| 1. |
- Abouhmad, Adel, et al.
(författare)
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Exploring the enzymatic and antibacterial activities of novel mycobacteriophage lysin b enzymes
- 2020
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Ingår i: International Journal of Molecular Sciences. - : MDPI AG. - 1661-6596 .- 1422-0067. ; 21:9
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Tidskriftsartikel (refereegranskat)abstract
- Mycobacteriophages possess different sets of lytic enzymes for disruption of the complex cell envelope of the mycobacteria host cells and release of the viral progeny. Lysin B (LysB) enzymes are mycolylarabinogalactan esterases that cleave the ester bond between the arabinogalactan and mycolic acids in the mycolylarabinogalactan-peptidoglycan (mAGP) complex in the cell envelope of mycobacteria. In the present study, four LysB enzymes were produced recombinantly and characterized with respect to their enzymatic and antibacterial activities. Examination of the kinetic parameters for the hydrolysis of para-nitrophenyl ester substrates, shows LysB-His6 enzymes to be active against a range of substrates (C4-C16), with a catalytic preference towards p-nitrophenyl laurate (C12). With p-nitrophenyl butyrate as substrate, LysB-His6 enzymes showed highest activity at 37◦C. LysB-His6 enzymes also hydrolyzed different Tween substrates with highest activity against Tween 20 and 80. Metal ions like Ca2+ and Mn2+ enhanced the enzymatic activity of LysB-His6 enzymes, while transition metal ions like Zn2+ and Cu2+ inhibited the enzymatic activity. The mycolylarabinogalactan esterase activity of LysB-His6 enzymes against mAGP complex was confirmed by LC-MS. LysB-His6 enzymes showed marginal antibacterial activity when tested alone against Mycobacterium smegmatis, however a synergetic activity was noticed when combined with outer membrane permealizers. These results confirm that LysB enzymes are lipolytic enzymes with potential application as antimycobacterials.
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| 2. |
- Abouhmad, Adel, et al.
(författare)
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Immobilization to Positively Charged Cellulose Nanocrystals Enhances the Antibacterial Activity and Stability of Hen Egg White and T4 Lysozyme
- 2017
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Ingår i: Biomacromolecules. - : American Chemical Society (ACS). - 1525-7797 .- 1526-4602. ; 18:5, s. 1600-1608
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Tidskriftsartikel (refereegranskat)abstract
- Antibacterial bionanostructures were produced from cellulose nanocrystals (CNC) with immobilized lysozyme from hen egg white (HEW) and T4 bacteriophage, respectively. The nanocrystals were prepared from microcrystalline cellulose by ammonium persulfate oxidation with a yield of 68% and having an average size of 250 nm and low polydispersity index. HEW lysozyme (HEWL) and T4 lysozyme (T4L) were immobilized to CNC by different mechanisms including adsorption and covalent coupling to carbodiimide-activated carboxylate groups and to glutaraldehyde-activated aminated CNC (Am-CNC), respectively. The effect of immobilization on the enzymatic activity (both lytic and hydrolytic) and antibacterial activity of the lysozymes was studied using different methods. Am-CNC-lysozyme conjugates retained the highest lytic activity, 86.3% and 78.3% for HEWL and T4L, respectively. They also showed enhanced bactericidal activity with high potency against Gram-positive as well as Gram-negative bacteria in a relatively shorter time as compared to the free enzymes and resulted in extensive cellular damage, as shown by transmission electron microscopy. The enhanced antibacterial activity was correlated with the increase in zeta potential of Am-CNC-lysozyme conjugates. The immobilized lysozyme preparations further exhibited enhanced storage stability at 4 and 22 °C.
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| 3. |
- Alkhalili, Rawana N., et al.
(författare)
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Antimicrobial protein candidates from the thermophilic Geobacillus sp. Strain ZGt-1 : Production, proteomics, and bioinformatics analysis
- 2016
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Ingår i: International Journal of Molecular Sciences. - : MDPI AG. - 1661-6596 .- 1422-0067. ; 17:8
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Tidskriftsartikel (refereegranskat)abstract
- A thermophilic bacterial strain, Geobacillus sp. ZGt-1, isolated from Zara hot spring in Jordan, was capable of inhibiting the growth of the thermophilic G. stearothermophilus and the mesophilic Bacillus subtilis and Salmonella typhimurium on a solid cultivation medium. Antibacterial activity was not observed when ZGt-1 was cultivated in a liquid medium; however, immobilization of the cells in agar beads that were subjected to sequential batch cultivation in the liquid medium at 60 °C showed increasing antibacterial activity up to 14 cycles. The antibacterial activity was lost on protease treatment of the culture supernatant. Concentration of the protein fraction by ammonium sulphate precipitation followed by denaturing polyacrylamide gel electrophoresis separation and analysis of the gel for antibacterial activity against G. stearothermophilus showed a distinct inhibition zone in 15-20 kDa range, suggesting that the active molecule(s) are resistant to denaturation by SDS. Mass spectrometric analysis of the protein bands around the active region resulted in identification of 22 proteins with molecular weight in the range of interest, three of which were new and are here proposed as potential antimicrobial protein candidates by in silico analysis of their amino acid sequences. Mass spectrometric analysis also indicated the presence of partial sequences of antimicrobial enzymes, amidase, and DD-carboxypeptidase.
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| 4. |
- Cavero-Olguin, Victor Hugo, et al.
(författare)
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Membrane-based continuous fermentation with cell recycling for propionic acid production from glycerol by Acidipropionibacterium acidipropionici
- 2023
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Ingår i: Microbial Cell Factories. - : Springer Science and Business Media LLC. - 1475-2859. ; 22
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Tidskriftsartikel (refereegranskat)abstract
- Background: Microbial production of propionic acid (PA) from renewable resources is limited by the slow growth of the producer bacteria and product-mediated inhibition. The present study evaluates high cell density continuous PA fermentation from glycerol (Gly) using Acidipropionibacterium acidipropionici DSM 4900 in a membrane-based cell recycling system. A ceramic tubular membrane filter of 0.22 μm pore size was used as the filtering device for cell recycling. The continuous fermentations were run sequentially at dilution rates of 0.05 and 0.025 1/h using varying glycerol concentrations and two different yeast extract concentrations.Results: PA volumetric productivity of 0.98 g/L.h with a product yield of 0.38 gPA/gGly was obtained with 51.40 g/L glycerol at a yeast extract concentration of 10 g/L. Increasing the glycerol and yeast extract concentrations to 64.50 g/L and 20 g/L, respectively, increased in PA productivity, product yield, and concentration to 1.82 g/L.h, 0.79 gPA/gGly, and 38.37 g/L, respectively. However, lowering the dilution rate to 0.025 1/h reduced the production efficiency. The cell density increased from 5.80 to 91.83 gCDW/L throughout the operation, which lasted for a period of 5 months. A tolerant variant of A. acidipropoinici exhibiting growth at a PA concentration of 20 g/L was isolated at the end of the experiment.Conclusions: Applying the current approach for PA fermentation can overcome several limitations for process industrialization.
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| 5. |
- Cavero-Olguin, Victor Hugo, et al.
(författare)
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Propionic acid production from glycerol in immobilized cell bioreactor using an acid-tolerant strain of Propionibacterium acidipropionici obtained by adaptive evolution
- 2021
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Ingår i: Process Biochemistry. - : Elsevier BV. - 1359-5113. ; 110, s. 223-230
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Tidskriftsartikel (refereegranskat)abstract
- Propionic acid (PA) production from agro-industrial residues using propionibacteria has gained interest as an alternative to fossil-based process. Microbial production is however characterized by product inhibition, lowering the product titers and productivity. In this study, Propionibacterium acidipropionici DSMZ4900 was subjected to adaptive evolution to tolerate higher acid concentrations. The strain adapted to growth in medium spiked with 20 g/L PA exhibited improved product titer (16.8 vs 8.72 g/L) and productivity (0.52 vs 0.17 g/L·h) with glycerol as carbon source in batch fermentations. It was immobilized on polyethyleneimine coated recycled glass beads Poraver® and used for fermentations in recycle batch mode with increasing glycerol concentration and decreasing pH, respectively. Doubling yeast extract concentration raised PA yield and productivity by >1.5 fold. Glycerol at 100 g/L was completely consumed to give ∼58 g/L PA at yield of 0.64 mol/mol and productivity of 0.28 g/L·h at pH 6.5. Decreasing fermentation pH to 5.0 increased PA productivity to 0.23 g/L·h from 0.14 g/L·h at pH 6.0 with 20 g/L glycerol, while immobilized cells exhibited no growth. The study shows combination of adaptive evolution and immobilization of cells to result in a robust system for PA fermentation at high glycerol concentration and lower pH.
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| 6. |
- Dishisha, Tarek
(creator_code:cre_t)
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3-hydroxypropionaldehyde detection and extraction
- 2018
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Patent (övrigt vetenskapligt/konstnärligt)abstract
- The present invention relates to a method of extg. 3-hydroxypropionaldehyde (3-HPA) and/or derivs. thereof from an aq. soln. comprising 3-HPA. This method comprises (a) contacting the aq. soln. comprising 3-HPA with chitosan and/or chitosan comprising polymers, (b) sepg. the 3-HPA bound chitosan and/or chitosan comprising polymers, and (c) washing the 3-HPA bound chitosan and/or chitosan comprising polymers at least once with a washing medium (wherein 3-HPA and/or derivs. thereof is in the washing medium).
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| 7. |
- Dishisha, Tarek, et al.
(författare)
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An economical biorefinery process for propionic acid production from glycerol and potato juice using high cell density fermentation.
- 2013
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Ingår i: Bioresource Technology. - : Elsevier BV. - 1873-2976 .- 0960-8524. ; 135, s. 504-512
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Tidskriftsartikel (refereegranskat)abstract
- An economically sustainable process was developed for propionic acid production by fermentation of glycerol using Propionibacterium acidipropionici and potato juice, a by-product of starch processing, as a nitrogen/vitamin source. The fermentation was done as high-cell-density sequential batches with cell recycle. Propionic acid production and glycerol consumption rates were dependent on initial biomass concentration, and reached a maximum of 1.42 and 2.30gL(-1)h(-1), respectively, from 50gL(-1) glycerol at initial cell density of 23.7g(CDW)L(-1). Halving the concentration of nitrogen/vitamin source resulted in reduction of acetic and succinic acids yields by ∼39% each. At glycerol concentrations of 85 and 120gL(-1), respectively, 43.8 and 50.8gL(-1) propionic acid were obtained at a rate of 0.88 and 0.29gL(-1)h(-1) and yield of 84 and 78mol%. Succinic acid was 13g% of propionic acid and could represent a potential co-product covering the cost of nitrogen/vitamin source.
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| 8. |
- Dishisha, Tarek, et al.
(författare)
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Batch- and continuous propionic acid production from glycerol using free and immobilized cells of Propionibacterium acidipropionici
- 2012
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Ingår i: Bioresource Technology. - : Elsevier BV. - 1873-2976 .- 0960-8524. ; 118C, s. 553-562
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Tidskriftsartikel (refereegranskat)abstract
- Propionicacid production from glycerol was studied using Propionibacterium acidipropionici DSM 4900 cells immobilized on polyethylenimine-treated Poraver (PEI-Poraver) and Luffa (PEI-Luffa), respectively. Using PEI-Luffa, the average productivity, yield and concentration of propionicacid from 40 g.L-1 glycerol were 0.29 g.L-1.h-1, 0.74 mol.mol-1 and 20.09 g.L-1, respectively, after four consecutive recycle-batches. PEI-Poraver supported attachment of 31 times higher amount of cells than PEI-Luffa and produced 20, 28 and 35 g.L-1propionicacid from 40, 65 and 85 g.L-1 glycerol, respectively (0.61 molPA.molGly-1). The corresponding production rates were 0.86, 0.43 and 0.35 g.L-1.h-1, which are the highest reported from glycerol via batch or fed-batch fermentations for equivalent propionicacid concentrations. Using a continuous mode of operation at a dilution rate of 0.1 h-1, cell washout was observed in the bioreactor with free cells; however, propionicacid productivity, yield and concentration were 1.4 g.L-1.h-1, 0.86 molPA.molGly-1, and 14.5 g.L-1, respectively, using immobilized cells in the PEI-Poraver bioreactor. The choice of the immobilization matrix can thus significantly influence the fermentation efficiency and -profile. The bioreactor using cells immobilized on PEI-Poraver allowed the fermentation of higher glycerol concentrations and provided stable and higher fermentation rates than that using free cells or the cells immobilized on PEI-Luffa.
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| 9. |
- Dishisha, Tarek, et al.
(författare)
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Bio-based 3-hydroxypropionic- and acrylic acid production from biodiesel glycerol via integrated microbial and chemical catalysis
- 2015
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Ingår i: Microbial Cell Factories. - : Springer Science and Business Media LLC. - 1475-2859. ; 14
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Tidskriftsartikel (refereegranskat)abstract
- Background 3-Hydroxypropionic acid (3HP) and acrylic acid (AA) are industrially important platform- and secondary chemical, respectively. Their production from renewable resources by environment-friendly processes is desirable. In the present study, both chemicals were almost quantitatively produced from biodiesel-derived glycerol by an integrated process involving microbial and chemical catalysis. Results Glycerol was initially converted in a fed-batch mode of operation to equimolar quantities of 3HP and 1,3-propanediol (1,3PDO) under anaerobic conditions using resting cells of Lactobacillus reuteri as a biocatalyst. The feeding rate of glycerol was controlled at 62.5 mg/gCDW.h which is half the maximum metabolic flux of glycerol to 3HP and 1,3PDO through the L. reuteri propanediol-utilization (pdu) pathway to prevent accumulation of the inhibitory intermediate, 3-hydroxypronionaldehyde (3HPA). Subsequently, the cell-free supernatant containing the mixture of 3HP and 1,3PDO was subjected to selective oxidation under aerobic conditions using resting cells of Gluconobacter oxydans where 1,3PDO was quantitatively converted to 3HP in a batch system. The optimum conditions for the bioconversion were 10 g/L substrate and 5.2 g/L cell dry weight. Higher substrate concentrations led to enzyme inhibition and incomplete conversion. The resulting solution of 3HP was dehydrated to AA over titanium dioxide (TiO2) at 230 °C with a yield of >95 %. Conclusions The present study represents the first report on an integrated process for production of acrylic acid at high purity and -yield from glycerol through 3HP as intermediate without any purification step. The proposed process could have potential for industrial production of 3HP and AA after further optimization.
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| 10. |
- Dishisha, Tarek, et al.
(författare)
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Flux analysis of the Lactobacillus reuteri propanediol-utilization pathway for production of 3-hydroxypropionaldehyde, 3-hydroxypropionic acid and 1,3-propanediol from glycerol
- 2014
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Ingår i: Microbial Cell Factories. - : Springer Science and Business Media LLC. - 1475-2859. ; 13
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Tidskriftsartikel (refereegranskat)abstract
- Background Lactobacillus reuteri converts glycerol to 3-hydroxypropionic acid (3HP) and 1,3-propanediol (1,3PDO) via 3-hydroxypropionaldehyde (3HPA) as an intermediate using enzymes encoded in its propanediol-utilization (pdu) operon. Since 3HP, 1,3PDO and 3HPA are important building blocks for the bio-based chemical industry, L. reuteri can be an attractive candidate for their production. However, little is known about the kinetics of glycerol utilization in the Pdu pathway in L. reuteri. In this study, the metabolic fluxes through the Pdu pathway were determined as a first step towards optimizing the production of 3HPA, and co-production of 3HP and 1,3PDO from glycerol. Resting cells of wild-type (DSM 20016) and recombinant (RPRB3007, with overexpressed pdu operon) strains were used as biocatalysts. Results The conversion rate of glycerol to 3HPA by the resting cells of L. reuteri was evaluated by in situ complexation of the aldehyde with carbohydrazide to avoid the aldehyde-mediated inactivation of glycerol dehydratase. Under operational conditions, the specific 3HPA production rate of the RPRB3007 strain was 1.9 times higher than that of the wild-type strain (1718.2 versus 889.0 mg/gCDW.h, respectively). Flux analysis of glycerol conversion to 1,3PDO and 3HP in the cells using multi-step variable-volume fed-batch operation showed that the maximum specific production rates of 3HP and 1,3PDO were 110.8 and 93.7 mg/gCDW.h, respectively, for the wild-type strain, and 179.2 and 151.4 mg/gCDW.h, respectively, for the RPRB3007 strain. The cumulative molar yield of the two compounds was ~1 mol/mol glycerol and their molar ratio was ~1 mol3HP/mol1,3PDO. A balance of redox equivalents between the glycerol oxidative and reductive pathway branches led to equimolar amounts of the two products. Conclusions Metabolic flux analysis was a useful approach for finding conditions for maximal conversion of glycerol to 3HPA, 3HP and 1,3PDO. Improved specific production rates were obtained with resting cells of the engineered RPRB3007 strain, highlighting the potential of metabolic engineering to render an industrially sound strain. This is the first report on the production of 3HP and 1,3PDO as sole products using the wild-type or mutant L. reuteri strains, and has laid ground for further work on improving the productivity of the biotransformation process using resting cells.
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