A FRET Approach to Detect Paraoxon among Organophosphate Pesticides Using a Fluorescent Biosensor

The development of faster, sensitive and real-time methods for detecting organophosphate (OP) pesticides is of utmost priority in the in situ monitoring of these widespread compounds. Research on enzyme-based biosensors is increasing, and a promising candidate as a bioreceptor is the thermostable enzyme esterase-2 from Alicyclobacillus acidocaldarius (EST2), with a lipase-like Ser–His–Asp catalytic triad with a high affinity for OPs. This study aimed to evaluate the applicability of Förster resonance energy transfer (FRET) as a sensitive and reliable method to quantify OPs at environmentally relevant concentrations. For this purpose, the previously developed IAEDANS-labelled EST2-S35C mutant was used, in which tryptophan and IAEDANS fluorophores are the donor and the acceptor, respectively. Fluorometric measurements showed linearity with increased EST2-S35C concentrations. No significant interference was observed in the FRET measurements due to changes in the pH of the medium or the addition of other organic components (glucose, ascorbic acid or yeast extract). Fluorescence quenching due to the presence of paraoxon was observed at concentrations as low as 2 nM, which are considered harmful for the ecosystem. These results pave the way for further experiments encompassing more complex matrices.

Production of Highly Thermostable Laccase from Penicillium spinulosum for Enhanced Delignification of Untreated Wheat Bran

Background: Lignin confers rigidity on plant cell wall and poses a challenge to hydrolysis of cellulose, which makes production of biofuels from lignocellulose an overwhelming problem. This prompts continuous search for novel ligninolytic enzymes, especially, laccases for delignification of lignocellulose for improved saccharification of biomass. Objective: This study reports production, physicochemical properties and delignification efficiency of laccases from Penicillium and Trichoderma species on untreated wheat bran. Methods: Fungal laccases were produced using different agroresidues (wheat bran, coconut shell and palm kernel shell) as substrates in submerged fermentation. Best substrate for laccase production was determined. Physicochemical properties of crude enzymes and delignification efficiency of the laccases were determined on untreated wheat bran using pure laccase as control. Results: Wheat bran supported maximum laccase production from fungi under study. Highest laccase yield of 22.5 U/mL was obtained from P. spinulosum. Laccase from P. spinulosum was optimally active at pH 7.0 and 50 °C and exhibited remarkable high thermostability with 61.6% residual activity at 90 °C after 2 h incubation. The activity of the thermostable enzyme was enhanced in the presence of Cu2+. Biodelignification efficiency of cell-free extract from P. spinulosum, T. koningii, and P. restrictum on wheat bran were 95%, 81.5% and 63.5%, respectively. Surprisingly, a much lower delignification efficiency of 33.42% was obtained with commercial laccase from Trametes versicolor. Conclusion: The high thermostability and striking delignification efficiency of laccase from P. spinulosum make the enzyme a good bioresource for biodelignification of untreated lignocellulose for biofuel production.

Development of a recombinant Taq DNA polymerase enzyme expressed using a synthetic gene and its comparison with a commercial enzyme

Taq DNA polymerase is a thermostable enzyme widely used for DNA amplification in the PCR technique. It was initially characterized and isolated from thermophilic bacteria, Thermus aquaticus. It was difficult to developed in this enzyme using a native host system. Therefore, the development of the recombinant Taq DNA polymerase expressed using a synthetic gene is important to improve production efficiency. In this study, we developed the in house Taq DNA polymerase recombinant based on a codon-optimized using E. coli expression system. We cloned 2685 bp of the Taq DNA polymerase gene in the pET151/D-TOPO vector. The gene was synthesized and the expression was analyzed with SDS-PAGE technique which indicated with a 100.9 kDa specific target protein. The concentration and activity of this purified enzyme were 5.17 mg/mL and 4.647 U/µL, respectively. The application of this enzyme to the PCR technique showed that this enzyme could amplify the target genes from 200 bp to 3500 bp amplicons with a minimum DNA concentration template 10 ng/µL. This assumes that the in house recombinant Taq DNA polymerase based on synthetic genes is successfully expressed, purified, and was functional and comparable to the commercial Taq polymerase.

Biotechnological Eminence of Chitinases: A Focus on Thermophilic Enzyme Sources, Production Strategies and Prominent Applications.

Background: Chitin, the second most abundant polysaccharide in nature, is a constantly valuable and renewable raw material after cellulose. Due to advancement in technology, industrial interest has grown to take advantage of the chitin. Objective: Now, biomass is being treated with diverse microbial enzymes or cells for the production of desired products under best industrial conditions. Glycosidic bonds in chitin structure are degraded by chitinase enzymes, which are characterized into number of glycoside hydrolase (GHs) families. Methods: Thermophilic microorganisms are remarkable sources of industrially important thermostable enzymes, having ability to survive harsh industrial processing conditions. Thermostable chitinases have an edge over mesophilic chitinases as they can hydrolyse the substrate at relatively high temperatures and exhibit decreased viscosity, significantly reduced contamination risk, thermal and chemical stability and increased solubility. Various methods are employed to purify the enzyme and increase its yield by optimizing various parameters such as temperature, pH, agitation, and by investigating the effect of different chemicals and metal ions etc. Results: Thermostable chitinase enzymes show high specific activity at elevated temperature which distinguish them from mesophiles. Genetic engineering can be used for further improvement of natural chitinases, and unlimited potential for the production of thermophilic chitinases has been highlighted due to advancement in synthetic biological techniques. Thermostable chitinases are then used in different fields such as bioremediation, medicine, agriculture and pharmaceuticals. Conclusion: This review will provide information about chitinases, biotechnological potential of thermostable enzyme and the methods by which they are being produced and optimized for several industrial applications. Some of the applications of thermostable chitinases have also been briefly described.

A thermostable bacterial lytic polysaccharide monooxygenase with high operational stability in a wide temperature range

Background Lytic polysaccharide monooxygenases (LPMOs) are oxidative, copper-dependent enzymes that function as powerful tools in the turnover of various biomasses, including lignocellulosic plant biomass. While LPMOs are considered to be of great importance for biorefineries, little is known about industrial relevant properties such as the ability to operate at high temperatures. Here, we describe a thermostable, cellulose-active LPMO from a high-temperature compost metagenome (called mgLPMO10). Results MgLPMO10 was found to have the highest apparent melting temperature (83 °C) reported for an LPMO to date, and is catalytically active up to temperatures of at least 80 °C. Generally, mgLPMO10 showed good activity and operational stability over a wide temperature range. The LPMO boosted cellulose saccharification by recombinantly produced GH48 and GH6 cellobiohydrolases derived from the same metagenome, albeit to a minor extent. Cellulose saccharification studies with a commercial cellulase cocktail (Celluclast®) showed that the performance of this thermostable bacterial LPMO is comparable with that of a frequently utilized fungal LPMO from Thermoascus aurantiacus (TaLPMO9A). Conclusions The high activity and operational stability of mgLPMO10 are of both fundamental and applied interest. The ability of mgLPMO10 to perform oxidative cleavage of cellulose at 80 °C and the clear synergy with Celluclast® make this enzyme an interesting candidate in the development of thermostable enzyme cocktails for use in lignocellulosic biorefineries.

Asymmetric Synthesis of Optically Pure Aliphatic Amines with an Engineered Robust ω-Transaminase

The production of chiral amines by transaminase-catalyzed amination of ketones is an important application of biocatalysis in synthetic chemistry. It requires transaminases that show high enantioselectivity in asymmetric conversion of the ketone precursors. A robust derivative of ω-transaminase from Pseudomonasjessenii (PjTA-R6) that naturally acts on aliphatic substrates was constructed previously by our group. Here, we explore the catalytic potential of this thermostable enzyme for the synthesis of optically pure aliphatic amines and compare it to the well-studied transaminases from Vibrio fluvialis (VfTA) and Chromobacterium violaceum (CvTA). The product yields indicated improved performance of PjTA-R6 over the other transaminases, and in most cases, the optical purity of the produced amine was above 99% enantiomeric excess (e.e.). Structural analysis revealed that the substrate binding poses were influenced and restricted by the switching arginine and that this accounted for differences in substrate specificities. Rosetta docking calculations with external aldimine structures showed a correlation between docking scores and synthetic yields. The results show that PjTA-R6 is a promising biocatalyst for the asymmetric synthesis of aliphatic amines with a product spectrum that can be explained by its structural features.

Counterbalance of Stability and Activity Observed for Thermostable Transaminase from Thermobaculum terrenum in the Presence of Organic Solvents

Pyridoxal-5’-phosphate-dependent transaminases catalyze stereoselective amination of organic compounds and are highly important for industrial applications. Catalysis by transaminases often requires organic solvents to increase the solubility of reactants. However, natural transaminases are prone to inactivation in the presence of water-miscible organic solvents. Here, we present the solvent tolerant thermostable transaminase from Thermobaculum terrenum (TaTT) that catalyzes transamination between L-leucine and alpha-ketoglutarate with an optimum at 75 °C and increases the activity ~1.8-fold upon addition of 15% dimethyl sulfoxide or 15% methanol at high but suboptimal temperature, 50 °C. The enhancement of the activity correlates with a decrease in the thermal denaturation midpoint temperature. The blue-shift of tryptophan fluorescence suggested that solvent molecules penetrate the hydration shell of the enzyme. Analysis of hydrogen bonds in the TaTT dimer revealed a high number of salt bridges and surface hydrogen bonds formed by backbone atoms. The latter are sensitive to the presence of organic solvents; they rearrange, conferring the relaxation of some constraints inherent to a thermostable enzyme at low temperatures. Our data support the idea that the counterbalance of stability and activity is crucial for the catalysis under given conditions; the obtained results may be useful for fine-tuning biocatalyst efficiency.

Cloning, Expression and Characterization of the α-glucuronidase from the Hyperthermophile DictyoglomusturgidumDSM 6724Ô

Conversion of biomass into fermentable sugars is a major requirement for successful and cost-effective biofuels production. The conversion of xylan to sugars requires multiple enzymes including α-glucuronidase. Here we report the cloning, expression, purification and characterization of the α-glucuronidase from Dictyoglomusturgidum(DtuAgu). DtuAgu is an intracellular protein of 685 amino acids and a predicted molecular weight of 79.4 kD. Enzymatic activity was optimum between pH 7.0 and 8.0 and at 85°C. The specific activity of the enzyme was 10 u/mg when measured using mixed aldouronic acids. The specific activity on isolated glucuronoxylan was approximately 20% of the value obtained with xylooligosaccharides. DtuAgu significantly improved xylan conversion to xylose when evaluated using two mixtures of thermostable bacterial enzymes and two sources of xylan. DtuAgu has the potential to be a key player in thermostable enzyme cocktails for the conversion to biomass to biofuels.α