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Title: The development of chromogenic substrates for microbial detection
Author: Davidson, Gwen
Awarding Body: University of Reading
Current Institution: University of Reading
Date of Award: 2011
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Various aspects in the field of chromogens were studied. In a multifaceted approach, investigations into their synthesis, coupling to less common sugars, and screening as substrates for biological activity, together with kinetic studies on the rate of hydrolysis of indoxyl substrates were carried out. A three-step route to synthesise substituted indoxyl chromogens was successfully developed. This method was subsequently used to prepare a number of halogen derivatives to study the effect of halogen type and position of substitution on the reaction yields. Overall yields range between 2 and 27 %. However attempts to make /V-alkyl indoxyl derivatives were unsuccessful. Studies were carried out to extend the range of target glycosidases by coupling indoxyl to a range of less common pentose and hexose sugars. The following sugars were successfully coupled; D- and L-glucose, D- and L-iyxose, D-galactose, L-rhamnose, 2-deoxy-D-ribose and acetyl-D-xylofuranose. The syntheses of indoxyl aminopeptidase substrates were successfully carried out using L- and D-alanine and L-pyroglutamic acid, with overall yields of between 1 and 3%. An ALDOL™ phosphatase substrate was synthesised with an overall yield of 18%. Studies determined that it was potentially suitable as both a fluorogenic and a chromogenic substrate. Agar based testing indicated that Clostridium perfringens could be successfully detected at a concentration 2.5 x 102 CFU/mL. The substrate was also found to fluoresce plastics. An agar method was developed to screen a range of key microorganism targets with indolyl substrates. The optimised method included the use of agar multipoint inoculators with 3mm pins, by inoculating 1.5 x 108 CFU/mL into nutrient agar containing chromogenic substrate (50mg/100mL), and incubating at 37 °C for 24 hours. Using quantitative UV-Vis and HPLC analyses, techniques to monitor the hydrolysis of indoxyl galactosides were developed and used to correlate molecular structure with rates of hydrolysis. Rates were observed to increase in the order unsubstituted
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available