Purification and properties of an polygalacturonic acid trans-eliminase produced by Bacillus pumilus.
A strain of Bacillus pumilus produced an extracellular pectic enzyme with polygalacturonic acid as the substrate. This enzyme, with optimal activity at pH 8.0 to 8.5, produced reaction products that strongly absorbed light at 232 nm, indicating the presence of a pectic acid trans-eliminase (PATE). Neither pectin esterase nor polygalacturonase was detected in the cell-free culture fluid. Chromatographic examination of the end products revealed the presence of large quantities of unsaturated oligouronides unlike those found with B. polymyxa. It was found that the PATE was produced extracellularly during the negative logarithmic death phase of the organism. The filtrate from sonically treated cells did not show any activity for PATE or hydrolases for lower oligogalacturonides at any time during the growth cycle. The enzyme was inducible. Pectin, National Formulary (NF) was the best inducer, followed by polygalacturonic acid and galacturonic acid. Enzyme activity was markedly stimulated by calcium and other divalent ions. Copper and cobalt ions were inhibitory. The partially purified enzyme showed no significant activity on pectin containing a high methoxyl content (96% esterified). However, pectin NF with a lower methoxyl content (68% esterified) was attacked to a degree by the partially purified and crude enzyme preparations. The initial rate of PATE activity increased up to 60 C, about 16-fold higher than that observed at room temperature. The activation energy was calculated as 12,183 cal/mole. A protective action of calcium chloride against heat inactivation of the PATE was observed. Degradation of polygalacturonic acid by this enzyme produced several unsaturated oligouronides soon after its addition to the substrate. The major endproduct was thought to be different from that of other known PATE enzymes. Paper chromatographic studies and viscosity measurements disclosed the random cleaving nature of the enzyme an endo-PATE.
D000645:Ammonium Sulfate; D001407:Bacillus / Q000201:enzymology* / Q000254:growth & development; D002122:Calcium Chloride / Q000494:pharmacology; D002130:Calcium Phosphates; D002474:Cell-Free System; D011232:Chemical Precipitation; D002848:Chromatography, DEAE-Cellulose; D002854:Chromatography, Paper; D003470:Culture Media; D004790:Enzyme Induction; D005782:Gels; D006863:Hydrogen-Ion Concentration; D008190:Lyases / Q000032:analysis / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D008722:Methods; D010368:Pectins / Q000378:metabolism; D013045:Species Specificity; D013696:Temperature; D014574:Uronic Acids / Q000378:metabolism; D014783:Viscosity
D002130:Calcium Phosphates; D003470:Culture Media; D005782:Gels; D014574:Uronic Acids; D010368:Pectins; D008190:Lyases; D002122:Calcium Chloride; D000645:Ammonium Sulfate