Culture filtrates prepared from strains of bacteria representing the genera Arthrobacter, Bacillus, Cytophaga, Erwinia, Flavobacterium and Xanthomonas were examined for their macerating activity at pH 6.0 and 50°C. A single strain of Bacillus (strain B3), identified as a growth factor requiring variant of B. subtilus, was selected for further examination. Potential ingredients for a commercially applicable medium for the cultivation of strain B3 were examined and a final formulation is recommended. The following activities were identified in culture filtrates prepared from cultures of strain B3 grown in the industrial medium, pectic acid lyase (PAL), pectin methyl esterase (PME), beta-arabino-furanosidase, xylopyranosidase, beta-galactopyranosidase and neutral protease. All of the enzymes identified were subject to catabolite repression. PAL was purified by ion-exchange chromatography and iso-electric focusing and its properties were examined. Optimum activity was shown at pH 8-9 and at 60-65°C. Calcium and to a lesser extent strontium, activated the enzyme, while EDTA inhibited activity. Time course experiments examining the end products and viscosity reduction of solutions of polygalacturonic acid indicated that PAL was an endo enzyme producing di and trigalacturonic acids as the major end products of activity. Endo-PAL proved to be a basic protein with a pi of 9.85 and a molecular weight of 33,000. Two peaks of maceration (pH 6-6.5 and 8-9) were produced by both culture filtrate and the purified lyase against carrot or potato. Evidence was obtained to show that the presence of lyase alone was responsible for the maceration of carrot at pH 6.0, but that it acted in conjunction with a heat-labile, high molecular weight factor extractable from carrot tissue. Carrot extracts were unable to macerate carrot but exhibited activity against polygalacturonic acid and it is suggested that the factor may be carrot exo-PG. Maceration at pH 8.5 was accounted for by the activities of PAL and PME.
|Date of Award||1975|