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Combined strategies for the improvement of heterologous expression of a His-tagged <i>Yarrowia lipolytica lipase</i> Lip2 in <i>Pichia pastoris</i>


X Wang
X Shen
H Zhao
Y Sun
T Liu
Y Liu
L Xu
Y Yan

Abstract

Yarrowia lipolytica lipase Lip2 (YlLip2) is an important biocatalyst for ester synthesis, biodiesel production and enantiomer resolution. The YlLip2 with an N-terminal  histidine-tag (His6-YlLip2) was successfully expressed in Pichia pastoris. Three different cultivation strategies had been compared for the production of His6-YlLip2 by P. pastoris using a 10-l bioreactor. The results showed that His6-YlLip2 activity and cell viability could be greatly improved by employing the combined strategies. Using a low salt medium (LSM) instead of the basal salt medium (BSM) and lowering the temperature from 28 to 25°C, the maximum His6-YlLip2 activity and volumetric productivity were respectively increased by 55.3 and 79.8%. The production of His6-YlLip2 and cell viability was further improved by combining sorbitol co-feeding with methanol. In this culture strategy, the maximum activity of His6-YlLip2 reached 15,600 U/ml after 114 h of induction. The cell mortality decreased by 11.2% (while the control decreased about 27.6%) after 120 h methanol induction. The N-terminal histidine-tag brought convenience to purification. The molecular weight of His6-YlLip2 was about 38 kDa. The pure His6-YlLip2 presented a specific activity of 4,830 U/mg when olive oil was used as the substrate.

Key words: Pichia pastoris, Yarrowia lipolytica, combined strategies, fed-batch culture, purification, lipase.


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eISSN: 1684-5315