Comparative characterisation of esterases and lipases for biosensors
Lind, P. A. (2002). Comparative characterisation of esterases and lipases for biosensors (Thesis, Doctor of Philosophy (PhD)). The University of Waikato, Hamilton, New Zealand. Retrieved from https://hdl.handle.net/10289/14057
Permanent Research Commons link: https://hdl.handle.net/10289/14057
Twenty four carboxylesterases and lipases from various microbial sources were compared with commercial pig liver esterase in order to determine which would be most appropriate for use in a biosensor application and in the elucidation of the relationship between protein hydration and gas phase activity. This involved comparison of appropriate kinetic parameters against the esters ethyl acetate, methyl butyrate and ethyl butyrate and their equivalent p-nitrophenylated synthetic model substrates. Comparison of functional pH ranges of the various esterases revealed Lipase B from Candida rugosa to be amongst the least inhibited when pH fell to between 4 and 5. C. rugosa Lipase B also exhibited the highest specific activities towards p-nitrophenyl acetate and butyrate, hydrolysed all three of the above esters and did not show protease activity against azocasein. For these reasons C. rugosa Lipase B was judged to be the best suited to the ester biosensor application, and so was partially purified and further characterised. Investigation of the effects of acyl chain length of the ester substrates on C. rugosa Lipase B activity revealed two peaks of preferential activity across the range C₂ − C₁₈. The first of these peaks, at C₄ indicative of true carboxylesterase activity, was more marked than that of pig liver esterase. The specificity of Lipase B (Yₘₐₓ/Kₘ) for ethyl acetate was 58 and 23 times lower than that for methyl butyrate and ethyl butyrate, respectively (compared to 4.7 and 2.5 times lower, respectively for pig liver esterase). The second, larger peak in the substrate size preference profile, for a C₁₂ acyl chain length, indicated a predominance of lipase activity. Concurrent analysis of thermal- and pH-stability showed C. rugosa Lipase B to be stable below 40°C and between pH 5.5 and 7.0, with optimal stability at pH 5.5. In these respects, it is superior to pig liver esterase, which is highly susceptible to denaturation by storage above 30°C or below pH 5.0. The influence of enzyme hydration on the hydrolysis of ethyl butyrate by C. rugosa Lipase B and pig liver esterase was investigated through the vapor-solid mode approach where each enzyme was studied as a hydrated solid phase in equilibrium with a substrate vapor phase. The effect of enzyme hydration was evaluated through measurements made in controlled atmospheres of known relative humidity. Although it was previously concluded that enzyme activity required a minimal water content of 0.2 gH₂O.g⁻¹ protein (0.2 h), we have shown that activity is possible at much lower (<0.03 h) hydration. C. rugosa Lipase B and pig liver esterase were hydrolytically active when exposed to mixtures of ethyl butyrate vapor over a range of relative humidities from 7 to 90%, equivalent to C. rugosa Lipase B and pig liver esterase water contents of between 0.054-0.47 h and 0.029-0.60 h, respectively. C.rugosa Lipase B hydrolytic activity was highly dependent on the degree of protein hydration and exhibited a linear relationship with respect to protein water content. For pig liver esterase activity, the effect of increasing hydration on activity was less marked where the increase in PLE activity began to stabilize at protein water contents over 0.17 h. Although no activity was seen at 0 h, since the reaction catalyzed (the hydrolysis of ethyl butyrate) requires water as a second substrate, this work does not discount the possibility of activity at lower hydration.
The University of Waikato
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