Perfusion chromatography separation of the tomato fruit-specific pectin methylesterase from a semipurified commercial enzyme preparation.

A rapid and simple method was developed, using perfusion chromatography media, to separate the fruit-specific pectin methylesterase (PME) isoform from the depolymerizing enzyme polygalacturonase (PG) and other contaminating pectinases present in a commercial tomato enzyme preparation. Pectinase activities were adsorbed onto a Poros HS (a strong cation exchanger) column in 20 M HEPES buffer at pH 7.5. The fruit-specific PME was eluted from the column with 80 mM NaCl, followed by a step to 300 mM NaCl to elute PG activity. Rechromatography of the PME activity peak with a linear gradient further resolved two PME isoenzymes and removed residual traces of PG activity. The PG activity peak was further treated with lectin affinity chromatography to provide purified PG enzyme, which was separated from a salt-dependent PME (tentatively identified as a "ubiquitous-type" isoform), and a pectin acetylesterase. The later enzyme has not been reported previously in tomato. This method provides monocomponent enzymes that will be useful for studying enzyme mechanisms and for modifying pectin structure and functional properties.

Hexose oxidase from Chondrus crispus: improved purification using perfusion chromatography(*).

1An improved method for purifying hexose oxidase (D-hexose: O(2) 1-oxidoreductase, EC 1.1.3.5) from the marine red alga Chondrus crispus is described for obtaining enzyme suitable for structural characterization and use in bioconversion of lactose to lactobionic acid. This involved extracting enzyme from finely ground lyophilized tissue in sodium phosphate buffer (pH 7) containing 20% ammonium sulfate, eliminating the previously used solvent extraction and protease treatments, and by applying Poros perfusion chromatography media to achieve rapid separations of high resolution. Primary separation of contaminating phycobiliproteins and carrageenans was achieved using Poros DEAE-50. Sequential HPLC purification steps using Poros HP2 and Poros HQ were followed by Sephacryl S200 h chromatography. Enzyme activity was determined with a peroxidase-coupled assay using 2,2'-azino-bis (3-ethylbenzthiazoline-6 sulfonic acid) substrate. A final specific activity of 69 U/mg was obtained, representing a 100-fold purification with an activity recovery of about 10%. A native size of approximately 117,000 Da was determined by size exclusion chromatography, and SDS-PAGE revealed the presence of 38,000 and 29,000 Da polypeptides that appear to be derived from a 65,000 Da subunit. Further properties of the enzyme are described.

Characterization of two novel type I ribosome-inactivating proteins from the storage roots of the andean crop Mirabilis expansa.

Two novel type I ribosome-inactivating proteins (RIPs) were found in the storage roots of Mirabilis expansa, an underutilized Andean root crop. The two RIPs, named ME1 and ME2, were purified to homogeneity by ammonium sulfate precipitation, cation-exchange perfusion chromatography, and C4 reverse-phase chromatography. The two proteins were found to be similar in size (27 and 27.5 kD) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and their isoelectric points were determined to be greater than pH 10.0. Amino acid N-terminal sequencing revealed that both ME1 and ME2 had conserved residues characteristic of RIPs. Amino acid composition and western-blot analysis further suggested a structural similarity between ME1 and ME2. ME2 showed high similarity to the Mirabilis jalapa antiviral protein, a type I RIP. Depurination of yeast 26S rRNA by ME1 and ME2 demonstrated their ribosome-inactivating activity. Because these two proteins were isolated from roots, their antimicrobial activity was tested against root-rot microorganisms, among others. ME1 and ME2 were active against several fungi, including Pythium irregulare, Fusarium oxysporum solani, Alternaria solani, Trichoderma reesei, and Trichoderma harzianum, and an additive antifungal effect of ME1 and ME2 was observed. Antibacterial activity of both ME1 and ME2 was observed against Pseudomonas syringae, Agrobacterium tumefaciens, Agrobacterium radiobacter, and others.

Biosynthesis of defense-related proteins in transformed root cultures of Trichosanthes kirilowii Maxim. var japonicum (Kitam.).

We have established transformed ("hairy") root cultures from Trichosanthes kirilowii Maxim. var japonicum Kitam. (Cucurbitaceae) and four related species to study the biosynthesis of the ribosome-inactivating protein trichosanthin (TCN) and other root-specific defense-related plant proteins. Stable, fast-growing root clones were obtained for each species by infecting in vitro grown plantlets with Agrobacterium rhizogenes American Type Culture Collection strain 15834. Each species accumulated reproducibly a discrete protein pattern in the culture medium. Analysis of the extracellular proteins from T. kirilowii var japonicum root cultures showed differential protein accumulation in the medium during the time course of growth in batch cultures. Maximum protein accumulation, approaching 20 micrograms/mL, was observed at mid-exponential phase, followed by a degradation of a specific protein subset that coincided with the onset of stationary phase. Two major extracellular proteins and one intracellular protein, purified by ion-exchange and reverse-phase high-performance liquid chromatography, were identified as class III chitinases (EC 3.2.1.14) based on N-terminal amino acid sequence and amino acid composition homologies with other class III chitinases. The Trichosanthes chitinases also showed reactivity with a cucumber class III chitinase antiserum and chitinolytic activity in a glycol chitin gel assay. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blot analysis of intracellular proteins showed that normal and transformed T. kirilowii var japonicum roots accumulated only low levels of TCN (approximately 0.5% total soluble protein). Storage roots from the plant displayed protein and antigen patterns different from root cultures and produced TCN as the dominant protein. Roots undergoing secondary growth and differentiation exhibited patterns similar to those of storage roots, including increased TCN levels, indicating that high production of TCN is associated with induction of secondary growth in roots.