Cryoprotective activity of mannoprotein from the cell membrane of Pichia anomala.

ABSTRACT

Some organisms like bacteria and plants have a cryoprotective protein to cryopreserve the freeze-labile enzyme under stable conditions having high activity. By screening of the cryoprotective activities of various food-industrial yeasts, we found that the cell membrane component that was a glucanase extractable component in Pichia anomala NBRC 141 had a high level of cryoprotective activity against freeze-labile lactate dehydrogenase (LDH). The absorption of the active compound in the crude extract by ConA-Sepharose chromatography suggested that this active compound might be a glycoprotein (COGP). Strain NBRC had the COGP constantly without the treatment of cold acclimation. The active compound, that is, a COGP, could be homogeneously purified using DEAE-TOYOPEARL and Sephacryl S-400 chromatography. The purified COGP had a cryoprotective activity of 50.9% at a sugar concentration of 17.9 microg/ml. The molecular weight of purified COGP was 83,000, which was composed of one protein with a molecular weight of 22,000 and polysaccharide. Furthermore, the constituent sugar of COGP was only D-mannose based on HPLC analysis of its acid hydrolysate. Also, we confirmed that the cryoprotective activity of COGP was higher than those of the commercial cell membrane components. The CP50 of COGP was 0.28 microM, which was half to the CP50 of BSA. This is the first report, to our knowledge, that the cell membrane component of Pichia anomala had a high level of cryoprotective activity against a freeze-labile enzyme.