| 1. |
- Lu, Xinnan, et al.
(författare)
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Ni2P Nanoparticles Embedded in Mesoporous SiO2 for Catalytic Hydrogenation of SO2 to Elemental S
- 2021
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Ingår i: ACS Applied Nano Materials. - : American Chemical Society (ACS). - 2574-0970. ; 4:6, s. 5665-5676
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Tidskriftsartikel (refereegranskat)abstract
- Highly active nickel phosphide (Ni2P) nanoclusters confined in a mesoporous SiO2 catalyst were synthesized by a two-step process targeting tight control over the Ni2P size and phase. The Ni precursor was incorporated into the MCM-41 matrix by one-pot synthesis, followed by the phosphorization step, which was accomplished in oleylamine with trioctylphosphine at 300 °C so to achieve the phase transformation from Ni to Ni2P. For benchmarking, Ni confined by the mesoporous SiO2 (absence of phosphorization) and 11 nm Ni2P nanoparticles (absence of SiO2) was also prepared. From the microstructural analysis, it was found that the growth of Ni2P nanoclusters was restricted by the mesoporous channels, thus forming ultrafine and highly dispersed Ni2P nanoclusters (<2 nm). The above approach led to promising catalytic performance following the order u-Ni2P@m-SiO2 > n-Ni2P > u-Ni@m-SiO2 > c-Ni2P in the selective hydrogenation of SO2 to S. In particular, u-Ni2P@m-SiO2 exhibited SO2 conversions of 94% at 220 °C and ∼99% at 240 °C, which are higher than the 11 nm stand-alone Ni2P particles (43% at 220 °C and 94% at 320 °C), highlighting the importance of the role played by SiO2 in stabilizing ultrafine nanoparticles of Ni2P. The reaction activation energy Ea over u-Ni2P@m-SiO2 is ∼33 kJ/mol, which is lower than those over n-Ni2P (∼36 kJ/mol) and c-Ni2P (∼66 kJ/mol), suggesting that the reaction becomes energetically favored over the ultrafine Ni2P nanoclusters.
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| 2. |
- Lu, Xinnan, et al.
(författare)
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Nickel Phosphide Nanoparticles for Selective Hydrogenation of SO2 to H2S
- 2021
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Ingår i: ACS Applied Nano Materials. - : American Chemical Society (ACS). - 2574-0970. ; 4:7, s. 6568-6582
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Tidskriftsartikel (refereegranskat)abstract
- Highly mesoporous SiO2-encapsulated NixPy crystals, where (x, y) = (5, 4), (2, 1), and (12, 5), were successfully synthesized by adopting a thermolytic method using oleylamine (OAm), trioctylphosphine (TOP), and trioctylphosphine oxide (TOPO). The Ni5P4@SiO2 system shows the highest reported activity for the selective hydrogenation of SO2 toward H2S at 320 degrees C (96% conversion of SO2 and 99% selectivity to H2S), which was superior to the activity of the commercial CoMoS@Al2O3 catalyst (64% conversion of SO2 and 71% selectivity to H2S at 320 degrees C). The morphology of the Ni5P4 crystal was finely tuned via adjustment of the synthesis parameters receiving a wide spectrum of morphologies (hollow, macroporous-network, and SiO2-confined ultrafine clusters). Intrinsic characteristics of the materials were studied by Xray diffraction, high-resolution transmission electron microscopy/scanning transmission electron microscopy-high-angle annular dark-field imaging, energydispersive X-ray spectroscopy, the Brunauer-Emmett-Teller method, H-2 temperature-programmed reduction, X-ray photoelectron spectroscopy, and experimental and calculated P-31 magic-angle spinning solid-state nuclear magnetic resonance toward establishing the structure-performance correlation for the reaction of interest. Characterization of the catalysts after the SO2 hydrogenation reaction proved the preservation of the morphology, crystallinity, and Ni/P ratio for all the catalysts.
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| 3. |
- Gaber, Yasser, et al.
(författare)
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An investigation of enzymatic kinetic resolution of racemic clopidogrel
- 2014
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Ingår i: Asian Journal of Microbiology, Biotechnology and Environmental Sciences. ; 16:2, s. 247-251
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Tidskriftsartikel (refereegranskat)abstract
- This paper presents a biocatalysis based approach for asymmetric hydrolysis of Clopidogrel, one of the most effective antiplatelet aggregators. Instead of the conventional "wet lab" screening method, we used an advanced chemical information retrieval tool, Scifinder®, to find suitable enzyme candidate(s) for the asymmetric hydrolysis. Scifinder® search for reactions similar to the target reaction retrieved three potential hits: horse liver esterase, chymotrypsin and Candida rugosa lipase. Among the three, horse liver esterase was experimentally found to hydrolyse the correct isomer (R) of the racemic clopidogrel. Investigation of the effect of different water miscible co-solvents showed DMSO to provide the highest enantiomeric excess (ee) value of 20% at 43% conversion of the racemic ester, while a dicyanamide based ionic liquid as a co-solvent showed 12% ee. This article demonstrates the potential of the chemical databases as a possible rapid, resource-efficient tool to find an appropriate selective biocatalyst.
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| 4. |
- Gaber, Yasser, et al.
(författare)
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Cross-linked enzyme aggregates of pig liver esterase evaluated in kinetic resolution of racemic clopidogrel
- 2017
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Ingår i: Biotechnology (Faisalabad). - : Science Alert. - 1682-296X. ; 16:3, s. 123-129
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Tidskriftsartikel (refereegranskat)abstract
- Background and Objective: Immobilization of enzymes as cross-linked aggregates is one of the cheapest, simplest and effective techniques for improving their stability and reusability and even avoiding contamination of the product with the catalyst. Clopidogrel is a widely used antiplatelet drug, only Sisomer is the biologically active enantiomer produced by resolution of the racemic compound. In the current study, cross-linked aggregates of pig liver esterase were prepared and evaluated for kinetic resolution of racemic clopidogrel. Materials and Methods: Cross-linked Enzyme Aggregates (CLEA) of the commercially available crude pig liver esterase cPLE were prepared using glutaraldehyde at concentrations of 12.5-125 mM as crosslinker either in presence or absence of Bovine serum albumin (BSA). cPLE-CLEA was used for kinetic resolution of racemic clopidogrel and compared to the performance of soluble cPLE. Light microscopy and scanning electron microscopy SEM were used to examine cPLE-CLEA. Results: Soluble cPLE showed ability to resolve racemic clopidogrel at enantioselectivity (E) of 9.2. The resolution of clopidogrel was found to be optimal at 30°C. The cPLE-CLEA preparations showed reduced enzymatic activity. The kinetic resolution experiments showed also lower E values (E = 1.3-4.5) compared to soluble cPLE. Microscopical examination of cPLE-CLEA showed wide size variation and SEM revealed the shape of cPLE-CLEA before and after use in the kinetic resolution experiments. Conclusion: Crude PLE was able to resolve racemic clopidogrel, the effects of different temperatures were studied and the highest E value recorded was 9.2 at 30°C. Increasing concentrations of glutaraldehyde as a cross-linker adversely affected PLE activity. The cPLE-CLEA showed lower enantioselectivity compared to the free cPLE.
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| 5. |
- Gaber, Yasser, et al.
(författare)
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Environmentally evaluated HPLC-ELSD method to monitor enzymatic synthesis of a non-ionic surfactant.
- 2014
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Ingår i: Chemistry Central Journal. - 1752-153X. ; 8
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Tidskriftsartikel (refereegranskat)abstract
- N-Lauroyl-N-methylglucamide is a biodegradable surfactant derived from renewable resources. In an earlier study, we presented an enzymatic solvent-free method for synthesis of this compound. In the present report, the HPLC method developed to follow the reaction between lauric acid/methyl laurate and N-methyl glucamine (MEG) and its environmental assessment are described.
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| 6. |
- Gaber, Yasser, et al.
(författare)
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Enzymatic synthesis of N-alkanoyl-N-methylglucamide surfactants: solvent-free production and environmental assessment
- 2010
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Ingår i: Green Chemistry. - : Royal Society of Chemistry (RSC). - 1463-9270 .- 1463-9262. ; 12:10, s. 1817-1825
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Tidskriftsartikel (refereegranskat)abstract
- A biocatalysis based method for the solvent-free production of N-alkanoyl-N-methylglucamide (MEGA) surfactants was developed and used as a case study for the evaluation of different environmental assessment tools, such as the freeware package EATOS (Environmental Assessment Tool for Organic Synthesis). In order to also consider energy usage and process facilities, e. g. heating, stirring and vacuum, a complementary tool was needed; hence the EcoScale method and the use of an energy monitoring socket were also exploited. The solvent-free method followed by a simple hydrolysis step gave a final amide yield of 99% and a product essentially free of remaining substrate, N-methylglucamine (MEG). The latter is important since MEG can potentially be converted to carcinogenic nitrosamines. The absence of solvent in the reaction medium was also found to result in a significantly reduced potential environmental impact. The environmental tools used in this study were further scrutinized, and even if they represent some of the best freely available tools for evaluation of early stage process development, some points for further improvements are suggested.
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| 7. |
- Gaber, Yasser, et al.
(författare)
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HPLC-EAT (Environmental Assessment Tool): A tool for profiling safety, health and environmental impacts of liquid chromatography methods
- 2011
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Ingår i: Green Chemistry. - : Royal Society of Chemistry (RSC). - 1463-9270 .- 1463-9262. ; 13:8, s. 2021-2025
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Tidskriftsartikel (refereegranskat)abstract
- A simple and efficient approach for profiling the greenness of high performance liquid chromatography (HPLC) methods is presented. This environmental assessment tool (EAT) takes into consideration the environmental, health and safety issues for all solvents involved in the chromatographic method, and calculates a total score that can be used for comparison of the greenness of different methods. A software, HPLC-EAT, has been designed to facilitate the calculation and can be downloaded free of charge at http://www.biotek.lu.se/hplc-eat/. HPLC-EAT was successfully applied for a set of different HPLC methods from the literature, including both analytical and preparative chromatography. The performance of the tool was validated and it was further combined with another free software Eco-solvent tool to perform life cycle assessments of waste disposal options of distillation or incineration. HPLC-EAT can be routinely used in method development to calculate the greenness beside the conventional standards of accuracy, robustness and reproducibility.
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| 8. |
- Gaber, Yasser
(författare)
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Hydrolases as Catalysts for Green Chemistry and Industrial Applications - Esterase, Lipase and Phytase
- 2012
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The use of enzymes in industrial applications has been recognised for providing clean processes with minimal impact on the environment. This thesis presents studies on engineering of enzymes and enzyme-based processes in the light of green chemistry and environmental sustainability, and focuses on three hydrolases: esterase, lipase and phytase. The use of esterase has been investigated to provide an alternative clean route for the synthesis of a chiral pharmaceutical compound, S-clopidogrel, by selective hydrolysis of the racemic precursor. Current production of the pure S- clopidogrel isomer involves the use of a resolving agent, L-camphorsulfonic acid, and organic solvents. Screening of different hydrolases revealed that crude pig liver esterase (PLE), a mixture of different isoenzymes, selectively acts on the R isomer, with E = 8.3. Two PLE isoenzymes, PLE-1 and PLE-3 were tested individually as catalysts for the reaction. Molecular modeling simulations indicated that a phenylalanine F407 residue is destabilizing for the R-isomer- PLE-1 tetrahedral intermediate. PLE-1 and its mutants were expressed in Escherichia coli with a chaperon system. Interestingly, mutations of F407 to alanine or leucine led to a dramatic increase in activity but with reversed selectivity (E=3.3 and E>100 respectively towards the S isomer). On the other hand, PLE-3 isoenzyme selectively hydrolysed the correct isomer, R, with E=10. Immobilized lipase B from Candida antarctica, was employed for the production of biodegradable specialty chemicals from renewable resources. N-alkanoyl-Nmethylglucamide, a bio-based surfactant, was produced in a solvent-free reaction. Engineering the molar ratio of the substrates in the reaction and adding a step involving hydrolysis of the by-product resulted in final yield of 99 %. Comparison with earlier reports based on green metrics showed the method to have a greener profile. Another product, trimethylolpropane-oleate, a biolubricant, was produced by lipase-catalysed esterification with high yield, better product quality and a greener profile compared to the process catalysed by other heterogeneous chemical catalysts. Reliable metrics of the greenness of a process are essential for the progress of green chemistry. As an important contribution in this area, a java-based software HPLC-EAT was developed to evaluate the greenness of liquid chromatographic methods. The tool is freely available at www.biotek.lu.se/hplc-eat. The enzyme phytase hydrolyses phytate (myo-inositol 1,2,3,4,5,6-hexakisphosphate), an anti-nutrient compound present in cereals and grains, and increases the bioavailability of phosphorus and other nutrients. A recombinant thermostable Bacillus sp. MD2 alkaline phytase, a metallo-enzyme, was characterized and the effect of various divalent metal ions on its stability and catalytic properties was studied. The presence of calcium ions on both the enzyme and the substrate was required for optimal activity and stability of the enzyme. Furthermore, site-directed mutagenesis of the enzyme was done to improve its activity and stability in the acidic environment. Mutation of a glutamate residue in the enzyme active site to serine E227S led to a slight decrease of the optimum pH and higher stability at low pH.
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| 9. |
- Gaber, Yasser, et al.
(författare)
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Rational mutagenesis of pig liver esterase (PLE-1) to resolve racemic clopidogrel
- 2015
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Ingår i: Journal of Molecular Catalysis B: Enzymatic. - : Elsevier BV. - 1873-3158 .- 1381-1177. ; 122, s. 156-162
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Tidskriftsartikel (refereegranskat)abstract
- Clopidogrel is an important and widely used antiplatelet drug; only the (S)-isomer is the active biological enantiomer. The industrial production of the active enantiomer involves resolution of the racemic compound using stoichiometric amounts of L-camphor sulphonic acid and crystallization in a process using large amounts of solvent. In the present study, the possibility of enzyme catalysed kinetic resolution was investigated. Screening of a number of hydrolytic enzymes showed the crude pig liver esterase (PLE) to exhibit modest enantioselectivity in the hydrolysis of racemic clopidogrel. Molecular modeling simulations were applied on the homology model of PLE-1, the major isoenzyme in the crude PLE, and distinct sites of mutations were proposed and 'produced in the laboratory. The PLE-1 variants with one mutation i.e. PLE-1-F407L and PLE-1-F407I resolved the racemic substrate yielding the (R)-isomer with E value >100. Contrarily, the isoenzyme PLE-3 resolved the racemic substrate yielding (S)-clopidogrel with E value of 10. (C) 2015 Elsevier B.V. All rights reserved.
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| 10. |
- Orellana Coca Åkerman, Cecilia, et al.
(författare)
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Clean synthesis of biolubricants for low temperature applications using heterogeneous catalysts
- 2011
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Ingår i: Journal of Molecular Catalysis B: Enzymatic. - : Elsevier BV. - 1873-3158 .- 1381-1177. ; 72:3-4, s. 263-269
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Tidskriftsartikel (refereegranskat)abstract
- Biolubricants derived from vegetable oils are environmentally compatible products due to their low toxicity and good biodegradability. Synthetic esters based on polyols and fatty acids possess suitable properties for lubricant applications, even at extreme temperatures. In this work, synthesis of esters from trimethylolpropane (TMP) and carboxylic acids from C5 to C18 has been studied and compared using different heterogeneous catalysts (silica-sulphuric acid, Amberlyst-15, and immobilised lipase B from Candida antarctica). Silica-sulphuric acid was found to be the most efficient catalyst followed by Amberlyst-15, especially when using short chain carboxylic acids. The reaction efficiency decreased with increasing alkyl chain length. On the other hand, the immobilised lipase (Novozym (R) 435) did not exhibit any activity with C5 acid and the activity increased with increase in length of the fatty acid chain. For synthesis of C18-ester, the biocatalytic production turned out to be comparable to silica-sulphuric acid, and moreover led to a better quality of the final product. The products showed suitable cold-flow properties for application at low temperature. A general trend of increasing pour point (-75 degrees C to -42 degrees C) and viscosity index (80-208) with increase in alkyl chain of the carboxylic acid from C5 to C18 was observed. The synthesis of TMP-trioleate using the solid acid catalysts and the biocatalyst was compared using the freeware package EATOS (environmental assessment tool for organic synthesis) and showed the enzymatic route to have the least environmental impact. (C) 2011 Elsevier B.V. All rights reserved.
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| 11. |
- Salama, Sara, et al.
(författare)
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Reviewing a plethora of oxidative-type reactions catalyzed by whole cells of Streptomyces species
- 2022
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Ingår i: RSC Advances. - : Royal Society of Chemistry (RSC). - 2046-2069. ; 12:12, s. 6974-7001
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Forskningsöversikt (refereegranskat)abstract
- Selective oxidation reactions represent a challenging task for conventional organic chemistry. Whole-cell biocatalysis provides a very convenient, easy to apply method to carry out different selective oxidation reactions including chemo-, regio-, and enantio-selective reactions. Streptomyces species are important biocatalysts as they can catalyze these selective reactions very efficiently owing to the wide diversity of enzymes and enzymatic cascades in their cell niche. In this review, we present and analyze most of the examples reported to date of oxidative reactions catalyzed by Streptomyces species as whole-cell biocatalysts. We discuss 33 different Streptomyces species and strains and the role they play in different oxidative reactions over the past five decades. The oxidative reactions have been classified into seven categories that include: hydroxylation of steroids/non-steroids, asymmetric sulfoxidations, oxidation of aldehydes, multi-step oxidations, oxidative cleavage, and N-oxidations. The role played by Streptomyces species as recombinant hosts catalyzing bio-oxidations has also been highlighted.
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| 12. |
- Sayed, Mahmoud, et al.
(författare)
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Multi-steps green process for synthesis of six-membered functional cyclic carbonate from trimethylolpropane by lipase catalyzed methacrylation and carbonation, and thermal cyclization.
- 2015
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Ingår i: Biotechnology Progress. - : Wiley. - 1520-6033 .- 8756-7938.
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Tidskriftsartikel (refereegranskat)abstract
- A highly functionalized six-membered cyclic carbonate, methacrylated trimethylolpropane (TMP) cyclic carbonate, which can be used as a potential monomer for bisphenol-free polycarbonates and isocyanate-free polyurethanes, was synthesized by two steps transesterifications catalyzed by immobilized Candida antarctica lipase B, Novozym®435 (N435) followed by thermal cyclization. TMP was functionalized as 70-80% selectivity of mono-methacrylate with 70% conversion was achieved, and the reaction rate was evaluated using various acyl donors such as methacrylic acid, methacrylate-methyl ester, -ethyl ester and -vinyl ester. As a new observation, the fastest rate obtained was for the transesterfication reaction using methacrylate methyl ester. By-products resulted from leaving groups were adsorbed on the molecular sieves (4Å) to minimize the effect of leaving group on the equilibrium. The difference of reaction rate was explained by molecular dynamic simulations on interactions between carbonyl oxygen and amino acid residues (Thr(40) and Gln(157) ) in the active site of lipase. Our docking studies revealed that as acyl donor, methyl ester was preferred for the initial conformation of the 1(st) tetrahederal intermediate with hydrogen bonding interactions. TMP-monomethacrylate (TMP-mMA) cyclic carbonate was obtained in 63% yield (74.1% calculated in 85% conversion) from the lipase-catalyzed carbonation reaction of TMP-mMA with dimethylcarbonate, and followed by thermal cyclization of the monocarbonate at 90°C. From the multiple reactions demonstrated in gram scale, TMP-mMA cyclic carbonate was obtained as a green process without using chlorinated solvent and reagent. This article is protected by copyright. All rights reserved.
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| 13. |
- Sayed, Mahmoud, et al.
(författare)
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Oxidation of 5-hydroxymethylfurfural with a novel aryl alcohol oxidase from Mycobacterium sp. MS1601
- 2022
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Ingår i: Microbial Biotechnology. - : Wiley. - 1751-7907 .- 1751-7915. ; 15:8, s. 2176-2190
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Tidskriftsartikel (refereegranskat)abstract
- Bio-based 5-hydroxymethylfurfural (HMF) serves as an important platform for several chemicals, among which 2,5-furan dicarboxylic acid (FDCA) has attracted considerable interest as a monomer for the production of polyethylene furanoate (PEF), a potential alternative for fossil-based polyethylene terephthalate (PET). This study is based on the HMF oxidizing activity shown by Mycobacterium sp. MS 1601 cells and investigation of the enzyme catalysing the oxidation. The Mycobacterium whole cells oxidized the HMF to FDCA (60% yield) and hydroxymethyl furan carboxylic acid (HMFCA). A gene encoding a novel bacterial aryl alcohol oxidase, hereinafter MycspAAO, was identified in the genome and was cloned and expressed in Escherichia coli Bl21 (DE3). The purified MycspAAO displayed activity against several alcohols and aldehydes; 3,5 dimethoxy benzyl alcohol (veratryl alcohol) was the best substrate among those tested followed by HMF. 5-Hydroxymethylfurfural was converted to 5-formyl-2-furoic acid (FFCA) via diformyl furan (DFF) with optimal activity at pH 8 and 30–40°C. FDCA formation was observed during long reaction time with low HMF concentration. Mutagenesis of several amino acids shaping the active site and evaluation of the variants showed Y444F to have around 3-fold higher kcat/Km and ~1.7-fold lower Km with HMF.
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| 14. |
- Thi Thuy, Tran, et al.
(författare)
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Site-directed mutagenesis of an alkaline phytase: Influencing specificity, activity and stability in acidic milieu
- 2011
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Ingår i: Enzyme and Microbial Technology. - : Elsevier BV. - 0141-0229. ; 49:2, s. 177-182
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Tidskriftsartikel (refereegranskat)abstract
- Site-directed mutagenesis of a thermostable alkaline phytase from Bacillus sp. MD2 was performed with an aim to increase its specific activity and activity and stability in an acidic environment. The mutation sites are distributed on the catalytic surface of the enzyme (P257R, E180N, E229V and S283R) and in the active site (K77R, K179R and E227S). Selection of the residues was based on the idea that acid active phytases are more positively charged around their catalytic surfaces. Thus, a decrease in the content of negatively charged residues or an increase in the positive charges in the catalytic region of an alkaline phytase was assumed to influence the enzyme activity and stability at low pH. Moreover, widening of the substrate-binding pocket is expected to improve the hydrolysis of substrates that are not efficiently hydrolysed by wild type alkaline phytase. Analysis of the phytase variants revealed that E229V and S283R mutants increased the specific activity by about 19% and 13%, respectively. Mutation of the active site residues K77R and K179R led to severe reduction in the specific activity of the enzyme. Analysis of the phytase mutant-phytate complexes revealed increase in hydrogen bonding between the enzyme and the substrate, which might retard the release of the product, resulting in decreased activity. On the other hand, the double mutant (K77R-K179R) phytase showed higher stability at low pH (pH 2.6-3.0). The E227S variant was optimally active at pH 5.5 (in contrast to the wild type enzyme that had an optimum pH of 6) and it exhibited higher stability in acidic condition. This mutant phytase, displayed over 80% of its initial activity after 3 h incubation at pH 2.6 while the wild type phytase retained only about 40% of its original activity. Moreover, the relative activity of this mutant phytase on calcium phytate, sodium pyrophosphate and p-nitro phenyl phosphate was higher than that of the wild type phytase. (C) 2011 Elsevier Inc. All rights reserved.
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| 15. |
- Thi Thuy, Tran, et al.
(författare)
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Thermostable alkaline phytase from Bacillus sp. MD2: Effect of divalent metals on activity and stability.
- 2011
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Ingår i: Journal of Inorganic Biochemistry. - : Elsevier BV. - 1873-3344 .- 0162-0134. ; 105:7, s. 1000-1007
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Tidskriftsartikel (refereegranskat)abstract
- Phytate, the major source of phosphorus in seeds, exists as a complex with different metal ions. Alkaline phytases are known to dephosphorylate phytate complexed with calcium ions in contrast to acid phytases that act only on phytic acid. A recombinant alkaline phytase from Bacillus sp. MD2 has been purified and characterized with respect to the effect of divalent metal ions on the enzyme activity and stability. The presence of Ca(2+) on both the enzyme and the substrate is required for optimal activity and stability. Replacing Ca(2+) with Ba(2+), Mn(2+), Mg(2+) and Sr(2+) in the phytase resulted in the expression of >90% of the maximal activity with calcium-phytate as the substrate, while Fe(2+) and Zn(2+) rendered the enzyme inactive. On the other hand, the calcium loaded phytase showed significant activity (60%) with sodium phytate and lower activity (17-20%) with phytate complexed with only Mg(2+), Sn(2+) and Sr(2+), respectively. On replacing Ca(2+) on both the enzyme and the substrate with other metal ions, about 20% of the maximal phytase activity was obtained only with Mg(2+) and Sr(2+), respectively. Only Ca(2+) resulted in a marked increase in the melting temperature (T(m)) of the enzyme by 12-21°C, while Ba(2+), Mn(2+), Sr(2+) or Cu(2+) resulted in a modest (2-3.5°C) increase in T(m). In the presence of 1-5mM Ca(2+), the optimum temperature of the phytase activity was increased from 40°C to 70°C, while optimum pH of the enzyme shifted by 0.4-1 pH unit towards the acidic region.
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