Departamento de Bioquimica e Microbiologia Instituto de Biociencias Universidade Estadual Paulista, UNESP, Avenida 24A, 1515, CEP 13506-900 Rio Claro, SP

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Pectin and Pectinases: Production, Characterization and Industrial Application of Microbial Pectinolytic Enzymes

A system was developed for the rapid characterization of microbial pectic enzyme complexes and then tested on Erwinia chrysanthemi and Sclerotium rolfsii. Pectic enzymes in minute samples of crude culture filtrates were resolved by ultrathin-layer polyacrylamide gel isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then assayed with an ultrathin pectate-agarose overlay stained with ruthenium red. The simple procedure can be completed within 30 min after isoelectric focusing, can detect extremely low levels of pectate lyase (6.4 x 10 mumol of product per min), and is sufficiently sensitive to determine the pectate lyase isozyme profile of a single bacterial colony with a diameter of 4 mm. Pectate lyases and polygalacturonases can be distinguished by altering buffer conditions in the overlays. The assay system revealed additional isozymes not resolved by classical techniques and generally corroborated the previously published isoelectric points and molecular weights of the pectate lyase isozymes and exo-poly-alpha-d-galacturonosidase produced by E. chrysanthemi and the endopolygalacturonase and exopolygalacturonase produced by S. rolfsii.

Activity stain for rapid characterization of pectic enzymes in isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gels.

Detection of depolymerase isoenzymes after electrophoresis or electrofocusing, or in titration curves

Pectin methyl esterase (PME) (EC 31111) catalyzes the hydrolysis of methylester groups of cell wall pectins We investigated the role of this enzyme in dormancy termination and germination of yellow cedar (Chamaecyparis nootkatensis [D Don] Spach) seeds PME activity was not detected in dormant seeds of yellow cedar but was induced and gradually increased during moist chilling; high activity coincided with dormancy breakage and germination PME activity was positively correlated to the degree of dormancy breakage of yellow cedar seeds The enzyme produced in different seed parts and in seeds at different times during moist chilling, germination, and early post-germinative growth consisted of two isoforms, both basic with isoelectric points of 87 and 89 and the same molecular mass of 62 kD The pH optimum for the enzyme was between 74 and 84 In intact yellow cedar seeds, activities of the two basic isoforms of PME that were induced in embryos and in megagametophytes following dormancy breakage were significantly suppressed by abscisic acid Gibberellic acid had a stimulatory effect on the activities of these isoforms in embryos and megagametophytes of intact seeds at the germinative stage We hypothesize that PME plays a role in weakening of the megagametophyte, allowing radicle emergence and the completion of germination

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An Increase in Pectin Methyl Esterase Activity Accompanies Dormancy Breakage and Germination of Yellow Cedar Seeds

The catalytic capacity of several excreted pectinolytic enzymes obtained from various yeast strains was examined using in vivo and biochemical techniques. Of the 33 yeast strains studied, 30 were isolated from champagne wine during alcoholic fermentation. Only one yeast strain was found to excrete pectinolytic enzymes and was identified as Saccharomyces cerevisiae and designated SCPP. Pulsed-field gel electrophoresis and the polymerase chain reaction technique were used to characterize further this specific strain. Three types of pectinolytic enzymes were found to be excreted by SCPP: polygalacturonase, pectin-lyase and pectin-esterase. These enzymes allow pectin hydrolysis during cell growth.

George C. Wade

Papers

Detection of pectic enzymes in pectin-acrylamide gels