Production of perillic acid from orange essential oil by Yarrowia lipolytica using a top-aerated bioreactor.

R-(+)-Perillic acid, a promising anticancer and immunomodulatory agent, is the major product from the biotransformation of R-(+)-limonene-rich orange essential oil by the yeast Yarrowia lipolytica. Due to the abundance and low cost of orange essential oil, which is a byproduct of the citrus industry, we attempted to improve the biotransformation process by optimizing yeast cell mass production. Then, the whole process was transposed and adapted to a 2-L instrumented bioreactor. Cell mass production was optimized in shaker flasks using a statistical experimental design. The optimized medium (g·L-1: 22.9 glucose, 7.7 peptone, 4.1 yeast extract and 1.0 malt extract) resulted in a 13.0 g·L-1 final cell concentration and 0.18 g cell·L-1·h-1 productivity. A further increase to 18.0 g·L-1 was achieved in a 2-L bioreactor upon fed-batch culture. High-purity limonene bioconversion was performed in the same bioreactor utilizing top aeration to diminish terpene volatilization; as a result, 839.6 mg·L-1 perillic acid accumulated after 48 h. Under the same conditions, industrial orange essential oil afforded 806.4 mg·L-1 perillic acid. The yeast growth medium optimization resulted in a twofold increase in biomass accumulation and a reduction in growth medium nitrogen sources, which lowered the catalytic biomass production cost. Compared with conventional bottom aeration, the bioreactor top aeration strategy resulted in higher bioconversion rates. The conditions developed for high-purity limonene bioconversion were successfully applied to low-cost orange essential oil, showing the robustness of Y. lipolytica yeast.

ASP-Enzymosomes with Saccharomyces cerevisiae Asparaginase II Expressed in Pichia pastoris: Formulation Design and In Vitro Studies of a Potential Antileukemic Drug.

The bacterial enzyme asparaginase is the main treatment option for acute lymphoblastic leukemia. However, it causes side effects, such as immunological reactions, and presents undesirable glutaminase activity. As an alternative, we have been studying asparaginase II from Saccharomyces cerevisiae, coded by ASP3 gene, which was cloned and expressed in Pichia pastoris. The recombinant asparaginase (ASP) presented antileukemic activity and a glutaminase activity 100 times lower in comparison to its asparaginase activity. In this work, we describe the development of a delivery system for ASP via its covalent attachment to functionalized polyethylene glycol (PEG) polymer chains in the outer surface of liposomes (ASP-enzymosomes). This new delivery system demonstrated antiproliferative activity against K562 (chronic myeloid leukemia) and Jurkat (acute lymphocytic leukemia) cell lines similar to that of ASP. The antiproliferative response of the ASP-enzymosomes against the Jurkat cells suggests equivalence to that of the free Escherichia coli commercial asparaginase (Aginasa®). Moreover, the ASP-enzymosomes were stable at 4 °C with no significant loss of activity within 4 days and retained 82% activity up to 37 days. Therefore, ASP-enzymosomes are a promising antileukemic drug.

Expression, purification, and characterization of asparaginase II from Saccharomyces cerevisiae in Escherichia coli.

l-asparaginase catalyzes the conversion of l-asparagine to l-aspartate and ammonium. This protein is an important therapeutic enzyme used for the treatment of acute lymphoblastic leukemia. In this study, the asparaginase II-encoding gene ASP3 from Saccharomyces cerevisiae was cloned into the expression vector pET28a in-fusion with a 6x histidine tag and was expressed in Escherichia coli BL21 (DE3) cells. The protein was expressed at a high level (225.6 IU/g cells) as an intracellular and soluble molecule and was purified from the supernatant by nickel affinity chromatography. The enzyme showed very low activity against l-glutamine. The denaturing electrophoresis analysis indicated that the recombinant protein had a molecular mass of ∼38 kDa. The native enzyme was a tetramer with a molecular mass of approximately 178 kDa. The enzyme preparation showed antitumor activity against the K562 and Jurkat cell lines comparable or even superior to the E. coli commercial asparaginase.

Saccharomyces cerevisiae asparaginase II, a potential antileukemic drug: Purification and characterization of the enzyme expressed in Pichia pastoris.

Asparaginase obtained from Escherichia coli and Erwinia chrysanthemi are used to treat acute lymphocytic leukaemia and non-Hodgkin's lymphoma. However, these agents cause severe adverse effects. Saccharomyces cerevisiae asparaginase II, encoded by the ASP3 gene, could be a potential candidate for the formulation of new drugs. This work aimed to purify and characterize the periplasmic asparaginase produced by a recombinant Pichia pastoris strain harbouring the ASP3 gene. The enzyme was purified to homogeneity with an activity recovery of 51.3%. The estimated molecular mass of the enzyme was 136 kDa (under native conditions) and 48.6 kDa and 44.6 kDa (under reducing conditions), suggesting an oligomeric structure. The recombinant asparaginase is apparently non-phosphorylated, and the major difference between the monomers seems to be their degree of glycosylation. The enzyme showed an isoelectric point of 4.5 and maximum activity at 46 °C and pH 7.2, retaining 92% of the activity at 37 °C. Circular dichroism and fluorescence analyses showed that the enzyme structure is predominantly α-helical with the contribution of ß-sheet and that it remains stable up to 45 °C and in the pH range of 6-10. In vitro tests indicated that the recombinant asparaginase demonstrated antitumoural activity against K562 leukaemic cells.

Bioconversion of R-(+)-limonene to perillic acid by the yeast Yarrowia lipolytica

Perillyl derivatives are increasingly important due to their flavouring and antimicrobial properties as well as their potential as anticancer agents. These terpenoid species, which are present in limited amounts in plants, may be obtained via bioconversion of selected monoterpene hydrocarbons. In this study, seventeen yeast strains were screened for their ability to oxidize the exocyclic methyl group in the p-menthene moiety of limonene into perillic acid. Of the yeast tested, the highest efficiency was observed for Yarrowia lipolytica ATCC 18942. The conversion of R (+)-limonene by Y. lipolytica was evaluated by varying the pH (3 to 8) and the temperature (25 to 30 ºC) in a reaction medium containing 0.5% v/v limonene and 10 gµL of stationary phase cells (dry weight). The best results, corresponding to 564 mgµL of perillic acid, were obtained in buffered medium at pH 7.1 that was incubated at 25 ºC for 48 h. The stepwise addition of limonene increased the perillic acid concentration by over 50%, reaching 855 mgµL, whereas the addition of glucose or surfactant to the reaction medium did not improve the bioconversion process. The use of Y. lipolytica showed promise for ease of further downstream processing, as perillic acid was the sole oxidised product of the bioconversion reaction. Moreover, bioprocesses using safe and easy to cultivate yeast cells have been favoured in industry.

Bioconversion of R-(+)-limonene to perillic acid by the yeast Yarrowia lipolytica.

Perillyl derivatives are increasingly important due to their flavouring and antimicrobial properties as well as their potential as anticancer agents. These terpenoid species, which are present in limited amounts in plants, may be obtained via bioconversion of selected monoterpene hydrocarbons. In this study, seventeen yeast strains were screened for their ability to oxidize the exocyclic methyl group in the p-menthene moiety of limonene into perillic acid. Of the yeast tested, the highest efficiency was observed for Yarrowia lipolytica ATCC 18942. The conversion of R (+)-limonene by Y. lipolytica was evaluated by varying the pH (3 to 8) and the temperature (25 to 30 °C) in a reaction medium containing 0.5% v/v limonene and 10 g/L of stationary phase cells (dry weight). The best results, corresponding to 564 mg/L of perillic acid, were obtained in buffered medium at pH 7.1 that was incubated at 25 °C for 48 h. The stepwise addition of limonene increased the perillic acid concentration by over 50%, reaching 855 mg/L, whereas the addition of glucose or surfactant to the reaction medium did not improve the bioconversion process. The use of Y. lipolytica showed promise for ease of further downstream processing, as perillic acid was the sole oxidised product of the bioconversion reaction. Moreover, bioprocesses using safe and easy to cultivate yeast cells have been favoured in industry.