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Title: Characterisation of thermophilic esterases
Author: Wood, Andrew N. P.
Awarding Body: University of London
Current Institution: University College London (University of London)
Date of Award: 1995
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Abstract:
Seventy two strains of thermophilic Bacilli (optimum growth temperatures 55[degrees]C-75[degrees]C) were screened for esterase activity. 23 strains showed some esterase activity when assayed with various p-NP substrates. The growth characteristics and nutritional requirements of six of these strains were investigated. An esterase activity obtained from one of the strains (Tok19 A1) was purified 5133-fold to electrophoretic homogeneity with 26% recovery. The purified esterase had a specific activity of 2032 [mu]molmin-1 mg-1 based on the hydrolysis of p-nitrophenyl caproate at pH 7.0 and 30[degrees]C. The apparent molecular mass was 50,000 +/- 2,000 Da from SDS/polyacrylamide gel electrophoresis and 45,000 +/- 3000 Da from gel filtration. Native polyacrylamide gels stained for esterase activity showed three bands. The isoelectric points were estimated to be 5.7, 5.8 and 6.0. Forty amino acid residues were sequenced at the N-terminus. The sequence showed no degeneracy suggesting that the three esterases are functionally identical carboxylesterases differing by a limited number of amino acids. The enzyme showed maximum activity at pH 7.0 and was very stable at pH 6.0-8.9 with optimum stability at pH 6.0. At this pH and 60[degrees]C the half life was 170 hours. Esterase activity was totally inhibited by PMSF, p-CMB, eserine and TPCK but not by EDTA. The esterase obeyed Michaelis-Menten kinetics in the hydrolysis of p-nitrophenyl esters, but both and Vmax and KM were protein-concentration dependent. The esterase was also stabilised by both homogeneous and heterogeneous protein-protein interactions. The stability of the esterases increased by more than 300 fold when the esterase concentration was increased from 2[mu]g mL-1 to 10[mu]g mL-1 or when BSA was added to a concentration of 1mg mL-1. The esterase was activated by up to 115% in 1-2% v/v propanol and the half-life in 35% methanol v/v was 20 hours at 30[degrees]C. In immiscible linear alcohols C5-C10 (50% v/v) the esterase retained 100% of its activity after 24 hours at 40[degrees]C. The esterase also appeared to exhibit high levels of transferase activity, even in the presence of low concentrations of aliphatic alcohols. The esterase was covalently immobilised to a glyoxyl agarose gel and was 10,000, 1,200 and 900 fold more stable than the soluble esterase at pHs 5, 7 and 9 respectively. The immobilised esterase also retained 70% of its initial activity after incubation in 50% DMF or DMSO at 30[degrees]C for 7 days. The esterase had a broad substrate specificity hydrolysing linear and branched esters, aromatic esters (including indoxyl and naphthyl esters) and sugar acetates such as [beta]-D-xylose tetraacetate.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.807216  DOI: Not available
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