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Competitive inhibition by substrates of the esterification reaction between l-phenylalanine and d-glucose catalysed by the lipases of Rhizomucor miehei and Candida rugosa

Abstract

A kinetic study on esterification between d-glucose and l-phenylalanine catalysed by lipases from Rhizomucor miehei (RML) and Candida rugosa (CRL) in organic media investigated in detail showed that both the lipases followed a Ping-Pong Bi-Bi mechanism with two distinct types of competitive inhibitions. Graphical double reciprocal plots and computer simulation studies showed that competitive double substrate inhibition took place at higher concentrations leading to dead-end inhibition in the case of RML and in the case of CRL, inhibition only by d-glucose at higher concentrations leading to dead-end lipase–d-glucose complexes. An attempt to obtain the best fit of these kinetic models through curve-fitting yielded in good approximation, the apparent values of important kinetic parameters, RML: k cat = 2.24 ± 0.23 mM h−1 (mg protein)−1, K m l-phenylalanine = 95.6 ± 9.7 mM, K m d-glucose = 80.0 ± 8.5 mM, K i l-phenylalanine = 90.0 ± 9.2 mM, K i d-glucose = 13.6 ± 1.42 mM; CRL: k cat = 0.51 ± 0.06 mM h−1 (mg protein)−1, K m l-phenylalanine = 10.0 ± 0.98 mM, K m d-glucose = 6.0 ± 0.64 mM, K i d-glucose = 8.5 ± 0.81 mM.

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Acknowledgements

Financial support from the Department of Science and Technology, India, is gratefully acknowledged. KL thanks the Council of Scientific and Industrial Research, India, for providing the Senior Research Fellowship. KL also thanks B.R. Somashekar for his help in several experiments.

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Correspondence to Soundar Divakar.

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Lohith, K., Manohar, B. & Divakar, S. Competitive inhibition by substrates of the esterification reaction between l-phenylalanine and d-glucose catalysed by the lipases of Rhizomucor miehei and Candida rugosa . World J Microbiol Biotechnol 23, 955–964 (2007). https://doi.org/10.1007/s11274-006-9320-4

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Keywords

  • Candida rugosa lipase
  • Competitive substrate inhibition
  • Dead-end lipase–substrate complex
  • d-Glucose
  • l-Phenylalanine
  • Ping-Pong Bi-Bi mechanism
  • Rhizomucor miehei lipase