Purification and partial characterization of an acidic polygalacturonase from Aspergillus kawachii.

An endo-polygalacturonase, named PGI, was purified to homogeneity from the culture filtrate of Aspergillus kawachii IFO 4033 grown in a glucose-tryptone medium. The molecular mass of PGI was estimated to be 60 kDa by SDS-PAGE and 40 kDa by gel filtration on Sephacryl S-100. The isoelectric point was 3.55 as determined by isoelectic focusing. PGI exhibited binding properties to ConA-Sepharose suggesting that the protein is glycosylated. The N-terminal amino acid sequence was also determined as S-T-C-T-F-T-D-A-A-T-A-S-E-S-K. The remarkable property of PGI was its high activity in the pH range 2.0-3.0 towards soluble and insoluble substrates, while being inactive at pH 5.0. Enzyme stability at low pHs was markedly enhanced by different compounds, such as proteins, polysaccharides, simple sugars and the substrate pectin. PGI was very efficient to extract pectin from lemmon protopectin and to macerate carrot tissues at pH 2.0. These properties make PGI an interesting biocatalyst for industrial applications under highly acidic conditions.

The proteolytic system of the yeast Kluyveromyces lactis.

Major proteolytic activities were characterized in the yeast K. lactis NRRL 1118, grown in chemostat cultures. This yeast expressed proteolytic activities similar to those found in S. cerevisiae. This fact was particularly evident in the case of proteases such as PrA, PrB and CpY with regard to substrate specificity, activation at pH 5. 0 and inhibition patterns. The presence of a CpS activity could not be detected in either fresh or activated cell-free extracts by using the dipeptide N-Cbz-Gly-Leu, even in the presence of Zn(+2). On the other hand, K. lactis exhibits at least two major intracellular Ap activities different from those reported in other yeasts, and these seem to be carried out by closely related proteins. These activities corresponded to molecular masses of about 60 kDa, close pI values, and a similar behaviour in non-denaturing polyacrylamide electrophoresis. Both activities were enhanced by Co(+2) and inhibited by EDTA. Among different aminoacyl-p-NAs, they preferentially hydrolysed Lys-p-NA. No increase of Ap activity was obtained by incubation of extracts at acid pH. The maximum PrA and PrB activities detected in N-limited cultures were six-fold higher than those expressed under C- or P-limitation. The effect of culture conditions on the Cp and Ap expression was much less pronounced in comparison with PrA and PrB activities, Ap levels even being slightly higher in C-limited cells. This fact suggests that hydrolysis of protein to peptides might be the limiting step in the pathway of general protein degradation in the vacuole.

Aspergillus kawachii produces an acidic pectin releasing enzyme activity.

A pectin-releasing (protopectinase, PPase) activity was found in a culture filtrate of Aspergillus kawachii IFO 4308. PPase activity was highest in the pH range of 2.0-2.5 and it was highly stable at 50 degrees C (85% of residual activity was found after a 10-h incubation in citrate-phosphate buffer, pH 3.0). Among other different enzyme activities, which are usually involved in plant cell-wall degradation, only polygalacturonase activity was detected. This result suggests that the PPase activity could correspond to a particular kind of polygalacturonase. Pectin extraction from lemon peels carried out at 50 degrees C for 2 h (pH 3.5) gave yields of ethanol-precipitated pectin equivalent to 17.4% of the initial total solids contained in the peels. Thus, this enzyme activity would allow carrying out a pectin extraction process at lower reaction pHs and higher temperatures in comparison with similar reports using other PPases. These properties seem to be very interesting from the practical point of view.