Cloning and application of epoxide hydrolases from yeasts

Epoxides are cyclic ethers that readily react with various nucleophilic compounds. Consequently, epoxides can be used in many chemical synthesis reactions. Two enantiomeric forms of an epoxide are possible if one of the carbon atoms is chiral. This means that the epoxide is actually a racemic mixture of its two enantiomers. Due to the universal presence of chirality in nature it is important to use the proper epoxide enantiomer in the synthesis of compounds such as pharmaceuticals and agrochemicals, which should affect biological processes. Therefore, enantiopure epoxides are valuable intermediates in the synthesis of biologically active compounds by the pharmaceutical and agrochemical industries.Epoxide hydrolases (EHs) catalyze the hydrolysis of an epoxide into its corresponding diol. Moreover, EHs can hydrolyze racemic epoxide mixtures in an enantioselective manner. This results in the hydrolysis of one enantiomer, while the other remains unaffected and thus enantiopure. Therefore, EHs might be valuable tools to obtain enantiopure epoxides from racemic mixtures.EHs from yeast species, Rhodotorula glutinis in particular, have been usedto hydrolyze various epoxides with high activity and enantioselectivity. Consequently, the EHs from yeast species are promising biocatalysts that can be used in the production of enantiopure epoxides.In chapter 1 the work that is presented in this thesis is introduced. The aims of the research project were to gain important fundamental knowledge on EHs from yeasts and to develop biotechnological processes based on the use of these enzymes for the production of enantiopure epoxides. To introduce EHs in general, a broad overview is given in chapter 2 dealing with the molecular biology, biochemistry and potential application of these enzymes.In order to determine the relationship of these yeasts EHs to other known EHs, the EH-encoding genes and cDNA sequences from the yeast strains Xanthophyllomyces dendrorhous and R. glutinis were isolated (chapters 3 and 4). The genes were denominated EPH1 . Whereas the X . dendrorhous EPH1 open reading frame (ORF) of 1236 bp was interrupted by 8 introns, the 1230 bp-large R. glutinis EPH1 ORF was interrupted by 9 introns. The genes encoded polypeptides of 411 and 409 amino acids respectively, with corresponding calculated molecular masses of 46 kDa. The deduced amino acid sequences were similar to that of mammalian microsomal epoxide hydrolases. These enzymes belong to thea/bhydrolase fold family of enzymes, which have similar enzymatic structures and mechanisms. The EPH1 cDNA sequences were expressed in Escherichia coli to demonstrate their function. The epoxides, 1,2-epoxyhexane and 1-methyl-cyclohexene oxide, were hydrolyzed in an enantioselective manner. The inactivation of the EPH1 gene of X. dendrorhous showed that it was not essential for growth in rich medium under laboratory conditions.The epoxide hydrolase of R. glutinis was overproduced in the heterologous host Escherichia coli BL21(DE3) in order to develop a highly effective epoxide hydrolysis system (chapter 5). A strong improvement in Eph1 activity was found in cell extracts of the recombinant E. coli when compared to cell extracts of Rhodotorula glutinis , despite the formation of inactive Eph1 inclusion bodies. Co-expression of genes encoding molecular chaperones (DnaK-DnaJ-GrpE, GroEL-GroES, and trigger factor) decreased the amount of Eph1 inclusion bodies. However, there was no equivalent increase in active soluble Eph1. An increase in the level of soluble Eph1 was demonstrated by lowering the cultivation temperature from 37ºC to 21ºC and by using a fermenter for cultivation. Compared to R. glutinis the total increase in Eph1 activity for the recombinant E . coli towards 1,2-epoxyhexane was over 200 times, without loss of enantioselectivity. The utility of this Eph1 overproduction system was demonstrated by the hydrolysis of 1-oxa-spiro[2.5]octane-2-carbonitrile, which is a new R. glutinis Eph1 substrate and a versatile building block in organic synthesis. Whereas the recombinant E. coli , expressing R. glutinisEPH1 , could be used to hydrolyze 1-oxa-spiro[2.5]octane-2-carbonitrile with high Eph1 activity in an enantioselective manner. This was not possible using R. glutinis itself.To explore the biological diversity and potential industrial use of EHs from yeasts the Eph1-encoding cDNA sequences were also isolated from the carotenoid producing yeast species Rhodosporidium toruloides CBS 349, Rhodosporidium toruloides CBS 14 and Rhodotorula araucariae CBS 6031. These cDNA sequences encoded polypeptides of 409, 409, and 410 amino acids large respectively with molecular masses of 46 kDa. The deduced amino acid sequences were similar to that of the epoxide hydrolases from R. glutinis , X. dendrorhous and Aspergillus niger , which all correspond to the microsomal epoxide hydrolase sequence. Consequently, these cloned Eph1s probably belong to thea/bhydrolase fold family of enzymes. The epoxide hydrolase encoding cDNAs of the Rhodosporidium and Rhodotorula species were expressed in Escherichia coli BL21(DE3). The recombinant strains were able to hydrolyze trans -1-phenyl-1,2-epoxypropane in an enantioselective manner. The recombinant counterpart of Rhodosporidium toruloides CBS 14 was found to be a highly active and enantioselective biocatalyst for this substrate, despite the low activity and enantioselectivity of Rhodosporidium toruloides CBS 14 itself

Olfaction in the Colorado beetle at the onset of host plant selection

Long-range olfactory orientation of the adult Colorado beetle was studied in a low- speed wind tunnel. The odour of fully grown potato plants elicits an upwind locomotory response in Colorado beetles (odour-conditioned positive anemotaxis), and increases the beetles' speed of locomotion (direct chemo-orthokinesis). The initial olfactory orientation of the Colorado beetle during its search for food, is mainly directed towards solanaceous plant species, thus restricting the final part of host plant selection.Chemical analysis of potato leaf essential oil revealed the complex of general green leaf volatiles. In the potato leaf odour this complex is composed of cis-3-hexen-1-ol, cis-3-hexenylacetate, trans-2-hexenal and trans-2-hexen-1-ol. The olfactory antennal receptor system of the Colorado beetle is sensitively tuned to the reception of these volatiles, and the single olfactory receptors respond differentially to these component stimuli of potato leaf odour. When their particular ratio in the potato leaf odour has been changed, the beetles no longer react with an odour-conditioned positive anemotaxis. The complex of volatiles, rather than one single compound, is essential for long-range olfactory orientation of the Colorado beetle towards its host plant. The general green leaf volatiles probably occur as important constituents of leaf odours attractive to various phytophagous insects

The Effect of Partial Defoliation, Leaf Position and Developmental Stage of the Vine on Leaf Chlorophyll Concentration in Relation to the Photosynthetic Activity and Light Intensity in the Canopy of Vitis vinif era L. cv. Cabernet Sauvignon

The effect of partial defoliation and leaf position on leaf chlorophyll concentration in relation to the photosynthetic activity and light intensity in the canopy of Vitis vinifera L. cv. Cabernet Sauvignon was investigated at berry set, pea size, veraison and ripeness stages. The leaves of the severely defoliated vines appeared to contain the highest chlorophyll concentration.In general, chlorophyll a decreased as the leaves were situated progressively deeper into the canopy. No consistent relationship between chlorophyll concentration, light intensity and photosynthetic activity could be found for the different leaf positions. However, to obtain leaves that photosynthesize optimally, the amount and time of leaf removal in the grapevine canopy must be carefully planned

Distribution of 14C-Photosynthetate in the Shoot of Vitis vinifera L. cv Cabernet Sauvignon I. The Effect of Leaf Position and Developmental Stage of the Vine

The distribution of photosynthetates, originating in leaves of different parts of the shoot of Vi tis vinifera L. cv Cabernet Sauvignon at berry set, pea size, veraison and ripeness stages, was investigated. Specific photosynthetic activity of the "CO,-treated leaves gradually decreased during the season. Photosynthetates were hoarded in the leaves at berry set, but were increasingly diverted to the bunches after that. The apical leaves displayed the Itlghes.t.photosynthesis. The leaves opposite and below the bunches accumulated very little photosynthetafes, especially from veraison to ripeness. Redistribution of photosynthetates among the basal, middle and apical leaves was generally very restricted at all stages. Multidirectional distribution from the site of application of 14CO, occurred at berry set stage, while from pea size to ripeness photosynthetates were mainly translocated basipetally. Highest accumulation in the bunches occurred at veraison, while the basal leaves were primarily used to nourish the bunch

The Effect of Partial Defoliation, Leaf Position and Developmental Stage of the Vine on the Photosynthetic Activity of Vitis vinifera L. cv Cabernet Sauvignon

The effect of partial defoliation, :eaf position and developmental stage of the vine on the photosynthesis, stomatal resistance and transpiration of Vitis vinifera L. cv Cabernet Sauvignon was investigated. Partially defoliated vines displayed a higher rate of photosynthesis, generally increasing with degree of defoliation. The highest photosynthetic rates were found for the apical leaves, while those of the leaves opposite and below the bunches were restricted. Generally, rate of photosynthesis declined as the season progressed. The course of transpiration rate and stomata! resistance correlated with that of the rate of photosynthesis. However, transpiration and photosynthesis correlated poorly in the case of the apical leaves. In general, photon flux density and relative humidity at the leaf surface increased with an increase in defoliation percentage for all leaf positions. Leaf temperature was not significantly affected by partial defoliation. The results of the investigation suggested that excess vegetative growth is detrimental to interior-canopy microclimate as well as the photosynthetic rate of the entire vine. Partial defoliation seemed to provide a means to reduce some of the deleterious effects of vigorous growth

Kennisbehoefte en wijze van informatievergaring bij multifunctionele ondernemers

Voor ondernemers die zich oriënteren op de multifunctionele landbouw of voor ondernemers die hun bestaande multifunctionele bedrijf verder willen ontwikkelen en professionaliseren is de juiste kennis en een goede toegang daartoe van groot belang. Om inzicht te krijgen in de kennisbehoefte van multifunctionele ondernemers en in de wijze waarop zij bij voorkeur kennis krijgen aangeboden of kunnen zoeken is een internetenquête gehouden onder ruim 600 multifunctionele landbouwbedrijven wat resulteerde in 117 ingevulde enquêtes. Het doel van de enquête was om een indruk te krijgen van een deel van de multifunctionele sector en welke vragen in dat deel van de sector leven

Morphological variation in the girdled lizard Cordylus mclachlani Mouton 1986

The girdled lizard Cordylus mclachlani, previously known only from the type locality in the south-western Cape, South Africa, was found at several new localities as far north as Nieuwoudtville. The external morphology of the 32 additional specimens thus obtained was investigated to establish the nature of geographical variation. Specimens from the type locality, which lies on the southern periphery of the known distribution range, differ from the rest in the number of suboculars, the shape of the interparietal scale and in the presence of a post-interparietal scale. Because of some overlap in these characters, separate taxonomie status for the specimens outside the type locality is not considered. The diagnostic character set for the species is updated and additional ecological information is supplied

The adoption of social media and social media marketing by dentists in South Africa

OBJECTIVES: The purpose of the study was to identify and understand social media usage behaviour of dentists in South Africa, in general and in particular as part of their marketing strategy and to consider the potential determinants associated with these behaviours. METHODS: Dentists who are members of the South African Dental Association were requested to anonymously complete an online questionnaire. Apart from demographic information, respondents were asked to report on their use of social media and their adoption of social media marketing. One-on-one interviews were also conducted with three dentists, to gain a deeper understanding of their adoption of this marketing option. RESULTS: South African dentists have started to embrace social media and 50.2% interact through these channels at least once a day. The most popular social media platforms are GooglePlus and Facebook. Respondents use social media mainly for personal purposes, including staying connected to family and friends. Only 13.2% of those responding currently use social media as a marketing tool, but the majority (83.5%) predict that such usage will increase in future. CONCLUSION: Social media marketing is a growing trend and will become more significant in future. Although respondents used social media regularly for personal purposes, most are only now starting to use it as a marketing tool.https://www.sada.co.za/the-sadj2015-07-30am201