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. Threedifferent cultivation strategies had been compared for the production of His₆-YlLip2 byP. 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 reached15,600 U/ml after 114 h of induction. The cell mortality decreased by 11.2% (while thecontrol 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