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- Harrysson, Hanna, 1987, et al.
(författare)
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Strategies for Improving the Protein Yield in pH-Shift Processing of Ulva lactuca Linnaeus: Effects of Ulvan Lyases, pH-Exposure Time, and Temperature
- 2019
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Ingår i: Acs Sustainable Chemistry & Engineering. - : American Chemical Society (ACS). - 2168-0485. ; 7:15, s. 12688-12691
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Tidskriftsartikel (refereegranskat)abstract
- Globally, there is a need for novel vegetarian protein sources. We recently showed that the pH-shift process, using alkaline protein solubilization followed by isoelectric precipitation, is an efficient way to produce extracts with high protein concentrations from Ulva lactuca (>50% on a dry matter basis). However, the total protein yield was low, and to improve this, the effects of adding ulvan lyase, preincubating the seaweed homogenate at pH 8.5 and using different protein extraction temperatures (8 degrees C, RT and 40 degrees C), were evaluated in this study. Addition of ulvan lyase reduced protein solubility but increased the precipitation. Incubation at pH 8.5, without ulvan lyase added, significantly increased both protein solubility and precipitation at 8 degrees C and RT. Temperature per se had no effect on protein solubility, while protein precipitation increased with decreasing temperature. Highest protein yield (29%) was achieved when keeping the process at 8 degrees C with a preincubation step at pH 8.5 for 1 h. By these process modifications, the yield was 3.2 times higher than achieved by the control process (9.2%).
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| 2. |
- Konasani, Venkat Rao, 1981, et al.
(författare)
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A novel ulvan lyase family with broad-spectrum activity from the ulvan utilisation loci of Formosa agariphila KMM 3901
- 2018
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 8
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Tidskriftsartikel (refereegranskat)abstract
- Ulvan, which is one of the major structural polysaccharides of the cell walls of green macroalgae, is degraded by ulvan lyases via the beta-elimination mechanism with the release of oligosaccharides that have unsaturated 4-deoxy-L-threo-hex-4-enopyranosiduronic acid (Delta) at the non-reducing end. These ulvan lyases belong to the PL24 or PL25 or PL28 family in the CAZy database. In this study, we identify and biochemically characterise a periplasmic novel broad-spectrum ulvan lyase from Formosa agariphila KMM 3901. The lyase was overexpressed in Escherichia coli, and the purified recombinant enzyme depolymerised ulvan in an endolytic manner with a K-m of 0.77 mg/ml, and displayed optimum activity at 40 degrees C and pH 8. This lyase also degraded heparan sulphate and chondroitin sulphate. Detailed analyses of the end-products of the enzymatic degradation of ulvan using H-1- and C-13-NMR and LC-MS revealed an unsaturated disaccharide (Delta Rha3S) and a tetrasaccharide (Delta Rha3S-Xyl-Rha) as the principal end-products. In contrast to the previously described ulvan lyases, this novel lyase is mostly composed of alpha-helices that form an (alpha/alpha)(6) incomplete toroid domain and displays a remarkably broad-spectrum activity. This novel lyase is the first member of a new family of ulvan lyases.
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| 3. |
- Konasani, Venkat Rao, 1981, et al.
(författare)
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A NOVEL ULVAN LYASE FAMILY WITH BROAD-SPECTRUM ACTIVITY FROM ULVAN UTILISATION LOCI OF FORMOSA AGARIPHILA KMM 3901
- 2018
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- The rapid proliferation of members of green macroalgae in eutrophicated waters causes algal blooms. The biomass of these blooms, which is rich in polysaccharides, is underutilised and majorly left to decompose. Ulvan is one of such polysaccharides which constitutes up to 30 % of dry weight in Ulva species. Ulvan and its oligosaccharides have potential applications in food and medicine. Ulvan lyases depolymerise ulvan via the β-elimination mechanism with the release of oligosaccharides that have unsaturated 4-deoxy-L-threo-hex-4-enopyranosiduronic acid at the non-reducing end. Ulvan lyases belong to the PL24, PL25 or PL28 family in the CAZy database [2-5]. In this study, we identified and biochemically characterised a periplasmic novel broad-spectrum ulvan lyase from Formosa agariphila KMM 3901. The lyase was heterologously overexpressed in Escherichia coli, and purified using a two-step purification. The purified recombinant enzyme depolymerised ulvan in an endolytic manner with a Km of 0.77 mg/ml. The enzyme was optimally active at 40 °C and pH 8. This lyase also degraded heparan sulphate and chondroitin sulphate. Detailed analyses of the endproducts of the enzymatic endolytic depolymerisation of ulvan using 1H- and 13C-NMR and LC-MS revealed an unsaturated disaccharide (∆Rha3S) and a tetrasaccharide (∆Rha3S-Xyl-Rha3S) as the principal end-products. This ulvan lyase is a member of the polysaccharide utilisation loci for ulvan. In contrast to the previously described ulvan lyases, this novel lyase is mostly composed of α-helices that form an (α/α)6 incomplete toroid domain and displays a remarkably broad-spectrum activity. This novel lyase is the first member of a new family, which sequence is not homologous to other ulvan lyases.
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| 4. |
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| 5. |
- Konasani, Venkat Rao, 1981, et al.
(författare)
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Ulvan lyase from Formosa agariphila and its applicability in depolymerisation of ulvan extracted from three different Ulva species
- 2018
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Ingår i: Algal Research. - : Elsevier BV. - 2211-9264. ; 36:December 2018, s. 106-114
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Tidskriftsartikel (refereegranskat)abstract
- Members of green macroalgae cause green tides in eutrophicated coastal waters. These green tides pose an environmental issue and an economic burden on coastal municipalities. The biomass from these green tides has a potential to be used as feedstock in biorefinery due to the content of interesting biomacromolecules. Ulvan, an anionic water-soluble polysaccharide, is one of such components, and its depolymerisation to high-value oligosaccharides or fermentable monosaccharides would bring value to green tide biomass which is otherwise left to decompose. However, only a few ulvan depolymerising enzymes are studied to date. Ulvan lyases depolymerise ulvan, via the β-elimination mechanism, leading to release of oligosaccharides with an unsaturated 4‑deoxy‑l‑threo‑hex‑4‑enopyranosiduronic acid at the non-reducing end. In this study, we have identified the presence of two different domains, a catalytic and a non-catalytic, in a putative ulvan lyase from Formosa agariphila KMM 3901. We overexpressed, purified, and biochemically characterised the full-length ulvan lyase, which was found to be most active at a temperature of 45 °C and pH 8.5. It exhibited high specificity for ulvan and did not degrade heparan sulphate, chondroitin sulphate, alginate, pectin or xanthan. Detailed analyses of end products of the enzymatic degradation of ulvan using 1H NMR and LC-MS revealed a disaccharide with an unsaturated uronic acid (∆) linked to 3‑sulphated rhamnose (Rha3S), trisaccharide with xylose (Xyl) flanked by Rha3S (Rha3S-Xyl-Rha3S), tetrasaccharide with an unsaturated uronic acid at the non-reducing end (∆Rha3S-Xyl-Rha3S) and pentasaccharides (Rha3S-Xyl-Rha3S-Xyl-Rha3S and branched ∆Rha3S-Xyl-(∆)Rha3S) as the principal end products. We also found that the catalytic domain that lacks the non-catalytic carbohydrate binding module exhibited higher affinity for the soluble ulvan and efficiently depolymerised it. This study reveals the characteristics of the endolytic ulvan lyase, which is a member of the ulvan utilisation loci in Formosa, and points towards the potential ulvan depolymerisation applications in Ulva biorefinery.
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