Relatively poor heterologous protein yields have limited the commerical applications of Galactomyces geotrichum lipase I (GGl I) efficacy trials. To address this， we have redesigned the GGl I gene to preferentially match codon frequencies of Pichia pastoris (P. pastoris) while retaining the same amino acid sequence. The wild type and codon optimised GGl I (GGl I-wt and GGl I-op) were synthesised and cloned into pPICZαA with an N-terminal 6 × His tag sequence and expressed in P. pastoris X 33. The hydrolytic activity of GGl I-op was 150 U/mL， whereas the activity of the GGl I-wt could not be detected. GGl I-op recombinant proteins were purified by Ni-affinity chromatography and then characterised. The ... More
Relatively poor heterologous protein yields have limited the commerical applications of Galactomyces geotrichum lipase I (GGl I) efficacy trials. To address this， we have redesigned the GGl I gene to preferentially match codon frequencies of Pichia pastoris (P. pastoris) while retaining the same amino acid sequence. The wild type and codon optimised GGl I (GGl I-wt and GGl I-op) were synthesised and cloned into pPICZαA with an N-terminal 6 × His tag sequence and expressed in P. pastoris X 33. The hydrolytic activity of GGl I-op was 150 U/mL， whereas the activity of the GGl I-wt could not be detected. GGl I-op recombinant proteins were purified by Ni-affinity chromatography and then characterised. The identity and purity of GGl I were confirmed by SDS-PAGE， MALDI-TOF mass spectrometry and Western blot analysis. Enzymatic deglycosylation was used to show that the lipase is a glycosylated protein， containing ∼10% sugar. The molecular weight (MW) of the GGl I secreted by recombinant P. pastoris was approximated at 63 kDa. The optimum pH and temperature of the recombinant lipase were 8.0 and 35 °C， respectively. The enzyme was active over a broad pH range (7.0-9.0) and temperature range (20 °C-45 °C). The lipase showed high activity toward medium- and long-chain fatty acid methyl esters (C8-C16) and retained much of its activity in the presence of Tween-80 and Trition X-100. Lipase activity was stimulated by Mg， Ca， Mn and Cu and inhibited by Fe， Fe， Zn and Co. This lipase may prove useful to the detergent industry and in organic synthesis reactions.
