Microbial membrane transport proteins and their biotechnological applications.

Because of the hydrophobic nature of the membrane lipid bilayer, the majority of the hydrophilic solutes require special transportation mechanisms for passing through the cell membrane. Integral membrane transport proteins (MTPs), which belong to the Major Intrinsic Protein Family, facilitate the transport of these solutes across cell membranes. MTPs including aquaporins and carrier proteins are transmembrane proteins spanning across the cell membrane. The easy handling of microorganisms enabled the discovery of a remarkable number of transport proteins specific to different substances. It has been realized that these transporters have very important roles in the survival of microorganisms, their pathogenesis, and antimicrobial resistance. Astonishing features related to the solute specificity of these proteins have led to the acceleration of the research on the discovery of their properties and the development of innovative products in which these unique properties are used or imitated. Studies on microbial MTPs range from the discovery and characterization of a novel transporter protein to the mining and screening of them in a large transporter library for particular functions, from simulations and modeling of specific transporters to the preparation of biomimetic synthetic materials for different purposes such as biosensors or filtration membranes. This review presents recent discoveries on microbial membrane transport proteins and focuses especially on formate nitrite transport proteins and aquaporins, and advances in their biotechnological applications.

Comparison of ion selectivities of nitrite channel NirC and water channel aquaporin.

nirC gene coding for the nitrite channel of E. coli K12 was cloned into the pET28a vector and expressed in E. coli BL21 cells. 28.5 kDa NirC monomer was purified from membrane components of E. coli. Selectivity of NirC for different ions including nitrite, nitrate, sulfate, formate, and acetate anions, and a divalent cation, magnesium, was compared with that of bacterial aquaporin from Halomonas elongata. Water and ion permeability values were determined by measuring the light scattering rates of proteoliposomes containing NirC and aquaporins during their water loss and gain. NirC shows a selective permeability to nitrite and is more resistant to the entry of other anions as compared to aquaporin. The single channel permeability of NirC for nitrite is about 10-fold that of a single aquaporin channel. Both aquaporin and NirC channel proteins were impermeable to MgCl2 and (NH4)2SO4 and their permeability to other tested ions was remarkably lower as compared to nitrite ions. The study also presents the 3D model and channel characteristics of NirC. The translocation channel of E. coli NirC is determined to be larger, and its length is shorter than aquaporin channels. Although the NirC channel throat is more hydrophobic than aquaporin, its water permeability is almost equal to that of aquaporin. The hydrophobic nature of the NirC channel might play an important role in the selective permeability of the channel for nitrite ions.

Affinity tag effect on the salt rejection potential of Halomonas elongata aquaporin incorporated in thin film nanocomposite membrane.

In this study, effect of affinity tags, Histidine (His) and Glutathione-S-Transferase (GST), on the activity of halophilic aquaporin was analyzed. The gene coding for H. elongata aquaporin was cloned into pET28a vector and expressed in E. coli BL21 successfully. Stopped flow light scattering measurements showed that His-tagged aquaporin is functional. The difference in the filtration parameters caused by affinity tags were determined by using thin film composite nano-filtration (NFC) membranes prepared with the aquaporins. At 100 mM salt concentration, water permeability (L/m2.h) and the % salt rejection of NFC membranes produced with the His-tagged aquaporin was found to be higher than that of the membrane with GST-tagged aquaporin. Salt rejection of His-tagged aquaporin-membrane was found to be 53% with a lower solute permeability value (B). Use of short affinity tag (His tag) for cloning resulted in higher solute rejection ability of TFC membranes prepared with H. elongata aquaporins.

Micropollutant removal capacity and stability of aquaporin incorporated biomimetic thin-film composite membranes.

Aquaporin incorporated nanofiltration membranes have high potential for future applications on separation processes. In this study, performance of biomimetic thin-film composite membranes containing Halomonas elongata and Escherichia coli aquaporins with different affinity tags for the removal of micropollutants was investigated.% rejection of the membranes for atrazine, terbutryn, triclosan, and diuron varied between 66.7% and 90.3% depending on the type of aquaporin and micropollutant. The highest removal rate was achieved with a membrane containing H. elongata aquaporin for atrazine and terbutryn which have methyl branching in their structure. Electrostatic interactions between micropollutants, thin-film layer of the membrane, and tags of aquaporins may also play important role in rejection of micropollutants. Stability experiments showed that biomimetic membranes can be used for six months period without a remarkable decrease in% rejection. Membrane used 24 times for atrazine removal for a year period lost most of its ability to repel atrazine.

Nitrite is reduced by nitrite reductase NirB without small subunit NirD in Escherichia coli.

The assimilatory nitrite reductase enzyme NirB and small subunit NirD genes encoded in nir operon in Escherichia coli were cloned into the pET28a vector, and the recombinant enzyme was characterized for the first time. Docking of NirB with NirD, NADH, NO2-, NO3-, and CHO2- was performed using docking modeling programs. Methyl viologen and sodium dithionite were used as electron couples, and the amount of reduced nitrite was measured to calculate enzyme activity. NirB is the main enzyme and shows high activity with or without NirD. However, the inclusion of NirD into the enzyme solution at a ratio of 1NirD:2NirB resulted in 10% higher nitrite reductase activity. The enzyme tends to aggregate in the absence of ß-mercaptoethanol, which causes the conversion of tetrameric NirB to monomeric form, and the NirB enzyme shows its highest activity in monomeric form. The optimum temperature for enzyme activity was 37 °C and the optimum pH was found to be 7.0. Km and Vmax values of NirB were calculated as 9833 µM and 416.67 µmol NO2- reduced min-1 mg-1. Enzyme activity decreased by 55% and 50% in the presence of 100 mM nitrate and formate, respectively. The presence of 25 mM Cd2+ protected the enzyme at room temperature and the enzyme showed 10% higher activity in the presence of cadmium.

Thermodynamically designed target-specific DNA probe as an electrochemical hybridization biosensor.

Applications of molecular techniques to elucidate identity or function using biomarkers still remain highly empirical and biosensors are no exception. In the present study, target-specific oligonucleotide probes for E. coli K12 were designed thermodynamically and applied in an electrochemical DNA biosensor setup. Biosensor was prepared by immobilization of a stem-loop structured probe, modified with a thiol functional group at its 5' end and a biotin molecule at its 3' end, on a gold electrode through self-assembly. Mercaptopropionic acid (MPA) was used to optimize the surface probe density of the electrode. Hybridization between the immobilized probe and the target DNA was detected via the electrochemical response of streptavidin-horseradish peroxidase in the presence of the substrate. The amperometric response showed a linear relationship with the target DNA concentration, ranging from 10 and 400 nM, with a correlation coefficient of 0.989. High selectivity and good repeatability of the biosensor showed that the thermodynamic approach to oligonucleotide probe design can be used in development of electrochemical DNA biosensors.

Nutritional and cultural parameters influencing antidipteran delta-endotoxin production.

In this study, various nutritional and cultural parameters influencing diptera-specific delta-endotoxin synthesis by Bacillus thuringiensis subsp. israelensis HD500 were investigated. Of various inorganic nitrogen sources, the highest yields of Cry11Aa and Cry4Ba proteins were obtained on (NH(4))(2)HPO(4). Among carbon sources, inulin, dextrin, maltose, lactose, sucrose, whey and glycerol were all stimulatory, while glucose, starch and molasses were suppressive. High concentrations of inorganic phosphate (50 to 100 mM K(2)HPO(4)) were required for an effective synthesis of Cry4Ba. Mn was the most critical element for the biosynthesis of both toxins at 10(-6) M concentration. Mg and Ca favored production when provided at 8 x 10(-3) M and 5.5 x 10(-4) M concentrations, respectively, while Fe, Zn and Cu negatively influenced biosynthesis. Cry4-toxin synthesis was best at neutral pH and also when the organism was grown at 25 degrees C. Throughout the study, the extent of growth and sporulation of the producer organism was also monitored.

Ethanol production from wheat straw by Saccharomyces cerevisiae and Scheffersomyces stipitis co-culture in batch and continuous system.

In this research, Scheffersomyces stipitis and Saccharomyces cerevisiae in immobilized and suspended state were used to convert pentose and hexose sugars to ethanol. In batch and continuous systems, S. stipitis and S. cerevisiae co-culture performance was better than S. cerevisiae. Continuous ethanol production was performed in packed bed immobilized cell reactor (ICR). In ICR, S. stipitis cells were found to be more sensitive to oxygen concentration and other possible mass transfer limitations as compared to S. cerevisiae. Use of co-immobilized S. stipitis and S. cerevisiae resulted in maximum xylose consumption (73.92%) and 41.68 g/L day ethanol was produced at HRT (hydraulic retention time) of 6h with wheat straw hydrolysate. At HRT of 0.75 h, the highest amount of ethanol with the values of 356.21 and 235.43 g/L day was produced when synthetic medium and wheat straw hydrolysate were used as feeding medium in ICR, respectively.

Evaluating pretreatment techniques for converting hazelnut husks to bioethanol.

This study examined the suitability of husk waste for bioethanol production and compared pretreatment techniques with regard to their efficiencies. Results showed that 4% NaBH4 (90 min) delignified the highest amount of lignin (49.1%) from the structure. The highest xylan solubility (77.9%) was observed when samples were treated with 4% NaOH for 90 min. Pretreatment with NaOH and NaBH4, compared to H2O2 and H2SO4, resulted in selective delignification. The highest glucan to glucose conversion (74.4%) and the highest ethanol yield (52.6 g/kg husks) were observed for samples treated with 2% NaOH for 90 min.

Alkaline peroxide pretreatment of rapeseed straw for enhancing bioethanol production by Same Vessel Saccharification and Co-Fermentation.

Alkaline peroxide pretreatment of rapeseed straw was evaluated for conversion of cellulose and hemicellulose to fermentable sugars. After pretreatment, a liquid phase called pretreatment liquid and a solid phase were separated by filtration. The neutralized pretreatment liquids were used in a co-fermentation process, with Saccharomyces cerevisiae and Pichia stipitis. The solid fraction was used for simultaneous saccharification and co-fermentation process in the same vessel. The effects of various operating variables were investigated. Pretreatment with 5% (v/v) H(2)O(2) at 50 °C for 1h was found to be the optimal pretreatment combination with respect to overall ethanol production. At this condition, 5.73 g ethanol was obtained from pretreatment liquid and 14.07 g ethanol was produced by co-fermentation of solid fraction with P. stipitis. Optimum delignification was observed when 0.5 M MgSO(4) was included in the pretreatment mixture, and it resulted in 0.92% increase in ethanol production efficiency.

The use of microporous divinyl benzene copolymer for yeast cell immobilization and ethanol production in packed-bed reactor.

Microporous divinyl benzene copolymer (MDBP) was used for the first time as immobilization material for Saccharomyces cerevisiae ATCC 26602 cells in a bed reactor and ethanol production from glucose was studied as a model system. A very homogenous thick layer of yeast cells were seen from the scanning electron micrographs on the outer walls of biopolymer. The dried weight of the cells was found to be approximately 2 g per gram of cell supporting material. Hydrophobic nature of polymer is an important factor increasing cell adhesion on polymer pieces. The dynamic flow conditions through the biomaterial due to its microporous architecture prevented exopolysaccharide matrix formation around cells and continuous washing out of toxic metabolites and dead and degraded cells from the reactor provided less diffusional limitations and dynamic living environment to the cells. In order to see the ethanol production performance of immobilized yeast cells, a large initial concentration range of glucose between 6.7 and 300 g/l was studied at 1 ml/min in continuous packed-bed reactor. The inhibition effect of glucose with increasing initial concentration was observed at above 150 g/l, a relatively high substrate concentration. The continuous fluid flow around the microenvironment of the attached cells and mass transferring ability of cell immobilized on MDBP can help in decreasing the inhibition effect of ethanol accumulation and high substrate concentration in the vicinity of the cells.

Thermostable amperometric lactate biosensor with Clostridium thermocellum L-LDH for the measurement of blood lactate.

The gene for Clostridium thermocellum L-lactate dehydrogenase enzyme was cloned into pGEX-4T-2 purification vector to supply a source for a thermostable enzyme in order to produce a stable lactate biosensor working at relatively high temperatures. The purified thermostable enzyme (t-LDH) was then immobilized on a gold electrode via polymerization of polygluteraldehyde and pyrrol resulting in a conductive co-polymer. t-LDH working electrode (t-LDHE) was used for determination of lactate in CHES buffer. Amperometric response of the produced electrodes was measured as a function of lactate concentration, at a fixed bias voltage of 200 mV in a three-electrode system. The linear range and sensitivity of the biosensor was investigated at various temperatures in the range of 25-60 degrees C. The sensitivity t-LDHE increased with increasing the temperature and reached its highest value at 60 degrees C. The calculated value was nearly 70 times higher as compared to the sensitivity value of the same electrode tested at 25 degrees C. The sensing parameters of t-LDHE were compared with the electrodes produced by commercially available rabbit muscle LDH (m-LDH). The sensitivity of t-LDHE was nearly 8 times higher than that of m-LDHE. t-LDHE was found to retain its activity for a week incubation at refrigerator (+5 degrees C), while m-LDHE lost its activity in this period. t-LDHE was also tested in the presence of human blood serum. The results showed that the current increased with increasing concentrations of lactate in the human blood serum and the biosensor is more sensitive to serum lactate as well as the commercial lactate dissolved in serum as compared to the commercial lactate dissolved in CHES buffer.

Cry3Aa11: a new Cry3Aa delta-endotoxin from a local isolate of Bacillus thuringiensis.

A local isolate of Bacillus thuringiensis Mm2 had insecticidal activity against the larvae of Melolontha melolontha, Agelastica alni, Leptinotarsa decemlineata and Amphimallon solstitiale and produced a 65 kDa protein. SDS-PAGE profile of B. thuringiensis Mm2 was compared with those of 29 different Cry3Aa producers which verified Cry3Aa biosynthesis by the isolate. The cry3Aa gene of Mm2 was cloned, sequenced and the deduced amino acid sequence was compared with the cry3Aa sequences of ten different quaternary ranks. Its identity to these sequences ranged between 97.4% and 99.2%. The gene was next cloned into E. coli-Bacillus shuttle vector pNW33N and expressed at a low level in B. subtilis 168.

Cloning and expression of the Clostridium thermocellum L-lactate dehydrogenase gene in Escherichia coli and enzyme characterization.

The structural gene for L-lactate dehydrogenase (LDH) (EC.1.1.1.27) from Clostridium thermocellum 27405 was cloned in Escherichia coli by screening the Lambda Zap II phage library of C. thermocellum genomic DNA. In one positive clone, an open reading frame of 948 base pairs corresponded to C. thermocellum ldh gene encoding for the predicted 315-residue protein. The ldh gene was successfully expressed in E. coli FMJ39 (ldh mutant) under the lac promoter. The recombinant enzyme was partially purified from E. coli cell extracts and its kinetic properties were determined. Clostridium thermocellum LDH was shown to catalyze a highly reversible reaction and to be an allosteric enzyme that is activated by fructose-1,6-diphosphate (FDP). For pyruvate, partially purified LDH had Km and Vmax values of 7.3 mmol/L and 87 micromol/min, respectively, and in the presence of FDP, a 24-fold decrease in Km and a 5.7-fold increase in Vmax were recorded. The enzyme exhibited no marked catalytic activity for lactate in the absence of FDP, whereas Km and Vmax values were 59.5 mmol/L and 52 micromol/min, respectively, in its presence. The enzyme did not lose activity when incubated at 65 degrees C for 5 min.