A novel high performance in-silico screened metagenome-derived alkali-thermostable endo-ß-1,4-glucanase for lignocellulosic biomass hydrolysis in the harsh conditions.

BACKGROUND: Lignocellulosic biomass, is a great resource for the production of bio-energy and bio-based material since it is largely abundant, inexpensive and renewable. The requirement of new energy sources has led to a wide search for novel effective enzymes to improve the exploitation of lignocellulose, among which the importance of thermostable and halotolerant cellulase enzymes with high pH performance is significant. RESULTS: The primary aim of this study was to discover a novel alkali-thermostable endo-ß-1,4-glucanase from the sheep rumen metagenome. At first, the multi-step in-silico screening approach was utilized to find primary candidate enzymes with superior properties. Among the computationally selected candidates, PersiCel4 was found and subjected to cloning, expression, and purification followed by functional and structural characterization. The enzymes' kinetic parameters, including Vmax, Km, and specific activity, were calculated. The PersiCel4 demonstrated its optimum activity at pH 8.5 and a temperature of 85 °C and was able to retain more than 70% of its activity after 150 h of storage at 85 °C. Furthermore, this enzyme was able to maintain its catalytic activity in the presence of different concentrations of NaCl and several metal ions contains Mg2+, Mn2+, Cu2+, Fe2+ and Ca2+. Our results showed that treatment with MnCl2 could enhance the enzyme's activity by 78%. PersiCel4 was ultimately used for enzymatic hydrolysis of autoclave pretreated rice straw, the most abundant agricultural waste with rich cellulose content. In autoclave treated rice straw, enzymatic hydrolysis with the PersiCel4 increased the release of reducing sugar up to 260% after 72 h in the harsh condition (T = 85 °C, pH = 8.5). CONCLUSION: Considering the urgent demand for stable cellulases that are operational on extreme temperature and pH conditions and due to several proposed distinctive characteristics of PersiCel4, it can be used in the harsh condition for bioconversion of lignocellulosic biomass.

Characterization of the Wild-Type and Truncated Forms of a Neutral GH10 Xylanase from Coprinus cinereus: Roles of C-Terminal Basic Amino Acid-Rich Extension in Its SDS Resistance, Thermostability, and Activity.

A neutral xylanase (CcXyn) was identified from Coprinus cinereus. It has a single GH10 catalytic domain with a basic amino acid-rich extension (PVRRK) at the C-terminus. In this study, the wild-type (CcXyn) and C-terminus-truncated xylanase (CcXyn-Δ5C) were heterologously expressed in Pichia pastoris and their characteristics were comparatively analyzed with aims to examine the effect of this extension on the enzyme function. The circular dichorism analysis indicated that both enzymes in general had a similar structure, but CcXyn-Δ5C contained less α-helices (42.9%) and more random coil contents (35.5%) than CcXyn (47.0% and 32.8%, respectively). Both enzymes had the same pH (7.0) and temperature (45°C) optima, and similar substrate specificity on different xylans. They all hydrolyzed beechwood xylan primarily to xylobiose and xylotriose. The amounts of xylobiose and xylotriose accounted for 91.5% and 92.2% (w/w) of total xylooligosaccharides (XOS) generated from beechwood by CcXyn and CcXyn-Δ5C, respectively. However, truncation of the C-terminal 5-amino-acids extension significantly improved the thermostability, SDS resistance, and pH stability at pH 6.0-9.0. Furthermore, CcXyn-Δ5C exhibited a much lower Km value than CcXyn (0.27 mg/ml vs 0.83 mg/ml), and therefore, the catalytic efficiency of CcXyn-Δ5C was 2.4-times higher than that of CcXyn. These properties make CcXyn-Δ5C a good model for the structure-function study of (α/ß)8-barrel-folded enzymes and a promising candidate for various applications, especially in the detergent industry and XOS production.

Effect of Flavonoid-Rich Extract of Glycyrrhiza glabra on Gut-Friendly Microorganisms, Commercial Probiotic Preparations, and Digestive Enzymes.

Flavonoid-rich extract prepared from Glycyrrhiza glabra has been found to be beneficial in patients with functional dyspepsia and was reported to possess some gut health-promoting properties such as antioxidant, anti-inflammatory and anti-Helicobacter pylori activities. In the present study, the flavonoid-rich extract of Glycyrrhiza glabra was evaluated for its compatibility with probiotic strains (Lactobacillus casei, Lactobacillus fermentum, Lactobacillus plantarum, and Streptococcus thermophilus), commercial probiotic drinks, and digestive enzymes (pancreatic α-amylase, α-glucosidase, phytase, xylanase, and pancreatic lipase). Results of this study indicated that the flavonoid-rich extract of Glycyrrhiza glabra is compatible with the tested probiotic strains, probiotic drinks and digestive enzymes.

Molecular characterization of a glycosyl hydrolase family 10 xylanase from Aspergillus niger.

A gene coding for an endo-ß-1,4-xylanase (XlnA) (glycosyl hydrolase family 10) from Aspergillus niger DSM 1957 was cloned and sequenced. The cDNA sequence (984 bp) and its putative endoxylanase (327 aa protein with a predicted molecular mass of 35.5 kDa and pI 6.23) showed 91.3-99.5% and 96.3-99.1% identities with cDNA sequences and their corresponding endoxylanases from A. niger strains from GenBank, respectively. The cDNA was expressed in Pichia pastoris GS115 under the control of AOX1 promoter at a level of 46.4 U/ml culture supernatant, after 144 h of growth at 30°C in YP medium induced with 0.5% (v/v) of methanol. The molecular mass of the purified XlnA determined by SDS-PAGE was 35.5k Da with a specific activity of 808.5 U/mg towards 1% (w/v) of birch wood xylan. Temperature and pH optimum were observed at 50°C and pH 7.0, respectively. The enzyme was stable over a temperature range of 25-40°C and at pH range of 4.5-8.5 and resistant to Tween 80 and acetone. The K(m) and V(max) value obtained for the purified xylanase were 25.5mg/ml and 5000 µmol/min/mg protein with birch wood xylan as substrate, respectively. The xylanase was free of cellulase and mannanase activity but highly active towards birch wood xylan. The major products of the birch wood xylan hydrolysis were predicted as xylotriose, xylotetraose, and xylopentose. The biochemical characteristics suggested that the recombinant xylanase has a potential application, including use as a feed enzyme.

Characteristics of thermostable endoxylanase and ß-xylosidase of the extremely thermophilic bacterium Geobacillus thermodenitrificans TSAA1 and its applicability in generating xylooligosaccharides and xylose from agro-residues.

An extremely thermophilic bacterial isolate that produces a high titer of thermostable endoxylanase and ß-xylosidase extracellularly in an inducible manner was identified as Geobacillus thermodenitrificans TSAA1. The distinctive features of this strain are alkalitolerance and halotolerance. The endoxylanase is active over a broad range of pH (5.0-10.0) and temperatures (30-100 °C) with optima at pH 7.5 and 70 °C, while ß-xylosidase is optimally active at pH 7.0 and 60 °C. The T 1/2 values of the endoxylanase and ß-xylosidase are 30 min at 80 °C, and 180 min at 70 °C, respectively. The endoxylanase activity is stimulated by dithiothreitol, but inhibited strongly by EDAC and Woodward's reagent K. N-BS and DEPC strongly inhibited ß-xylosidase. MALDI-ToF (MS/MS) analysis of tryptic digest of ß-xylosidase revealed similarity with that of G. thermodenitrificans NG 80-2, and suggested that this belongs to the GH 52 glycosyl hydrolase super family. The action of endoxylanase on birch wood xylan and agro-residues such as wheat bran and wheat straw liberated xylooligosaccharides similar to endoxylanases of the family 10 glycoside hydrolases, while the enzyme preparation having both endoxylanase and ß-xylosidase liberated xylose as main hydrolysis product.

Purification and characterization of a thermostable-cellulase free xylanase from Syncephalastrum racemosum Cohn.

Syncephalastrum racemosum Cohn. produces an extracellular xylanase that was shown to potentially bleach pulp at pH 10 and 50 degrees C. The enzyme was found to be a dimer with an apparent molecular weight of 29 kDa as determined by SDS-PAGE. The optimum activity was found at two pH values 8.5 and 10.5; however the activity sharply decreased below pH 6 and above pH 10.5. The enzyme was stable for 72 h at pH 10.5 and at 50 degrees C. Kinetic experiments at 50 degrees C gave V(max) and K(m) of 1,400 U/ml min(-1) mg(-1) protein and 0.05 mg/ml respectively. The enzyme had no apparent requirement for cofactors, and its activity was strongly inhibited by group II b metal ions like Zn2+, Hg2+, etc. Xylan completely protected the enzyme from being inactivated by N-bromosuccinimide.