The aminoacyl-tRNA synthetases are a family of enzymes involved in the process of translation, more specifically, ligating amino acids to their cognate tRNA molecules. Recent evidence suggests that aminoacyl-tRNA synthetases are capable of aminoacylating proteins, some of which are involved in the autophagy pathway. Here, we test the conditions under which E. coli and human threonyl-tRNA synthetases, as well as hisidyl-tRNA synthetase aminoacylate themselves. These reactions are ATP dependent, stimulated by Mg2+, and are inhibited by increasing cognate tRNA concentrations. These data represent the foundation for future aminoacylation experiments, specifically delving into the relationship between the autophagy pathway and the aminoacylation of proteins. Additionally, we provide evidence of the inhibitory abilities of the compound EHTS-0 on both E. coli and human threonyl-tRNA synthetases. Further, we also show that an EHTS-0 analog, EHTS-1, also significantly inhibits E. coli threonyl-tRNA synthetase but not the human enzyme. These data could be useful in determining the potential for EHTS-0 and EHTS-1 as possibly anti-angiogenic drugs.

Finding alternatives to petroleum-based energy sources is of interest since it could reduce the emissions of net carbon dioxide to the atmosphere by increasing the usage of renewable energy sources. To do so improvements are needed in the renewable energy production sector. Biogas production is of interest since the anaerobic digestion process can degrade many different biomolecules and is, contrary to e.g. bioethanol and biodiesel, not dependent on specific molecules. Thus, many wastes such as slaughterhouse waste, sludge from waste water treatment and lignocellulose residual material etc. can be used as substrates for biogas production. However, there are limitations in the degradation process depending on the composition of the selected substrate. To overcome these limitations such as inhibition of different microorganisms, or recalcitrant substrate, different methods can be used to increase the biogas production.  In this study different substrates were selected and analyzed/treated for remedies of early stage rate limiting problems of the anaerobic digestion process. Different analyzes and techniques were selected depending on the limitations correlated to the main problematic component of the specific substrate.  Improvements could be reached for the degradation of slaughterhouse waste by augmentation with the clay mineral zeolite. Addition of different enzymes to the substrate environment of different waste water treatment plant sludges resulted in limited life time of the selected enzymes. However, certain enzymes proved to be promising candidates with an effect of increased biogas production rate and yield for the time that the enzyme remained active. In an additional experiment, cellulolytic enzymes, naturally produced by a biogas producing microbial community, were induced, collected and added to a biogas experiment of ensiled forage ley, by which it was shown that these cellulases led to an increase in biogas production rate and yield. Thus, the studies demonstrate different techniques for improving the anaerobic digestion process of different types of substrates. / <p>Handledare saknas</p>

Graduation date: 1999

Chitin, a homopolymer of β (1-4)-linked N-acetylglucosamine, is one of the most abundant biopolymers in nature. It is widely distributed in the exoskeleton of crustaceans and insects, in the cell walls of most fungi and some algae. Chitin is an extremely insoluble material with limited industrial applicability. The deacetylated derivative of chitin, chitosan, is a water soluble cationic biopolymer having a broad range of applications (Hirano, 1999). Chitosan is naturally found in the cell wall of Zygomycetes, in the ascospore of Saccharomyces cerevisiae (Briza et al., 1988) and in the cyst wall of Entamoeba invadens (Das et al., 2006). Chitosan biosynthesis requires the coordinated action of chitin synthase (E.C.2.4.1.16) and chitin deacetylase (E.C.3.5.1.41) (Davis & Bartnicki, 1984). Chitin synthase polymerizes N-acetyl glucosamine precursor molecules into chitin and chitin deacetylase catalyzes the deacetylation of the nascent chitin chains. The chitin deacetylase enzymes are members of the family 4 of carbohydrate esterases (CE-4s) as defined by the CAZY database [http://afmb.cnrs-mrs.fr/~cazy/CAZY] (Couthino et al., 1999), which includes several members sharing a conserved region in the primary structure assigned as the NodB homology domain(Caufrier et al., 2003) or polysaccharide deacetylase domain. Chitin deacetylase was first identified and partially purified from extracts of the fungus Mucor rouxii. Since then, chitin deacetylase has been purified from several fungi and chitin deacetylase open reading frames have been cloned from a few microorganisms including M. rouxii (Kafetzopoulos et al., 1993), Colletotrichum lindemuthianum (Tokuyasu et al., 1999; Shresta et al., 2004), Phycomyces blakesleeanus (GenBank AB046690), Schizophillum commune (GenBank AF271216), Blumeria graminis (GenBank AAK84438), Saccharomyces cerevisiae (Christodoulidou et al., 1999) and Schizosaccharomyces pombe (Matsuo et al., 2005). The structure and the catalytic mechanism of chitin deacetylase from C. lindemuthianum were recently studied (Blair et al., 2006). Chitin deacetylase plays a role in the cell wall biosynthesis in M. rouxii and Absidia coerulea (Gao et al., 1995). In C. lindemuthianum and Aspergillus nidulans, it was suggested that chitin deacetylase participates in plant-pathogen interactions to promote plant invasion (Tsigos et al., 2000). In S. cerevisiae, chitin deacetylase is essential for the ascospore cell wall rigidity and the resistance against lytic enzymes (Christodoulidou et al., 1996). The use of chitin deacetylase enzyme for the industrial deacetylation of chitin awaked a great interest. Different fungal strains were screened and compared for their ability to produce a chitin deacetylase secreted, active on insoluble substrates and showing low inhibition with acetate, a product of reaction. Rhizopus circinans proved to be a good chitin deacetylase producer with the targeted characteristics. The second part of the work was to isolate the cDNA encoding for the chitin deacetylase of R. circinans. The native enzyme was purified to homogeneity for sequencing the N-Terminal extremity. The enzyme was purified in only two steps from the culture supernatant of R. circinans. Then, the purified enzyme was sequenced and the first nine amino acids were identified. In the same way, a R. circinans cDNA library was also constructed. The cDNA library was screened using two approaches: on the one hand with radiolabeled homologous probe and on the other hand by PCR with primers designed for the 5 extremity, on the basis of the deduced sequence of the N-Terminal extremity of the native enzyme and for the 3 extremity, from the deduced R. oryzae chitin deacetylase. Two cDNA sequences (D2 and I3/2) with homology to fungal chitin deacetylase genes were isolated with the radiolabeled probe and one sequence (RC+) by PCR approach. The sequences were analyzed and characterized. The three sequences possessed several characteristics of chitin deacetylase sequence: homology with known chitin deacetylase cDNA, the presence of the deacetylase polysaccharide domain, and the same potential glycosylation sites than M. rouxii chitin deacetylase. The cDNA D2, I3/2 and RC (RC sequence is the mature protein sequence of RC+ sequence) were expressed in the yeast Pichia pastoris to confirm their potential chitin deacetylase activity. Numerous constructions were tested. A poly-histidine tag was cloned to facilitate the further purification of the recombinant enzyme. Only the RC sequence showed a high chitin deacetylase activity. Several hypotheses were emitted to explain the low chitin deacetylase activity level measured with the inserts D2 and I3/2. The recombinant RC protein was purified to homogeneity in one step, and partially characterized.

Thesis (M. Med. Sc.)--University of Hong Kong, 2001. / Includes bibliographical references (leaves 33-36).

Thesis (Ph. D.)--School of Chemistry and Biochemistry, Georgia Institute of Technology, 2004. Directed by James C. Powers. / Vita. Includes bibliographical references (leaves 132-151).

Thesis (M. Phil.)--University of Hong Kong, 1986.

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 108-114).

Directed evolution has been explored for a long time. Various ideas, methods, have been shown to be feasible and successful in the enzyme field. We were interested in evolving enzymes for applications. Therefore, we evolved human cystathionine gamma-lyase (hCGL) and E. coli biotin ligase for therapeutic and biotechnology applications. Wild-type human cystathionine gamma-lyase does not have any methionine-degrading activity, unlike the high methionine-degrading abilities of bacterial methionine gamma-lyase (MGL) found in Pseudomonas putida. The ability to engineer hCGL to breakdown methionine can be a potential cancer treatment by targeting the methionine-dependent cancer cells. However, the methionine-degrading activity of previously engineered hCGL has only shown 1% activity compared to MGL, too low to be useful in practical cancer therapeutics. By using a combination of protein design and phylogenetic analysis, we further evolved hCGL to achieve a higher methionine-degrading activity, with one variant displaying as much as 7% activity compared to bacterial MGL, making it a more likely candidate in cancer treatment.In addition, it has been shown that new orthogonal pairs of biotin protein ligase and biotin have many biotechnology applications. Therefore, we have developed selection scheme for directing the evolution of E. coli biotin protein ligase (BPL, gene: BirA) via in vitro compartmentalization, and have altered the substrate specificity of BPL towards the utilization of the biotin analogue desthiobiotin. Following just 6 rounds of selection and amplification several variants that demonstrated higher activity with desthiobiotin were identified. The best variants from Round 6, BirA₆₋₄₀ and BirA₆₋₄₇, showed 17-fold and 10-fold higher activity, respectively, their abilities to use desthiobiotin as a substrate. Further characterization of BirA₆₋₄₀ and the single substitution variant BirA[subscript M157T] revealed that they had 2- to 3-fold higher kcat values for desthiobiotin, and 3- to 4-fold higher K[subscript M] values. The k[subscript cat]/K[subscript M] values for these enzymes were around 0.7-fold that of BirA[subscript wt-]. It is interesting the selections did not lower the K[subscript M] for desthiobiotin and actually led to a less efficient enzyme. This is an example of how "you get what you select for". Because peptide:DNA conjugates were distributed such that there was on average one template or less per emulsion compartment there was selection only for the catalytic rate (k[subscript cat]) of desthiobiotinylation and not for turnover. Given these conditions, it might be anticipated that the peptide substrate, rather than desthiobiotin, should be bound better by the winning variants, and in fact BirA₆₋₄₀ showed a reduced K[subscript M] value for BAP. / text

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