximum protease production was duckweed 43.9 g/L, wheat flour 20 g/L, sodium chloride 0.08 M, Tween 80 1% v/v, initial pH 11.0, and inoculum size 7% v/v. The maximum protease activity reached 1922.8 U/mL in the optimized medium, with about 18.3‐fold higher than that in the unoptimized medium. Most importantly, the protease from Serratia sp. SYBC H has successfully catalyzed the specific acylation of sucrose in a two‐solvent medium consisting of pyridine and n‐hexane (1:1, v/v), and non‐specific acylation of sucrose in anhydrous DMSO. These results demonstrated that the protease from Serratia sp. SYBC H is a solvent‐stable protease and it could be an ideal biocatalyst for sugar esters syntheses in non‐aqueous media.     

Degradation of starchy substrates by a crude enzyme preparation and utilization of the hydrolysates for lactic fermentation

An enzyme preparation obtained from Aspergillus ustus, possessing cellulase, α-amylase, amyloglucosidase, proteinase and d-xylanase activities, was used along with commercial bacterial α-amylase and amyloglucosidase for the degradation of ragi (Eleusine coracana) flour and wheat (Triticum vulgare) bran. Lactic acid yield from ragi hydrolysate, adjusted to 5% reducing sugars (w/v), was 25% when fermented with Lactobacillus plantarum. The yields increased to 78% and 94% when the ragi hydrolysate was fortified with 20% and 60% (v/v) wheat bran hydrolysate, respectively. When commercial α-amylase and amyloglucosidase were used for the hydrolysis of ragi and wheat bran and L. plantarum was employed to ferment the hydrolysates containing 5% reducing sugars (w/v), lactic acid yields were 10% in ragi hydrolysate and 57% and 90% when the ragi hydrolysate was fortified with 20% and 60% (v/v) of wheat bran hydrolysate, respectively. α-Amylase and amyloglucosidase hydrolysed wheat bran added at 20% (v/v) as the sole source of nutrient to soluble starch hydrolysate (5% reducing sugars) gave 22% yield of lactic acid. The yield increased to 55% by the utilization of A. ustus enzyme preparation in addition to α-amylase and amyloglucosidase for wheat bran hydrolysis.     

Hydrophobicity of stored (15, 35 °C), or dry-heated (120 °C) rice flour and deteriorated breadmaking properties baked with these treated rice flour/fresh gluten flour

Rice flour was stored at 15 °C/9 months, at 35 °C/14 days, or dry-heated at 120 °C/20 min. The breadmaking properties baked with this rice flour/fresh gluten flour deteriorated. In addition, the rice flour was mixed with oil in water vigorously, and oil-binding ability was measured. Every rice flour subjected to storage or dry-heated at 120 °C showed higher hydrophobicity, owing to changes in proteins. Then, proteins in the stored rice flour were excluded with NaOH solution, and bread baked with the deproteinized rice flour showed the same breadmaking properties as unstored rice flour/fresh gluten flour. The viscoelasticity of wheat glutenin fraction decreased after the addition of dry-heated rice flour in a mixograph profile. DDD staining increased Lab in color meter, which suggested an increase in SH groups in rice protein. The increase in SH groups caused a reduction in wheat gluten protein resulting in a deterioration of rice bread quality.     

Optimization of phytase production by solid substrate fermentation

The production of phytase by three feed-grade filamentous fungi (Aspergillus ficuum NRRL 3135, Mucor racemosus NRRL 1994 and Rhizopus oligosporus NRRL 5905) on four commonly used natural feed ingredients (canola meal, cracked corn, soybean meal, wheat bran) was studied in solid substrate fermentation (SSF). A. ficuum NRRL 3135 had the highest yield [15 IU phytase activity/g dry matter (DM)] on wheat bran. By optimizing the supplementation of wheat bran with starch and (NH₄)₂SO₄, phytase production increased to 25 IU/g DM. Optimization was carried out by Plackett-Burman and central composite experimental designs. Using optimized medium, phytase, phosphatase, alpha-amylase and xylanase production by A. ficuum NRRL 3135 was studied in Erlenmeyer flask and tray SSF. By scaling up SSF from flasks to stationary trays, activities of 20 IU phytase activity/g DM were reproducibly obtained. Electronic Publication     

Optimization of nutrient medium containing agricultural waste for xylanase production by Bacillus pumilus B20

We aimed to optimize a nutrient medium containing agricultural waste for xylanase production by Bacillus pumilus B20. Xylanase production with lignocellulosic material was optimized in two steps using DeMeo’s fractional factorial design. A 3.4-fold increase in xylanase production (313.3 U/mL) was achieved using the optimized culture medium consisting of (g/L): K₂HPO₄, 2; MgSO₄·7H₂O, 0.3; CaCl₂·2H₂O, 0.01; NaCl, 2; peptone, 5 yeast extract, 4; and wheat bran, 50. _{B. pumilus} B20 produced a high level of xylanase, which may have potential industrial application.     

Optimization of xylanase production using inexpensive agro-residues by alkalophilic Bacillus subtilis ASH in solid-state fermentation

Alkalophilic Bacillus subtilis ASH produced high levels of xylanase using easily available inexpensive agricultural waste residues such as wheat bran, wheat straw, rice husk, sawdust, gram bran, groundnut and maize bran in solid-state fermentation (SSF). Among these, wheat bran was found to be best substrate. Xylanase production was highest after 72 h of incubation at 37 °C and at a substrate to moisture ratio of 1:2 (w/v). The inoculum level of 15% resulted in maximum production of xylanase. The enzyme production was stimulated by the addition of nutrients such as yeast extract, peptone and beef extract. In contrast, addition of glucose and xylose repressed the production of xylanase. The extent of repression by glucose (10%, w/v) was 81% and it was concentration-dependent. Supplementation of the medium with 4% xylose caused 59% repression. Under optimized conditions, xylanase production in SSF (8,964 U of xylanase/g dry wheat bran) was about twofold greater than in submerged fermentation. Thus, B. subtilis produced a very high level of xylanase in SSF using inexpensive agro-residues, a level which is much higher than that reported by any other bacterial isolate. Furthermore, the enzyme was produced at room temperature and with tap water without the addition of any mineral salt in SSF, leading to a marked decrease in the cost of xylanase production, which enhances its industrial potential.     

Improvement of direct ethanol fermentation from woody biomasses by the Antarctic basidiomycetous yeast, Mrakia blollopis, under a low temperature condition

The Antarctic basidiomycetous yeast Mrakia blollopis SK-4 can quite uniquely ferment various sugars under low temperature conditions. When strain SK-4 fermented lignocellulosic biomass using the direct ethanol fermentation (DEF) technique, approximately 30% to 65% of the theoretical ethanol yield was obtained without and with the addition of the non-ionic surfactant Tween 80, respectively. Therefore, DEF from lignocellulosic biomass with M. blollopis SK-4 requires the addition of a non-ionic surfactant to improve fermentation efficiency. DEF with lipase converted Eucalyptus and Japanese cedar to 12.6 g/l, and 14.6 g/l ethanol, respectively. In the presence of 1% (v/v) Tween 80 and 5 U/g-dry substrate lipase, ethanol concentration increased about 1.4- to 2.4-fold compared to that without Tween 80 and lipase. We therefore consider that the combination of M. blollopis SK-4 and DEF with Tween 80 and lipase has good potential for ethanol fermentation in cold environments.     

Enzymatic bleaching of kraft pulp by xylanase from Aspergillus sydowii SBS 45

Crude xylanase from Aspergillus sydowii SBS 45 was tested for enzymatic bleaching of kraft (Decker) pulp. After optimization of three parameters, consistency of pulp, retention time and enzyme dose, considerable increase in the release of UV and visible absorbance spectra of materials and reducing sugars was observed, which clearly indicated the action of xylanase on pulp. Final brightness of pulp was increased from 29.42 to 70.42% and kappa number was reduced from 15.93 to 1.61, when 25 U of xylanase was given with a retention time of 5 h and at a consistency of 10%. When 10 U g⁻¹ xylanase was given, 14.3% elemental chlorine and 14.3% H₂O₂ could be reduced and when 25 U g⁻¹ xylanase was given 14.3% elemental chlorine and 28.6% H ₂O₂ could be reduced thereby retaining the brightness at control level.     

Improved hydrolase activity in barley and reduced malting time by adding phytase as an activator during malting steeping

Objectives

Exogenous phytase improved the activity of hydrolases to decrease the malting time.

Results

Treatment with phytase during barley steeping increased activity of hydrolases (α-/β-amylase, proteinase, β-glucanase and xylanase) in green malt. Maximal activity was observed for α-/β-amylase, β-glucanase and xylanase with 0.8 U phytase/g and proteinase with 1.2 U phytase/g. Phytase promoted acrospire growth of green malt and reduced malting process with 0.8 U phytase/g in 96 h, which is 24 h less than the control. No significant variation of malt quality was found between control malt and malt treated with 0.8 U/g or 1.2 U phytase/g (P > 0.05), which makes application of exogenous phytase during steeping process as a good option for reducing malting time.

Conclusion

Adding phytase during steeping process increases the activity of hydrolases, which reduces malting time without impacting on malt quality.

     

Recombinant xylanase from Streptomyces coelicolor Ac-738: characterization and the effect on xylan-containing products

A xylanase gene was isolated from the genomic DNA of Streptomyces coelicolor Ac-738. The 723-bp full-length gene encoded a 241-amino acid peptide consisting of a 49-residue putative TAT signal peptide and a glycoside hydrolase family-11 domain. The mature enzyme called XSC738 was expressed in Escherichia coli M15[pREP4]. The electrophoretically homogeneous protein with a specific activity of 167 U/mg for beechwood xylan was purified. The pH optimum of XSC738 was at pH 6; a high activity was retained within a pH range of 4.5–8.5. The enzyme was thermostable at 50–60 °C and retained an activity at pH 3.0–7.0. Xylanase XSC738 was activated by Mn²⁺, Co²⁺ and largely inhibited by Cd²⁺, SDS and EDTA. The products of xylan hydrolysis were mainly xylobiose, xylotriose, xylopentaose and xylohexose. Xylotetraose appeared as a minor product. Processing of such agricultural xylan-containing products as wheat, oats, soy flour and wheat bran by xylanase resulted in an increased content of sugars.