ABSTRACT White-rot fungi and their enzymes are receiving increasing attention for biotechnological applications in the pulp and paper industry as alternatives to conventional bleaching. Laccase has been identified as one of the enzymes that plays a major role in lignin degradation. Laccase only attacks phenolic subunits of lignin, but its substrate range can be extended to non-phenolic subunits by the inclusion of a mediator. The use of this enzyme was, therefore, not successful in pulp bleaching trials until the discovery of mediators. Although the existence of natural mediators has not been confirmed, various components have been identified that are able to act as mediators. Improved methods of laccase production could benefit the industrial utilisation of the enzyme. White-rot fungi constitutively produce low concentrations of laccase, but higher concentrations can be obtained with the inclusion of inducers in the cultivation media. The enzyme is mainly produced during the stationary growth phase of the fungi, but various factors such as glucose, nitrogen and pH can influence levels of laccase production. The enzyme does not only hold potential for biological pulp bleaching operations, but also has application in bioremediation, the textile dye industry as well as the food and beverage industries.

YEAST DIVERSITY IN BLUE MOULD RIPENED CHEESES. ABSTRACT During the ripening process of blue veined cheese, different microbial groups interact and contribute to the final product. One of the most important of these groups are yeast. Further studies are needed to clarify their specific contribution to the ripening process. In order to accomplish this, a suitable and satisfactory enumeration medium is needed. Consequently, ten selective media were evaluated for their potential to inhibit and suppress the growth of moulds and bacteria without affecting the yeasts. Based on statistically compared data, no significant difference could be found amongst the ten media, except for one. Further studies were performed on the three media considered to be the most effective, MEA + Ox, MEA + NaCl and MEA + BP based on qualitative results. Accordingly, the three selected media were evaluated based on their ability to support the growth of the five most frequently occurring yeast species in blue veined cheese. No significant difference was obtained between two of the three media. MEA + NaCl however, was unable to support the growth of two of the five most dominant yeast species. MEA + Ox and MEA + BP proved to be superior for the enumeration and isolation of yeasts from blue veined cheese. DRBC, RBC and DG18 proved satisfactory regarding the enumeration of yeasts, whereas OGGY, MEA + SP and molybdate containing media are not recommended. Key words: Selective media, Yeasts, Moulds, Enumeration

Laboratory canning and evaluation of dry beans are common practices for testing canning quality of cultivars before commercial release to canning industries. Suitable laboratory canning and evaluation procedures for small white beans in tomato sauce were identified. Standard values for choice and standard grade beans for laboratory evaluation of canning quality were defined, using four small white bean cultivars from nine localities during the 2000/01 season. The cultivar Teebus was used as reference standard for choice grade beans and its canning quality complied with international guidelines when the modified canning technique (MCT) was used. From the laboratory and modified canning evaluation procedures hydration coefficient, percentage washed drained weight, visual appearance (scale 1 to 10), splits (scale 1 to 10), texture (kg.100 g -1 .12 s -1 ), size, clumping, L-values, aL-values and bL-values were identified as suitable canning parameters for small scale evaluation of beans. Beans canned with the MCT were also canned and evaluated industrially and results compared. The interpretation of the different canning parameters with laboratory and industrial canning were simplified by the use of canonical variate analysis (CVA). Canonical variate analysis indicated the same groupings for cultivars according to choice and standard grade canning quality for laboratory and industrial canned beans. Laboratory canning and evaluation could be used in the evaluation of the canning quality of beans intended for industrial canning. Canning quality of seven small white bean cultivars from 33 localities and two seasons was determined with the MCT and CVA. Cultivars with acceptable and unacceptable canning quality were identified using laboratory evaluation and CVA. The CVA resulted in a prediction model for canonical variates 1 and 2 (CV 1 and CV 2) by identifying two discriminative equations for CV 1 and CV 2 scores. The CVA for environments identified differences in the canning quality of beans from different regions, while also indicating seasonal differences. The canning quality of dry bean cultivars from different environments can be determined using CVA. The model equations for the prediction of the canning quality of small white beans were validated on four cultivar samples from four regions (2000/01 season) and 24 breeding samples from three localities (2002/03 season) that were not included in the development of the model. The CVA and the model identified the same entries from breeding trials over localities not to be significantly different from Teebus (P > 0.05) in canning quality, but were unable to group cultivars statistically correct according to choice grade. The model was however capable of grouping standard and choice grade cultivars separately. The model could be applied to identify breeding trial entries as choice grade and to identify entry x locality interactions. The use of small-scale canning and evaluation procedures in combination with CVA could be employed to classify cultivar canning quality as either choice- or standard grade and to determine environmental canning quality. These techniques could be used, with the assistance of the prediction model to compare samples from a breeding program with a reference standard.

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.