[show abstract][hide abstract] ABSTRACT: The artificial implementation of the Darwinian theory of evolution to create new variants of functional proteins, a process referred to as directed evolution, has acquired many applications in biochemical engineering. Directed evolution is a handy tool in the nascent science of glycobiology, where it is used in the conversion of glycosyl hydrolases into transglycosidases or for improving the transglycosylation behaviour of glycosynthases. This review focuses on recent applications of the directed evolution approach to harness the transglycosidase potential of glycosidases and to enhance the functional fitness of glycosynthases.
Engineering the functional fitness of transglycosidases and glycosynthases by directed evolution. 04/2011; 10:1727-1735.
[show abstract][hide abstract] ABSTRACT: A Gram-negative obligate alkaliphilic bacterium (BOG6 T) that secretes carotenoids was isolated from the outflow of Lake Bogoria hot spring located in the Kenyan Rift Valley. The bacterium is motile by means of a polar flagellum, and forms red colonies due to the production of xanthophyll carotenoid pigments. 16S rRNA gene sequence analysis showed this strain to cluster phylogenetically within the genus Paracoccus. Strain BOG6 T is aerobic, positive for both catalase and oxidase, and non-methylotrophic. The major fatty acid of the isolate is C18: 1ω ω ω ω7c. It accumulated polyhydroxybutyrate granules. Strain BOG6 T gave astaxanthin yield of 0.4 mg/g of wet cells indicating a potential for application in commercial production of carotenoids. On the basis of its genotypic characteristics, fatty acid composition and physiological reaction profiles, it is proposed that the isolate may be assigned to the genus Paracoccus as Paracoccus bogoriensis sp. nov. The type strain is BOG6 T (=DSM16578 =LMG22798). The GenBank 16S rDNA nucleotide sequence accession number is AJ580352.
African journal of microbiology research 09/2009; 3:426-433. · 0.54 Impact Factor
[show abstract][hide abstract] ABSTRACT: Differentiation of bloodstream-form trypanosomes into procyclic (midgut) forms is an important first step in the establishment of an infection within the tsetse fly. This complex process is mediated by a wide variety of factors, including those associated with the vector itself, the trypanosomes and the bloodmeal. As part of an on-going project in our laboratory, we recently isolated and characterized a bloodmeal-induced molecule with both lectin and trypsin activities from midguts of the tsetse fly, Glossina longipennis [Osir, E.O., Abubakar, L., Imbuga, M.O., 1995. Purification and characterization of a midgut lectin-trypsin complex from the tsetse fly, Glossina longipennis. Parasitol. Res. 81, 276-281]. The protein (lectin-trypsin complex) was found to be capable of stimulating differentiation of bloodstream trypanosomes in vitro. Using polyclonal antibodies to the complex, we screened a G. fuscipes fuscipes cDNA midgut expression library and identified a putative proteolytic lectin gene. The cDNA encodes a putative mature polypeptide with 274 amino acids (designated Glossina proteolytic lectin, Gpl). The deduced amino acid sequence includes a hydrophobic signal peptide and a highly conserved N-terminal sequence motif. The typical features of serine protease trypsin family of proteins found in the sequence include the His/Asp/Ser active site triad with the conserved residues surrounding it, three pairs of cysteine residues for disulfide bridges and an aspartate residue at the specificity pocket. Expression of the gene in a bacterial expression system yielded a protein (M(r) approximately 32,500). The recombinant protein (Gpl) bound d(+) glucosamine and agglutinated bloodstream-form trypanosomes and rabbit red blood cells. In addition, the protein was found to be capable of inducing transformation of bloodstream-form trypanosomes into procyclic forms in vitro. Antibodies raised against the recombinant protein showed cross-reactivity with the alpha subunit of the lectin-trypsin complex. These results support our earlier hypothesis that this molecule is involved in the establishment of trypanosome infections in tsetse flies.
[show abstract][hide abstract] ABSTRACT: We have previously described a bloodmeal-induced molecule (lectin-trypsin complex) from the midgut of the tsetse fly, Glossina longipennis, with both lectin and trypsin activities (Osir et al., 1995). In this paper, we report on the isolation of a similar molecule from the midguts of Glossina fuscipes fuscipes and provide direct evidence for its involvement in the development of African trypanosomes. The molecule (native Mr ~65,700) has two non-covalently linked subunits, Mr ~28,800 and Mr ~35,700. The native molecule was found to be capable of inducing differentiation of bloodstream-form trypanosomes into procyclic (midgut forms) in vitro. In the assays, specific antibodies against procyclin were used to monitor the transformation of the bloodstream-form trypanosomes into procyclic forms. This induction was specifically inhibited by D-glucosamine. Cis-aconitate was also capable of inducing the transformation process with the same efficiency as that of the lectin-trypsin complex. While increasing the concentrations of the lectin-trypsin complex (100 g protein/ml) in the incubation assays resulted into higher transformation rates, it also led to high parasite mortality. These results provide evidence for the involvement of the midgut lectin-trypsin complex in the differentiation of bloodstream-form trypanosomes within tsetse midgut.
Insect Science and Its Application. 08/2003; 23(3):197-205.