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ISOLATION OF MUCOR MIEHEI AND M. PUSILLUS ASPARTIC PROTEINASES FROM PARTIALLY PURIFIED SOURCES USING PREPARATIVE ISOELECTRIC FOCUSING
Author(s) -
WYNNE M.G.,
YADA R.Y.
Publication year - 1991
Publication title -
journal of food biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.507
H-Index - 47
eISSN - 1745-4514
pISSN - 0145-8884
DOI - 10.1111/j.1745-4514.1991.tb00420.x
Subject(s) - chromatography , isoelectric point , isoelectric focusing , size exclusion chromatography , chemistry , fractionation , yield (engineering) , enzyme , biochemistry , materials science , metallurgy
Dialysis, preparative isoelectric focusing (IEF) and gel filtration were employed to obtain final enrichments of 2.2‐fold and 1.4‐fold for Mucor miehei ( CBS 360.75) proteinase (MMP) and M. pusillus (var. Lindt) proteinase (MPP), respectively, from partially purified sources. Recovery of total activity (percent yield) of MMP was 38% following preparative IEF and 6.7% after gel filtration. Percent yield of MPP was 61% following preparative IEF and 15% after gel filtration. Preparative IEF fractionation with a lowpH (3–5) ampholyte produced most of the increase, and partitioned brown pigments into fractions of pH 2.0 to 3.5. The protocol produced proteinases of high purity as indicated by SDS‐PAGE. The isoelectric point, molecular weight, extinction coefficient and amino acid composition of MMP and MPP proteinases were in agreement with previously determined values. Secondary structure analysis confirmed previous findings that these enzymes contain a high proportion of β‐sheet structure. Purifications starting with 4 to 5 g of crude partially purified MMP preparation (at least 50% NaCl) yielded between 18 and 28 mg of purified protein.