This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Abstract 12Directed enzyme prodrug therapy is a form of cancer chemotherapy in which bacterial prodrug-13 activating enzymes, or their encoding genes, are directed to the tumour before administration of a 14 2 prodrug. The prodrug can then be activated into a toxic drug at the tumour site, reducing off-target 1 effects. The bacterial Nitroreductases are a class of enzymes used in this therapeutic approach and 2 although very promising, the low turnover rate of prodrug by the most studied nitroreductase enzyme, 3NfnB from E. coli (NfnB_Ec), is a major limit to this technology. There is a continual search for 4 enzymes with greater efficiency, and as part of the search for more efficient bacterial nitroreductase 5 enzymes, two novel enzymes from Bacillus cereus (strain ATCC 14579) have been identified and 6shown to reduce the CB1954 (5-(Aziridin-1-yl)-2,4-dinitrobenzamide) prodrug to its respective 2'-and 7 4'-hydroxylamine products. Both enzymes shared features characteristic of the Nitro-FMN-reductase 8Superfamily including non-covalently associated FMN, requirement for the NAD(P)H cofactor, 9 homodimeric, could be inhibited by Dicoumarol (3,3'-methylenebis(4-hydroxy-2H-chromen-2-one), 10 and displayed ping pong bi bi kinetics. Based on the biochemical characteristics and nucleotide 11 alignment with other nitroreductase enzymes, one enzyme was named YdgI_Bc and the other 12YfkO_Bc. Both B. cereus enzymes had greater turnover for the CB1954 prodrug compared with 13 NfnB_Ec, and in the presence of added NADPH cofactor, YfkO_Bc had superior cell killing ability, 14 and produced mainly the 4'-hydroxylamine product at low prodrug concentration. The YfkO_Bc was 15 identified as a promising candidate for future enzyme prodrug therapy. 16

A toxin is a poisonous substance produced within living cells or organisms. One of the most potent groups of toxins currently known are the Botulinum Neurotoxins (BoNTs). These are so deadly that as little as 62 ng could kill an average human; to put this into context that is approximately 200,000 × less than the weight of a grain of sand. The extreme toxicity of BoNTs leads to the need for methods of determining their concentration at very low levels of sensitivity. Currently the mouse bioassay is the most widely used detection method monitoring the activity of the toxin; however, this assay is not only lengthy, it also has both cost and ethical issues due to the use of live animals. This review focuses on detection methods both existing and emerging that remove the need for the use of animals and will look at three areas; speed of detection, sensitivity of detection and finally cost. The assays will have wide reaching interest, ranging from the pharmaceutical/clinical industry for production quality management or as a point of care sensor in suspected cases of botulism, the food industry as a quality control measure, to the military, detecting BoNT that has been potentially used as a bio warfare agent.

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Mutant strains of Pseudomonas aeruginosa, blocked in the pathway of aromatic amino acid biosynthesis, were isolated to determine the branchpoint of pyocyanine biosynthesis. Studies of the enzyme complement of these mutants and determination of pyocyanine production indicated that chorismic acid is an intermediate in the pathway and is the branchpoint compound.

Botulinum neurotoxin is one of the deadliest biological toxins known to mankind and is able to cause the debilitating disease botulism. The rapid detection of the different serotypes of botulinum neurotoxin is essential for both diagnosis of botulism and identifying the presence of toxin in potential cases of terrorism and food contamination. The modes of action of botulinum neurotoxins are well-established in literature and differ for each serotype. The toxins are known to specifically cleave portions of the SNARE proteins SNAP-25 or VAMP; an interaction that can be monitored by electrochemical impedance spectroscopy. This study presents a SNAP-25 and a VAMP biosensors for detecting the activity of five botulinum neurotoxin serotypes (A–E) using electrochemical impedance spectroscopy. The biosensors are able to detect concentrations of toxins as low as 25 fg/mL, in a short time-frame compared with the current standard methods of detection. Both biosensors show greater specificity for their compatible serotypes compared with incompatible serotypes and denatured toxins.