| 1. |
- Hassan, Noor, 1977-
(författare)
-
Characterization and engineering of carbohydrate-active enzymes for biotechnological applications
- 2015
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Extremozymes are enzymes produced by microorganisms that live in extreme habitats. Due to their higher stability, extremozymes is attracting interest as biocatalysts in various industrial processes. In this context, carbohydrate-active extremozymes can be used in various processes relevant to the paper, food and feed industry.In this thesis, the crystal structure, biochemical characterization and the capacity to synthesize prebiotic galacto-oligosaccharides (GOS) were investigated for a β-glucosidase (HoBGLA) from the halothermophilic bacterium Halothermothrix orenii. The wild-type enzyme displays favorable characteristics for lactose hydrolysis and produces a range of prebiotic GOS, of which β-D-Galp-(1→6)-D-Lac and β-D-Galp-(1→3)-D-Lac are the major products (Paper I).To further improve GOS synthesis by HoBGLA, rational enzyme engineering was performed (Paper II). Six enzyme variants were generated by replacing strategically positioned active-site residues. Two HoBGLA variants were identified as potentially interesting, F417S and F417Y. The former appears to synthesize one particular GOS product in higher yield, whereas the latter produces a higher yield of total GOS.In Paper III, the high-resolution crystal structure and biochemical characterization of a hemicellulase (HoAraf43) from H. orenii is presented. HoAraf43 folds as a five-bladed β-propeller and displays α-Larabinofuranosidase activity. The melting temperature of HoAraf43 increases significantly in the presence of high salt and divalent cations, which is consistent with H. orenii being a halophile.Furthermore, the crystal structures of a thermostable tetrameric pyranose 2-oxidase from Phanerochaete chrysosporium (PcP2O) were determined to investigate the structural determinants of thermostability (Paper IV). PcP2O has an increased number of salt links between subunits, which may provide a mechanism for increased stability. The structures also imply that the N-terminal region acts as an intramolecular chaperone during homotetramer assembly.
|
|
| 2. |
- Jonsson Rudsander, Ulla, 1973-
(författare)
-
Functional studies of a membrane-anchored cellulase from poplar
- 2007
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Cellulose in particular and wood in general are valuable biomaterials for humanity, and cellulose is now also in the spotlight as a starting material for the production of biofuel. Understanding the processes of wood formation and cellulose biosynthesis could therefore be rewarding, and genomics and proteomics approaches have been initiated to learn more about wood biology. For example, the genome of the tree Populus trichocarpa has been completed during 2006. A single-gene approach then has to follow, to elucidate specific patterns and enzymatic details. This thesis depicts how a gene encoding a membrane-anchored cellulase was isolated from Populus tremula x tremuloides Mich, how the corresponding protein was expressed in heterologous hosts, purified and characterized by substrate analysis using different techniques. The in vivo function and modularity of the membrane-anchored cellulase was also addressed using overexpression and complementation analysis in Arabidopsis thaliana. Among 9 genes found in the Populus EST database, encoding enzymes from glycosyl hydrolase family 9, two were expressed in the cambial tissue, and the membrane-anchored cellulase, PttCel9A1, was the most abundant transcript. PttCel9A1 was expressed in Pichia pastoris, and purified by affinity chromatography and ion exchange chromatography. The low yield of recombinant protein from shake flask experiments was improved by scaling up in the fermentor. PttCel9A1 was however highly heterogenous, both mannosylated and phosphorylated, which made the protein unsuitable for crystallization experiments and 3D X-ray structure determination. Instead, a homology model using a well-characterized, homologous bacterial enzyme was built. From the homology model, interesting point mutations in the active site cleft that would highlight the functional differences of the two proteins could be identified. The real-time cleavage patterns of cello-oligosaccharides by mutant bacterial enzymes, the wildtype bacterial enzyme and PttCel9A1 were studied by 1H NMR spectroscopy, and compared with results from HPAEC-PAD analysis. The inverting stereochemistry for the hydrolysis reaction of the membrane-anchored poplar cellulase was also determined by 1H NMR spectroscopy, and it was concluded that transglycosylation in vivo is not a possible scenario. The preferred in vitro polymeric substrates for PttCel9A1 were shown to be long, low-substituted cellulose derivatives, and the endo-1,4--glucanase activity was not extended to branched or mixed linkage substrates to detectable levels. This result indicates an in vivo function in the hydrolysis of “amorphous” regions of cellulose, either during polymerization or crystallization of cellulose. In addition, overexpressing PttCel9A1 in A. thaliana, demonstrated a correlation with decreased crystallinity of cellulose. The significance of the different putative modules of PttCel9A1 was investigated by the construction of hybrid proteins, that were introduced into a knock-out mutant of A. thaliana, and the potential complementation of the phenotype was examined. A type B plant cellulase catalytic domain could not substitute for a type A plant cellulase catalytic domain, although localization and interaction motifs were added to the N- and C-terminus.
|
|
| 3. |
- Nordberg Karlsson, Eva
(författare)
-
Xylan degradation by the thermophilic bacterium Rhodothermus marinus. Characterization and function of a thermostable xylanase
- 1999
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- A gene encoding a multidomain xylanase from the thermophilic bacterium Rhodothermus marinus has been cloned, sequenced and produced in Escherichia coli . The gene product was found to be a 109 kDa protein consisting of five domains. The catalytic domain and the two polysaccharide binding domains (PBD, previously called carbohydrate binding domains) have been cloned and produced separately for functional studies. The catalytic domain is classified under glycosyl hydrolase family 10, a family in which 3D-structure determined representatives exhibit 8-fold alpha/beta(TIM)-barrels. It catalyses endo-hydrolysis of xylans and to a lower extent mixed linkage (1-3,4) ß-glucans. Hydrolysis has been observed using oligosaccharides with a degree of polymerisation of 4 or higher. The two polysaccharide binding domains are likely displaying folds based on ß-strands. Their binding specificities to soluble substrates are corresponding well to observed substrate specificity of the catalytic domain. Among insoluble substrates they display binding to both amorphous cellulose and xylan. Both the binding level and thermal stability were markedly increased by additions of calcium ions. The three functionally characterized domains have transition temperatures for unfolding above, or long half-lives within, the growth temperatures (55-77 °C) of the organism R. marinus. The efficiency in enzyme aided bleaching of kraft pulps was tested for three catalytically active forms of the enzyme. All forms lead to brightness gains although at different extents. The gain was found to be dependent on both enzyme construction and pulp characteristics. The trial with an enzyme construct lacking the two N-terminal PBDs was most successful. A strategy for production in E. coli was also developed for two enzyme forms. The results show that production in the selected host-vector system was dependent on both cultivation mode, medium composition and type of inducer. Most efficient production was obtained in fed-batch cultivations in the presence of complex nutrients. Under these conditions the two alternative inducers, IPTG/lactose, resulted in approximately equal amounts of the produced recombinant proteins.
|
|
