A method for the preparation ofTetrahymena thermophilaphospholipase A₁suitable for large‐scale production

Abstract

A rapid and economical method for the purification of phospholipase A1 (PLA1) from the extracellular medium of the ciliate Tetrahymena thermophila is presented. Essentially, the procedure, here designated as purification by selective interaction (PSI), entails the incubation of media containing PLA1 with liposomes made of soy bean phospholipids. The PLA1‐lipid complexes are precipitated by the addition of CaCl2 and collected by centrifugation. Elution of the PLA1 is effected by treating the complexes with 40% dimethylformamide, a reversible inhibitor of this enzyme, which is easily removed by dialysis. In combination with DEAE cellulose ion exchange chromatography, PSI yielded homogeneous PLA1 preparations with a 14% recovery and a 416‐fold increase in specific activity. This procedure, which can be completed within 1 day, may prove useful for the isolation of phospholipases from other sources. This practical method for the purification of a microbial PLA1 opens the way to large‐scale production of these types of enzyme, which are not as yet commercially available.

Screening of phospholipase A activity and its production by new actinomycete strains cultivated by solid-state fermentation

“…Similarly, a native PLA 1 from Streptomyces albidoflavus produced 1.5 U/mL of phospholipase activity in the culture supernatant (Sugimori, Kano & Matsumoto, 2012) determined using a fluorometric method with a BODIPY(R) fluorescent dye-labeled substrate. In other hand, a microbial PLA 1 was isolated from a mutant strain of the ciliated protozoan Tetrahymena thermophile and had 0.068 U/mL of phospholipase activity in the crude extract (Guberman et al, 1999) determined with the radioactive substrate 1-palmitoyl-2-[1- 14 C]linoleoylphosphatidylcholine. Despite the differences in activity quantification, comparison of these values with the PLA activities obtained here with the cHTS-PLA assay using PC as a substrate (2.59 U/gS (3.45 U/mL) for 2F-1, 1.93 U/gS (2.57 U/mL) for 6C-3 and 1.53 U/gS (2.04 U/mL) for 2F-2 strain), clearly demonstrates that their production by SSF with sugar-cane bagasse at the conditions employed here, appears to improve profiles for native microbial PLA production.…”

Phospholipases A1

“…Lyso-PLs are currently produced commercially via their chemical synthesis [103] or via the action of extracts from porcine pancreas, which contains a PLA 2 . Viable alternative processes produce lyso-PLs enzymatically and with greater efficiency are being investigated, recently reviewed in [104]. …”

Phospholipases Used in Lipid Transformations

“…Enzymes possessing PLA 1 activity have been described as intracellular, membranebound or soluble enzymes from rat liver lysosomes (Waite et al, 1981), bovine testis (Higgs et al, 1998), Aspergillus oryzae , E. coli (Scandella and Kornberg, 1971;Nakagawa et al, 1991), Corticium centrifugum (Uehara et al, 1979), hornet venom (Soldatova et al, 1993), tobacco hornworm (Nieder and Law, 1983) and other sources (references in Van den Bosch, 1980) as well as extracellular forms from Tetrahymena pyriformis (Arai et al, 1986) or Tetrahymena thermophila (Guberman et al, 1999). Hitherto, PLA 1 , however, has been poorly studied systematically and scarcely applied in biocatalysis (see Section 12.5.…”