Expression and functional analysis of a recombinant aquaporin Z from Antarctic Pseudomonas sp. AMS3.

Aquaporin (AQP) is a water channel protein from the family of transmembrane proteins which facilitates the movement of water across the cell membrane. It is ubiquitous in nature, however the understanding of the water transport mechanism, especially for AQPs in microbes adapted to low temperatures, remains limited. AQP also has been recognized for its ability to be used for water filtration, but knowledge of the biochemical features necessary for its potential applications in industrial processes has been lacking. Therefore, this research was conducted to express, extract, solubilize, purify, and study the functional adaptations of the aquaporin Z family from Pseudomonas sp. AMS3 via molecular approaches. In this study, AqpZ1 AMS3 was successfully subcloned and expressed in E. coli BL21 (DE3) as a recombinant protein. The AqpZ1 AMS3 gene was expressed under optimized conditions and the best optimized condition for the AQP was in 0.5 mM IPTG incubated at 25°C for 20 h induction time. A zwitterionic mild detergent [(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate was the suitable surfactant for the protein solubilization. The protein was then purified via affinity chromatography. Liposome and proteoliposome was reconstituted to determine the particle size using dynamic light scattering. This information obtained from this psychrophilic AQP identified provides new insights into the structural adaptation of this protein at low temperatures and could be useful for low temperature application and molecular engineering purposes in the future.

Molecular dynamics simulation and structural analysis of aquaporin Z from an Antarctic Pseudomonas sp. strain AMS3.

Aquaporin is a water channel protein that facilitates the movement of water across the cell membrane. Aquaporin from the Antarctic region has been noted for its psychrophilic properties and its ability to perform at a lower temperature but there remains limited understanding of the water mechanism of Antarctic Pseudomonas sp. strain AMS3 However, studies regarding aquaporin isolated from psychrophilic Pseudomonas sp. are still scattered. Recently, the genome sequence of an Antarctic Pseudomonas sp. strain AMS3 revealed a gene sequence encoding for a putative aquaporin designated as AqpZ1 AMS3. In this study, structure analysis and a molecular dynamics (MD) simulation of a predicted model of a fully hydrated aquaporin tetramer embedded in a lipid bilayer was performed at different temperatures for structural flexibility and stability analysis. The MD simulation results revealed that the structures were able to remain stable at low to medium temperatures. The protein was observed to have high flexibility in the loop region as compared to the helices region throughout the simulated temperatures. The selectivity filter and NPA motifs play a major role in solute selectivity and the pore radius of the protein. The structural and functional characterization of this psychrophilic aquaporin provides new insights for the future applications of this protein.Communicated by Ramaswamy H. Sarma.

The effect of emergency department pharmacists on drug overuse and drug underuse in patients with an ADE-related hospitalisation: a controlled intervention study.

BACKGROUND: Drug overuse or drug underuse are the most common causes of adverse drug events and can lead to hospital admissions. Using clinical pharmacists in the emergency department may improve patient safety as they are specialised in recognising of adverse drug events and tackling drug overuse and drug underuse. This study tested the effect of an emergency department pharmacist on the number of medication changes for drug overuse and drug underuse taking place in patients with an adverse drug event-related hospitalisation following an emergency department visit. METHODS: A multicenter prospective non-randomized controlled intervention study was conducted in a university hospital and a general teaching hospital. Trained emergency department pharmacists included patients in the intervention group with a hospital admission related to an adverse drug event. The interdisciplinary intervention consisted of a pharmacist-led medication review, patient counselling regarding medication, and information transmission to general practitioners and community pharmacies after discharge. The control patients were also admitted after an emergency department visit and received the usual care. The primary outcome was the number of medication changes for drug overuse and drug underuse that took place during hospital admission and persisted 6 months thereafter. Poisson regression analysis was used to estimate the difference in these medication changes between the intervention group and the control group. RESULTS: A total of 216 patients were included (intervention group 104, control group 112). In the intervention group, 156 medication changes for drug overuse and drug underuse persisted 6 months after admission compared to 59 in the control group (adjusted rate ratio 1.22 [95%CI 1.01-1.49] p = 0.039). CONCLUSION: Emergency department pharmacists do contribute to reduction of drug overuse and drug underuse of medication in patients with a hospitalisation related to adverse drug events after an emergency department visit.

Ocular Manifestation and Correlation with CD4+ T Cell Count among Adult HIV/AIDS Patients in Bangladesh.

Patients with HIV or AIDS suffer from wide varieties of complications that are related to infection. The eye as an organ is not spared from HIV-related manifestations. The ocular manifestations can be the presenting sign of a systemic infection in an otherwise asymptomatic HIV-positive person. The disease can have adnexal, anterior segment, posterior segment, orbital and neuro-ophthalmic manifestations. The objective of the study was to evaluate the ophthalmological manifestations among adult HIV infected patients of Bangladesh and co-relate the findings with CD-4+ T cell count. This cross sectional study was conducted in the department of Community Ophthalmology, Bangabandhu Sheikh Mujib Medical University (BSMMU), Dhaka, Bangladesh from January 2013 to September 2015. Purposive sampling technique was applied to enroll the patients. Total 110 patients were enrolled regardless of their immunological status by inclusion and exclusion criteria. Relevant clinical evaluation including history & physical examinations, laboratory investigations and some ocular examinations like- visual acuity, slit lamp biomicroscopy, IOP, indirect ophthalmoscopy with +90D (diopter) and +20D were done. The age of the study population ranged from 20-58 years with mean±SD 37.63±8.16 years. Among the study population 67(60.9%) were male and 43(39.1%) were female. According to ART status, 58(52.7%) were on ART and 52(47.3%) were ART naive. The mean CD4+ T- cells count was 410±281.65 with minimum to maximum was 6-1266 cells/µl. Among them 53(48.2%) had HIV related ocular findings and 57(51.8%) had no HIV related ocular manifestation. In relation with CD+ T- cells count, highly significant relation was found with lower CD4+ T- cells count and ocular manifestation (p=0.001).

Scale-up synthesis of lipase-catalyzed palm esters in stirred-tank reactor.

Lipase-catalyzed production of palm esters by alcoholysis of palm oil with oleyl alcohol in n-hexane was performed in 2L stirred-tank reactor (STR). Investigation on the performance of reactor operation was carried out in batch mode STR with single impeller mounted on the centrally located shaft. Rushton turbine (RT) impellers provide the highest reaction yield (95.8%) at lower agitation speed as compared to AL-hydrofoil (AL-H) and 2-bladed elephant ear (EE) impellers. Homogenous enzyme particles suspension was obtained at 250 rpm by using RT impeller. At higher impeller speed, the shear effect on the enzyme particles caused by agitation has decreased the reaction performance. Palm esters reaction mixture in STR follows Newtons' law due to the linear relation between the shear stress (tau) and shear rate (dupsilon/dy). High stability of Lipozyme RM IM was observed as shown by its ability to be repeatedly used to give high percentage yield (79%) of palm esters even after 15 cycles of reaction. The process was successfully scale-up to 75 L STR (50 L working volume) based on a constant impeller tip speed approach, which gave the yield of 97.2% after 5h reaction time.

Cloning and expression of a novel lipase gene from Bacillus sphaericus 205y.

A Bacillus sphaericus strain (205y) that produces an organic solvent-tolerant lipase was isolated in Port Dickson, Malaysia. The gene for the lipase was recovered from a genomic library and sequenced. Phylogenetic analysis was performed based on an alignment of thirteen microbial lipase sequences obtained from the NCBI database. The analysis suggested that the B. sphaericus lipase gene is a novel gene, as it is distinct from other lipase genes in Families I.4 and I.5 reported so far. Expression in Escherichia coli under the control of the lacZ promoter resulted in an eight-fold increase in enzyme activity after a 3-h induction with 1 mM IPTG. The crude enzyme thus obtained showed a slight (10%) enhancement in activity after a 30-min incubation in 25% (v/v) n-hexane at 37 degrees C, and retained 90% of its activity after a similar period in 25% (v/v) p-xylene.

Modified enzymes for reactions in organic solvents.

Recent studies on biocatalysis in water-organic solvent biphasic systems have shown that many enzymes retain their catalytic activities in the presence of high concentrations of organic solvents. However, not all enzymes are organic solvent tolerant, and most have limited and selective tolerance to particular organic solvents. Protein modification or protein tailoring is an approach to alter the characteristics of enzymes, including solubility in organic solvents. Particular amino acids may play pivotal roles in the catalytic ability of the protein. Attaching soluble modifiers to the protein molecule may alter its conformation and the overall polarity of the molecule. Enzymes, in particular lipases, have been chemically modified by attachment of aldehydes, polyethylene glycols, and imidoesters. These modifications alter the hydrophobicity and conformation of the enzymes, resulting in changes in the microenvironment of the enzymes. By these modifications, newly acquired properties such as enhancement of activity and stability and changes in specificity and solubility in organic solvents are obtained. Modified lipases were found to be more active and stable in organic solvents. The optimum water activity (a(w)) for reaction was also shifted by using modified enzymes. Changes in enantioselective behavior were also observed.

Ion pairs involved in maintaining a thermostable structure of glutamate dehydrogenase from a hyperthermophilic archaeon.

Intersubunit ion pairs are considered to be involved for maintaining a stable structure of the glutamate dehydrogenase (GDH) from hyperthermophiles. In order to demonstrate an effect of intersubunit ion pairs on the structural stability, two kinds of mutation (T138E, Thr at position 138 was replaced by Glu; E158Q, Glu at position 158 was replaced by Gln) which add and remove ion pairs, respectively, were introduced into Pk-gdhA gene encoding GDH from Pyrococcus kodakaraensis KOD1. Addition of one ion pair (Pk-GDHA-T138E) increased the optimum temperature and thermostability. In contrast, Pk-GDH-E158Q showed lower optimum temperature and less thermostability than wild type GDH. Structure analysis of GDHs was performed by circular dichroism (CD) and indicated that all recombinant enzymes (Pk-GDH, Pk-GDH-T138E, Pk-GDH-E158Q) possess different structures from that of natural GDH. Upon heat treatment (60 degrees C, 2 h), the structures of Pk-GDH and Pk-GDH-T138E were converted to another form close to the natural structure. However, no structural conversion by heat treatment was observed in Pk-GDH-E158Q. These results indicate that intersubunit ion pairs play an important role in forming thermostable structure of Pk-GDH.

Sequence analysis of glutamate dehydrogenase (GDH) from the hyperthermophilic archaeon Pyrococcus sp. KOD1 and comparison of the enzymatic characteristics of native and recombinant GDHs.

The gdhA gene encoding glutamate dehydrogenase (GDH) from the hyperthermophilic archaeon Pyrococcus sp. KOD1 was cloned and sequenced. Phylogenetic analysis was performed on an alignment of 25 GDH sequences including KOD1-GDH, and two protein families were distinguished, as previously reported. KOD1-GDH was classified as new member of the hexameric GDH Family II. The gdhA gene was expressed in Escherichia coli, and recombinant KOD1-GDH was purified. Its enzymatic characteristics were compared with those of the native KOD1-GDH. Both enzymes had a molecular mass of 47,300 Da and were shown to be functional in a hexameric form (284 kDa). The N-terminal amino acid sequences of native KOD1-GDH and the recombinant GDH were VEIDPFEMAV and MVEIDPFEMA, respectively, indicating that native KOD1-GDH does not retain the initial methionine at the N-terminus. The recombinant GDH displayed enzyme characteristics similar to those of the native GDH, except for a lower level of thermostability, with a half-life of 2 h at 100 degrees C, compared to 4 h for the native enzyme purified from KOD1. Kinetic studies suggested that the reaction is biased towards glutamate production. KOD1-GDH utilized both coenzymes NADH and NADPH, as do most eukaryal GDHs.

Characterization of recombinant glutamine synthetase from the hyperthermophilic archaeon Pyrococcus sp. strain KOD1.

The glnA gene encoding glutamine synthetase was cloned from the hyperthermophilic archaeon Pyrococcus sp. strain KOD1, and its nucleotide sequence was determined. The glnA gene was expressed in Escherichia coli ME8459 (glnA mutant strain), and the protein was purified to homogeneity and shown to be functional in a dodecameric from (637,000 Da), exhibiting both transferase and synthetase activities. However, kinetic studies indicated that the enzyme possessed low biosynthetic activity, suggesting that the reaction was biased towards glutamate production. The optimum temperature for both activities was 60 degrees C, which was lower than the optimal growth temperature of KOD1. Recombinant KOD1 GlnA exhibited different optimum pHs depending on the reaction employed (pH 7.8 for the synthetase reaction and pH 7.2 for the transferase reaction). Of the various nucleoside triphosphates tested, GTP as well as ATP was involved in the synthetase reaction.

Gene cloning, sequencing and enzymatic properties of glutamate synthase from the hyperthermophilic archaeon Pyrococcus sp. KOD1.

The gltA gene encoding a glutamate synthase (GOGAT) from the hyperthermophilic archaeon Pyrococcus sp. KOD1 was cloned as a 6.6 kb HindIII-BamHI fragment. Sequence analysis indicates that gltA encodes a 481- amino acid protein (53,269 Da). The deduced amino acid sequence of KOD1-GltA includes conserved regions that are found in the small subunits of bacterial GOGAT: two cysteine clusters, an adenylate-binding consensus sequence and an FAD-binding consensus sequence. However, no sequences homologous to the large subunit of bacterial GOGAT were found in the upstream or downstream regions. In order to examine whether GltA alone can act as a functional GOGAT, GltA was overexpressed in Escherichia coli BL21 (DE3) cells using an expression plasmid. GltA was purified to homogeneity and shown to be functional as a homotetramer of approximately 205 kDa, which is equivalent to the molecular weight of the native GOGAT from KOD1, thus indicating that KOD1-GOGAT is the smallest known active GOGAT. GltA is capable of both glutamine-dependent and ammonia-dependent synthesis of glutamate. Synthesis of glutamate by KOD1-GltA required NADPH, indicating that this enzyme is an NADPH-GOGAT (EC 1.4.1.13). The optimum pH for both activities was 6.5. However, GltA exhibited different optimum temperatures for activity depending on the reaction assayed (glutamine-dependent reaction, 80 degrees C; ammonia-dependent reaction, 90 degrees C).

Effect of heat treatment on proper oligomeric structure formation of thermostable glutamate dehydrogenase from a hyperthermophilic archaeon.

Natural glutamate dehydrogenase (Pk-GDH) was purified from hyperthermophilic archaeon Pyrococcus sp. KOD1 to homogeneity and its activity and structure were compared with those of recombinant enzyme, which was expressed in Escherichia coli. Determination of the molecular weight of these enzymes by SDS-PAGE and gel filtration revealed that the natural enzyme was purified only as a hexameric form, whereas the recombinant enzyme was purified as both monomeric and hexameric forms. Determination of the enzymatic activities indicated that only the enzyme in a hexameric form is active. Moreover, it is noted that the specific activity of the hexameric form of the recombinant enzyme is much lower than that of the natural enzyme and that circular dichroism spectra of these enzymes are distinctly different from each other. These results suggest that the structure of the hexameric form of the recombinant enzyme with low specific activity (Type I) is different from that of the natural enzyme with high specific activity (Type II). Upon heat treatment (80 degrees C, 15 min), the Type I structure was effectively converted to Type II structure and the specific activity of the enzyme was increased by 2.6-fold. Likewise, upon heat treatment (70 degrees C for 15 min), the inactive monomeric form of the recombinant enzyme was at least partially associated with the hexameric form. These results indicate that high temperature plays an important role for proper folding and oligomerization of Pk-GDH.