U529343.pdf (16.84 MB)
Physiology and biochemistry of amidases from Methylophilus methylotrophus.
thesisposted on 2015-11-19, 09:07 authored by James. Mills
Physiological regulation of acetamidase, formamidase and urease expression in M. methylotrophus was investigated using continuous culture under various nutrient limitations. Acetamidase was maximally induced by acetamide (acetamide>formamide> urea), formamidase by urea (urea>formamide>acetamide) and urease by urea and formamide (urea/formamide>acetamide). All three enzymes were repressed by ammonia. The ability of acetamidase to undergo a rapid, heat-reversible loss of activity (switch-off) was extensively investigated. No switch-off was observed during growth under any steady- state continuous culture growth regime. However, significant switch-off (a decrease in kcat from 147s-1 to 66s-1) was observed following the addition of ammonia pulses to dual acetamide/methanol-limited cultures. Similar decreases in kcat were also seen when the growth regime was transiently changed from dual acetamide/methanol limitation to dual ammonia/methanol limitation, while maintaining a constant nitrogen input. Switched-off acetamidase was purified from an ammonia-pulsed culture and found to remain in the low- activity form during purification. Increased switch-off was observed during purification and also in response to various in vitro manipulations. Experiments carried out with nongrowing cultures in an attempt to further investigate the biochemistty of the switch-off event were inconclusive. Formamidase also appeared to undergo putative switch-off in response to similar physiological signals as acetamidase. Prolonged growth of wild-type M. methylotrophus in formamide-limited continuous culture led to the selection of a mutant strain (MM25). This strain produced formamidase at the wild-type concentration but with a significantly increased specific activity. Purification of the enzyme showed that this was due to an increased kcat. MM25 also exhibited an increased rate of formate oxidation and over-expressed two proteins with subunit MrS of approximately 40,000, and another with a subunit Mr of approximately 32,000. The Mr 40,000 proteins were identified as the products of the fmdC and fmdD genes. FmdD was purified from MM25 and shown to be a monomeric urea/formamide-binding protein (U/F- BP) with an Mr of 41,000 (41,870), which bound urea stoichiometricaly with a KD of 7.15uM. U/F-BP was found to belong to a structurally related family of amino acid- and amide-binding proteins. Microbial amidases were classified into three groups using specific antibodies raised against M. methylotrophus acetamidase and formamidase.
Date of award1995-01-01
Awarding institutionUniversity of Leicester