High level production of a recombinant acid stable exoinulinase from Aspergillus kawachii

BARUQUE, DIEGO J.; GHIRINGHELLI, PABLO D.; WAGNER, EVELYN; CAVALITTO, SEBASTIÁN F.; WAGNER, EVELYN; CAVALITTO, SEBASTIÁN F.; CHESINI, MARIANA; VITA, CAROLINA E.; ROJAS, NATALIA L.; CHESINI, MARIANA; VITA, CAROLINA E.; ROJAS, NATALIA L.; BARUQUE, DIEGO J.; GHIRINGHELLI, PABLO D.

PROTEIN EXPRESSION AND PURIFICATION

ACADEMIC PRESS INC ELSEVIER SCIENCE

Año: 2018 vol. 149 p. 29 - 37

1046-5928

Exoinulinases, enzymesextensively studied in recent decades because of their industrial applications,should be produced in suitable quantities to support that demand. In thispaper, an acid stable recombinant inulinase from Aspergillus kawachii isproduced in the Pichia pastoris system and its biochemicalcharacteristics and potential application in industrial processes arepresented. After an appropriate cloning strategy, this recombinant enzyme issuccessfully overproduced in fed batch fermentations, reaching up to 840 U/mlafter 72 h cultivation. The protein was purified to homogeneity usingchromatographic techniques, obtaining 42% in yield. Biochemical characteristicswere determined: the enzyme had an optimal pH of 3; it is stable at least 3 hat 55 ºC, and Hg+2 inhibits almost completely its activity. Kmand Vmax for the recombinant inulinase using inulin as substrate were1.35 mM and 2673 U/min/mg. It was found that the recombinant enzyme is anexoinulinase, and it also shows synthetic activity (fructosyltransferase). Thehigh level of production of the enzyme plus its interesting biochemicalproperties, led to consider the process presented in this paper as a possibleresource for industrial applications in carbohydrate processing.