Improved pH Stability Research Articles

Engineering a highly active thermophilic β-glucosidase to enhance its pH stability and saccharification performance.

Backgroundβ-Glucosidase is an important member of the biomass-degrading enzyme system, and plays vital roles in enzymatic saccharification for biofuels production. Candidates with high activity and great stability over high temperature and varied pHs are always preferred in industrial practice. To achieve cost-effective biomass conversion, exploring natural enzymes, developing high level expression systems and engineering superior mutants are effective approaches commonly used.ResultsA newly identified β-glucosidase of GH3, Bgl3A, from Talaromyces leycettanus JCM12802, was overexpressed in yeast strain Pichia pastoris GS115, yielding a crude enzyme activity of 6000 U/ml in a 3 L fermentation tank. The purified enzyme exhibited outstanding enzymatic properties, including favorable temperature and pH optima (75 °C and pH 4.5), good thermostability (maintaining stable at 60 °C), and high catalytic performance (with a specific activity and catalytic efficiency of 905 U/mg and 9096/s/mM on pNPG, respectively). However, the narrow stability of Bgl3A at pH 4.0–5.0 would limit its industrial applications. Further site-directed mutagenesis indicated the role of excessive O-glycosylation in pH liability. By removing the potential O-glycosylation sites, two mutants showed improved pH stability over a broader pH range (3.0–10.0). Besides, with better stability under pH 5.0 and 50 °C compared with wild type Bgl3A, saccharification efficiency of mutant M1 was improved substantially cooperating with cellulase Celluclast 1.5L. And mutant M1 reached approximately equivalent saccharification performance to commercial β-glucosidase Novozyme 188 with identical β-glucosidase activity, suggesting its great prospect in biofuels production.ConclusionsIn this study, we overexpressed a novel β-glucosidase Bgl3A with high specific activity and high catalytic efficiency in P. pastoris. We further proved the negative effect of excessive O-glycosylation on the pH stability of Bgl3A, and enhanced the pH stability by reducing the O-glycosylation. And the enhanced mutants showed much better application prospect with substantially improved saccharification efficiency on cellulosic materials.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0560-8) contains supplementary material, which is available to authorized users.

Efficacy of a Phosphate-Charged Soil Material in Supplying Phosphate for Plant Growth in Soilless Root Media

A soil material high in crystalline Fe hydrous oxides and noncrystalline Al hydrous oxides collected from the Bw horizon of a Hemcross soil containing allophane from the state of Oregon was charged with phosphate-P at rates of 0, 2.2, and 6.5 mg·g−1, added to a soilless root medium at 5% and 10% by volume, and evaluated for its potential to supply phosphate at a low, stable concentration during 14 weeks of tomato (Solanum esculentumL.) seedling growth. Incorporation of the soil material improved pH stability, whether it was charged with phosphate or not. Bulk solution phosphate-P concentrations in the range of 0.13 to 0.34 mg·dm−3were associated with P deficiency. The only treatment that sustained an adequate bulk solution concentration of phosphate-P above 0.34 mg·dm−3for the 14 weeks of testing contained 10% soil material charged with 6.5 mg·g−1P, but initial dissolved P concentrations were too high (>5 mg·g−1phosphate-P) from the standpoint of phosphate leaching. The treatment amended with 10% soil material charged with 2.2 mg·g−1P maintained phosphate-P within an acceptable range of 0.4 to 2.3 mg·dm−3for 48 d in a medium receiving no postplant phosphate fertilization.

Preparation and characterization of κ-carrageenase immobilized onto magnetic iron oxide nanoparticles

Background: Carboxyl-functionalized magnetic nanoparticles were synthesized via chemical co-precipitation method and modified with oleic acid which was oxidized by potassium permanganate, and κ-carrageenase from Pseudoalteromonas sp. ASY5 was subsequently immobilized onto them. The immobilization conditions were further optimized, and the characterizations of the immobilized κ-carrageenase were investigated. Results: The κ-carrageenase was immobilized onto magnetic iron oxide nanoparticles, and the bonding was verified by Fourier transform infrared spectroscopy. The optimal conditions for κ-carrageenase immobilization were 2.5% (w/v) glutaraldehyde, 13.9 U κ-carrageenase for 20 mg of magnetic nanoparticles, a 2-h cross-linking time, and a 2-h immobilization time at 25°C. Under these conditions, the activity of the immobilized enzyme and the enzyme recovery rate were 326.0 U · g - 1 carriers and 46.9%, respectively. The properties of the immobilized κ-carrageenase were compared with those of the free enzyme. The optimum temperatures of the free and immobilized κ-carrageenase were 60 and 55°C, respectively, and the optimum pH of κ-carrageenase did not change before and after immobilization (pH 7.5). After immobilization, κ-carrageenase exhibited lower thermal stability and improved pH stability, as well as better storage stability. The immobilized κ-carrageenase maintained 43.5% of the original activity after being used 4 times. The kinetic constant value (Km) of κ-carrageenase indicates that the immobilized enzyme had a lower binding affinity for the substrate. Conclusions: Under optimal conditions, the activity of the immobilized enzyme and enzyme recovery rate were 326.0 U · g - 1 ·κ-carrageenase-CMNPs and 46.9%, respectively. The thermal, pH, and storage stabilities of κ-carrageenase-CMNPs were relatively higher than those of free κ-carrageenase.

Purification, immobilization, and characterization of protease from local Bacillus subtilis M‐11

AbstractProtease from Bacillus subtilis M‐11 was produced and purified using ammonium sulfate precipitation, gel filtration chromatography with 15.8‐fold. The purified enzyme showed a single band on sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, and it was determined that its molecular weight was 32 kDa. The purified enzyme was immobilized on polysulfone membrane containing Silica gel‐3 aminopropyltriethoxysilane (Si‐APTS) by physical adsorption. The optimal immobilization conditions (pH, temperatures, and incubation time) were investigated. The properties of immobilized protease were determined and compared with free enzyme. The investigations on immobilized enzyme indicated that the optimum pH was shifted 0.5 units to alkaline pH (pH 8.0–8.5) after immobilization with physical adsorption. Immobilization improved pH stability of enzyme between pH 7.5 and 8.5. Immobilized enzyme sustained 77.3% of its activity after usage of 10 times. However, there is no change on optimum temperature (50 °C) after immobilization.All these results indicated that polysulfone membrane containing Si‐APTS could be a proper support for protease immobilization, and immobilized protease by using physical adsorption could be used for industrial applications efficiently. © 2015 Curtin University of Technology and John Wiley & Sons, Ltd.

Dispersible composites of exfoliated graphite and polyaniline with improved electrochemical behaviour for solid-state chemical sensor applications

The PANI–graphene/graphite composites show improved pH stability and electrochemical behaviour in aqueous electrolyte solutions at pH ≤ 8.

The Single T65S Mutation Generates Brighter Cyan Fluorescent Proteins with Increased Photostability and pH Insensitivity

Cyan fluorescent proteins (CFP) derived from Aequorea victoria GFP, carrying a tryptophan-based chromophore, are widely used as FRET donors in live cell fluorescence imaging experiments. Recently, several CFP variants with near-ultimate photophysical performances were obtained through a mix of site-directed and large scale random mutagenesis. To understand the structural bases of these improvements, we have studied more specifically the consequences of the single-site T65S mutation. We find that all CFP variants carrying the T65S mutation not only display an increased fluorescence quantum yield and a simpler fluorescence emission decay, but also show an improved pH stability and strongly reduced reversible photoswitching reactions. Most prominently, the Cerulean-T65S variant reaches performances nearly equivalent to those of mTurquoise, with QY = 0.84, an almost pure single exponential fluorescence decay and an outstanding stability in the acid pH range (pK1/2 = 3.6). From the detailed examination of crystallographic structures of different CFPs and GFPs, we conclude that these improvements stem from a shift in the thermodynamic balance between two well defined configurations of the residue 65 hydroxyl. These two configurations differ in their relative stabilization of a rigid chromophore, as well as in relaying the effects of Glu222 protonation at acid pHs. Our results suggest a simple method to greatly improve numerous FRET reporters used in cell imaging, and bring novel insights into the general structure-photophysics relationships of fluorescent proteins.

Fabrication of polyelectrolyte multilayer films comprising nanoblended layers.

Polyelectrolyte multilayer thin films were prepared via the alternate deposition of poly(allylamine hydrochloride) (PAH) and a blend of poly(acrylic acid) (PAA) and poly(styrenesulfonate) (PSS). When the pH of the blend solution was 3.5, the presence of PAA in this solution significantly increased the total film thickness. With only 10 wt % PAA in the blend adsorption solution, a large increase in film thickness was observed (92 nm cf. 18 nm). It was also demonstrated that the total amount of PSS adsorbed was enhanced by the presence of PAA in the blend solution, showing that the blend solution composition influenced that of the multilayer films. Thin films prepared with nanoblended layers also showed improved pH stability, because they exhibited reduced film rearrangement upon exposure to acidic conditions (pH = 2.5).

SAR and pH stability of cyano-substituted epothilones.

[formula: see text] 3-Cyano epothilones 15-18 are the only examples of non-hydroxy C-3-substituted analogues. Their tubulin binding affinity and cytotoxicity provide meaningful structure-activity relationship information on the dependence of C-1/C-3 conformation upon activity. 12-Cyano epothilone 24 has improved pH stability over epothilone B, and its activity further supports the hypothesis that C-12 stereochemistry is not critical for tubulin affinity.

453 Evaluation of a Phosphate-charged Soil Material as a Source of Phosphorus for Soilless Root Media

A soil material high in metal oxides collected from the Bw horizon of a Hemcross soil in the state of Oregon was charged with phosphate, added to a soilless root medium, and evaluated for its potential to supply phosphate at a low, stable concentration during 14 weeks of tomato cropping (three successive crops). Three rates of phosphate were charged on the soil material, 0, 2.2, and 6.5 m P/g soil material and the soil material was incorporated into a 3 peatmoss: 1 perlite (v:v) medium at 5 % (40 g) and 10 % (80 g) of the volume of a 13.6-cm pot (1.0 L of medium). Uncharged soil material incorporated into soilless root medium at 5% and 10% reduced soil solution phosphate to deficient levels for 2 and 7 weeks, respectively. Phosphate was adequately supplied for 7, 10, 12, and more than 14 weeks in the 2.2P-5%, 2.2P-10%, 6.5P-5%, and 6.5P-10% treatment, respectively, as determined by symptoms of P deficiency. Phosphate and K levels in soil solution were highest at the beginning of crop 1 and tended to decline thereafter. Incorporation of soil material into soilless root medium improved pH stability whether it was charged with phosphate or not. The loss of the phosphate-charged soil material was negligible, 0.3% for the 6.5P-5% treatment and 1.2% for the 6.5P-10% treatment. The minimum critical concentration of soil solution phosphate for tomato in a 3 peatmoss: 1 perlite (v:v) medium as determined by the pour-through extraction procedure was found to be 0.3 mg·L–1 or slightly less.

Pantoprazole: a novel H +/K +-ATPase inhibitor with an improved pH stability

The action of the H +/K +-ATPase inhibitors pantoprazole and omeprazole was compared in different in vitro test systems. In gastric membrane vesicles under conditions shown to result in acidification of the vesicle interior, pantoprazole and omeprazole inhibited H +/K +-ATPase activity with IC 50 values of 6.8 and 2.4 μM, respectively. When intravesicular acidification was reduced by inclusion of imidazole (5 mM), a membrane permeable weak base, the inhibitory action of omeprazole was partially lost (IC 50 30 μM) and that of pantoprazole almost completely lost. After incubation for 40 min with pumping membrane vesicles, a half-maximal reduction in intravesicular H + concentration occurred at pantoprazole and omeprazole concentrations of 1.1 and 0.6 μM, respectively. Again, when the intravesicular H + concentration was reduced by inclusion of imidazole (2.5 mM), pantoprazole (20 and 60 μM) did not reduce the remaining intravesicular proton concentration, whereas omeprazole (10 and 30 μM) did. Both drugs inhibited, with similar potency, papain activity at pH 3.0 and inactivated the enzyme in a similar time-dependent manner; at pH 5.0 omeprazole (IC 50 17 μM) was more potent than pantoprazole (IC 50 37 μM) and enzyme inhibition was faster than with pantoprazole. These results indicate that pantoprazole is a potent inhibitor of H +/K +-ATPase under highly acidic conditions and that it is more stable than omeprazole at a slightly acidic pH such as pH 5.0.