Alkali-tolerant Xylanase Research Articles

Microwave-assisted xylanase reaction: impact in the production of prebiotic xylooligosaccharides.

The enzymatic production of prebiotic xylooligosaccharides (XOS) has become an attractive way to valorise lignocellulosic biomass. However, despite numerous xylanases reported for potential use in the production of XOS, most of the family GH10 also produce xylose. This monosaccharide can negatively affect the selectivity to stimulate the growth of intestinal microorganisms beneficial to human health. In this work, thermostable alkali-tolerant xylanase (BhXyn10A) from Bacillus halodurans S7 has been used to produce XOS under conventional convective heat transfer and microwave radiation. The microwave-assisted reaction markedly decreases the xylose content in the hydrolysates and significantly increases the yield of XOS, compared to conventional heating. Molecular dynamics simulations of BhXyn10A have shown increased fluctuations of the amino acids of the aglycone subsites suggesting that these subsites can determine the production of xylose. Thus, microwave heating could affect the amino acid fluctuations in the aglycone subsites reducing the xylose formation. These findings open up new avenues in enzyme technology for the production of XOS.

Valorization of untreated rice bran towards bioflocculant using a lignocellulose-degrading strain and its use in microalgal biomass harvest

BackgroundMicroalgae are currently considered as a promising feedstock for the production of biofuels and high-value products. However, the efficient harvest of microalgal biomasses from their culture broth is a major challenge. The harvesting of algal biomass by flocculation combined with gravity sedimentation is more convenient and cost-effective than traditional methods such as centrifugation and filtration. Compared to inorganic and chemically synthetic flocculants, bioflocculants are a suitable choice for microalgal harvest due to their biodegradable and nontoxic properties. Nonetheless, the high production costs associated with expensive substrates hinder the commercial applications of bioflocculants. Previous studies have shown that the hydrolysates of lignocellulosic biomasses from dilute acid hydrolysis can be utilized as an inexpensive carbon source for the production of bioflocculants. However, the toxic by-products generated in the dilute acid hydrolysis step limit the efficiency of subsequent fermentation. The strains that produce bioflocculants by using untreated lignocellulosic materials can circumvent the pretreatment process, as well as promote the application of bioflocculants in microalgal harvest.ResultsUnder alkaline fermentation conditions, the alkaliphilic strain Bacillus agaradhaerens C9 secreted 1.69 IU/mL of alkali-tolerant xylanase and 0.06 IU/mL of cellulase, indicating that this particular strain can efficiently convert untreated rice bran into bioflocculant (RBBF-C9), thereby circumventing rice bran pretreatment for downstream fermentation. The optimal fermentation conditions that result in the highest bioflocculant yield (12.94 g/L) were as follows: 20 g/L of untreated rice bran, 3 g/L of yeast extract, and 20 g/L of Na2CO3 at 37 °C for 24 h. RBBF-C9 contained 74.12% polysaccharides and 4.51% proteins, and was estimated to be 137 kDa. Furthermore, the bioflocculant RBBF-C9 exhibited good flocculating efficiency (91.05%) of oil alga Chlorella minutissima UTEX2341 when 60 mg/L of RBBF-C9 was added into the algal culture broth.ConclusionsThis study demonstrated that untreated rice bran is a suitable inexpensive substrate for the production of bioflocculants, and thus provides a novel approach in utilizing rice bran. The extracted bioflocculants may be potentially used in biomass harvesting of the oil algae C. minutissima UTEX2341 from the culture broth.

Extracellular expression of alkali tolerant xylanase from Bacillus subtilis Lucky9 in E. coli and application for xylooligosaccharides production from agro-industrial waste

An alkali tolerant xylanase gene from Bacillus subtilis Lucky9 was cloned and extracellular expressed in E. coli BL21. Xylanase amino acid sequence showed 99% identity with xylanase sequence from Bacillus subtilis 168, and was belonged to glycoside hydrolase family 11. The recombinant E. coli (pET-pelB-xynLC9) containing pelB signal peptide produced extracellular xylanase of 436.5U/mL for 8h, which was used arabinose as extra carbon source and inducer for enhancing extracellular production. The extracellular xylanase was determined by SDS–PAGE with a relative molecular mass of 21kDa. The recombinant xylanase was optimally activity at pH 6.5 and 60°C. The xylanase exhibited 80% residual activity over a broad pH range of 6.0–9.0 for 24h. Thermostability studies showed that xylanase retained 60% residual activity after 2h at 60°C. The main end-products of hydrolysis of beech-wood xylan and corncob by the extracellular xylanase were xylobiose and xylotriose. This extracellular xylanase without purification is a suitable candidate for application in the industrial production of xylooligosaccharides from agro-industrial waste for use as prebiotics.

Investigation on the underlying mechanism: How fusion xylanase-ELPs self-assembles into insoluble active aggregates

We have successfully obtained a recombinant xylanase by fusing with elastin-like polypeptides (ELPs), the xylanase SoxB underwent a sharp irreversible phase transition, and self-assembled into an insoluble but more catalytically operative particle. This was analogous to the immobilized xylanase to a large extent and aroused our interest to gain new insights into the determinant factor that may cause this phenomenon. We herein listed several candidate factors including the length of ELPs, linker sequence, buffer properties, and the target protein, subsequently we evaluated their contributions to the formation of the active aggregates. The results suggested that SoxB was fused with ELPs as desired protein partners, neither ELPs length nor the linker type made crucial contribution to the formation of active aggregates. However, when Na2CO3 was chosen as the salt to trigger the phase transition, the catalytic activities detected in aggregates accounted for more than 87.7% of total activity, whereas above 83.8% of the activity remained in supernatant when using Na2SO4. Then we introduced an alkali-tolerant xylanase termed as Xyl and compared it with SoxB, and found that the activity ratio in insoluble particle dropped to 15.3% in Na2CO3 and 19.3% in Na2SO4 respectively, only a few insoluble aggregates observed during the purification. Therefore, we speculated the property of xylanase partner fused to ELPs should be the predominant factor to form the catalytically active insoluble aggregates, and this provides a promising not yet reported perspective for industrial application of “immobilized” SoxB.

Optimal secretion of alkali-tolerant xylanase in Bacillus subtilis by signal peptide screening.

Xylanases are industrially important enzymes for xylan digestion. We experimentally screened over 114 Sec and 24 Tat pathway signal peptides, with two different promoters, for optimal production of an alkaline active xylanase (XynBYG) from Bacillus pumilus BYG in a Bacillus subtilis host. Though both promoters yielded highly consistent secretion levels (0.97 Pearson correlation coefficient), the Sec pathway was found to be more efficient than the Tat pathway for XynBYG secretion. Furthermore, the optimal signal peptide (phoB) for XynBYG secretion was found to be different from the optimal peptides for cutinase and esterase reported in previous studies. A partial least squares regression analysis further identified several statistically important variables: helical properties, amino acid composition bias, and the discrimination score in Signal P. These variables explain the observed 23% variance in the secretion yield of XynBYG by the different signal peptides. The results also suggest that the helical propensity of a signal peptide plays a significant role in the beta-rich xylanase, but not in the helix-rich cutinase, suggesting a coupling of the conformations between the signal peptide and its cargo protein for optimal secretion.

Application of pure, thermostable, alkali-tolerant xylanase in bleaching of oxygen-delignified pine kraft pulp

The effect of oxygen (O2)-delignified pine kraft pulp pretreatment by high-purity, thermostable, and alkaline-tolerant xylanases on elemental chlorine free (ECF) bleaching of O2-delignification kraft pulp was studied. The study found that xylanase pretreatment preserved the intrinsic viscosity and yield of O2-delignified pulp while causing about 7% of delignification with high delignification selectivity. The xylanases with high purity, higher thermostability (75°C~80°C) in highly alkaline media (pH 8.0~9.5) could be applied on an industrial scale. Pulp pretreatment by the high-purity, thermostable, and alkaline tolerant xylanases could improve pulp brightness or reduce the chlorine dioxide (ClO2) consumption. In a D0ED1D2 bleaching sequence using the same amount of ClO2, the xylanase-pretreated pulp obtained a higher brightness (88.2% vs. 89.7% ISO) at the enzyme dose of 2 U/g pulp; or for the same brightness as control (88.2% ISO), the ClO2 dosage in the D0 stage was reduced by 27%, which represents a 16% savings in total ClO2 used for bleaching.

Production of prebiotic xylooligosaccharides from alkaline extracted wheat straw using the K80R-variant of a thermostable alkali-tolerant xylanase

Agricultural by-products are raw materials of importance for increased utilization of renewable biomass. Wheat straw is a raw material of significant production volume and is in this work used for production of xylooligosaccharides (XOS). Extraction of xylan by dilute alkali was followed by hydrolysis using a variant of the alkali-tolerant Bacillus halodurans S7 endoxylanase A mutated at K80R. The xylan yield was on average 56.5g xylose equivalents per kg dried, ground wheat straw, with 1 arabinose per 12 xylose residues. The K80R variant, which displayed higher specific activity than the wild type enzyme, was added at a load of 96U/g extracted xylan. The XOS-yield (xylobiose–xylopentaose) was evaluated at time intervals in the temperature range of 50–65°C, at pHs from 7 to 10. The enzyme was optimally active at 60°C up to pH 9. Hydrolysis was completed within 7h, resulting in 36% conversion of the xylan to predominantly xylobiose. Xylose content was low (2.4%) despite extended incubation, which is desirable for XOS-production. The XOS-containing hydrolysate was confirmed as a suitable carbon source for the putative probiotic strain Lactobacillus brevis DSM 1269, showing the applicability of the method to obtain prebiotic XOS.

Cloning, expression, and characterization of a novel alkali-tolerant xylanase from alkaliphilic Bacillus sp. SN5.

A xylanase gene (xyn11A) was cloned from the genomic library of alkalophilic Bacillus sp. SN5. It encoded a polypeptide of 366 amino acids, consisting of a family 11 glycoside hydrolase, a short linker region, and a family 36 carbohydrate-binding module (CBM). The intact xylanase Xyn11A and the CBM-linker-truncated Xyn11A-LC were expressed in Escherichia coli BL21 (DE3). Both purified recombinant proteins exhibited the highest activity at 55 °C. The optimal pH for Xyn11A activity was 7.5, whereas Xyn11A-LC showed a broad pH profile (>80% activity at pH 5.5-8.5) with optimal activity at pH 5.5 and 7.5-8.0. They had high alkali tolerance, retaining over 80% residual activity after preincubation at pH 8.5-11.0 at 37 °C for 1 H. Xyn11A-LC showed better thermal stability, lower affinity, and lower catalytic activity to insoluble xylan than Xyn11A, whereas its specific activity for soluble beechwood xylan (4,511.9 U/mg) was greater than that of Xyn11A (3,136.4 U/mg). These results implied that the CBM of Xyn11A could change the enzymatic properties and play a role in degrading insoluble xylan. Xyn11A-LC is a family 11 alkali-tolerant cellulase-free xylanase with high specific activity, which qualifies it as a potential candidate for industrial applications, especially in the paper industry.

An alkali-tolerant xylanase produced by the newly isolated alkaliphilic Bacillus pumilus from paper mill effluent

An alkaline active xylanase, XynBYG, was purified from an alkaliphilic Bacillus pumilus BYG, which was newly isolated from paper mill effluent. It had an optimum pH of 8.0-9.0, and showed good stability after incubated at pH 9.0 for 120 min. The optimum temperature for the activity was 50°C, and the enzyme retained below 55% of its original activity for 30 min at 55°C. The gene coding for XynBYG consists of 687 bp and encodes 229 amino acids. Similarity analysis indicated that XynBYG belong to family 11 glycosyl hydrolases. Site-directed mutagenesis was performed to replace five sites (Tyr/Ser) to Arg/Glu and the results demonstrated that the optimum temperature of the mutant Y7 (S39R-T146E) increased 5°C and the half-life of inactivation (T1/2) at 60 and 65°C was 1 h and 25 min, respectively. Thus, it provides a potential xylanase that can meet the harsh conditions in the industrial applications.

Parthenium sp. as a plant biomass for the production of alkalitolerant xylanase from mutant Penicillium oxalicum SAUE-3.510 in submerged fermentation

Abstract The use of congress grass ( Parthenium sp.) and water hyacinth ( Eichhornia crassipes ) as low cost raw materials for xylanase production from mutant Penicillium oxalicum SAU E -3.510 in submerged fermentation was investigated. For development of mutant from wild type P. oxalicum SA-8 ITCC 6024, a strategy of mixed mutagenesis was followed using UV-irradiation and ethidium bromide, which had resulted into 1.87 fold increases in the activity of the enzyme. For enzyme production, the fungus was cultivated in mineral medium containing congress grass as carbon source. Considerably higher levels of production (475.2 ± 6.0 IU ml −1 ) were achieved in media containing congress grass, although it was slightly less than that was obtained (488.5 ± 6.5 IU ml −1 ) in presence of commercial oat spelt xylan. This fact confirms the feasibility of using this low cost non-food resource as an alternative carbon source to save costs of the enzyme production process. Maximum xylanase activity was reported at 55 °C with its stability at 80 °C for 2 h. The highest activity of xylanase at pH 9.0 and its stability at similar pH for 24 h denote the alkalitolerant nature of enzyme.

Application of crude xylanase from Bacillus licheniformis 77-2 to the bleaching of eucalyptus Kraft pulp

Alkalophilic Bacillus licheniformis 77-2 produced an extracellular alkali-tolerant xylanase with negligible cellulase activity in medium containing corn straw. The effectiveness of crude xylanase on treatment of eucalyptus Kraft pulp was evaluated. A biobleaching experiment was carried out to compare the chlorine saving with pulp treated and untreated by the enzyme. Two-stage bleaching was employed, using a ClO2 chlorination and NaOH extraction (DE sequence). With the enzymatic treatment, in order to obtain the same value of Kappa number and brightness, respectively 28.5 and 30% less ClO2 was required in comparison to the enzymatically untreated samples.

A cellulase-poor, thermostable, alkalitolerant xylanase produced by Bacillus circulans AB 16 grown on rice straw and its application in biobleaching of eucalyptus pulp

Bacillus circulans AB 16 isolated from a garbage dump produced appreciable quantities (19.28 IU/ml) of extracellular thermophilic xylanase, but negligible quantities of cellulase, when grown on 0.3% xylan. The optimum pH for the enzyme was 6.0–7.0, but it was stable over a wide range of pH (5.0–9.0). The optimum temperature was 80°C. The organism produced 20.6 IU/ml of xylanase in shake flask on rice straw, an inexpensive lignocellulosic biomass. Glucose, fructose, xylose and other sugars induced enzyme levels only in the range 0.82–2.52 IU/ml. The crude enzyme produced on rice straw showed good thermal and pH stability, retaining 67% activity after 1 h at 70°C, pH 9 and 84.5% activity after 2 h at 65°C, pH 9. The enzyme had a half-life of 24 h at 70°C, pH 7. When the xylanase from B. circulans AB 16 was used in the prebleaching of eucalyptus Kraft pulp the amount of chlorine was reduced by 20% without any decrease in brightness. The viscosity of xylanase-treated pulp was 9.5–9.7 cp, whereas that of the pulp treated exclusively with chlorine was 9.2 cp.

Enhanced production, purification and characterisation of a novel cellulase-poor thermostable, alkalitolerant xylanase from Bacillus circulans AB 16

A thermophilic Bacillus circulans AB 16, isolated from a garbage dump produced novel extracellular cellulase-poor, thermostable xylanase in basal medium containing rice straw. The xylanase yield was enhanced more than two and a half fold in the presence of tryptone and other media modifications. The highest xylanase activity obtained in liquid culture was 55 IU/ml. Two xylanases Xyl A and Xyl B were purified to homogenity by Q Sepharose and Sepharose 6B chromatograph. Xyl A ( M r 30 000) had a pH optima of 6 and a temperature optima of 75–80°C, while Xyl B ( M r 22 000) also had a pH optima of 6 but with a temperature optima of 65–70°C. Both Xyl A and Xyl B had a pH optima of 6 but retained 46% activity at pH 8. Xyl A retained 70% activity at 65°C, pH 9 and 2 h incubation while Xyl B retained 34% under the similar conditions. Both Xyl A and Xyl B showed 90% inhibition in activity by 1 mM Hg 2+. The hydrolysis pattern of Xyl A yielded mainly xylobiose with lower amount of xylo-oligosaccharides and xylose while Xyl B yielded mainly higher oligosaccharides with lesser amounts of xylobiose and negligible amounts of xylose. The crude enzyme from B. circulans AB 16 showed higher chromophore release of 0.360 U as compared to Xyl A and Xyl B with a chromophore release of 0.115 and 0.069 units and thus would be more useful for pulp bleaching than purified xylanases.

Production of a thermostable alkali-tolerant xylanase from Bacillus circulans AB 16 grown on wheat straw.

Bacillus circulans AB 16 was able to produce 50 IU/ml of xylanase, with negligible cellulase activity when grown on untreated wheat straw. The pH optimum of the crude enzyme was 6–7 with a temperature optimum of 80 ∘C. The enzyme showed high pH and thermal stability retaining 100% activity at 60 ∘C, pH 8 and 9 after 2.5 h of incubation. The residual activity at 70 ∘C after 2.5 h was 62% and 45% at pH 8 and 9, respectively. At 75 ∘C only 22.2% activity remained at pH 8 after 1 h incubation. Since Kraft pulp is alkaline this enzyme could be used for prebleaching of pulp at temperatures up to 70 ∘C without pH adjustment.