Enhanced activity of hyperthermostable Pyrococcus horikoshii endoglucanase in superbase ionic liquids

Objectives Ionic liquids (ILs) that dissolve biomass are harmful to the enzymes that degrade lignocellulose. Enzyme hyperthermostability promotes a tolerance to ILs. Therefore, the limits of hyperthemophilic Pyrococcus horikoschii endoglucanase (PhEG) to tolerate 11 superbase ILs were explored. Results PhEG was found to be most tolerant to 1-ethyl-3-methylimidazolium acetate ([EMIM]OAc) in soluble 1% carboxymethylcellulose (CMC) and insoluble 1% Avicel substrates. At 35% concentration, this IL caused an increase in enzyme activity (up to 1.5-fold) with CMC. Several ILs were more enzyme inhibiting with insoluble Avicel than with soluble CMC. K-m increased greatly in the presence ILs, indicating significant competitive inhibition. Increased hydrophobicity of the IL cation or anion was associated with the strongest enzyme inhibition and activation. Surprisingly, PhEG activity was increased 2.0-2.5-fold by several ILs in 4% substrate. Cations exerted the main role in competitive inhibition of the enzyme as revealed by their greater binding energy to the active site. Conclusions These results reveal new ways to design a beneficial combination of ILs and enzymes for the hydrolysis of lignocellulose, and the strong potential of PhEG in industrial, high substrate concentrations in aqueous IL solutions.Peer reviewe

Optimization and partial characterization of endoglucanase produced by Streptomyces sp. B-PNG23

Streptomyces sp. B-PNG23 was selected as a promising cellulolytic strain and tested for its ability to produce cellulases from agroindustrial residues. A pH value of 7 and temperature of 28°C were found to be optimal for maximum enzyme production. The highest endoglucanase activity was obtained in a medium comprised of wheat bran (2 g/l), yeast extract (2 g/l), NaCl (2 g/l), NH4Cl (2.5 g/l), and (0.4 g/l) of MgSO4. The enzyme was active at a broad range of pH (5-8) and temperatures (40-70°C). The optimum pH and temperature were 6 and 50°C, respectively. In the presence of metal ions Mn2+, Cu2+ and NH4 + the activity of the enzyme increased significantly. The enzyme retained 50% of its activity after heating at 50°C for 6 h. This enzyme could be considered as a thermotolerant biocatalyst that could be utilized in biotechnological applications

Optimization and partial characterization of endoglucanase produced by Streptomyces SP B-PNG23

Streptomyces sp. B-PNG23 was selected as a promising cellulolytic strain and tested for its ability to produce cellulases from agroindustrial residues. A pH value of 7 and temperature of 28 C were found to be optimal for maximum enzyme production. The highest endoglucanase activity was obtained in a medium comprised of wheat bran (2 g/l), yeast extract (2 g/l), NaCl (2 g/l), NH4Cl (2.5 g/l), and (0.4 g/l) of MgSO4. The enzyme was active at a broad range of pH (5-8) and temperatures (40-70 degrees C). The optimum pH and temperature were 6 and 50 degrees C, respectively. In the presence of metal ions Mn2+, Cu2+ and NH4+ the activity of the enzyme increased significantly. The enzyme retained 50% of its activity after heating at 50 degrees C for 6 h. This enzyme could be considered as a thermotolerant biocatalyst that could be utilized in biotechnological applications

Optimization of β-1,4-endoxylanase production by an Aspergillus niger strain growing on wheat straw and application in xylooligosaccharides production

20 p.-6 fig.-6 tab.Plant biomass constitutes the main source of renewable carbon on the planet. Its valorization has traditionally been focused on the use of cellulose, although hemicellulose is the second most abundant group of polysaccharides on Earth. The main enzymes involved in plant biomass degradation are glycosyl hydrolases, and filamentous fungi are good producers of these enzymes. In this study, a new strain of Aspergillus niger was used for hemicellulase production under solid-state fermentation using wheat straw as single-carbon source. Physicochemical parameters for the production of an endoxylanase were optimized by using a One-Factor-at-a-Time (OFAT) approach and response surface methodology (RSM). Maximum xylanase yield after RSM optimization was increased 3-fold, and 1.41- fold purification was achieved after ultrafiltration and ion-exchange chromatography, with about 6.2% yield. The highest activity of the purified xylanase was observed at 50 °C and pH 6. The enzyme displayed high thermal and pH stability, with more than 90% residual activity between pH 3.0–9.0 and between 30–40 °C, after 24 h of incubation, with half-lives of 30 min at 50 and 60 °C. The enzyme was mostly active against wheat arabinoxylan, and its kinetic parameters were analyzed (Km = 26.06 mg·mL−1 and Vmax = 5.647 U·mg−1). Wheat straw xylan hydrolysis with the purified β-1,4 endoxylanase showed that it was able to release xylooligosaccharides, making it suitable for different applications in food technology.This work has been funded by Projects GLYSUS RTI2018-093683-Β-I00 (MCIU/AEI/FEDER) and RETOPROSOST2 S2018/EMT-4459 (Comunidad de Madrid).Peer reviewe

Biochemical properties of a new thermo- and solvent-stable xylanase recovered using three phase partitioning from the extract of Bacillus oceanisediminis strain SJ3

The present study investigates the production and partial biochemical characterization of an extracellular thermostablexylanase from the Bacillus oceanisediminis strain SJ3 newly recovered from Algerian soil using three phasepartitioning (TPP). The maximum xylanase activity recorded after 2 days of incubation at 37 °C was 20.24 U/ml in thepresence of oat spelt xylan. The results indicated that the enzyme recovered in the middle phase of TPP system usingthe optimum parameters were determined as 50% ammonium sulfate saturation with 1.0:1.5 ratio of crude extract:t-butanol at pH and temperature of 8.0 and 10 °C, respectively. The xylanase was recovered with 3.48 purificationfold and 107% activity recovery. The enzyme was optimally active at pH 7.0 and was stable over a broad pH range of5.0–10. The optimum temperature for xylanase activity was 55 °C and the half-life time at this temperature was of 6 h.At this time point the enzyme retained 50% of its activity after incubation for 2 h at 95 °C. The crude enzyme resist tosodium dodecyl sulfate and β-mercaptoethanol, while all the tested ions do not affect the activity of the enzyme. Therecovered enzyme is, at least, stable in tested organic solvents except in propanol where a reduction of 46.5% wasobserved. Further, the stability of the xylanase was higher in hydrophobic solvents where a maximum stability wasobserved with cyclohexane. These properties make this enzyme to be highly thermostable and may be suggested asa potential candidate for application in some industrial processes. To the best of our knowledge, this is the first reportof xylanase activity and recoverey using three phase partitioning from B. oceanisediminis

Isolation and Screening of Fungal Culture Isolated From Algerian Soil for the Production of Cellulase and Xylanase

Lignocellulolytic enzymes constitute a very large group of extracellular proteins secreting by fungi who is ecologically involved in the degradation of a variety of complex materials, a property that is attributed to a battery of enzymes produced by these microorganisms like cellulases and xylanases who are of significant industrial value and relevance. Forty fungal isolated from rich soil in organic matter were screened for lignocellulolytic enzymes production, its organized on the basis of their hydrolytic potential of cellulose and xylan. The isolates strains presented enzymatic activity which was ranked as follows: cellulolytic (56%), xylanolytic (44%). Some selected strains that produce high levels of enzymes (cellulase, xylanase) grown in submerged fermentation (SmF) and were quantitatively evaluated. The fermentation experiments were carried out in shake flasks. The highest CMCase (5,10 IU/ml) and xylanase (98,25 IU/ml) activities were obtained from Trichoderma sp strain Mtr6 isolate. Keywords: Fungi, Trichoderma sp, lignocellulolytic enzymes, soil, screening, organic matter

Three phase partitioning, a scalable method for the purification and recovery of cucumisin, a milk-clotting enzyme, from the juice of Cucumis melo var . reticulatus

Cucumisin [EC 3.4.21.25] was first purified from Cucumis melo var. reticulatus juice by three-phase parti-tioning (TPP). Optimum purification parameters of the TPP system were determined as 60% ammoniumsulfate saturation with 1.0:1.25 ratio of crude extract: t-butanol at pH and temperature of 8.0 and 20◦C,respectively. Cucumisin was purified with 4.61 purification fold and 156% activity recovery. The molecularweight of the recovered cucumisin was determined as 68.4 kDa and its isoelectric point is 8.7. OptimumpH and temperature of cucumisin were pH 9.0 and 60–70◦C, respectively. The protease was very stable at20–70◦C and a pH range of 2.0–12.0. Km and Vmax constants were 2.24 ± 0.22 mgmL−1and 1048 ± 25 Mmin−1, respectively. The enzyme was stable against numerous metal ions and its activity was highlyenhanced by Ca2+, Mg2+, and Mn+2. Cucumisin activity was 2.35-folds increased in the presence of 5 mM ofCaCl2. It was inactivated by Co2+, Cd2+, Zn2+and Fe2+and dramatically by PMSF. Cucumisin milk-clottingactivity was highly stable when stored under freezing (−20◦C) compared at 4◦C and 25◦C. Finally, TPPrevealed to be a useful strategy to concentrate and purify cucumisin for its use as a milk-clotting enzymefor cheese-making

Partial Characterization of Xylanase Produced by Caldicoprobacter algeriensis, a New Thermophilic Anaerobic Bacterium Isolated from an Algerian Hot Spring

To date, xylanases have expanded their use in many processing industries, such as pulp, paper, food, and textile. This study aimed the production and partial characterization of a thermostable xylanase from a novel thermophilic anaerobic bacterium Caldicoprobacter algeriensis strain TH7C1T isolated from a northeast hot spring in Algeria. The obtained results showed that C. algeriensis xylanase seems not to be correlated with the biomass growth profile whereas the maximum enzyme production (140.0 U/ml) was recorded in stationary phase (18 h). The temperature and pH for optimal activities were 70 °C and 11.0, respectively. The enzyme was found to be stable at 50, 60, 70, and 80 °C, with a half-life of 10, 9, 8, and 4 h, respectively. Influence of metal ions on enzyme activity revealed that Ca+2 enhances greatly the relative activity to 151.3 %; whereas Hg2+ inhibited significantly the enzyme. At the best of our knowledge, this is the first report on the production of xylanase by the thermophilic bacterium C. algeriensis. This thermo- and alkaline-tolerant xylanase could be used in pulp bleaching process

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