| 1. |
- Ara, Kazi Zubaida Gulshan, et al.
(författare)
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Engineering CGTase to improve synthesis of alkyl glycosides
- 2021
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Ingår i: Glycobiology. - : Oxford University Press (OUP). - 1460-2423. ; 31:5, s. 603-612
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Tidskriftsartikel (refereegranskat)abstract
- Alkyl glycoside surfactants with elongated carbohydrate chains are useful in different applications due to their improved biocompatibility. Cyclodextrin glucanotransferases can catalyse the elongation process through the coupling reaction. However, due to the presence of a hydrophobic tail, the interaction between an alkyl glycoside acceptor and the active site residues is weaker than the interaction with maltooligosaccharides at the corresponding site. Here we report the mutations of F197, G263 and E266 near the acceptor subsites in the CGTase CspCGT13 from Carboxydocella sp. The results showed that substitutions of both F197 and G263 were important for the binding of acceptor substrate dodecyl maltoside during coupling reaction. The double mutant F197Y/G263A showed enhanced coupling activity and displayed a 2-fold increase of the primary coupling product using γ-cyclodextrin as donor when compared to wildtype CspCGT13. Disproportionation activity was also reduced, which was also the case for another double mutant (F197Y/E266A) that however not showed the corresponding increase in coupling. A triple mutant F197Y/G263A/E266A maintained the increase in primary coupling product (1.8-fold increase) using dodecyl maltoside as acceptor, but disproportionation was approximately at the same level as in the double mutants. In addition, hydrolysis of starch was slightly increased by the F197Y and G263A substitutions, indicating that interactions at both positions influenced the selectivity between glycosyl and alkyl moieties.
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| 2. |
- Barchan, Nikolina, et al.
(författare)
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Synthesis of glycophospholipid conjugates with mono- and disaccharides by enzymatic transphosphatidylation
- 2024
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Ingår i: European Journal of Lipid Science and Technology. - 1438-7697. ; 126:3
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Tidskriftsartikel (refereegranskat)abstract
- Phospholipids, PLs, are interesting and highly abundant amphiphilic molecules, which self-assemble into 3D nanostructures that have big interest as formulation excipients in, for example, pharma industry. However, the structures that are formed by naturally occurring PLs usually suffer from rigidity problems, and the nanostructures have to be modified in various ways for improved stability. One such approach is by the conjugation of saccharides to the PL head group. In this study, we investigate reaction conditions for the scalable phospholipase D–catalyzed transphosphatidylation reaction for the synthesis of glycophospholipids. Biphasic reaction systems with different solvents are compared with a purely aqueous system with PLs dispersed as vesicles. The investigations showed that use of the biphasic system containing chloroform and a glucose/phosphatidylcholine ratio of 50, in combination with carefully selected enzyme concentration and reaction time, led to an optimized process without any hydrolytic side reaction for the synthesis of phosphatidyl glucose. The reaction system was then applied to a variety of different mono- and disaccharides for the synthesis of a range of different glycophospholipids, resulting in yields up to 85% of phosphatidyl monosaccharides and 35% of disaccharides. Practical Application: Phospholipids and other polar lipids are of great scientific interest as formulation excipients. The chemical structures of lipids used for such applications have major impact on the properties of the self-aggregated systems. Synthesis of new phospholipids with modified head groups can tremendously widen the portfolio of available choices of formulation excipients and make it possible to make customized formulations with the desired properties. The introduction of saccharides in the hydrophilic part of the phospholipid alters the chemistry of head group and its interaction with surrounding water in vesicle systems and should therefore have a significant effect on its formulating properties compared to natural phospholipids.
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| 3. |
- Berger, Karin, et al.
(författare)
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Xylooligosaccharides Increase Bifidobacteria and Lachnospiraceae in Mice on a High-Fat Diet, with a Concomitant Increase in Short-Chain Fatty Acids, Especially Butyric Acid
- 2021
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Ingår i: Journal of Agricultural and Food Chemistry. - : American Chemical Society (ACS). - 0021-8561 .- 1520-5118. ; 69:12, s. 3617-3625
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Tidskriftsartikel (refereegranskat)abstract
- Effects of xylooligosaccharides (XOSs) as well as a mixture of XOS, inulin, oligofructose, and partially hydrolyzed guar gum (MIX) in mice fed a high-fat diet (HFD) were studied. Control groups were fed an HFD or a low-fat diet. Special attention was paid to the cecal composition of the gut microbiota and formation of short-chain fatty acids, but metabolic parameters were also documented. The XOS group had significantly higher cecum levels of acetic, propionic, and butyric acids than the HFD group, and the butyric acid content was higher in the XOS than in the MIX group. The cecum microbiota of the XOS group contained more Bifidobacteria, Lachnospiraceae, and S24-7 bacteria than the HFD group. A tendency of lower body weight gain was observed on comparing the XOS and HFD groups. In conclusion, the XOS was shown to be a promising prebiotic candidate. The fiber diversity in the MIX diet did not provide any advantages compared to the XOS diet.
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| 4. |
- Causevic, Ariana, et al.
(författare)
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Effects of Lipase Immobilization Conditions and Support Materials for the Production of Structured Triacylglycerols
- 2023
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Ingår i: European Journal of Lipid Science and Technology. - : Wiley. - 1438-7697 .- 1438-9312. ; 125:2
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Tidskriftsartikel (refereegranskat)abstract
- Structured triacylglycerols (STAG) with desired properties can be synthesized by transesterification using immobilized lipases. Herein, the effect of immobilization conditions and support material on the immobilization yield, specific activity and regioselectivity of lipases from Rhizomucor miehei (RML) and Rhizopus oryzae (ROL) in the production of STAG, are evaluated. Four different support materials utilizing adsorption and one with covalent binding are investigated. Ammonium sulfate is found to significantly increase the activity-based immobilization yield (12% and 38% for RML and ROL, respectively) and specific activity on Accurel MP1000 (MP1000) when used as the immobilization buffer. Furthermore, the immobilization principle and support material influenced both the activity and regiospecificity. Immobilization by adsorption is found to result in higher catalytic activity, while covalent binding resulted in lipase inactivation. For RML, the highest specific activity of 43 µmol STAG min−1 g−1 (U) is obtained on MP1000, while ROL, which exhibited higher activities in general, results in a maximum activity of 120 U on Lifetech ECR8806. The obtained specific activites are comparable to the commercial preparations Novozym 40086 (45 U) and Lipase DF “Amano” IM (147 U) while the regiospecificity of the developed preparations is even higher, forming at least 64% less byproduct. Practical applications: There is an increasing need for lipids with specific nutritional and physical properties in health, nutrition, and food applications. In this context, STAG are highly promising products due to the possibility to tailor the composition to obtain the desired properties. Immobilized lipases are the catalyst of choice for the production of STAG, in which activity and regioselectivity are particularly important parameters for the process performance. In this study, it is shown that these parameters can be affected by the immobilization conditions and support material. Immobilized preparations with high activity and excellent regiospecificity are created on commercially available supports. This shows the possibility of STAG synthesis with high purity and beneficial properties.
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| 5. |
- Causevic, Ariana, et al.
(författare)
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Impact of critical parameters influencing enzymatic production of structured lipids using response surface methodology with water activity control
- 2022
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Ingår i: Biochemical Engineering Journal. - : Elsevier BV. - 1369-703X. ; 187
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Tidskriftsartikel (refereegranskat)abstract
- Structured lipids with desired properties can be produced by enzymatic transesterification. This is a multistep reaction with many factors influencing both the product yield and quality. In this study, the single and combined effects of water activity, temperature and substrate ratio were studied on the reaction between high oleic sunflower oil and ethyl stearate producing 1,2-stearin-3-olein (SOS) using immobilized lipase from Rhizopus oryzae (Lipase DF “Amano” IM). Product quality was assessed as the ratio between SOS and the unwanted isomer SSO. Response surface methodology was used to create models at several time points, for increased understanding and showing the importance of the factors studied, for efficient production of structured lipids. To set the water activity, a control system based on dry/humid nitrogen gas sparging was developed. The models were found to adequately describe the effect of the factors on product yield and quality as well as being robust. For the yield, it was found that the factors influencing the enzymatic activity were highly important initially whereas factors affecting the thermodynamic equilibrium were dominating later. Increased temperature and water activity had initial positive influence that shifted to substrate ratio and a negative influence of water activity as equilibrium was approached. Product quality was mostly affected by temperature negatively, as an increased temperature promoted acyl migration.
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| 6. |
- Causevic, Ariana, et al.
(författare)
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Non-aqueous reversed phase liquid chromatography with charged aerosol detection for quantitative lipid analysis with improved accuracy
- 2021
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Ingår i: Journal of Chromatography A. - : Elsevier BV. - 0021-9673. ; 1652
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Tidskriftsartikel (refereegranskat)abstract
- There is a great need for efficient analysis of the composition of vegetable oils and fats, since it affects the physical and technical properties. However, due to the complex nature of these kind of samples, it is often difficult and costly. In the present study, we developed a Non-Aqueous Reversed-Phase HPLC method that can be used to separate and quantify different free fatty acids, fatty acid esters, monoacylglycerides, diacylglycerides and triacylglycerides, including regioisomers such as SOS/SSO and 1,2- and 1,3-diolein. Two 25 cm Nucleodur C18 Isis columns in series, sub-ambient column temperature and a mobile phase gradient composed of acetonitrile, acetic acid, isopropanol and heptane were used for the separation. The lipids were detected and quantified using a charged aerosol detector and it was found that the peak shape highly affected the detector response as well as the response uniformity, even when inverse gradient compensation was employed. Thus, calibration and determination of response factors were necessary for reliable quantification. A correlation between response factors and peak width at half peak height was found and used for quantification of non-calibrated components. A quantification approach was suggested including an appropriate selection of calibrated components, depending on sample composition and the accuracy required. It was shown in a complex oil sample that the reduced calibration approach, using only 6 instead of 33 calibrated components, resulted in virtually the same composition, but yielded a more accurate result compared to using relative area that neglects response factors. The method validation showed good reproducibility and accuracy, making it an excellent tool for extensive analysis of complex lipid mixtures.
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| 7. |
- Hoffmann, Christian, et al.
(författare)
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Improved Alkyl Glycoside Synthesis by trans-Glycosylation through Tailored Microenvironments of Immobilized β-Glucosidase
- 2020
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Ingår i: ChemPlusChem. - : Wiley. - 2192-6506. ; 85:1, s. 137-141
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Tidskriftsartikel (refereegranskat)abstract
- We present how the microenvironment can directly improve biocatalytic selectivity of immobilized β-glucosidase. β-Glucosidase from Thermotoga neapolitana was immobilized on a variety of functionalized off-stoichiometric thiol-ene (OSTE) particles, where highest activities were observed for thiol and imidazole functional particles. Compared to the soluble enzyme, the selectivity (rs/rh) between trans-glycosylation of p-nitrophenyl β-D-glucopyranoside (pNPG) with 1-propanol over hydrolysis was increased by a factor of 2–3 using particles containing imidazole (rs/rh of 6.7) and carboxylic acid moieties (rs/rh of 9.2), respectively. These results demonstrate clearly that enzyme selectivity depends directly on the local environment of the enzyme with the support.
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| 8. |
- Linares-Pastén, Javier A, et al.
(författare)
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Novel xylan-degrading enzymes from polysaccharide utilizing loci of Prevotella copri DSM18205
- 2021
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Ingår i: Glycobiology. - : Oxford University Press (OUP). - 1460-2423. ; 31:10, s. 1330-1349
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Tidskriftsartikel (refereegranskat)abstract
- Prevotella copri is a bacterium that can be found in the human gastrointestinal tract (GIT). The role of P. copri in the GIT is unclear, and elevated numbers of the microbe have been reported both in dietary fiber-induced improvement in glucose metabolism but also in conjunction with certain inflammatory conditions. These findings raised our interest in investigating the possibility of P. copri to grow on xylan, and identify the enzyme systems playing a role in digestion of xylan-based dietary fibers. Two xylan degrading polysaccharide utilizing loci (PUL10 and 15) were found in the genome, with three and eight glycoside hydrolase (GH) -encoding genes, respectively. Three of them were successfully produced in Escherichia coli: One extracellular enzyme from GH43 (subfamily 12, in PUL10, 60 kDa) and two enzymes from PUL15, one extracellular GH10 (41 kDa), and one intracellular GH43 (subfamily 137 kDa). Based on our results, we propose that in PUL15, GH10 (1) is an extracellular endo-1,4-β-xylanase, that hydrolazes mainly glucuronosylated xylan polymers to xylooligosaccharides (XOS); while, GH43-1 in the same PUL, is an intracellular β-xylosidase, catalyzing complete hydrolysis of the XOS to xylose. In PUL10, the characterized GH43-12 is an arabinofuranosidase, with a role in degradation of arabinoxylan, catalyzing removal of arabinose-residues on xylan.
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| 9. |
- Lindberg Yilmaz, Jenny, et al.
(författare)
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Polar Lipids Reduce In Vitro Duodenal Lipolysis Rate of Oat Oil and Liquid Oat Base Products
- 2020
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Ingår i: European Journal of Lipid Science and Technology. - : Wiley. - 1438-9312 .- 1438-7697.
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Tidskriftsartikel (refereegranskat)abstract
- Alternative ways for increased appetite control are today widely sought for due to the growing global health issues connected to obesity. In in vivo studies, oat has been proven an attractive candidate for inducing satiety. Oat is rich in polar lipids, of which the galactolipids are especially interesting, and a hypothesis is that these lipids play an important role for the ileal brake mechanism. In this study, the aim is to investigate the role of polar oat lipids on pancreatic lipolysis rate, using a pH‐stat based in vitro digestion model of the duodenum. Lipolysis of oat oil, a mix of oat oil/rapeseed oil (RSO), as well as a liquid oat base (OB) simulating an oat drink with different polar lipid content are investigated, and compared with RSO as control. Increasing the polar lipid content of the product digested leads to a significantly decreased lipolysis rate, and this effect is even observed when mixing RSO with a low amount of oat oil (10%). The results support the hypothesis that polar lipids can delay lipolysis also in a complex, natural system like the liquid OB, and even a minor amount of oat lipids can have large effect on lipolysis rates.Practical applications: The number of studies connecting galactolipids with a decreasing effect on duodenal lipolysis is growing; however, the mechanism behind this phenomenon is still not clarified. Here, the same effect is seen in a complex, natural food system. These findings open up for interesting future food products, where inclusion of oat oil, even at low concentrations, can have a prolonging effect on satiety. Oat for human consumption is an increasing market, thanks to the positive health benefits oat has been connected to, in combination with the current trend toward climate‐friendly plant‐based options for meat and dairy products. It is believed that oat oil can be attractive as an ingredient in various food products, for example, protein bars and spreads. More studies are needed to confirm the results in vivo. However, a great potential is seen for the use of oat oil to enhance appetite control.
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| 10. |
- Mohammadi, Milad, et al.
(författare)
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Xylanases and high-degree wet milling improve soluble dietary fibre content in liquid oat base
- 2024
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Ingår i: Food Chemistry. - 0308-8146. ; 442
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Tidskriftsartikel (refereegranskat)abstract
- The growth of plant-based food and drink substitutes has led to increased interest in oat-based milk substitute as a dairy milk alternative. Conventional liquid oat base (LOB) production results in a fibre-rich insoluble by-product and loss of valuable macronutrients. This study investigates the use of xylanase enzymes to release insoluble arabinoxylan (AX) fibre and employs different degrees of milling in the LOB manufacturing process, with the aim to reduce insoluble waste and simultaneously increase soluble dietary fibre in oat-based milk substitutes. The combination of decreased mill gap space from 1 to 0.05 mm and addition of GH10 xylanase, resulted in a homogenous LOB product and solubilization of all available AX. Potential prebiotic arabinoxylooligosaccharides of DP3-7 from GH10 hydrolysis were identified using HPAEC-PAD and MS analysis. These findings demonstrate the value of utilizing xylanases and fine-milling in LOB manufacturing, offering a sustainable approach to maximize health benefits of oat-based beverages.
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