Endo-pectate Lyase Research Articles

Cloning and expression in Escherichia coli of pectinase genes of Erwinia carotovora subsp. carotovora.

Genes coding for an endo-pectate lyase, an exo-pectate lyase, and an endopolygalacturonase of Erwinia carotovora subsp. carotovora Ecc71 were cloned in Escherichia coli HB101, using the cosmid pHC79. The products of the cloned pectinase genes paralleled their counterparts in strain Ecc71 in isoelectric mobility, mode of substrate degradation, and ability to macerate potato tuber tissue.

Biochemical pulping. Part XV. Strain dependence of pI-isozyme distribution of endo-pectate lyases secreted by Erwinia carotovora.

Biochemical pulping. Part XV. Strain dependence of pI-isozyme distribution of endo-pectate lyases secreted by Erwinia carotovora.

Cell wall-degrading enzymes produced by Penicillium oxalicum Curie et Thom

Penicillium oxalicum Curie et Thom produced an exo-polygalacturonase, an endo-polygalacturonase and an endo-pectate lyase. It also produced cellulase Cx, cellobiase, B-glucosidase, xylanase, galactanase and arabinanase in culture and in infected yam tissues. The exo-polygalacturonase was unstable. It has a molecular weight of about 38 000 daltons. The endopolygalacturonase and endo-pectate lyase had an optimal pH of 5.0 and 8.5 and an isoelectric point (pI) at pH 3.6 and 4.9 respectively. Molecular weights of both enzymes were about 28 500 and 30 000 daltons respectively. The end products of the reaction were oligogalacturonides and revealed that cellulose was degraded to glucose, with cellobiose as an intermediate product. The end products of the degradation of hemicelluloses were xylose, galactose and arabinose.

Biochemical pulping. Part XI. Isolation and fundamental properties of endo-pectate lyase pI-isozymes from Erwinia carotovora.

A strain of Erwinia carotovora was found to produce extracellularly four kinds of endo-pectate lyase pl-isozyme (PATE-I, -II, -III and -IV) having similar properties. The four pl-isozymes were purified to homogenous states by ion exchange chromatography, gel filtration and isoelectric focusing. The approximate molecular weights were 28,000 for PATE-I and -III, 32,000 for PATE-II and 33,000 for PATE-IV, respectively, and they had isoelectric points of 10~11. The optimum pHs for the reaction catalyzed by these pl-isozymes were 9.3 for PATE-II, 9.5 for PATE-IV and 9.7 for PATE-I and III, respectively. There were no differences in the optimum Ca2+ concentration (0.5~0.6 mM) or Km and Vmax among these four pl-isozymes. The mode of action of all the pl-isozymes was determined to be the endo-type.

Cell wall plant polysaccharide-degrading enzymes in autolysis of Botrytis cinerea

The mycelium of Botrytis cinerea undergoes a loss of 80% of its dry weight during the autolytic phase of growth in culture. The activities of lytic enzymes (polygalacturonase, endo-polygalacturonase, polymethylgalacturonase, endo-polymethylgalacturonase, pectin lyase, endo-pectin lyase, pectin esterase, xylanase, exo-cellulase and endo-cellulase) directly implicated in the degradation of plant cell walls were always higher in the culture liquid than in the mycelial extracts. Enzyme activities in the culture liquid increased during autolysis then decreased slightly at the end of this period, except for pectin esterase and polymethylgalacturonase which disappeared. With the exception of endopectin lyase, activities in the mycelial extract decreased and eventually disappeared during or at the end of the autolysis. Pectate lyase and endo-pectate lyase were not evident. The enzymatic complex obtained from autolyzed cultures of B. cinerea produced maceration of tissues, death of the cells and attack on bean plants.

Studies on the Pectic Substances of Plant Cell Walls

Pectate lyase was isolated from the cell extract of Erwinia aroideae. The enzyme was further purified to a high degree by a procedure involving ammonium sulfate fractionation and chromatography on CM-Sephadex C-50 and on Sephadex G-200. The enzyme attacked its substrate in an endo fashion and was more active on the sodium salt of acid-insoluble polygalacturonate or pectic acid than it was on the methoxylated pectin. The enzyme had an optimum pH at 9.3, was stimulated by calcium ions, and was completely inhibited by ethylenediaminetetraacetic acid. In addition, the reaction products showed an absorption maximum between 230 and 235 nm and reacted with thiobarbituric acid. These results indicate that the purified enzyme is an endopectate lyase. The endopectate lyase also had the ability to solubilize effectively the pectic fraction from the cell walls of carrot (Daucus carota) root tissue. The enzyme released 30.5% of the wall as soluble products and also liberated all of the galacturonic acid present in the walls. The total neutral sugars released by the enzyme were 10.6% of the walls, which corresponded to 71.5% of noncellulosic neutral sugars. The soluble products were separated into five fractions by DEAE-Sephadex A-50 column chromatography. Based on the analysis of sugar composition of each fraction, the pectic fraction of carrot cell wall is presented.

The degradation of polygalacturonic acid by rumen ciliate protozoa.

The depolymerase activity of cell-free extracts of nine species of rumen ciliate protozoa and two mixed protozoal preparations, grown in vivo and in vitro, towards polygalacturonic acid was examined. The highest activity was found with Eremoplastron bovis and Ostracodinium obtusum bilobum while there was none in the spined or spineless forms of Entodinium caudatum and little in Polyplastron multivesticulatum. On the basis of the rapid drop in viscosity, inhibition by EDTA and the production of u.v.-absorbing material, the enzymes from all active species were designated as endopectate lyases (EC4.2.2.2) although some polygalacturonase may be present. Neither pectin nor polygalacturonic acid supported the survival or growth of any of the protozoal species tested.

Ultrastructural and biochemical effects of endopectate lyase on cell walls from cell suspension cultures of bean and rice

Cell walls prepared from suspension cell cultures of bean and rice in log-phase growth were used to examine the effects of endopectate lyase (PL) on the solubilization of cell wall carbohydrates and concomitant ultrastructural alterations. Cell wall preparations from both plant sources were heterogeneous and contained a range of wall types from primary walls to xylem elements with spiral, secondary wall thickenings. Marked differences in wall thickness and number of wall laminations typified both preparations.Bean cell walls were more susceptible to degradation by PL than were those of rice. Upon treatment of the former with 2.3 × 10−3 units/mL of PL (1 unit released 1 μmol of unsaturated uronide/min at 30 °C from polygalacturonic acid at pH 8.5), 27% of the noncellulosic wall carbohydrate was solubilized in 1 h. This represented 50% of the PL susceptible carbohydrate in the preparation. Only 3% of the noncellulosic carbohydrate was released from rice cell walls in 1 h when treated with 115 × 10−3 units of PL/mL. This accounted for 60% of the PL susceptible wall fractions. Only uronic acid, rhamnose, galactose, and arabinose were solubilized from both preparations by PL.Cell walls in the bean and rice preparations were affected differentially by the PL. Those walls with secondary thickenings did not appear to be degraded, while the distinct fibrillar appearance of both bean and rice walls tended to fade or disappear. The middle lamella tended to dissolve to varying degrees in the presence of PL. Bean walls were more severely degraded than were the rice walls and many exhibited swelling, separation of wall layers, markedly reduced staining intensity, and (or) a granular ultrastructure.This study has demonstrated that as PL acts on susceptible cell walls there are major changes evoked in cell wall structure which suggest that the rhamnogalacturonan fraction of the higher plant cell wall contributes significantly to cell wall integrity. This study also emphasizes the need for cell wall material of proven uniformity for investigations of both cell wall composition and effects of specific polysaccharide degrading enzymes on cell wall integrity. Preliminary studies indicate that tobacco pith may provide more uniform cell walls than do cell suspension cultures.

Another pectate lyase produced by Streptomyces nitrosporeus.

Another pectate lyase was purified to a nearly homogeneous state from the culture filtrate of Streptomyces nitrosporeus. The molecular weight was estimated to be about 41,000. Iso-electric point was pH 4.6. The enzyme was most active at pH 10.0 and 50°C, and was relatively stable at a pH range of 4–11 (at 2°C for 48 hr) and below 40°C (at pH 7.0 for 10 min). Ca2+ was required for maximum activity. The enzyme was an endo-pectate lyase which was more active on low methoxyl pectin than on polygalacturonic acid and had macerating activity on potato tissue and Ganpi bark.

SOME CHARACTERISTICS OF AN ENDO‐PECTATE LYASE PRODUCED BY A THERMOPHILIC BACILLUS ISOLATED FROM OLIVES

ABSTRACTAn endo pectate lyase (PAL), or endo‐pectic acid transeliminase secreted by a thermophilic Bacillus sp., responsible for softening in pickled olives, was purified 30‐fold by ammonium sulfate precipitation and successive chromatography on Bio‐gel P‐100, DEAE‐cellulose, and CM‐cellulose columns. It degraded both pectic acid and pectin N.F. in a random manner, but did not attack fully esterified Link pectin. The end products of its action on polygalacturonic acid were largely unsaturated di‐ and tri‐galacturonic acids, with smaller amounts of saturated di‐ and tri‐galacturonic acids. Both saturated and unsaturated pentra‐ and tetra‐galacturonic acids were cleaved by the enzyme. Addition of either the culture or the isolated enzyme to the pickled olives caused softening of the tissue within a week at 37°C when a Chatillon spring punch (0.064 in.) was applied. The activation energy of this heat‐stable pectate lyase enzyme on 0.5% pectic acid was 17.1 kcal/mole, and the Q10 value was 2.35 between 60 and 70°C. The enzyme had a Vmax at 0.56 μmoles reducing groups/min and Km at 0.34% polygalacturonic acid.

Pectic Enzymes of Pigmented Strains of Clostridium

The pectic enzymes of six strains of pigmented, pectolytic clostridia isolated from potatoes were examined and compared with those strains of Clostridium felsineum and Clostridium aurantibutyricum using cup-plate assays. The organisms could be divided into two groups. Group 1 consisted of the potato isolates and the strain of C. aurantibutyricum; these formed pectic lyase enzymes and pectinesterase but no pectic hydrolase. Group 2 consisted of eight strains of C. felsineum (and one culture labelled Clostridium roseum); these formed pectic lyase and pectic hydrolase but no detectable pectinesterase. Viscometric studies and analyses of degradation products of pectate formed by enzymes of four representative strains from group 1 and two strains from group 2 showed that all these clostridia formed endopectate lyase enzymes; studies of one enzyme preparation showed that the pH optimum was about 9.5. In addition, the group 2 organisms formed endopolygalacturonase and probably also exopolygalacturonase.

Multiple Forms of Endo-Pectate Lyase Formed in Culture and in Infected Squash Hypocotyls by Hypomyces solani f. sp. cucurbitae

Multiple Forms of Endo-Pectate Lyase Formed in Culture and in Infected Squash Hypocotyls by Hypomyces solani f. sp. cucurbitae

Relationship of cell death in plant tissue treated with a homogeneous endopectate lyase to cell wall degradation

A homogeneous endopectate lyase (EC ef 1 lyase) from Erwinia chrysanthemi solubilized the pectic fraction in isolated potato and tobacco pith cell walls. This enzyme rapidly released unsaturated uronide from intact plant tissue, and caused both tissue maceration and cell death. The ratio of the rate of electrolyte loss from tissue (cell injury) to the rate of uronide release from tissue (cell wall breakdown) was constant when lyase concentration, tissue (substrate) concentration or temperature was varied. Both cell injury and breakdown were greatly decreased upon removal of lyase from tissue at intervals prior to death of all the cells in treated tissue. Plasmolysis of tissue protected the protoplasts from injury by EC ef 1 lyase although cell wall breakdown was not retarded. When EC ef 1 lyase was removed from wall-degraded, plasmolyzed tissue and the tissue subjected to deplasmolysis, cell death occurred immediately; all electrolytes in the tissue were lost at a rate ofequal to the rate of depletion of electrolytes from free space. Mere contact of the plasmalemma with lyase-degraded cell walls did not result in membrane damage; damage to protoplasts in lyase-treated tissue occurred only when the protoplasts were subjected to osmotic stress. The results of this study support the hypothesis that injury to cells in plant tissue treated with pectic enzymes results from a loss in the ability of enzymatically degraded cell walls to support the limiting membrane (plasmalemma) of living cells.

Maceration of plant tissues by microbial enzymer. IX. Purification and properties of pectate lyase produced by Streptomyces fradiae IFO 3439.

A pectate lyase (EC 4. 2. 2. 2) was purified from the culture filtrate of Streptomyces fradiae by precipitation with ammonium sulfate, followed by column chromatography on DEAE-Sephadex A-50, SP-Sephadex C-50 and by gel-filtration on Sephadex G-100. The enzyme was an endo-pectate lyase with an optimum activity at pH 9.0_??_9.2, and required only calcium ion for the activity. The enzyme had macerating activity and was active on both native pectin and polygalacturonic acid, but the activity toward oligogalacturonides was small.