The global rise in urbanization and industrial activity has led to the production and incorporation of foreign contaminant molecules into ecosystems, distorting them and impacting human and animal health. Physical, chemical, and biological strategies have been adopted to eliminate these contaminants from water bodies under anthropogenic stress. Biotechnological processes involving microorganisms and enzymes have been used for this purpose; specifically, laccases, which are broad spectrum biocatalysts, have been used to degrade several compounds, such as those that can be found in the effluents from industries and hospitals. Laccases have shown high potential in the biotransformation of diverse pollutants using crude enzyme extracts or free enzymes. However, their application in bioremediation and water treatment at a large scale is limited by the complex composition and high salt concentration and pH values of contaminated media that affect protein stability, recovery and recycling. These issues are also associated with operational problems and the necessity of large-scale production of laccase. Hence, more knowledge on the molecular characteristics of water bodies is required to identify and develop new laccases that can be used under complex conditions and to develop novel strategies and processes to achieve their efficient application in treating contaminated water. Recently, stability, efficiency, separation and reuse issues have been overcome by the immobilization of enzymes and development of novel biocatalytic materials. This review provides recent information on laccases from different sources, their structures and biochemical properties, mechanisms of action, and application in the bioremediation and biotransformation of contaminant molecules in water. Moreover, we discuss a series of improvements that have been attempted for better organic solvent tolerance, thermo-tolerance, and operational stability of laccases, as per process requirements.

The thermal denaturation of the dimeric enzyme triosephosphate isomerase (TIM) from Saccharomyces cerevisiae was studied by spectroscopic and calorimetric methods. At low protein concentration the structural transition proved to be reversible in thermal scannings conducted at a rate greater than 1.0 degrees C min(-1). Under these conditions, however, the denaturation-renaturation cycle exhibited marked hysteresis. The use of lower scanning rates lead to pronounced irreversibility. Kinetic studies indicated that denaturation of the enzyme likely consists of an initial first-order reaction that forms thermally unfolded (U) TIM, followed by irreversibility-inducing reactions which are probably linked to aggregation of the unfolded protein. As judged from CD measurements, U possesses residual secondary structure but lacks most of the tertiary interactions present in native TIM. Furthermore, the large increment in heat capacity upon denaturation suggests that extensive exposure of surface area occurs when U is formed. Above 63 degrees C, reactions leading to irreversibility were much slower than the unfolding process; as a result, U was sufficiently long-lived as to allow an investigation of its refolding kinetics. We found that U transforms into nativelike TIM through a second-order reaction in which association is coupled to the regain of secondary structure. The rate constants for unfolding and refolding of TIM displayed temperature dependences resembling those reported for monomeric proteins but with considerably larger activation enthalpies. Such large temperature dependences seem to be determinant for the occurrence of kinetically controlled transitions and thus constitute a simple explanation for the hysteresis observed in thermal scannings.

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Thermostable phytases, which are active over broad pH ranges, may be useful as feed additives, since they can resist the temperatures used in the feed-pelleting process. We designed new beta-propeller phytases, using a structure-guided consensus approach, from a set of amino acid sequences from Bacillus phytases and engineered Pichia pastoris strains to overproduce the enzymes. The recombinant phytases were N-glycosylated, had the correct amino-terminal sequence, showed activity over a pH range of 2.5 to 9, showed a high residual activity after 10 min of heat treatment at 80°C and pH 5.5 or 7.5, and were more thermostable at pH 7.5 than a recombinant form of phytase C from Bacillus subtilis (GenBank accession no. AAC31775). A structural analysis suggested that the higher thermostability may be due to a larger number of hydrogen bonds and to the presence of P257 in a surface loop. In addition, D336 likely plays an important role in the thermostability of the phytases at pH 7.5. The recombinant phytases showed higher thermostability at pH 5.5 than at pH 7.5. This difference was likely due to a different protein total charge at pH 5.5 from that at pH 7.5. The recombinant beta-propeller phytases described here may have potential as feed additives and in the pretreatment of vegetable flours used as ingredients in animal diets.

BackgroundThe GTPase KRas4B has been utilized as a principal target in the development of anticancer drugs. PDE6δ transports KRas4B to the plasma membrane, where it is released to activate various signaling pathways required for the initiation and maintenance of cancer. Therefore, identifying new small molecules that prevent activation of this GTPase by stabilizing the KRas4B-PDE6δ molecular complex is a practical strategy to fight against cancer.MethodsThe crystal structure of the KRas4B-PDE6δ heterodimer was employed to locate possible specific binding sites at the protein-protein interface region. Virtual screening of Enamine-database compounds was performed on the located potential binding sites to identify ligands able to simultaneously bind to the KRas4B-PDE6δ heterodimer. A molecular dynamics approach was used to estimate the binding free-energy of the complex. Cell viability and apoptosis were measured by flow cytometry. G-LISA was used to measure Ras inactivation. Western blot was used to measure AKT and ERK activation. MIA PaCa-2 cells implanted subcutaneously into nude mice were treated with D14 or C22 and tumor volumes were recorded.ResultsAccording to the binding affinity estimation, D14 and C22 stabilized the protein-protein interaction in the KRas4B-PDE6δ complex based on in vitro evaluation of the 38 compounds showing antineoplastic activity against pancreatic MIA PaCa-2 cancer cells. In this work, we further investigated the antineoplastic cellular properties of two of them, termed D14 and C22, which reduced the viability in the human pancreatic cancer cells lines MIA PaCa-2, PanC-1 and BxPC-3, but not in the normal pancreatic cell line hTERT-HPNE. Compounds D14 and C22 induced cellular death via apoptosis. D14 and C22 significantly decreased Ras-GTP activity by 33% in MIA PaCa-2 cells. Moreover, D14 decreased AKT phosphorylation by 70% and ERK phosphorylation by 51%, while compound C22 reduced AKT phosphorylation by 60% and ERK phosphorylation by 36%. In addition, compounds C22 and D14 significantly reduced tumor growth by 88.6 and 65.9%, respectively, in a mouse xenograft model.ConclusionsWe identified two promising compounds, D14 and C22, that might be useful as therapeutic drugs for pancreatic ductal adenocarcinoma treatment.Electronic supplementary materialThe online version of this article (10.1186/s12885-018-5142-7) contains supplementary material, which is available to authorized users.

We have isolated a cDNA clone of the glycolytic enzyme, triosephosphate isomerase (TPI) from Entamoeba histolytica. Degenerate oligonucleotides obtained by reverse translation of conserved polypeptide sequences, derived from TPIs of other organisms, were used to amplify a 450-bp fragment using E. histolytica cDNA as a template. The fragment was used to screen a cDNA library. The isolated cDNA, encoding a protein of 261 amino acids, shares 43-52.6% positional identity with other known protozoan TPIs. The catalytic residues were conserved ; nevertheless, several indels occurred at other regions in the protein sequence. The complete coding sequence of the E. histolytica TPI gene was cloned into the expression vector pRSET and expressed as a wild-type TPI enzyme (E. histolytica TPI) and as a fusion protein with an N-terminal tail of six histidine residues E. histolytica TPI-His,) ; both recombinant proteins were purified.Molecular modeling of E. histolytica TPI showed an identical topology to the known structures of other TPI molecules, but with a remarkable feature; more than 10 inserted residues are located in the same region of the molecular surface. Studies were performed to detect possible changes that might be caused by the inserted amino acids. The catalytic activity and oligomeric state of the purified protein were similar to that reported for TPI from other sources. In contrast, stability towards dilution, as well as thermal inactivation and unfolding assays, showed that E. histolytica TPI is significantly more stable towards denaturation than Trypanosoma brucei TPI.