Extreme halophilic alcohol dehydrogenase mediated highly efficient syntheses of enantiopure aromatic alcohols.

Enzymatic synthesis of enantiopure aromatic secondary alcohols (including substituted, hetero-aromatic and bicyclic structures) was carried out using halophilic alcohol dehydrogenase ADH2 from Haloferax volcanii (HvADH2). This enzyme showed an unprecedented substrate scope and absolute enatioselectivity. The cofactor NADPH was used catalytically and regenerated in situ by the biocatalyst, in the presence of 5% ethanol. The efficiency of HvADH2 for the conversion of aromatic ketones was markedly influenced by the steric and electronic factors as well as the solubility of ketones in the reaction medium. Furthermore, carbonyl stretching band frequencies ν (C[double bond, length as m-dash]O) have been measured for different ketones to understand the effect of electron withdrawing or donating properties of the ketone substituents on the reaction rate catalyzed by HvADH2. Good correlation was observed between ν (C[double bond, length as m-dash]O) of methyl aryl-ketones and the reaction rate catalyzed by HvADH2. The enzyme catalyzed the reductions of ketone substrates on the preparative scale, demonstrating that HvADH2 would be a valuable biocatalyst for the preparation of chiral aromatic alcohols of pharmaceutical interest.

Effect of organic solvents on the activity and stability of halophilic alcohol dehydrogenase (ADH2) from Haloferax volcanii.

The effect of various organic solvents on the catalytic activity, stability and substrate specificity of alchohol dehydrogenase from Haloferax volcanii (HvADH2) was evaluated. The HvADH2 showed remarkable stability and catalysed the reaction in aqueous-organic medium containing dimethyl sulfoxide (DMSO) and methanol (MeOH). Tetrahydrofuran and acetonitrile were also investigated and adversely affected the stability of the enzyme. High concentration of salt, essential to maintain the enzymatic activity and structural integrity of the halophilic enzyme under standard conditions may be partially replaced by DMSO and MeOH. The presence of organic solvents did not induce gross changes in substrate specificity. DMSO offered a protective effect for the stability of the enzyme at nonoptimal pHs such as 6 and 10. Salt and solvent effects on the HvADH2 conformation and folding were examined through fluorescence spectroscopy. The fluorescence findings were consistent with the activity and stability results and corroborated the denaturing properties of some solvents. The intrinsic tolerance of this enzyme to organic solvent makes it highly attractive to industry.

A comparison of two novel alcohol dehydrogenase enzymes (ADH1 and ADH2) from the extreme halophile Haloferax volcanii.

Haloarchaeal alcohol dehydrogenases are exciting biocatalysts with potential industrial applications. In this study, two alcohol dehydrogenase enzymes from the extremely halophilic archaeon Haloferax volcanii (HvADH1 and HvADH2) were homologously expressed and subsequently purified by immobilized metal-affinity chromatography. The proteins appeared to copurify with endogenous alcohol dehydrogenases, and a double Δadh2 Δadh1 gene deletion strain was constructed to prevent this occurrence. Purified HvADH1 and HvADH2 were compared in terms of stability and enzymatic activity over a range of pH values, salt concentrations, and temperatures. Both enzymes were haloalkaliphilic and thermoactive for the oxidative reaction and catalyzed the reductive reaction at a slightly acidic pH. While the NAD(+)-dependent HvADH1 showed a preference for short-chain alcohols and was inherently unstable, HvADH2 exhibited dual cofactor specificity, accepted a broad range of substrates, and, with respect to HvADH1, was remarkably stable. Furthermore, HvADH2 exhibited tolerance to organic solvents. HvADH2 therefore displays much greater potential as an industrially useful biocatalyst than HvADH1.

Characterization of alcohol dehydrogenase (ADH12) from Haloarcula marismortui, an extreme halophile from the Dead Sea.

Haloarchaeal alcohol dehydrogenases are of increasing interest as biocatalysts in the field of white biotechnology. In this study, the gene adh12 from the extreme halophile Haloarcula marismortui (HmADH12), encoding a 384 residue protein, was cloned into two vectors: pRV1 and pTA963. The resulting constructs were used to transform host strains Haloferax volcanii (DS70) and (H1209), respectively. Overexpressed His-tagged recombinant HmADH12 was purified by immobilized metal-affinity chromatography (IMAC). The His-tagged protein was visualized by SDS-PAGE, with a subunit molecular mass of 41.6 kDa, and its identity was confirmed by mass spectrometry. Purified HmADH12 catalyzed the interconversion between alcohols and aldehydes and ketones, being optimally active in the presence of 2 M KCl. It was thermoactive, with maximum activity registered at 60°C. The NADP(H) dependent enzyme was haloalkaliphilic for the oxidative reaction with optimum activity at pH 10.0. It favored a slightly acidic pH of 6.0 for catalysis of the reductive reaction. HmADH12 was significantly more tolerant than mesophilic ADHs to selected organic solvents, making it a much more suitable biocatalyst for industrial application.

Stabilization of insulin using low molecular weight chitosan carbonate nanocarrier.

The current study aims to design a nanoparticulate system that could encapsulate insulin and improve its stability. Nanoparticles were formulated by ionic cross-linking of chitosan (CS) with carbonate divalent anions. The interaction between the two moieties was evidenced by AFM, FTIR and surface tension measurements. CS carbonate nanoparticles were prepared with different mole fractions. The mole fraction of carbonate that produced the smallest size nanoparticles and highest zeta potential (40 nm and +39 mV, respectively) was determined. Circular dichroism (CD) studies revealed that insulin conformation was not affected by CS at 20 °C. However, the studies at elevated temperatures demonstrated that CS had a role in insulin stabilization. Fluorescence spectroscopy indicated the interaction between insulin and CS carbonate. The findings from this investigation showed the potential use of CS carbonate as an insulin stabilizer and at the same time as an insulin nanocarrier system.

A promising antimicrobial bionanocomposite based poly(3-hydroxybutyrate-co-3-hydroxyvalerate) reinforced silver doped zinc oxide nanoparticles.

A bionanocomposite based on biosynthesized poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and reinforced with silver@zinc oxide (Ag-ZnO) was synthesized in variable loadings of Ag-ZnO using the in-situ casting dissolution technique. The degradable biopolymer PHBV had been biosynthesized from date waste as a renewable carbon source. The fabricated products were investigated as promising antibacterial materials. The Ag-ZnO nanoparticles were also synthesized using the green method in the presence of Gum Arabic. The Ag-ZnO nanoparticles were loaded within the PHBV biopolymer backbone at concentration of 1%, 3%, 5% and 10%, PHBV/Ag-ZnO(1,3,5,10%). The chemical structure, morphology, physical and thermal properties of the PHBV/Ag-ZnO bionanocomposites were assessed via common characterization tools of FTIR, TGA, XRD, SEM and EDX. One step of the degradation process was observed in the range of 200-220 °C for all the obtained materials. The onset degradation temperature of the bionanocomposites have been noticeably increased with increasing the nanofiller loading percentage. In addition, fabricated products were investigated for their interesting antibacterial performance. A detailed biological screening for the obtained products was confirmed against some selected Gram-positive and Gram-negative strains S. aureus and E. coli, respectively. Overall, the bionanocomposite PHBV/Ag-ZnO(10%) was the most potent against both types of the selected bacteria. The order of bacterial growth inhibition on the surface of the fabricated bionanocomposites was detected as follows: PHBV/Ag-ZnO(10%) > PHBV/Ag-ZnO(5%) > PHBV/Ag-ZnO(3%) > PHBV/Ag-ZnO(1%).

Biosynthesized poly(3-hydroxybutyrate-co-3-hydroxyvalerate) as biocompatible microcapsules with extended release for busulfan and montelukast.

An extended release dosage form based on encapsulating the challenging drug busulfan within microspheres of the biodegradable, biocompatible and biosynthesized poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) polyester was achieved. The used (PHBV) polymer was biosynthesized by the halophilic archaeon Haloferax mediterranei from date waste biomass as feed-stock. PHBV microspheres of 1.2-2.1 µm diameter were successfully fabricated and loaded with busulfan with an encapsulation efficiency of 29.2 ± 0.2%. In addition, PHBV microspheres of 1.5-3.5 µm diameter and loaded with montelukast sodium (MK) drug were also fabricated with an encapsulation efficiency of 16.0 ± 0.4%. The double-emulsion solvent evaporation method was used to fabricate the drug-loaded microspheres. The drug-loaded microspheres have been characterized by XRD, FTIR and SEM, and confirmed to be successfully fabricated. The drugs in vitro release profiles have shown extended release for up to 3 days in case of busulfan and 8 h in case of montelukast sodium. The in vitro release profiles for busulfan and montelukast suggest that these drug-loaded microcapsules can be efficiently used as new dosage forms to solve the current issues of busulfan administration protocols, and to introduce a new dosage form for montelukast with extended release performance.

Optimization of nitrogen source supply for enhanced biosynthesis and quality of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by extremely halophilic archaeon Haloferax mediterranei.

The extreme halophilic archaeon, Haloferax mediterranei can accumulate polyhydroxyalkanoate (PHA) from different renewable resources. To enhance the biosynthesis and quality of PHA, H. mediterranei cultivation media was optimized at different C/N ratios using glucose as the main carbon source. Three sets of media (yeast extract [YE], NH4 Cl and combination of YE and NH4 Cl) were prepared at different nitrogen concentrations to achieve C/N ratios of 9, 20, and 35, respectively. The media containing YE (organic nitrogen source) produced a higher growth rate of H. mediterranei than NH4 Cl (inorganic source) at all tested C/N ratios. The highest PHA accumulation (18.4% PHA/cell dry mass) was achieved in a media that combined YE with NH4 Cl at a C/N ratio of 20. Analysis of the produced polymers revealed the production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBHV) with different 3-hydroxyvalerate (3HV) content. The polymers produced from YE and the combined media have greater 3HV content (10 mol%) than those polymers recovered from NH4 Cl (1.5 mol%). Resultingly, PHBHV from YE and the combined media displayed reduced melting points at 144°C. The nitrogen type/concentration was found to also have an impact on the molecular weights and polydispersity indices of the produced biopolymers. Furthermore, the tensile strengths were found to vary with the best tensile strength (14.4 MPa) being recorded for the polymer recovered from YE at C/N = 9. Interestingly, the tensile strength of PHBHV was significantly higher than petroleum-based polyethylene (13.5 MPa), making it a much more suitable bioplastic for industrial application.

Utilizing the crop waste of date palm fruit to biosynthesize polyhydroxyalkanoate bioplastics with favorable properties.

Polyhydroxyalkanoate (PHA), a family of biodegradable and renewable biopolymers that could potentially play a significant role in bioeconomy. In this study we investigated the potential of date waste (DW) biomass as feedstock to produce PHA by the halophilic archaeon Haloferax mediterranei. The concentration of essential trace elements for H. mediterranei cells during growth and PHA biopolymer accumulation was optimized. A maximum cell dry mass of (CDM) (12.8 g L-1) and PHA concentration of (3.20 g L-1) were achieved in DW extract media that was not supplemented with trace elements, indicating that DW is a promising source for trace elements. The cultivation was scaled-up to fed-batch bioreactor fermentations under non-sterile conditions and resulted in CDM and PHA content of 18.0 g L-1 and 25%, respectively. The produced PHA was confirmed to be poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) with high 3-hydroxyvalerate (3 HV) content of 18.0 mol%. This 3 HV molar percent was achieved without the addition of expensive precursors. The PHBV is of high molecular weight (746.0 kDa) and narrow polydispersity (PDI = 1.5), and displayed reduced melting at 148.1 °C. The X-ray diffraction (XRD) analysis showed that the PHBV has amorphous nature which increases the degradation rates and workability of the biopolymer. The isotopic ratio 13C/12C (δ 13C) for PHBV was found to be - 19.1‰, which indicated that H. mediterranei prefers lighter bonds to break and uses the lighter atoms for the biosynthesis of PHBV.

Purification and biochemical characterization of photo-active membrane protein bacteriorhodopsin from Haloarcula marismortui, an extreme halophile from the Dead Sea.

Bacteriorhodopsin (BR) is an exciting photo-active retinal protein with many potential industrial applications. In this study, BR from the extremely halophilic archaeon Haloarcula marismortui (HmBR) was purified successfully using aqueous two phase extraction method. Absorption spectroscopy analysis showed maximum absorption peak of HmBR retinal protein (λmax) at 415 nm. The purified HmBR was visualized by SDS-PAGE, with a subunit molecular mass of 27 kDa, and its identity was confirmed by resonance Raman spectroscopy, Fourier transform infrared spectroscopy and atomic force microscopy. The effect of pH and salt concentration on the absorption spectrum of HmBR was evaluated. Red-shifted in λmax of HmBR was recorded at acidic condition (pH 5) and HmBR showed remarkable optical activity under high salinity condition. The photoelectric activity of HmBR was evaluated by measuring the DC-voltage generated from HmBR coated on indium tin oxide (ITO) glass when light illumination was applied.

A one-stage cultivation process for the production of poly-3-(hydroxybutyrate-co-hydroxyvalerate) from olive mill wastewater by Haloferax mediterranei.

Olive mill wastewater (OMW), a highly polluting waste from the olive oil industry, was utilized as sole carbon source for the production of polyhydroxyalkanoate (PHA) by extremely halophilic Haloferax Mediterranei (H. mediterranei) in a one stage cultivation step. H. mediterranei showed remarkable cell growth and tolerated the inhibitory effect of polyphenols present in medium containing 25% of OMW. H. mediterranei cultivation conditions were optimized in medium containing 15% OMW by investigating several parameters that affect the production of PHA. The highest polymer yield (0.2g/L) and PHA content (43% PHA/cell dry mass) were achieved at 37°C, 170rpm and 22% salt concentration. Analysis of the produced PHA revealed the production of copolyester poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBHV) containing 6.5mol% 3-hydroxyvalerate (3HV). The production of PHBHV was observed without the need for fermentation step or adding external carbon source. The PHBHV displayed reduced melting points at 140.1°C and 154.4°C when compared to homopolymer polyhydroxybutyrate.

Covalent immobilization of alcohol dehydrogenase (ADH2) from Haloferax volcanii: how to maximize activity and optimize performance of halophilic enzymes.

Alcohol dehydrogenase from halophilic archaeon Haloferax volcanii (HvADH2) was successfully covalently immobilized on metal-derivatized epoxy Sepabeads. The immobilization conditions were optimized by investigating several parameters that affect the halophilic enzyme-support interaction. The highest immobilization efficiency (100 %) and retention activity (60 %) were achieved after 48 h of incubation of the enzyme with Ni-epoxy Sepabeads support in 100 mM Tris-HCl buffer, pH 8, containing 3 M KCl at 5 °C. No significant stabilization was observed after blocking the unreacted epoxy groups with commonly used hydrophilic agents. A significant increase in the stability of the immobilized enzyme was achieved by blocking the unreacted epoxy groups with ethylamine. The immobilization process increased the enzyme stability, thermal activity, and organic solvents tolerance when compared to its soluble counterpart, indicating that the immobilization enhances the structural and conformational stability. One step purification-immobilization of this enzyme has been carried out on metal chelate-epoxy Sepabeads, as an efficient method to obtain immobilized biocatalyst directly from bacterial extracts.