Cloning and Expression of Phytase appA Gene from Shigella sp. CD2 in Pichia pastoris and Comparison of Properties with Recombinant Enzyme Expressed in E. coli

The phytase gene appA_S was isolated from Shigella sp. CD2 genomic library. The 3.8 kb DNA fragment contained 1299 bp open reading frame encoding 432 amino acid protein (AppA_S) with 22 amino acid signal peptide at N-terminal and three sites of N-glycosylation. AppA_S contained the active site RHGXRXP and HDTN sequence motifs, which are conserved among histidine acid phosphatases. It showed maximum identity with phytase AppA of Escherichia coli and Citrobacter braakii. The appA_S was expressed in Pichia pastoris and E. coli to produce recombinant phytase rAppA_P and rAppA_E, respectively. Purified glycosylated rAppA_P and nonglycosylated rAppA_E had specific activity of 967 and 2982 U mg^-1, respectively. Both had pH optima of 5.5 and temperature optima of 60°C. Compared with rAppA_E, rAppA_P was 13 and 17% less active at pH 3.5 and 7.5 and 11 and 18% less active at temperature 37 and 50°C, respectively; however, it was more active at higher incubation temperatures. Thermotolerance of rAppA_P was 33% greater at 60°C and 24% greater at 70°C, when compared with rAppA_E. Both the recombinant enzymes showed high specificity to phytate and resistance to trypsin. To our knowledge, this is the first report on cloning and expression of phytase from Shigella sp.     

Selective cellular imaging with lanthanide‐based upconversion nanoparticles

Upconversion nanoparticles (UCNPs) with sodium yttrium fluoride, NaYF₄ (host lattice) doped with Yb³⁺ (sensitizer) and Er³⁺ (activator) were synthesized via hydrothermal route incorporating polyethyleneimine (PEI) for their long‐term stability in water. The cationic PEI‐modified UCNPs with diameter 20 ± 4 nm showed a zeta potential value of +36.5 mV and showed an intense, visible red luminescence and low‐intensity green emission with 976 nm laser excitation. The particles proven to be nontoxic to endothelial cells, with a 3‐(4,5‐dimethylthiazol‐2yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay, showing 90% to 100% cell viability, across a wide range of UCNP concentrations (0.3 ng/mL‐0.3 mg/mL) were used in multiphoton imaging. Multiphoton cellular imaging and emission spectroscopy data reported here prove that the UCNPs dispersed in cell culture media are predominantly concentrated in the cytoplasm than the cell nucleus. The energy transfer from PEI‐coated UCNPs to surrounding media for red luminescence in the biological system is also highlighted with spectroscopic measurements. Results of this study propose that UCNPs can, therefore, be used for cytoplasm selective imaging together with multiphoton dyes (eg, 4′,6‐diamidino‐2‐phenylindole (DAPI)) that are selective to cell nucleus.      

Natural Variation for Nutrient Use and Remobilization Efficiencies in Switchgrass

Nutrient management in biomass production systems serves to maximize yield and minimize production costs and environmental impact. Loss of soil nutrients with harvested biomass can be reduced by the judicious choice of genotype and harvest time. Sustainable production of switchgrass for biofuel will depend, in part, on breeding of varieties that are conservative in their use of soil nutrients to produce biomass. To aid such breeding programs, we assessed the natural variation in nutrient-use and remobilization efficiencies of 31 accessions of Panicum virgatum by measuring the concentration of 20 elements (N, P, K, Li, B, Na, Mg, Ca, Mn, Fe, Co, Ni, Cu, Zn, As, Se, Rb, Sr, Mo, and Cd) in shoots of field-grown plants harvested at two different stages of development. Significant differences between accessions were found for elemental composition at maturity and after senescence. The concentration of several elements (N, P, K, and Rb) decreased in the shoots of all accessions during senescence, although the efficiency of remobilization ranged from 20% to 61% for N, 31% to 65% for P, 25% for 84% for K, and 33% to 84% for Rb. The accessions/cultivars with the greatest nutrient-use efficiency (smallest loss of nutrient per unit biomass) were BN-14668-65, Kanlow, Caddo from the point of view of N content, and Kanlow, Cave-in-Rock, and Blackwell from the point of view of P content in senescent shoots. Finally, differences in elemental composition between upland and lowland ecotypes were also found. The information presented here will help to guide future breeding programs and nutrient management practices.     

Diversification of function in the haloacid dehalogenase enzyme superfamily: The role of the cap domain in hydrolytic phosphoruscarbon bond cleavage

Phosphonatase functions in the 2-aminoethylphosphonate (AEP) degradation pathway of bacteria, catalyzing the hydrolysis of the CP bond in phosphonoacetaldehyde (Pald) via formation of a bi-covalent Lys53ethylenamine/Asp12 aspartylphosphate intermediate. Because phosphonatase is a member of the haloacid dehalogenase superfamily, a family predominantly comprised of phosphatases, the question arises as to how this new catalytic activity evolved. The source of general acid-base catalysis for Schiff-base formation and aspartylphosphate hydrolysis was probed using pH-rate profile analysis of active-site mutants and X-ray crystallographic analysis of modified forms of the enzyme. The 2.9 Å X-ray crystal structure of the mutant Lys53Arg complexed with Mg²⁺ and phosphate shows that the equilibrium between the open and the closed conformation is disrupted, favoring the open conformation. Thus, proton dissociation from the cap domain Lys53 is required for cap domain–core domain closure. The likely recipient of the Lys53 proton is a water-His56 pair that serves to relay the proton to the carbonyl oxygen of the phosphonoacetaldehyde (Pald) substrate upon addition of the Lys53. The pH-rate profile analysis of active-site mutants was carried out to test this proposal. The proximal core domain residues Cys22 and Tyr128 were ruled out, and the role of cap domain His56 was supported by the results. The X-ray crystallographic structure of wild-type phosphonatase reduced with NaBH₄ in the presence of Pald was determined at 2.4 Å resolution to reveal Nε-ethyl-Lys53 juxtaposed with a sulfate ligand bound in the phosphate site. The position of the C(2) of the N-ethyl group in this structure is consistent with the hypothesis that the cap domain Nε-ethylenamine-Lys53 functions as a general base in the hydrolysis of the aspartylphosphate bi-covalent enzyme intermediate. Because the enzyme residues proposed to play a key role in PC bond cleavage are localized on the cap domain, this domain appears to have evolved to support the diversification of the HAD phosphatase core domain for catalysis of hydrolytic PC bond cleavage.     

Characterization of thermostable cyclodextrinase from Clostridium thermohydrosulfuricum 39E.

Clostridium thermohydrosulfuricum 39E produced a cell-bound cyclodextrin (CD)-degrading enzyme (cyclodextrinase). It was partially purified 205-fold (specific activity, 14.5 U/mg of protein) by solubilizing with Triton X-100, ammonium sulfate treatment, and DEAE-Sepharose CL-6B column chromatography. The enzyme activity was found to be stable at pH 5.5 and 60 degrees C and optimally active at pH 6.0 and 65 degrees C. The enzyme preparation hydrolyzed CDs, with alpha-CD greater than beta-CD greater than gamma-CD, and displayed a putative multiple attack pattern. The enzyme activity was inhibited by p-chloromercuribenzoate but not by N-bromosuccinimide.     

Some blood genetic markers of the Nuba and Hawazma tribes of western Sudan

Two hundred eighty subjects comprising 112 Nuba and 168 Hawazma of the Sudan were tested for the distribution of hemoglobins, eight red cell enzymes, and four serum proteins. The Nuba, the indigenous negroid tribe, had no HbS, HbO-Arab, or GdB(Khartoum) compared to the Hawazma tribe of Negro-Arab descent. The gene frequencies of the above polymorphic systems in the latter were as follows: HbS, 0.13; HbO-Arab, 0.01; GdB(Khartoum), 0.03. The frequency of GdA was higher in the Hawazma than in the Nuba. A high frequency of glucose 6-phosphate dehydrogenase (G6PD) deficiency and HpO was present in both the tribes. Essentially similar gene frequencies of Hp1, TfD, PGDC, pC, and PGM1 were observed in both Nuba and Hawazma. The average heterozygosity at five polymorphic loci was the same (0.23) in both the tribes. The above results agree with the social practice whereby people of mixed Hawazma and Nuba descent are considered members of the Hawazma tribe and confirm that racial admixture between the two groups can be seen as a process of gene flow from the Nuba to the Hawazma, even though the Nuba are the indigenous group, while the Hawazma are the new settlers.     

Multiphasic denaturation of the lambda repressor by urea and its implications for the repressor structure.

Urea denaturation of the lambda repressor has been studied by fluorescence and circular dichroic spectroscopies. Three phases of denaturation could be detected which we have assigned to part of the C-terminal domain, N-terminal domain and subunit dissociation coupled with further denaturation of the rest of the C-terminal domain at increasing urea concentrations. Acrylamide quenching suggests that at least one of the three tryptophan residues of the lambda repressor is in a different environment and its emission maximum is considerably blue-shifted. The transition in low urea concentration (midpoint approximately 2 M) affects the environment of this tryptophan residue, which is located in the C-terminal domain. Removal of the hinge and the N-terminal domain shifts this transition towards even lower urea concentrations, indicating the presence of interaction between hinge on N-terminal and C-terminal domains in the intact repressor.