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| 2. |
- Hassan, Noor, et al.
(författare)
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Engineering a thermostable Halothermothrix orenii beta-glucosidase for improved galacto-oligosaccharide synthesis
- 2016
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Ingår i: Applied Microbiology and Biotechnology. - : Springer. - 0175-7598 .- 1432-0614. ; 100:8, s. 3533-3543
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Tidskriftsartikel (refereegranskat)abstract
- Lactose is produced in large amounts as a by-product from the dairy industry. This inexpensive disaccharide can be converted to more useful value-added products such as galacto-oligosaccharides (GOSs) by transgalactosylation reactions with retaining beta-galactosidases (BGALs) being normally used for this purpose. Hydrolysis is always competing with the transglycosylation reaction, and hence, the yields of GOSs can be too low for industrial use. We have reported that a beta-glucosidase from Halothermothrix orenii (HoBGLA) shows promising characteristics for lactose conversion and GOS synthesis. Here, we engineered HoBGLA to investigate the possibility to further improve lactose conversion and GOS production. Five variants that targeted the glycone (-1) and aglycone (+1) subsites (N222F, N294T, F417S, F417Y, and Y296F) were designed and expressed. All variants show significantly impaired catalytic activity with cellobiose and lactose as substrates. Particularly, F417S is hydrolytically crippled with cellobiose as substrate with a 1000-fold decrease in apparent k(cat), but to a lesser extent affected when catalyzing hydrolysis of lactose (47-fold lower k(cat)). This large selective effect on cellobiose hydrolysis is manifested as a change in substrate selectivity from cellobiose to lactose. The least affected variant is F417Y, which retains the capacity to hydrolyze both cellobiose and lactose with the same relative substrate selectivity as the wild type, but with similar to 10-fold lower turnover numbers. Thin-layer chromatography results show that this effect is accompanied by synthesis of a particular GOS product in higher yields by Y296F and F417S compared with the other variants, whereas the variant F417Y produces a higher yield of total GOSs.
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| 3. |
- Kittl, Roman, et al.
(författare)
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Molecular cloning of three pyranose dehydrogenase-encoding genes from Agaricus meleagris and analysis of their expression by real-time RT-PCR
- 2008
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Ingår i: Current Genetics. - : Springer Science and Business Media LLC. - 0172-8083 .- 1432-0983. ; 53:2, s. 117-127
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Tidskriftsartikel (refereegranskat)abstract
- Sugar oxidoreductases such as cellobiose dehydrogenase or pyranose oxidase are widespread enzymes among fungi, whose biological function is largely speculative. We investigated a similar gene family in the mushroom Agaricus meleagris and its expression under various conditions. Three genes (named pdh1, pdh2 and pdh3) putatively encoding pyranose dehydrogenases were isolated. All three genes displayed a conserved structure and organization, and the respective cDNAs contained ORFs translating into polypeptides of 602 or 600 amino acids. The N-terminal sections of all three genes encode putative signal peptides consistent with the enzymes extracellular secretion. We cultivated the fungus on different carbon sources and analyzed the mRNA levels of all three genes over a period of several weeks using real-time RT-PCR. The glyceraldehyde-3-phosphate dehydrogenase gene from A. meleagris was also isolated and served as reference gene. pdh2 and pdh3 are essentially transcribed constitutively, whereas pdh1 expression is upregulated upon exhaustion of the carbon source; pdh1 appears to be additionally regulated under conditions of oxygen limitation. These data are consistent with an assumed role in lignocellulose degradation.
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| 4. |
- Mate, Diana, et al.
(författare)
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Blood tolerant laccase by directed evolution
- 2013
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Ingår i: Chemistry and Biology. - : Elsevier. - 1074-5521 .- 1879-1301. ; 20:2, s. 223-231
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Tidskriftsartikel (refereegranskat)abstract
- High-redox potential laccases are powerful biocatalysts with a wide range of applications in biotechnol. We have converted a thermostable laccase from a white-rot fungus into a blood tolerant laccase. Adapting the fitness of this laccase to the specific compn. of human blood (above neutral pH, high chloride concn.) required several generations of directed evolution in a surrogate complex blood medium. Our evolved laccase was tested in both human plasma and blood, displaying catalytic activity while retaining a high redox potential at the T1 copper site. Mutations introduced in the second coordination sphere of the T1 site shifted the pH activity profile and drastically reduced the inhibitory effect of chloride. This proof of concept that laccases can be adapted to function in extreme conditions opens an array of opportunities for implantable nanobiodevices, chem. syntheses, and detoxification.
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| 5. |
- Osipov, Evgeny, et al.
(författare)
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Effect of the L499M mutation of the ascomycetous Botrytis aclada laccase on redox potential and catalytic properties
- 2014
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Ingår i: Acta Crystallographica Section D. - : International Union of Crystallography. - 0907-4449 .- 1399-0047. ; 70:11, s. 2913-2923
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Tidskriftsartikel (refereegranskat)abstract
- Laccases are members of a large family of multicopper oxidases that catalyze the oxidn. of a wide range of org. and inorg. substrates accompanied by the redn. of dioxygen to water. These enzymes contain four Cu atoms per mol. organized into three sites: T1, T2 and T3. In all laccases, the T1 copper ion is coordinated by two histidines and one cysteine in the equatorial plane and is covered by the side chains of hydrophobic residues in the axial positions. The redox potential of the T1 copper ion influences the enzymic reaction and is detd. by the nature of the axial ligands and the structure of the second coordination sphere. In this work, the laccase from the ascomycete Botrytis aclada was studied, which contains conserved Ile491 and nonconserved Leu499 residues in the axial positions. The three-dimensional structures of the wild-type enzyme and the L499M mutant were detd. by X-ray crystallog. at 1.7 Å resoln. Crystals suitable for X-ray anal. could only be grown after deglycosylation. Both structures did not contain the T2 copper ion. The catalytic properties of the enzyme were characterized and the redox potentials of both enzyme forms were detd.: E 0 = 720 and 580 mV for the wild-type enzyme and the mutant, resp. Since the structures of the wild-type and mutant forms are very similar, the change in the redox potential can be related to the L499M mutation in the T1 site of the enzyme.
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| 6. |
- Scheiblbrandner, Stefan, et al.
(författare)
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Evolving stability and pH-dependent activity of the high redox potential Botrytis aclada laccase for enzymatic fuel cells
- 2017
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Ingår i: Scientific Reports. - : Nature Publishing Group. - 2045-2322. ; 7
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Tidskriftsartikel (refereegranskat)abstract
- Fungal high redox potential laccases are proposed as cathodic biocatalysts in implantable enzymatic fuel cells to generate high cell voltages. Their application is limited mainly through their acidic pH optimum and chloride inhibition. This work investigates evolutionary and engineering strategies to increase the pH optimum of a chloride-tolerant, high redox potential laccase from the ascomycete Botrytis aclada. The laccase was subjected to two rounds of directed evolution and the clones screened for increased stability and activity at pH 6.5. Beneficial mutation sites were investigated by semi-rational and combinatorial mutagenesis. Fourteen variants were characterised in detail to evaluate changes of the kinetic constants. Mutations increasing thermostability were distributed over the entire structure. Among them, T383I showed a 2.6-fold increased half-life by preventing the loss of the T2 copper through unfolding of a loop. Mutations affecting the pH-dependence cluster around the T1 copper and categorise in three types of altered pH profiles: pH-type I changes the monotonic decreasing pH profile into a bell-shaped profile, pH-type II describes increased specific activity below pH 6.5, and pH-type III increased specific activity above pH 6.5. Specific activities of the best variants were up to 5-fold higher (13 U mg(-1)) than BaL WT at pH 7.5.
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| 7. |
- Tan, Tien-Chye, et al.
(författare)
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Structural basis for cellobiose dehydrogenase action during oxidative cellulose degradation
- 2015
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Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 6, s. 7542-7542
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Tidskriftsartikel (refereegranskat)abstract
- A new paradigm for cellulose depolymerization by fungi focuses on an oxidative mechanism involving cellobiose dehydrogenases (CDH) and copper-dependent lytic polysaccharide monooxygenases (LPMO); however, mechanistic studies have been hampered by the lack of structural information regarding CDH. CDH contains a haem-binding cytochrome (CYT) connected via a flexible linker to a flavin-dependent dehydrogenase (DH). Electrons are generated from cellobiose oxidation catalysed by DH and shuttled via CYT to LPMO. Here we present structural analyses that provide a comprehensive picture of CDH conformers, which govern the electron transfer between redox centres. Using structure-based site-directed mutagenesis, rapid kinetics analysis and molecular docking, we demonstrate that flavin-to-haem interdomain electron transfer (IET) is enabled by a haem propionate group and that rapid IET requires a closed CDH state in which the propionate is tightly enfolded by DH. Following haem reduction, CYT reduces LPMO to initiate oxygen activation at the copper centre and subsequent cellulose depolymerization.
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