Thermostable Xylanase Research Articles

Utility of Thermostable Xylanases of Mycothermus thermophilus in Generating Prebiotic Xylooligosaccharides

Xylooligosaccharides as emerging prebiotics are able to promote the growth of probiotic bacteria. In the present study, four neutral, thermostable xylanases (MtXyn11A, MtXyn11At, MtXyn11B, and MtXyn11C) from compost fungus Mycothermus thermophilus CGMCC3.18119 were overexpressed in Pichia pastoris GS115 and used to produce xylooligosaccharides from beechwood xylan. The enzymes showed similar enzymatic properties (maximal activities at pH 6.0-6.5 and 65 °C) but varied in catalytic efficiency and cleaving actions. MtXyn11A, MtXyn11At, and MtXyn11C mainly produced xylobiose (59-62%), xylose (16-20%), and xylotriose (16-19%), while MtXyn11B released xylobiose (51%), xylotriose (32%), and xylose (12%) as the main products. When using the xylan hydrolysates of different xylanases as the carbon source, four probiotic Lactobacillus strains Lactobacillus brevis 1.2028, Lactobacillus rhamnosus GG, Lactobacillus casei BL23, and Lactobacillus plantarum WCSF1 were confirmed to use the xylooligosaccharides efficiently (83.8-98.2%), with L. brevis 1.2028 as the greatest.

Droplet-based microfluidic high-throughput screening of heterologous enzymes secreted by the yeast Yarrowia lipolytica

BackgroundDroplet-based microfluidics is becoming an increasingly attractive alternative to microtiter plate techniques for enzymatic high-throughput screening (HTS), especially for exploring large diversities with lower time and cost footprint. In this case, the assayed enzyme has to be accessible to the substrate within the water-in-oil droplet by being ideally extracellular or displayed at the cell surface. However, most of the enzymes screened to date are expressed within the cytoplasm of Escherichia coli cells, which means that a lysis step must take place inside the droplets for enzyme activity to be assayed. Here, we take advantage of the excellent secretion abilities of the yeast Yarrowia lipolytica to describe a highly efficient expression system particularly suitable for the droplet-based microfluidic HTS.ResultsFive hydrolytic genes from Aspergillus niger genome were chosen and the corresponding five Yarrowia lipolytica producing strains were constructed. Each enzyme (endo-β-1,4-xylanase B and C; 1,4-β-cellobiohydrolase A; endoglucanase A; aspartic protease) was successfully overexpressed and secreted in an active form in the crude supernatant. A droplet-based microfluidic HTS system was developed to (a) encapsulate single yeast cells; (b) grow yeast in droplets; (c) inject the relevant enzymatic substrate; (d) incubate droplets on chip; (e) detect enzymatic activity; and (f) sort droplets based on enzymatic activity. Combining this integrated microfluidic platform with gene expression in Y. lipolytica results in remarkably low variability in the enzymatic activity at the single cell level within a given monoclonal population (<5%). Xylanase, cellobiohydrolase and protease activities were successfully assayed using this system. We then used the system to screen for thermostable variants of endo-β-1,4-xylanase C in error-prone PCR libraries. Variants displaying higher thermostable xylanase activities compared to the wild-type were isolated (up to 4.7-fold improvement).Conclusions Yarrowia lipolytica was used to express fungal genes encoding hydrolytic enzymes of interest. We developed a successful droplet-based microfluidic platform for the high-throughput screening (105 strains/h) of Y. lipolytica based on enzyme secretion and activity. This approach provides highly efficient tools for the HTS of recombinant enzymatic activities. This should be extremely useful for discovering new biocatalysts via directed evolution or protein engineering approaches and should lead to major advances in microbial cell factory development.

English

Filamentous fungi have been investigated as producer of xylanases with relevant characteristics for application in different industrial sectors, such as bakery, beverage, biofuel, textile, animal feed, pharmaceutical, pulp and paper. Thus, this review will focus on biochemical properties and industrial use of thermostable xylanases produced by different filamentous fungi, as well as mechanisms of adaptation of thermophilic organisms to tolerate in high-temperature environments. These enzymatic properties of thermal and pH stability are crucial, especially in processes such as the manufacture of animal feed, pulp and paper industry. Reports on changes in enzyme structure, such as site-directed mutagenesis, insertion or substitution of amino acids, addition of disulfide bonds in the alpha helix or beta-sheet structure for improving the thermal stability will also be reported. However, strains of Thermomyces lanuginosus has been described as good producers of thermostable xylanases, as well as promising enzymes, because it does not require any change in structure to increase the tolerance to high temperatures. Key words: Hemicellulase, thermostability, Thermomyces.

The thermophilic biomass-degrading fungus Thielavia terrestris Co3Bag1 produces a hyperthermophilic and thermostable β-1,4-xylanase with exo- and endo-activity.

A hyperthermophilic and thermostable xylanase of 82kDa (TtXynA) was purified from the culture supernatant of T. terrestris Co3Bag1, grown on carboxymethyl cellulose (CMC), and characterized biochemically. TtXynA showed optimal xylanolytic activity at pH 5.5 and at 85°C, and retained more than 90% of its activity at a broad pH range (4.5-10). The enzyme is highly thermostable with a half-life of 23.1days at 65°C, and active in the presence of several metal ions. Circular dichroism spectra strongly suggest the enzyme gains secondary structures when temperature increases. TtXynA displayed higher substrate affinity and higher catalytic efficiency towards beechwood xylan than towards birchwood xylan, oat-spelt xylan, and CMC. According to its final hydrolysis products, TtXynA displays endo-/exo-activity, yielded xylobiose, an unknown oligosaccharide containing about five residues of xylose and a small amount of xylose on beechwood xylan. Finally, this report represents the description of the first fungal hyperthermophilic xylanase which is produced by T. terrestris Co3Bag1. Since TtXynA displays relevant biochemical properties, it may be a suitable candidate for biotechnological applications carried out at high temperatures, like the enzymatic pretreatment of plant biomass for the production of bioethanol.

Purification, characterization, gene cloning and expression of GH-10 xylanase (Penicillium citrinum isolate HZN13).

An extracellular thermostable xylanase (Xyl-IIb) produced by Penicillium citrinum isolate HZN13 was purified to homogeneity using DEAE-Sepharose, Sephadex G-100 and Bio-Gel P-60 chromatography with specific activity of 6272.7 U/mg and 19.6-fold purification. The purification revealed the occurrence of multiple forms of xylanases (Xyl-I, Xyl-IIa, Xyl-IIb and Xyl-III). The molecular mass of highly purified Xyl-IIb was ~31 kDa with SDS-PAGE. The enzyme was cellulase-free, thermostable (55–75 °C) and acidophilic (3.5–5.0). It was activated by Ca2+, Ba2+, DTT and β-mercaptoethanol, whereas inhibited by Hg2+, Pb2+, Ni2+ and p-CMB. Purified Xyl-IIb exhibited highest specificity toward birchwood and oat spelts xylan. Kinetics of Xyl-IIb revealed a K m of 10 mg/ml and 16.7 mg/ml and V max of 9523g and 15,873 U/mg with birchwood and oat spelts xylan, respectively, indicating high affinity toward birchwood xylan. The xylanase (Xyl-IIb) belongs to glycosyl hydrolase (GH) family 10 based on conserved regions. Xylanase-encoding gene (xynB) consists of 1501 bp with an open reading frame of 264 bp which was predicted to encode a protein having 87 amino acids and shared homology with endo-1,4-beta-xylanase (xynB) gene from Penicillium citrinum. Cloned xynB gene was expressed in E. coli BL21 (DE3) with xylanase activity (80 U/mg) and confirmed to be GH-10 Xyl-IIa based on molecular mass (~40 kDa). These properties of xylanase make it promising for their applications in biofuel industries.Electronic supplementary materialThe online version of this article (doi:10.1007/s13205-016-0489-4) contains supplementary material, which is available to authorized users.

Construction of Thermophilic Xylanase and Its Structural Analysis.

The glycoside hydrolase family 11 xylanase has been utilized in a wide variety of industrial applications, from food processing to kraft pulp bleaching. Thermostability enhances the economic value of industrial enzymes by making them more robust. Recently, we determined the crystal structure of an endo-β-1,4-xylanase (GH11) from mesophilic Talaromyces cellulolyticus, named XylC. Ligand-free XylC exists to two conformations (open and closed forms). We found that the "closed" structure possessed an unstable region within the N-terminal region far from the active site. In this study, we designed the thermostable xylanase by the structure-based site-directed mutagenesis on the N-terminal region. In total, nine mutations (S35C, N44H, Y61M, T62C, N63L, D65P, N66G, T101P, and S102N) and an introduced disulfide bond of the enzyme contributed to the improvement in thermostability. By combining the mutations, we succeeded in constructing a mutant for which the melting temperature was partially additively increased by >20 °C (measured by differential scanning calorimetry) and the activity was additively enhanced at elevated temperatures, without loss of the original specific activity. The crystal structure of the most thermostable mutant was determined at 2.0 Å resolution to elucidate the structural basis of thermostability. From the crystal structure of the mutant, it was revealed that the formation of a disulfide bond induces new C-C contacts and a conformational change in the N-terminus. The resulting induced conformational change in the N-terminus is key for stabilizing this region and for constructing thermostable mutants without compromising the activity.

CLONING, PURIFICATION AND CHARACTERIZATION OF HALOTOLERANT XYLANASE FROM Geobacillus Thermodenitrificans C5

High levels of extracellular xylanase activity (994.50 IU/ml) produced by Geobacillus thermodenitrificans C5 originated gene was detected when it was expressed in E. coli BL21 host. Thermostable xylanase (GthC5Xyl) was purified to homogeneity and showed a molecular mass of approximately 44 kDa according to SDS-PAGE. The specific activity of the purified GthC5Xyl was up to 1243.125IU/mg with a 9.89-fold purification. The activity of GthC5Xyl was stimulated by CoCl2, MnSO4, CuSO4, MnCl2 but was inhibited by FeSO4, Hg, CaSO4. GthC5Xyl showed resistant to SDS, Tween 20, Triton X-100, β- Mercaptoethanol, PMSF, DTT, NEM and DEPC, SDS, and EDTA. A greater affinity for oat spelt xylan was exhibited by GthC5Xyl with maximum enzymatic activity at 60°C and 6.0 pH. The activity portrayed by GthC5Xyl was found to be acellulytic with stability at high temperature (70°C-80°C) and low pH (4.0 to 8.0). Xylobiose and xylopentose were the end products of proficient oat spelt xylanase hydrolysis by GthC5Xyl. High SDS resistance and broader stability of GthC5Xyl proves it to be worthy of impending application in numerous industrial processes especially textile, detergents and animal feed industry.

Exploration of a N-terminal disulfide bridge to improve the thermostability of a GH11 xylanase from Aspergillus niger.

To improve the thermostability of xylanase XynZF-2 from Aspergillus niger XZ-3S, a disulfide bridge was introduced in the N-terminal domains by site-directed mutagenesis (V1C and E27C). Simultaneously, the active sites of XynZF-2 were predicted by bioinformatics software and verified by site-directed mutagenesis (E103D and E194D). The mutated active sites xynED- and the mutated disulfide bridge xynDC-encoding genes were constructed and expressed in Escherichia coli BL21 (DE3). Compared to the native xylanase, it was found that the residual activity of the mutated XynED was 0.17%. The optimum temperature of the variant XynDC was increased to 45°C from 40°C of XynZF-2. After treatment at 40°C for 60 min, the variant XynDC retained 66.77% of their original activity, while the XynZF-2 retained about 44.36% residual activity. t1/2(45°C) of the variant XynDC also increased from 7 min to 14 min. The results of the mutated xylanases indicated that the active center of XynZF-2 mainly consisted of two catalytic residues (Glu103 and Glu194), and the introduction of a disulfide bridge in the N-terminal domains can improve the thermostability of XynZF-2.

Optimization, Isolation and Characterization of Cellulase–Free Thermostable Xylanase from &amp;lt;i&amp;gt;Paenibacillus sp.&amp;lt;/i&amp;gt;

Xylanases are hydrolytic enzymes that cleave the &beta;-1,4-linkage ofwheat bran xylan. For screening of xylanase producing bacteria soil samples were diluted by serial dilution and cultured on selective wheat bran xylan agar media. Two bacterial strains showing clear transparent zone around the colony on xylan agar plate were selected as xylanase producing bacteria. The strain <i>Paenibacillus sp.</i> showed highest xylanolytic activity. The strain was thermophile and produced highly active cellulase free xylanase. The enzyme secretion was enhanced when the medium was supplemented with 0.5% wheat bran xylan, peptone and Ca²⁺ salt. The peak in xylanase production was achieved within 48-60 hours at temperature 50°-55°C and at pH 7.0.The cellulase free xylanase was partially purified by ammonium sulfate fractionation and heat treatment at 50°C. The xylanase was optimally active at pH 7.0 and 55°C; and showed high substrate activity to wheat bran xylan but no activity towards carboxymethylcellulose, cellulose and starch.In future we want to know the structure function relationship of the purified enzyme and also want to known the molecular biological study using highly purified xylanase. For this purpose we have to determine the N-terminal & C-terminal amino acid sequence.

Characterization of Crude Xylanase Produced by Edible Mushroom Pleurotus eryngii

Xylanase has been increasingly forthcoming in recent years because of their possible involvement in numerous industrial processes including bioconversion of lignocellulose derived sugars in to fuels, processing food and the paper and fiber industries. Edible mushrooms are emerging as important source of xylanolytic enzymes and this study has concentrated to produce and characterize xylanases by Pleurotus eryngii. The crude enzyme was characterized on the basis of various parameters such as incubation time, substrate specificity, substrate concentration, enzyme volume, buffer, pH, pH stability, temperature, temperature stability, and effect of various metal ions or compounds. The xylanase activity was noted maximum at 15 minutes of incubation time, 2.0% xylan and 0.5 ml enzyme volume. The highest enzyme activity was found at pH 4.5, whereas xylanase exhibited maximum stability in the range of pH 4.0 to 10.0. The maximum xylanase activity was noted at 60°C, while enzyme was most active and retains more than 40% activity at 90°C within 10 minutes of incubation. ZnCl2 (10mM) stimulated the xylanase activity as compare to other metal ions or compounds. It is concluded that Pleurotus eryngii is capable to produce pH stable and thermostable xylanase for industrial purposes.

English

The production of xylanase without cellulase is required for prebleaching of pulp in pulp and paper industry. Aspergillus flavus produced high levels of xylanase on agricultural residues with wheat bran and sugarcane bagasse (4.17 U/mg), and wheat bran and corncob (2.97 U/mg). Xylanase was found to be stable at 45°C with 100% of its original activity remaining after 2 h incubation. At 50°C, xylanase was stable for the first twenty minutes, and had half-life of 50 min. The pH stability for the xylanase from A. flavus was most stable in the range of pH 3.0-8.0 retaining more that 100% activity after 1 h. The addition of 5% glycerol, mannitol or xylitol protected the xylanase from thermal inactivation at 50°C. The protective effect by glycerol, xylitol and mannitol resulted in increases of 162, 262.5 and 150% when compared with the control at 120 min, approximately. Increasing the polyols concentration up to 20% (w/v) further improved the thermostability of xylanase after 120 min at 50°C by 300% when compared with the control (no additive). The kappa number reduced 2.56 points, which corresponds to 18.34 kappa efficiency. This xylanase is an attractive enzyme for potential future application in the pulp and paper industries, since industrial application requires a cellulase-free activity, maintenance of high temperature and enzyme stability are desirable. Key words: Aspergillus flavus, polyols, xylanase, biobleaching cellulose pulp.

Characterization of Two GAS1 Genes and Their Effects on Expression and Secretion of Heterologous Protein Xylanase B in Kluyveromyces lactis.

β-1,3-glucanosyltransferases play essential roles in cell wall biosynthesis in yeast. Kluyveromyces lactis has six putative β-1,3-glucanosyltransferase genes. KlGAS1-1 and KlGAS1-2 are homologs of Saccharomyces cerevisiae gene GAS1. RT-qPCR indicated the transcription level of KlGAS1-1 was significantly reduced while heterologous protein (thermostable xylanase B) secretion was enhanced during medium optimization. To evaluate if these two events were related, and to improve xylanase B secretion in K. lactis, we constructed KlGAS1-1 and KlGAS1-2 single deletion strains and double deletion strain, respectively. KlGAS1-1 gene deletion resulted in the highest xylanase B activity among the three mutants. Only the double deletion strain showed morphology similar to that of the GAS1 deletion mutant in S. cerevisiae. The two single deletion strains differed in terms of cell wall thickness and xylanase B secretion. Transcription levels of β-1,3-glucanosyltransferase genes and genes related to protein secretion and transport were assayed. The β-1,3-glucanosyltransferase genes displayed transcription complementation in the cell wall synthesis process. KlGAS1-1 and KlGAS1-2 affected transcription levels of secretion- and transport-related genes. Differences in protein secretion ratio among the three deletion strains were associated with changes of transcription levels of secretion- and transport-related genes. Our findings indicate that KlGAS1-1 deletion is an effective tool for enhancing industrial-scale heterologous protein secretion in K. lactis.

Characterization of a purified thermostable xylanase from Caldicoprobacter algeriensis sp. nov. strain TH7C1T

The present study investigates the purification and biochemical characterization of an extracellular thermostable xylanase (called XYN35) from Caldicoprobacter algeriensis sp. nov., strain TH7C1T, a thermophilic, anaerobic strain isolated from the hydrothermal hot spring of Guelma (Algeria). The maximum xylanase activity recorded after 24 h of incubation at 70 °C and in an optimized medium containing 10 g/L mix birchwood- and oats spelt-xylan was 250 U/mL. The pure protein was obtained after heat treatment (1 h at 70 °C), followed by sequential column chromatographies on Sephacryl S-200 gel filtration and Mono-S Sepharose anion-exchange. Matrix assisted laser desorption ionization–time of flight mass spectrometry (MALDI–TOF/MS) analysis indicated that the purified enzyme is a monomer with a molecular mass of 35,075.10 Da. The results from amino-acid sequence analysis revealed high homology between the 21 NH2-terminal residues of XYN35 and those of bacterial xylanases. The enzyme showed optimum activity at pH 11 and 70 °C. While XYN35 was activated by Ca2+, Mn2+, and Mg2+, it was completely inhibited by Hg2+ and Cd2+. The xylanase showed higher specific activity on soluble oat-spelt xylan, followed by beechwood xylan. This enzyme was also noted to obey the Michaelis–Menten kinetics, with Km and kcat values on oat-spelt xylan being 1.33 mg/mL and 400 min−1, respectively. Thin-layer chromatography soluble oat-spelt xylan (TLC) analysis showed that the final hydrolyzed products of the enzyme from birchwood xylan were xylose, xylobiose, and xylotriose. Taken together, the results indicated that the XYN35 enzyme has a number of attractive biochemical properties that make it a potential promising candidate for future application in the pulp bleaching industry.

Production of thermostable xylanase by thermophilic fungal strains isolated from maize silage

The search for microorganisms able to produce thermostable xylanases with high yield and characteristics desired for industrial applications has been strongly encouraged since such enzymes are widely used in large-scale processes. In the present study, thermophilic fungal strains able to grow at high temperatures (≥55 °C) were isolated from maize silage. The strains were molecularly identified and used for the production of extracellular xylanase by solid-state fermentation using corn cobs as support-substrate material. Species from the genera Rhizomucor and Aspergillus were identified among the isolated strains and these species demonstrated good ability to produce xylanase under solid-state fermentation conditions. Maximal values of enzymatic activity (824 U/g) and productivity (8.59 U/g.h) were obtained with Rh. pusillus SOC-4A (values per g dry weight of fermented medium). The xylanase produced by this fungus presented thermal stability at 75 °C, with maximum activity at 70 °C and pH 6.0, revealing, therefore, great potential for application in different areas.

Comparison and Optimization of Thermostable Xylanase Production by Bacillus Pumilus and Bacillus Cereus using Corn Husk

One of the most studied and widely used commercial Xylanase is endo-1, 4-s-xylanases.It is useful because the specific activity of Xylanase is higher than the other hydrolytic xylanases. Therefore, xylanases are commonly used in processes involved in degradation of plant materials, paper and pulp bleaching. The present study investigate the production and optimization of thermostable Xylanase by the bacteria Bacillus pumilus (MTCC 10209) and Bacillus cereus (MTCC 10202) under solid state fermentation using corn husk as carbon source since corn husk contain Xylan (28%) and Xylose (17.2%). Maximum production of Xylanase was observed to be 2.2019 (U/ml) and 2.5010 (U/ml) for B.cereus and B.pumilus respectively. The Response Surface Methodology revealed that the optimum temperature, pH and inoculums size for the enzyme activity were 65⁰C, pH 7.0 and 1 (%v/w) for B.cereus, whereas for B.pumilus it was 65⁰C, pH 7.0 and 1.50 (%v/w) respectively. The increasing concentration of metal ion (FeSo₄) ranging from 1mM- 10mM increases the enzyme activity of B.pumilus. Meanwhile, the enzyme activity of B.cereus was strongly inhibited by increasing metal ion concentration.

Highly Thermostable Xylanase Production from A Thermophilic Geobacillus sp. Strain WSUCF1 Utilizing Lignocellulosic Biomass.

Efficient enzymatic hydrolysis of lignocellulose to fermentable sugars requires a complete repertoire of biomass deconstruction enzymes. Hemicellulases play an important role in hydrolyzing hemicellulose component of lignocellulose to xylooligosaccharides and xylose. Thermostable xylanases have been a focus of attention as industrially important enzymes due to their long shelf life at high temperatures. Geobacillus sp. strain WSUCF1 produced thermostable xylanase activity (crude xylanase cocktail) when grown on xylan or various inexpensive untreated and pretreated lignocellulosic biomasses such as prairie cord grass and corn stover. The optimum pH and temperature for the crude xylanase cocktail were 6.5 and 70°C, respectively. The WSUCF1 crude xylanase was found to be highly thermostable with half-lives of 18 and 12 days at 60 and 70°C, respectively. At 70°C, rates of xylan hydrolysis were also found to be better with the WSUCF1 secretome than those with commercial enzymes, i.e., for WSUCF1 crude xylanase, Cellic-HTec2, and AccelleraseXY, the percent xylan conversions were 68.9, 49.4, and 28.92, respectively. To the best of our knowledge, WSUCF1 crude xylanase cocktail is among the most thermostable xylanases produced by thermophilic Geobacillus spp. and other thermophilic microbes (optimum growth temperature ≤70°C). High thermostability, activity over wide range of temperatures, and better xylan hydrolysis than commercial enzymes make WSUCF1 crude xylanase suitable for thermophilic lignocellulose bioconversion processes.

Direct cloning, expression of a thermostable xylanase gene from the metagenomic DNA of cow dung compost and enzymatic production of xylooligosaccharides from corncob.

To acquire a thermostable xylanase, that is suitable for xylooligosaccharide production from pretreated corncobs, the metagenomic method was used to obtain the gene from an uncultured environmental microorganism. A thermostable xylanase-encoding gene (xyn10CD18) was cloned directly from the metagenomic DNA of cow dung compost. When xyn10CD18 was expressed in Bacillus megaterium MS941, extracellular xylansae activity at 106 IU/ml was achieved. The purified recombinant Xyn10CD18 was optimally active at pH 7 and 75 °C as measured over 10 min. It retained over 55% of its initial activity at 70 °C and pH 7 after 24 h. Its action on birchwood xylan for 18 h liberated xylooligosaccharides with 2°-4° of polymerization, with xylobiose and xylotetraose as the main products. When pretreated corncobs were hydrolyzed by Xyn10CD18 for 18 h, the xylooligosaccharides (DP 2-4) products increased to 80% and the xylose was just increased by 3%. Xyn10CD18 is a thermostable endoxylanase and is a promising candidate for biomass conversion and xylooligosaccharide production.

Characterization of a thermostable, specific GH10 xylanase from Caldicellulosiruptor bescii with high catalytic activity

Xylanase (EC 3.2.1.8) is one of the most important enzymes for the biodegradation of xylan. Since many industrial processes utilizing xylanase are operated at elevated temperatures, thermostable xylanases are highly desirable. In the present study, xyn10B gene from thermophilic bacterium Caldicellulosiruptor bescii that encodes a glycoside hydrolase (GH) family 10 xylanase was overexpressed in Escherichia coli and systematically characterized. CbXyn10B exhibited optimal activity at pH 7.2 and 70°C. It had a half-life of about 7.7h at 60°C, and retained over 85% of maximal activity after incubation at pH 4.0–12.0. The activity of this xylanase was not affected by most divalent cations, but inhibited by Fe3+ and Zn2+. CbXyn10B exhibited high activity on beech wood xylan, oat spelt xylan, and birch wood xylan, with specific activities of about 450Umg−1. Compared with other GH10 xylanases, CbXyn10B was highly specific for xylan and showed low catalytic efficiency toward sodium carboxymethyl cellulose and p-nitrophenyl-β-d-xylopyranoside. HPLC analysis of the products released from xylo-oligosaccharides and xylan revealed that xylobiose was the predominant hydrolytic product. The action mode of the enzyme was studied by product analysis, homology modeling and molecular docking to gain an insight into the structural basis for its substrate recognition mechanism.

Production by Tobacco Transplastomic Plants of Recombinant Fungal and Bacterial Cell-Wall Degrading Enzymes to Be Used for Cellulosic Biomass Saccharification.

Biofuels from renewable plant biomass are gaining momentum due to climate change related to atmospheric CO2 increase. However, the production cost of enzymes required for cellulosic biomass saccharification is a major limiting step in this process. Low-cost production of large amounts of recombinant enzymes by transgenic plants was proposed as an alternative to the conventional microbial based fermentation. A number of studies have shown that chloroplast-based gene expression offers several advantages over nuclear transformation due to efficient transcription and translation systems and high copy number of the transgene. In this study, we expressed in tobacco chloroplasts microbial genes encoding five cellulases and a polygalacturonase. Leaf extracts containing the recombinant enzymes showed the ability to degrade various cell-wall components under different conditions, singly and in combinations. In addition, our group also tested a previously described thermostable xylanase in combination with a cellulase and a polygalacturonase to study the cumulative effect on the depolymerization of a complex plant substrate. Our results demonstrate the feasibility of using transplastomic tobacco leaf extracts to convert cell-wall polysaccharides into reducing sugars, fulfilling a major prerequisite of large scale availability of a variety of cell-wall degrading enzymes for biofuel industry.

Bioenergy and Biomass Utilization.

Bioenergy is of increasing interest as a renewable, environmentally friendly alternative to energy derived from fossil fuels. Through a variety of processes, biomass can be converted to solid, liquid, or gaseous biofuels. However, various challenges persist to the maintenance and development of biomass-to-energy utilization. This special issue focuses on the most recent advances in the frontier research of bioenergy and biomass utilization and features six selected papers with high quality. The paper titled “Nanofibrillated Cellulose and Copper Nanoparticles Embedded in Polyvinyl Alcohol Films for Antimicrobial Applications” by T. Zhong et al. develops a method to produce hybrids of TEMPO nanofibrillated cellulose (TNFC) and copper nanoparticles. The hybrid material is embedded in polyvinyl alcohol thermoplastic resin and the films are produced using a solvent casting method. The authors evaluate the films in terms of its morphology and thermal and antimicrobial properties and prove that TNFC-copper nanoparticles as antimicrobial nanofillers are valuable for PVA applications. The paper titled “Response of Arbuscular Mycorrhizal Fungi to Hydrologic Gradients in the Rhizosphere of Phragmites australis (Cav.) Trin ex. Steudel Growing in the Sun Island Wetland” by L. Wang et al. assesses the variations of hydrologic gradients in the relationships among AM fungi, reed, and rhizospheric microorganisms. The authors discover water content in soil and reed growth parameters are both positively associated with AM fungi colonization, but only the positive correlations between reed biomass parameters and the colonization could be expected, or both the host plant biomass and the AM fungi could be beneficial. This study could shed light on the mechanisms inside AM fungi-reed symbioses and would be referred to for optimizing the combined phytorhizoremediation. The paper titled “Production by Tobacco Transplastomic Plants of Recombinant Fungal and Bacterial Cell-Wall Degrading Enzymes to Be Used for Cellulosic Biomass Saccharification” by L. Paolo et al. presents work on saccharification of plant biomass. The authors express in tobacco chloroplasts microbial genes encoding five cellulases and a polygalacturonase. Leaf extracts containing the recombinant enzymes show the ability to degrade various cell-wall components under different conditions. In addition, a thermostable xylanase is also tested in combination with a cellulase and a polygalacturonase to study the cumulative effect on the depolymerization of a complex plant substrate. Results demonstrate the feasibility of using transplastomic tobacco leaf extracts to convert cell-wall polysaccharides into reducing sugars, fulfilling a major prerequisite of large scale availability of a variety of cell-wall degrading enzymes for biofuel industry. The paper titled “Enzymatic Saccharification of Lignocellulosic Residues by Cellulases Obtained from Solid State Fermentation Using Trichoderma viride” by T. Sartori et al. presents an approach that uses cellulolytic complex produced by Trichoderma viride in solid state fermentation to hydrolyze lignocellulosic residues. This produced enzyme shows viability in comparison with the commercial cellulase enzyme. The synthesis of cellulases by microorganisms from lignocellulosic residues is a process of great interest, representing the search for renewable sources to replace the fossil energetic matrix. The paper titled “Improving Biomethane Production and Mass Bioconversion of Corn Stover Anaerobic Digestion by Adding NaOH Pretreatment and Trace Elements” by C. Liu et al. describes an approach to improve biomethane production from corn stover. The authors use NaOH pretreatment and trace elements supplementation as a strategy to enhance the biodegradability of corn stover for biomethane production. The approach is compared with only NaOH-pretreated and untreated corn stover, and the paper highlights the supplementation of trace elements. The paper titled “Biochemical Modulation of Lipid Pathway in Microalgae Dunaliella sp. for Biodiesel Production,” by A. F. Talebi et al., explores the use of myoinositol to modulate LP and biodiesel quality. Inclusion of myoinositol in the media indeed improves the total lipid accumulation and biodiesel quality parameters, and this work highlights the fact that the biochemical modulation strategies should be progressively considered in the hope of finding more efficient and economically feasible strategies leading to more viable production systems.