Genetic and biochemical studies of microbial peptidase enzymes

Nathan, P.B., 1989. Genetic and biochemical studies of microbial peptidase enzymes. PhD, Nottingham Trent University.

Preview

Text 10183123.pdf - Published version Download (37MB) | Preview

Abstract

Investigations were undertaken on the microbial peptidase enzymes catalysing hydrolysis of L-a-aspartyl peptides, particularly those peptidases synthesised by the bacterium Escherichia coli K12. Bacterial strains, though not all fungal strains tested, used peptides as sources of required nutrients. All microbial strains, however, possessed aspartyl peptidase activities, demonstrable using a PAGE based activity stain. E. coli K12 produced 2 bands of aspartyl peptidase activity, mobilities 0.3 and 0.5 on 7.5% polyacrylamide gels, the former corresponding to the previously described peptidase B, and the latter an aspartyl peptide specific activity previously undescribed in this organism, designated peptidase E. Growth tests on peptidase recombinants indicated a third enzyme, peptidase Q, a prolyl peptidase, catalysed hydrolysis of Asp-Pro. Peptidase B and E activities, along with an Acinetobacter calcoaceticus aspartyl- ?-napthylamide (ANA) hydrolysing peptidase were partially purified by ammonium sulphate fractionnation, ion-exchange chromatography and lastly gel filtration, which was used to estimate molecular weights (M ) at 230kd, 35kd and 480kd respectively. These peptidases catalysed hydrolysis of N-terminal aspartyl peptides but not C-terminal aspartyl, N-terminal asparaginyl or glutamyl peptides, and all showed optimal activity at pH 7.8 with both peptidase E and ANA hydrolysing activities remaining high at pH 10. Sulphydryl and serine protease inhibitors did not significantly affect peptidase activities whereas the metal-ion chelator EDTA inhibited peptidase B and ANA hydrolysing activities though not peptidase E. Isolation of E. coli K12 mutants lacking aspartyl peptidase activity or unable to utilise aspartyl peptides proved impossible, however the gene encoding peptidase B was mapped at 57.5 minutes on the genome by interrupted mating and PI transduction. Levels of peptidase B and E activities were higher in stationary phase than exponential cells, and higher in glycerol grown than glucose grown cells, but activity levels were unaffected by inclusion of aspartyl peptides in growth media. No synthesis of the aspartyl dipeptide sweetener, aspartame (Asp-PheOMe), was observed when whole bacterial cells, crude cell extracts and partially purified peptidases were used as catalysts for sweetener synthesis under various experimental conditions.

Item Type:	Thesis
Creators:	Nathan, P.B.
Date:	1989
ISBN:	9781369314045
Identifiers:	Number Type PQ10183123 Other
Divisions:	Schools > School of Science and Technology
Record created by:	Jeremy Silvester
Date Added:	18 Sep 2020 08:45
Last Modified:	19 Jul 2023 10:45
URI:	https://irep.ntu.ac.uk/id/eprint/40790

Actions (login required)

Edit View

Views

Downloads