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Biotechnology and Applied Biochemistry (2000) 32, (179–187) (Printed in Great Britain)

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Characterization of recombinant invertase expressed in methylotrophic yeasts

Niuris Acosta*^1,2, Alejandro Beldarraín†², Luis Rodríguez* and Yamirka Alonso*

*Bioindustry Department, Center for Genetic Engineering and Biotechnology, P.O. Box 6162, Havana, Cuba, and †Processes and Systems Evaluation Department, Production Plant, Center for Genetic Engineering and Biotechnology, P.O. Box 6162, Havana, Cuba

Abbreviations used: DSC, differential scanning calorimetry; InvHp, recombinant invertase from Hansenula polymorpha; InvPp, recombinant invertase from Pichia pastoris; InvSc, invertase from Saccharomyces cerevisiae; T_m, melting temperature; DH^cal, calorimetric enthalpy of denaturation; C_p, heat capacity.

¹ To whom correspondence should be addressed (e-mail niury.acosta@cigb.edu.cu).

² These authors contributed equally to this paper.

We studied, for the first time, characterization of the invertase expressed in the methylotrophic yeasts Hansenula polymorpha and Pichia pastoris in terms of enzyme conformational stability and structural behaviour induced by temperature as a function of pH using enzymic assays, differential scanning calorimetry, fluorescence and CD. The enzyme produced in both hosts was very stable over a broad range of pH values, keeping its enzymic activity and structure above 60 °C. Thermal denaturation, as measured by differential scanning calorimetry, was always irreversible. However, the fact that scanning rate had no effect on the calorimetric curves gave us the chance to analyse the data from a thermodynamic point of view. The conformational stabilities were essentially identical under the experimental conditions studied, but stability was always slightly higher in the enzyme expressed in H. polymorpha. This fact indicates that the greater degree of glycosylation of this enzyme form contributed to its increased global stability. Reactivation upon heating at 80 °C depends on protein concentration, suggesting that irreversibility could be associated with slow refolding kinetics at high protein concentration.

Received 21 August 2000; accepted 4 September 2000

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