. "Structure and Function of Pectic Enzymes: Virulence Factors of Plant Pathogens." (NAS Colloquium) Virulence and Defense in Host--Pathogen Interactions: Common Features Between Plants and Animals. Washington, DC: The National Academies Press, 2001.
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COLLOQUIUM ON Virulence and Defense in Host—Pathogen Interactions: Common Features Between Plants and Animals
Table 1. Specific activities and kinetic parameters of wild-type and mutatedPLB
Specific activity, units/mg
PLB wild type
DNA Manipulations. Cloning experiments for the preparation of PLB mutants were performed in E. coli DH5α (36) by using standard protocols (37). Restriction enzymes were used as described by the supplier (GIBCO/BRL). Nucleotide sequences were determined by using a Cy5 AutoCycle Sequencing Kit (Amersham Pharmacia) with universal and reverse primers or gene specific primers. The reactions were analyzed with an ALFred DNA sequencer (Amersham Pharmacia). Computer analysis was done by using the program GENERUNNER (Hastings Software). The cloning of A. niger N400 pelB PLB was described previously (38). A translational promoter gene fusion was constructed by using the pyruvate kinase promoter (39). The pki-pelB fusion (pPK-PLB) was described previously (38).
Site-Directed Mutagenesis. Site-directed mutagenesis of PLB was carried out by using the Altered Sites II kit (Promega) and synthetic oligonucleotides (Isogen, Maarsen, The Netherlands). The procedure was performed as described (40). The pki-pelB promoter gene fusion was excised with pPK-PLB by using restriction endonucleases BamHI and HindIII and was ligated into BamHI- and HindIII-digested pALTER I, resulting in plasmid pIM3550. Plasmid DNA was isolated and sequenced to confirm the desired mutations and to check the gene for undesired mutations. Those plasmids showing the correct sequence and the expected mutation were used to transform A. niger strain NW1888 (cspAI, pyrA6, leu-13, prtF28), a derivative of A. niger N400 (CBS. 120.49). Transformations were carried out as described (41).
Culture Conditions and Enzyme Purifications. Mutant PLB producing transformants were selected by growing individual transformants in minimal medium as described (42). Large scale cultivation of transformants producing mutant PLB was performed as outlined (43) in multiple 300 milliliter batches in one liter Erlenmeyer flasks incubated in an orbital shaker (250 revolutions per minute) at 30 °C. Wild-type and mutant PLB were purified essentially as described by Kester and Visser (43) with the following modification. After dialysis of the solubilized ammonium sulfate precipitate, the dialysate was loaded onto a Source Q anion exchanger (15.5-milliliter bed volume) (Pharmacia) preequilibrated with 10 mM Tris·HCl (pH 7.5). Proteins were eluted with a 0–200 mM sodium chloride linear gradient in 10 mM Tris ·HCl (pH 7.5). PLB-containing fractions were pooled and exhaustively dialyzed against 20 mM sodium phosphate (pH 6.0). Enzyme solutions were stored at − 20°C. The purity of the mutant enzyme was confirmed by SDS/PAGE and Coomassie brilliant blue staining. The concentration of purified mutant enzymes was determined by measuring the absorbance at a wavelength of 280 nm, using a molar absorption coefficient ( ε) of 50,220 M−1·cm−1 for PLB, as calculated from the tryptophan, tyrosine, and cysteine content (44).
Enzyme Assay and Determination of Kinetic Parameters. Standard PLB assays were carried out in 50 mM Tris·HCl and 0.06 M sodium chloride (pH 8.5), containing 3 mg/ml (wt/vol) lime pectin with 75% methyl esterification (Copenhagen Pectin Factory, Lille Skensved, Denmark) in a total volume of 1.0 ml. The assay buffer was equilibrated at 30°C, and the reaction was started by the addition of 20 µl of enzyme solution. The activity was determined by measuring the increase in absorbance at 235 nm (ε = 5200 M−1·cm−1). The kinetic traces were corrected for spontaneous chemical β-elimination. Km and Vmax values were determined from triplicate initial rate measurements in the same way as described for the standard assays, with the exception that the pectin concentrations varied from 0.5 to 7.0 mg/ml.
The research was supported by the U.S. Department of Agriculture (Grant 98-35304). The research was conducted in part at the Stanford Synchrotron Radiation laboratory, which is operated by the Office of Basic Energy Science of the U.S. Department of Energy.
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