Yasuhide Ota

Isolation and Identification of Alkaline Lipase Producing Microorganisms, Cultural Conditions and Some Properties of Crude Enzymes

Two bacterial strains 26.IB (I) and 22.39B (II) were isolated from soil as alkaline lipase producing microorganisms. Strain 26. IB (I) was identified as Pseudomonas nitroreducens nov. var. thermotolerans, and strain 22.39B (II) was identified as Ps. fragi. When they were cultivated aerobically in a 20-liter jar fermentor in medium containing 2.0% soybean meal, they secreted a large amount of alkaline lipases. The enzymes were recovered efficiently as precipitates by adjusting the pH of the culture fluids to 4.0 with HC1. The enzymatic characteristics included: optimum pH, 9.5 (I,II); stable pH range, 5~11 (I,II); optimum temperatures, 50°C (I) and 75~80°C (II); and more than 95% of the enzyme activity remained after incubated at 70°C for 20 min (I,II). Lipase activities were inhibited remarkably in the presence of anionic surfactants (I,II) and bile salts (I,II).

Lipase from Candida paralipolytica: Part I. Anionic Surfactants as the Essential Activator in the Systems Emulsified by Polyvinyl Alcohol

The extracellular lipase from Candida paralipolytica required bile salts as the essential activator in the reaction mixture emulsified by polyvinyl alcohol. It has been found that some anionic surfactants can be used as the activator. The effect of bile salts and anionic surfactants on lipase activity has been studied in detail. Some cationic and non-ionic surfactants were not the activator but the inhibitor of the lipase.

Studies on Lipase from Candida cylindracea :Part I. Purification and Properties

Lipase from Candida cylindracea has been purified by ammonium sulfate precipitation, sodium deoxycholate treatment, ethanol-ether precipitation and chromatography on SESephadex and Sephadex G-100 columns. The purification of the enzyme was 33.4-fold with a yeild of 18.0 % on the basis of activity per weight of protein. The purified enzyme was homogeneous on ultracentrifugation and electrophoresis. Optimum pH for the hydrolysis of olive oil was 7.2 by the assay method using a polyvinylalcohol-emulsified system and 5.2 by the assay method using a shaken system without a macromolecular emulsifier. Optimum temperature was 45°C. The enzyme was stable up to 15°C and in the range of pH from 2.0 to 8.5. Sodium taurocholate showed either an activating or an inhibiting effect at pH 7.0, depending on the sodium taurocholate concentration and on the assay system.

Lipase from Candida paralipolytica:Part II. Alkaline Earth Metal Ions as the Cofactor in the Shaken System Containing no Emulsifier

The extracellular lipase from Candida paralipolytica required alkaline earth metal ion as the cofactor*2 in the reaction mixture not emulsified but dispersed by shaking, contradicting the fact that it required bile salt or anionic surfactant as the essential activator*3 in the systems emulsified with polyvinyl alcohol, as previously reported.1) The two kinds of factors necessary to activate both reaction systems respectively were unexchangeable for each other. These facts would be direct evidences of the difference of interfacial nature between two substrate forms prepared from the same substrate.The zero-order reaction has been observed under the non-emulsified conditions and the activation mechanism by alkaline earth metal ions has been studied partly.

Lipase from Candida paralipolytica :Part III. Further Studies on the Activation of the Enzyme Systems with Bile- or Calcium Salts

The extracellular lipase from Candida paralipolytica required essential activators* (usually bile- or calcium salts) for the in vitro hydrolysis of triglycerides. The reaction systems emulsified with gum arabic, gelatin, lecithin, methyl cellulose, pectin, polyvinyl alcohol, sodium cholate, or without emulsifier were compared concerning requirement for essential activator, inhibition with sodium chloride and maximum reaction rate, and the following findings have been obtained. (1) The emulsions used can be classified into five groups by the essential activator requirement. (2) The inhibition with sodium chloride depended on reaction system. (3) Each reaction system gave a similar reaction rate at pH 8.2. (4) Long-chain fatty acid dissolved in substrate was necessary to the activation with calcium salts.