<p>Alternative sources of fuel are required worldwide, and bio-ethanol is the leading candidate. Lignocellulosic biomass, a waste component of the agricultural industry, is a promising renewable source. Due to its complex structure it is highly recalcitrant, requiring the synergistic action of a battery of enzymes to achieve complete digestion. These enzymes include cellulases, hemicellulase and the accessory enzymes acetyl xylan esterase (AXE) and ferulic acid esterase (FAE). Thermpohilic Actinomyces isolates with the ability to hydrolyze xylan were screened for esterase activity. Two isolates (ORS10 and GSIV1), identified as Streptomyces spp, were positive for AXE activity. A cosmid library representative of isolate ORS10 was composed and screened for AXE activity using -naphthyl acetate as substrate. An 18 kb cosmid clone, 18D7, tested positive for AXE activity. Intracellular fractions extracted from ORS10 were precipitated with ammonium sulphate and partially purified 161-fold. Specific activity was measured after dialysis and ion-exchange chromatography. Overall yield of the partially purified enzyme was 34 %. Two protein bands of molecular masses 40 kDa and 60 kDa have been subjected to trypsin digestion and MALDI-TOF mass spectrometry analysis. The partially purified AXE displayed optimum activity at pH 9 and at 50&deg / C. AXE activity was stable for at least 1.5 hours between 30&deg / C and 40&deg / C, and for 24 hours between pH 6-9. The kM and Vmax values were 16.93 mg/ml and 1645 units/mg enzyme, respectively. The stability of the partially purified AXE at 30&deg / C-40&deg / C suggests potential for industrial applications that utilise mesophilic fermentations.</p>

The objective of this research was the selection of the most efficient arbuscular mycorrhizal (AM) fungus on pepper (Capsicum frutescens L. cv. North star) and sweet corn (Zea mays L. cv. Bicolor) growing under controlled and field conditions. The inoculum treatments consisted of 9 different AM inocula and an autoclaved mix of roots and sand used as a control. Plants were inoculated and planted in a pasteurized growth medium (greenhouse) and non-fumigated soil in 4 different field locations for each crop. Glomus intraradices (GinA) and both the same strain (GinA) and a mix of Sclerocysitis rubiformis and G. fasciculatum (Sru$+$) developed the highest AM colonization in sweet corn and pepper, respectively, under controlled conditions. However, no significant increases in growth were found compared with non-mycorrhizal plants. Only a mix of G. microagreggatum, G. mosseae and G. fasciculatum (Gmi+) produced a greater shoot dry mass compared with the control treatment in sweet corn under controlled conditions. None of the mycorrhizal strains used in the field experiments increased the growth of sweet corn or pepper compared with non-inoculated plants under field conditions.

No description available.

No description available.

Chitinases have been implicated in aspects of growth and morphology of chitin containing fungi such as hyphal branching, anastamosis and, possibly, apical growth itself. Cloning of <i>Neurospora crassa</i> chitinase genes would allow a molecular genetic analysis of the involvement of chitinases in such phenomena through the study of regulation, subcellular location and the use of specific mutagenesis. The cytosolic chitinase was partially purified to a high specific activity and characterised. Allosamidin, the first specific inhibitor of chitinase to be described, was shown to be a potent inhibitor of the cytosolic chitinase. Direct sequencing of the chitinase protein was not possible due to the inability to purify the protein to homogeneity. However, protein sequence information was obtained from protein electroblotted onto Immobilon polyvinylide difluoride membrane, after first identifying the chitinase protein band <i>in situ</i> after polyacrylamide gel electrophoresis using a specific activity stain. Protein sequence data was used to design an oligonucleotide probe for the screening of a genomic library in the cosmid vector pSV50. Screening of the library resulted in the identification of a single positive cosmid. The region of cosmid 23D6 that showed hybridisation to the oligonucleotide was subcloned and sequenced. The oligonucleotide was shown to hybridise to a sequence that had a single mis-match. However, when the sequence surrounding the oligonucleotide binding site was converted to an amino acid sequence, using the reading frame for the chitinase protein sequence, no homology with the remaining chitinase protein sequence was seen.

The aim of this work was to establish the relative contribution of genetic regulation of the <I>PYK1</I>, <I>PFK1</I> and <I>PFK2</I> genes to the control of glycolysis. A series of isogenic mutant strains were constructed where the promoters and 5' untranslated sequences of the <I>PYK1, PFK1</I> and <I>PFK2</I> genes were replaced with those from <I>PGK1</I>. In addition , a second series of mutant strains were constructed where synthesis of Pyk1p and Pflkp was driven by the <I>PGK1_Δuas</I> promoter. These latter series of mutants were designed to contain weak expression of Pyklp and Pflkp. Analysis of UKC1 (<I>PGK::PYK1)</I> in shake flask cultures revealed similar growth rates on glucose and on lactate and similar rates of ethanol production and glucose consumption to those of the wild-type strain. This suggested that the native genetic regulation did not appear to play a significant role in the control of glycolysis. Nonetheless, analysis of this strain in the fermentor revealed that genetic regulation of <I>PYK1</I> may be important in co-ordinating Pyk1p synthesis, under the conditions studied. Analysis of YKC11 (<I>PGK_Δuas::PYK1)</I> in both shake flask and fermentor experiments showed that genetic control was important in maintaining Pyk1p levels in order to sustain glycolytic flux. Shake flask analysis of the single and double <I>PFK</I> mutants under the control of the <I>PGK1</I> promoter revealed that the genetic regulation of the <I>PFK1</I> and <I>PFK2</I> genes did not appear to be important in the control of glycolysis. Weak expression of the <I>PFK1</I> and <I>PFK2</I> genes, under the control of the <I>PGK_Δuas</I> promoter showed the importance of genetic regulation in maintaining Pflkp levels to support glycolytic flux, under the conditions studied.

No description available.

No description available.

In host defence, pattern recognition plays an essential role by enabling the immune system to discriminate self from pathogenic non-self. Pattern recognition is mediated by leukocyte expressed pattern recognition molecules (PRMs), which recognise pathogen associated molecular patterns (PAMPs) on pathogens. Phagocytosis is a critical event for anti-microbial defence and its contribution is not limited to the clearance and killing of pathogens, but extends to the activation of adaptive immunity through production of pro-inflammatory mediators and antigen presentation. Anti- fungal immunity is extremely efficient and operates via recognition, phagocytosis and killing of fungal pathogens by leukocytes. We have examined Dectin-1, a non-opsonic pattern recognition receptor that recognises live fungi and fungal derived particles and that is highly expressed on various leukocyte populations. We wanted to establish whether Dectin-1 contributes to anti-fungal defence by analysing various aspects of the receptor biology. Using both confocal microscopy and flow-cytometry, we demonstrate that Dectin-1 is a phagocytic receptor. Furthermore, using cell lines expressing receptor mutants, we show that this capacity is mediated by the membrane proximal tyrosine residue located in the ITAM-like motif. This makes Dectin-1 the first described phagocytic leukocyte expressed receptor for unopsonised fungi and fungal derived particles, and the first pattern recognition receptor that mediates phagocytic uptake through a tyrosine based motif. We demonstrate that the mechanisms by which Dectin-1 mediates cytoskeletal activation and actin polymerisation are novel, and not shared with the canonical IT AM containing Fc(gamma)Rs. In particular the observation that Syk kinase plays not role in Dectin- 1 mediated phagocytosis in macrophages. We show that Dectin-1 mediates cellular activation in response to zymosan particles and that these (beta)-glucan dependent biological effects require collaboration with toll-like receptors (TLRs) at the cell surface. We also show that ligand size determines intracellular receptor trafficking following internalisation. Furthermore, we show that when biologically active soluble glucans are internalised by Dectin-1, the receptor is retained intracellularly yet, when biologically silent glucans are used, Dectin-1 is recycled. Dectin-1 is thus established as both an important phagocytic fungal pattern recognition receptor with pro- inflammatory abilities and an additional tool with which to study the diversity of signalling processes associated with leukocyte expressed receptors.

No description available.