Tannin Acyl Hydrolase (E.C. 3.1.1.20) is commonly referred to as tannase. Teighem accidentally discovered this unique enzyme in 1867 (Teighem, 1867). He reported the formation of gallic acid when two fungal species were exposed to an aqueous solution of tannins. The fungal species were later identified as Penicillium glaucum and Aspergillus niger (Lekha & Lonsane, 1997). Tannase is responsible for the hydrolysis of ester and depside linkages in tannins to liberate gallic acid and glucose. This was a very interesting observation due to the usual complexation of proteins with tannic acid and naturally occurring tannins to form water insoluble complexes that inactivates enzymes (Haworth et al., 1985). Tannins have since been shown to be the natural substrate for the tannase enzyme. The enzyme also attacks gallic acid methyl esters, but it possesses high specificity towards the acyl moiety of the substrate. It has been known that certain moulds and fungi belonging to the species Aspergillus and Penicillium produce the enzyme (Rajakumar & Nandy, 1983). According to the work done by Yamada et al., (1968) the enzyme was mainly found intracellularly although the culture broth also contained the enzyme. Aspergillus niger, A. flavus and A. oryzae were found to be the best tannase producers on tannic acid as a sole source of carbon. From these growth studies it became evident that the tannase enzyme was an inducible enzyme (Gupta et al., 1997, Jean et al., 1981 and Mattiason & Kaul, 1994).

Hemicelluloses are non-cellulosic low-molecular-mass polysaccharides that are found together with cellulose in plant tissues. Xylan is the major component of the plant cell wall and the most abundant renewable hemicellulose (Timell, 1967). Heteropolysaccharides, based on a backbone structure of �� -1,4-linked D-xylose residues, are collectively referred to as �� -1,4-xylans and constitute the main polymeric compound of the hemicellulose fraction (Coughlan and Hazlewood, 1993). �� -Xylanase (1,4-�� -D-xylan-xylanohydrolase, EC 3.2.1.8) is capable of degrading xylans and has received considerable attention in the food, feed and paper industries (Graham and Inborr, 1992; Maat et al., 1992; Nissen et al., 1992; Wong and Saddler, 1993). A thermostable, cellulase-free xylanase from the filamentous fungus Thermomyces lanuginosus was isolated by Singh et al. (2000b). The xylanase from this fungus is not only remarkably thermostable, but is also active over a wide pH range (Singh et al., 2000a). The yeast Saccharomyces cerevisiae has several properties which have established it as a host for the expression of heterologous proteins of biotechnological interest, and several studies have been conducted on the secretion of heterologous xylanases by S. cerevisiae (Crous et al., 1995; P��rez-Gonzalez et al., 1996; La Grange et al., 1996).

Haemophilus paragallinarum , the causative agent of infectious coryza (IC), an acute respiratory disease in chickens and fowl, was first isolated in 1931 by De Blieck (1932). The first serious, documented outbreak in South Africa occurred in 1968 (Buys, 1982) on a multi-age layer-farm, soon the bacterium spread to most large production sites and established itself as the most common bacterial infection in layers (Bragg, 1995). The disease has a low mortality rate but leads to a drop in egg production of up to 40 % in layer hens and increased culling in broilers and thus poses significant financial liability to chicken farmers (Arzay, 1987; Bragg, 1995). One of the reasons for the success of survival for this bacterium is that after recovering from infection, birds become carriers of the bacterium, therefore aiding the spread of H. paragallinarum (De Blieck, 1948). Secondly, the bacterial strain belongs to one of nine serovars, which makes combating the spread of the disease through inactivated vaccination ineffective especially due to low cross protection among these serovars. (Rimler et al., 1977; Kume et al., 1980a). Various potential factors have been identified as potential virulence factors, e.g. the haemagglutinin protein. This protein plays a crucial role in adherence of the bacteria to the host's cells and is considered a possible virulence factor (Sawata et al., 1982; Yamaguchi et al., 1989). Sawata and co-workers (1982) reported at least three different haemagglutinins from H. paragallinarum strain 221 with one, HA-L, being serovar specific with the other common types shared by the different serovars in one serogroup.It would therefore be important to understand the working and interaction of the various virulence factors of H. paragallinarum, especially the haemagglutinins, in order to combat this bacterium.

Haemophilus paragallinarum causes an acute respiratory disease of chickens known as infectious coryza (IC), a disease first recognized as a distinct entity in the late 1920's. Since the disease proved to be infectious and primarily affected nasal passages, the name "infectious coryza" was adopted (Blackall, 1989). Infectious coryza may occur in both growing chickens and layers. The major economic effect of the disease is an increased culling rate in meat chickens and a reduction in egg production (10-40%) in laying and breeding hens. The disease is limited primarily to chickens and has no public health significance (Yamamoto, 1991). The most common clinical signs are a nasal discharge, conjunctivitis, and swelling of the sinuses and face. Various sulfonamides and antibiotics are useful in alleviating the severity and course of infectious coryza; however, none of the therapeutic agents has been found to be bactericidal. Relapse often occurs after treatment is discontinued, and the carrier state is not eliminated (Yamamoto, 1991). All the commercially available bacterins against IC, consist of inactivated broth cultures of a combination of two or three different serotypes. Although vaccines against IC have been used in South Afr ica since 1975, it became apparent in the 1980s that the vaccines were becoming less effective in controlling the disease (Bragg et al., 1996). This could have been due to the emergence of a previously unknown serovar, or even serogroup and the possibility of changes in the population dynamics. Vaccine efficiency is therefore a problem and an alternative to available vaccines is needed. Capsules have long been associated with virulence properties of bacteria. The role that the capsule play in the virulence of bacterial species related to H. paragallinarum has been investigated by several workers (Kroll et al., 1988; Inzana et al., 1993; Boyce and Adler, 2000). Mutation, deletion or allelic exchange of gene/s involved in the transport of capsule polysaccharides in related species like Haemophilus influenza, Actinobacillus pleuropneumoniae and Pasteurella multocida, resulted in organisms with reduced virulence. The noncapsulated mutants of Actinobacillus pleuropneumoniae reported by Inzana et al. (1993) showed extreme stability and induceda protective immune response without any symptoms of disease. This not only proves the capsule�s involvement in virulence of bacteria but also offers the opportunity to investigate the possibility of producing live vaccines. The aim of this study was an attempt to understand the genetic organization of the capsular genes of H. paragallinarum in comparison to related HAP organisms and the possibility of producing a mutant lacking the capsule. The goals were: 1. Isolation and cloning of the capsule transport gene locus. 2. Sequencing and characterization of the locus 3. Transplacement of a gene/s to produce a noncapsulated mutant of H. paragallinarum.

The food spoilage potential of the genus Chryseobacterium is the ability of a pure culture of this genus to produce the metabolites that are associated with the spoilage of a particular food product. A careful combination of microbial, sensory and chemical analyses are required to determine the food spoilage potential of the genus Chryseobacterium. The role and significance of the genus Chryseobacterium in food and their proven and potential significance as food spoilage bacteria have not been studied in equal detail as the taxonomy and nomenclature of this genus and this has been the main reason for the present research project. It was regarded as necessary to obtain a better understanding of the characteristics of these organisms pertaining to their food spoilage potential. The purpose of this work would be to add to the knowledge on this genus and in the process inform the food scientist of the practical implications of food contamination by this group of microorganisms. The ability to utilise carbon sources by Chryseobacterium species tested in this study does not directly reflect the probability of food spoilage defects, but the BIOLOG system can be used as an effective screening method for identifying the carbon sources that could be investigated further for their potential to produce food spoilage defects. Phenotypic tests on Chryseobacterium species can be used as an alternative method to investigate the hydrolysis of food components and the production of metabolites, which could result in potential food spoilage defects such as putrefaction and alkalinisation, which are usually associated with disagreeable odours. Some Chryseobacterium species had the ability to decarboxylate some of the precursors of biogenic amines at different temperatures and in the presence of different sodium chloride concentrations. This results in the formation of biogenic amines which could cause amine poisoning and these organisms should consequently be regarded as significant spoilage organisms in food products. In this study it was observed that Chryseobacterium species were able to grow at 4°C, at a pH of 5 to 10 and at sodium chloride concentrations of 1 to 3%. It is known that Chryseobacterium species have the potential to spoil milk. For these reasons a decrease in temperature (below 4°C ), in pH (below 5) and an increase in sodium chloride concentration (above 4%) will inhibit the growth of this genus and have a preservative effect in products with these characteristics. Optimum growth was observed at 25°C, and it could be expected that spoilage defects in food products, kept at this temperature would develop most rapidly. The genus Chryseobacterium has the potential ability to produce spoilage defects due to proteolytic and lipolytic activity. Such activity could result in off-flavours and off-odours. Similarly the production of phospholipase C could enhance lipolysis and rancidity defects. Chryseobacterium species were able to produce volatile compounds in milk. The primary alcohols produced were not likely to contribute to flavour, while the carboxylic acids can be responsible for the production of a variety of flavours (e.g. fruity). Sensory evaluation on inoculated milk samples resulted in the identification of odour descriptors, such as âputridâ and âsmelly feetâ. It is therefore advantageous to use a technique such as gas chromatography to identify volatile compounds produced by the genus Chryseobacterium. This will help in evaluating the spoilage potential of this genus in a product such as milk more accurately. The role and significance as well as the potential food spoilage defects of Chryseobacterium species should be investigated in more detail in more practical scenarios.

The cytochrome P-450 monooxygenase and ï¢-oxidation systems of alkane-utilizing yeasts have been studied extensively, whereas very little is known about the fatty (long chain) alcohol and fatty aldehyde oxidizing enzymes. With the recent completion of sequencing of the genome of Yarrowia lipolytica, an alkane-degrading yeast, several putative aldehyde dehydrogenases (ALDHs) have been identified. Four of these were identified as fatty ALDHs (FALDHs). Northern blot analysis and RT-PCR showed that one of the FALDH genes, labelled FALDH4, is induced during growth of Y. lipolytica on alkanes, whereas another aldehyde dehydrogenase gene, labelled ALDH1, was constitutively expressed. Functional analysis of the four FALDH isogenes was initiated by single gene deletion of the four fatty aldehyde dehydrogenase isogenes in all possible combinations. The Cre-loxP recyclable tools system was used for gene disruption. Growth properties of the triple and quadruple deletion strains on alkanes were investigated. A slightly arrested growth in hexadecane was observed in two strains, the triple deletion mutant with intact FALDH2 isogene and the quadruple deletion mutant with all four FALDH isogenes deleted. Very strong hydrophobicity during growth of these mutants in hexadecane was also observed. At this stage one can only say that disruption of FALDH isogenes had a slight negative effect on growth of this yeast on alkanes.; However, it is not yet clear which individual isogenes are the most important for alkane metabolism in this organism. Although fatty aldehyde dehydrogenase (FALDH) activity has been detected in fungi no FALDH genes have yet been cloned, sequenced and expressed. Through BLAST searches using the human FALDH sequence as query we have identified 28 FALDH/FALDH-like gene sequences of which nine are from molds and 19 from yeast species. A comparative study of these sequences showed that fungal FALDH sequences may fall into several different subclasses of the ALDH3 family. Unique features of these proteins included presence of several transmembrane domains and in particular relatively long C- and N-termini. Searches of the sequenced Y. lipolytica genome for fatty alcohol oxidase (FAOD) and fatty alcohol dehydrogenase (FADH) encoding genes, which could be involved in the oxidation of fatty alcohols to aldehydes, yielded only one putative FADH encoding gene. However, FADH activity during growth on n-alkanes was very low and Northern-blot analyses showed that this gene was only weakly expressed during growth on hydrocarbon and non-hydrocarbon substrates.

A variety of microorganisms, including yeasts, are capable of utilizing n- alkanes as carbon source (Schmitz et al., 2000; Watkinson & Morgan, 1990). The over expression of P450 genes such as the CYP52 family coding for the alkane hydroxylases may lead to an increase in activity and increased formation of possible useful products from hydrocarbon metabolism (Iida et al., 2000). Disruption of the -oxidation pathway by deleting the genes coding for acyl CoA-oxidases, also leads to the accumulation of products that would normally be broken down (Picataggio et al., 1991). The genetic engineering of these two points of control opens up many possibilities for the accumulation of different products from hydrocarbons. Although some work was done concerning these systems in Candida tropicalis very little work has been done in Yarrowia lipolytica. It was the aim of the project to investigate the biotransformation of alkanes, alkylbenzenes and their derivatives by different groups of genetically engineered Y. lipolytica strains in order to investigate a number of questions. The possible accumulation of monocarboxylic acids in Yarrowia lipolytica was inestigated by using substrates such as undecene and hexylbenzene. Y. lipolytica MTLY37 a -oxidation disrupted strain with POX2, POX3, POX4 and POX5 genes deleted could not accumulate any monocarboxylic acid from undecene. The undecene was however fully utilized indicating that this strain still had some -oxidation activity. Little phenylacetic acid was formed (0.4 mM) from hexylbenzene. Another product that could not be positively identified at the time, but which might have been phenylhexanoic acid accumulated (4mM). No monocarboxylic acids other than phenylacetic acid could also be accumulated from alkylbenzenes in strains with blocked - oxidation expressing CPR and CYP genes, leading to the conclusion that Y. lipolytica can not accumulate monocarboxylic acids. Y. lipolytica strains with disrupted -oxidation as well as a strain with functional -oxidation expressing additional YlCPR and CYP52F1 genes accumulated the full-length dioic acid from 5-methylundecane. All these strains also sequentially broke down the 5-methylundecanedioic acid to 5- methylnonanedioic acid, 3-methylheptanedioic acid and 3-methylpentanedioic acid. Y. lipolytica MTLY76 was the only strain that did not degrade the 5- methylundecanedioic acid completely. Using hexylbenzene as substrate it was possible to establish that ethanol delayed the induction of both the native ALK genes as well as the inserted CYP genes. However, the cloned genes were later induced quite strongly (probably by the phenylalkanoic acids formed from hexylbenzene) for an extended period, while the native genes were only weekly induced. The maximum activity of Y. lipolytica was slightly lower when ethanol was used as inducer (13µmol.min -1 l -1 ) than when oleic acid was used as inducer (19µmol.min -1 l -1 ). The alkane hydroxylase activity was however maintained for a longer time when ethanol was used as inducer. When dodecane was used as inducer native genes were strongly induced for a relatively long period, but not as long as the cloned genes after ethanol. Alkylbenzenes as substrate was also useful to distinguish between alkane hydroxylase activity of native and cloned monooxygenases. A significant difference in the activity of Y. lipolytica TVN356 expressing CPR together with CYP557A1 (putative fatty acid hydroxylase from Rhodotorula retinophila) and Y. lipolytica TVN91 expressing CPR together with CYP53 (benzoate para- hydroxylase from R. minuta) could be observed (14µmol.min -1 l -1 and 8µmol.min -1 l -1 respectively) when decylbenzene was used as substrate. To better study the hydroxylase activity of inserted P450s, it may be better to use the ICL1 promoter to drive the expression of the inserted CYP genes and use ethanol as inducer.

Psittacine beak and feather disease (PBFD) is a readily recognisable dermatologic condition in wild and captive psittacines worldwide. It is caused by Beak and feather disease virus (BFDV) which is classified in the family Circoviridae and the genus Circovirus. BFDV has a circular ss-DNA genome consisting of seven open reading frames (ORFs), three being conserved in all BFDV isolates, ORF 1 which encodes the Rep protein, ORF 2 which encodes the coat or capsid protein (CP) and ORF 5 which encodes a protein whose function is as yet unknown. General symptoms of the disease include the symmetrical loss of feathers, feather abnormalities, beak and claw deformities, weight loss, anorexia and immunosuppression. The inability to grow BFDV in tissue culture or in embryonated eggs has hindered the routine diagnosis of PBFD affected birds and the development of reliable diagnostic tests and an effective vaccination program. PBFD is widespread in South Africa, leading to a loss of at least 10% of psittacine breeding stocks annually. The disease is also a major threat to the already endangered Cape Parrot (Poicephalus robustus) and the black-cheeked lovebird (Agapornis nigrigenis) and it is only a matter of time before we may see the extinction of these and other parrot species due to the lack of a preventative vaccine. The economical and natural implications of the attack by PBFD led to the aims of the present study which were to develop a potential DNA vaccine candidate, develop an expression system for production of recombinant CP as antigenic protein and establish an enzyme linked immunosorbent assay for the detection of BFDV-specific antibodies in parrots. The entire CP gene which has been suggested to encode for the epitopic protein of the virus was amplified by polymerase chain reaction (PCR) and ligated into a bacterial vector, pBAD/His B or a yeast vector, pKOV136 for expression of recombinant CP in Escherichia coli or Yarrowia lipolytica, respectively. Alternatively, CP gene PCR products were ligated into the mammalian expression vector pcDNAâ¢3.1D/V5-His-TOPO® which was the vector of choice for DNA vaccine design and used to transiently transfect Chinese hamster ovary cells. Subsequently, the candidate DNA vaccine was used in a basic vaccine trial where budgerigars (Melopsittacus undulatus) were vaccinated either with the DNA vaccine candidate or a sub-unit vaccine consisting of purified recombinant CP. Expression of recombinant CP was monitored using polyacrylamide gel electrophoresis (PAGE), chemiluminescent and colorimetric detection on Western blots and ELISAs. While expression of the recombinant CP was unsuccessful in the yeast system using pKOV136, expression of recombinant CP was achieved in E. coli cells using the pBAD vector. Recombinant CP was partially purified and applied in both indirect and indirect competitive ELISAs as coating antigen for the detection of BFDV specific antibodies. Using the established ELISAs, BFDV specific antibodies could be detected in naturally infected parrots as well as in budgerigars vaccinated with the DNA vaccine and sub-unit vaccine. Comparable results were obtained when nonpurified recombinant CP was applied in the ELISAs in lieu of partially purified recombinant CP. Vaccinated budgerigars formed BFDV specific antibodies in response to the DNA vaccine and sub-unit vaccine that were detected using the indirect competitive ELISA established in the study. The antibody responses to the sub-unit vaccine were higher than those in response to vaccination with the DNA vaccine candidate. Although the indirect competitive ELISA could not provide an indication of whether these antibody responses are protective, the results obtained during the trial are a preliminary indication that both the DNA vaccine and sub-unit vaccine may be functional in parrots and safe to use as no adverse reactions were observed.

Mannan polysaccharides occur in hemicellulose fraction of the plant cell walls. The hydrolysis of these polymers involves the action of enzymes such as β-mannanase, β-mannosidase and α- galactosidase which are produced by both fungi and bacteria. The current study reports on the production of β-mannanase, β-mannosidase and α-galactosidase by newly isolated Scopulariopsis candida strains LMK004 and LMK008. The effect of medium composition and carbon source on growth and enzyme production was evaluated in a liquid culture. A combination of Vogelâs medium and locust bean gum was found to stimulate growth and increase β-mannanase production. Optimal β-mannanase production of 7800 nkat/g biomass for LMK004 and 13300 nkat/g biomass for LMK008 was achieved in media containing 10% NaCl, 1X Vogelâs medium, 1% yeast extract and 1% locust bean gum. Both strains secreted trace amounts (less than 1 nkat/ml) of β-mannosidase and α-galactosidase indicating that these enzymes may be retained intracellularly. Native-PAGE and SDS-PAGE were used together with the zymogram to assess purity and to estimate the molecular weight of the proteins. The molecular weight of LMK004 β-mannanase was estimated to be â41 kDa whereas that of LMK008 β-mannanase could not be determined due to excessive loss of protein material during dialysis. The β-mannanase from LMK004 was most active at pH 5 and 50 °C, and retained ⥠80% of its activity at pH 5 â 6.5 after 24 hrs of incubation at 4 °C. In contrast, the LMK008 β- mannanase retained ⥠60% activity between pH 6 â 7. Both enzymes remained stable for 3 hrs at temperature between 30 °C and 40 °C, and showed loss of activity at higher temperatures. The two enzymes displayed different degrees of halotolerance. The LMK008 β-mannanase tolerated high NaCl concentrations with 60% activity remaining after incubation for 2 hrs at 20% NaCl, whereas the LMK004 β-mannanase was only active between 0% - 10% NaCl. It is clear from the current study that the two strains of S. candida produce distinct β-mannanases which may be useful candidates in low water activity reactions.

The genus Dipodascopsis was extensively studied with regards to reproductive cycles as well as the presence, distribution and function of 3-OH oxylipins. Most of this research was carried out on D. uninucleata var. uninucleata (Canadian strain) as well as D. tóthii. However, little is known concerning D. uninucleata var. uninucleata isolated from South African soil, as well as D. uninucleata var. wickerhamii. Consequently, using gas chromatography-mass spectrometry, electron microscopy and confocal laser scanning microscopy, the two varieties were compared regarding their morphologies, oxylipin production, mitochondrial activity as well as life cycles and ascospore release. According to literature, the two varieties differ only in their ability to assimilate certain carbon sources. During this study, differences in ascospore size as well as differences in ascospore clustering, after release, was observed. Furthermore, differences in the type of 3-OH oxylipin produced by the two varieties, also existed. 3-OH oxylipin production was found to be associated mainly with the sexual stage and concentrated in the ascus surrounding the ascospores, in both varieties. Furthermore, increased mitochondrial activity was also observed during the sexual stage and found to be concentrated in close vicinity of the ascospores. Since mitochondria produce 3-OH oxylipins, it is suggested that the increased activity during sexual development would be to aid in the production and release of the ascospores, as well as the accumulation of these 3-OH oxylipins. In addition, acetylsalicylic acid was found to inhibit the production of 3-OH oxylipins by probably decreasing mitochondrial activity resulting in the inhibition of ascospore release.