BREAKDOWN OF PECTIC SUBSTANCES BY A NEW ENZYME FROM NEUROSPORA*

BY ELIZABETH ROBOZ, R. W. BARRATT, AND E. L. TATUM

(Prom the Department of Biological Sciences, Stanford University, Stanford, California)

(Received for publication, August 21, 1951)

The enzymatic hydrolysis of pectic substances has been under investi- gation for a considerable period of time but the precise mechanism of this reaction is not yet fully understood. It is clear that two general types of enzymes are involved in the breakdown of pectin; pectinesterase, which cleaves the methyl ester linkages in the molecule to yield pectic acid, and polygalacturonase, which hydrolyzes the 1,4 glycosidic bonds and yields galacturonic acid (l-4). Most of the work to date on the splitting of the glycosidic linkages has involved the use of enzyme preparations from fungi, particularly commercial preparations. There is so far no definitive evidence that mold polygalacturonase preparations contain only a single enzyme. All the preparations examined, including partially purified ones from Pectinol,’ show qualitatively the same type of reaction, characterized by the production of n-galacturonic acid, as the only end-product (5). However, some transitory compounds intermediate between pectic acid and n-galacturonic acid have been detected in the course of enzymatic hydroly- sis with chromatographic methods (6, 7). Other pectic enzymes have been reported which do not break down pectic acid completely (8-10). Ki- netic evidence (11-13) suggests that more than one enzyme may be in- volved in the hydrolysis of the glycosidic linkages by polygalacturonase or that a single enzyme may have different affinities for the various inter- mediate products (14).

An extracellular enzyme has been found in Neurospora crassa which rapidly reduces the viscosity of pectic acid and pectin with the hydrolysis of some of the glycosidic bonds. This enzyme differs from other reported enzymes in that it yields lower polyuronides as end-products rather than n-galacturonic acid, and also in that it degrades pectin without preliminary demethylation. This extracellular enzyme has been given the name pectin

* This work was supported by research grants from the Jane Coffin Childs Me- morial Fund for Medical Research, the National Institutes of Health, United States Public Health Service, and from the American Cancer Society, recommended by the Committee on Growth of the National Research Council. Presented in part at the meeting of the American Chemical Society, Chicago, 1950.

1 Commercial pectic enzyme preparation manufactured by Rohm and Haas Com- pany, Philadelphia, Pennsylvania.

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460 PECTIN DEPOLYMERASE

depolymerase.2 The present paper describes the properties of Neurospora pectin depolymerase as well as the isolation and partial characterization of the polyuronide resulting from its action on pectic acid.

EXPERIMENTAL

Several strains of Neurospora crassa of different origin were tested for pectin utilization and enzyme production. Wild type strain SY7A was selected as most suitable and was used in the experiments reported in this paper.

The culture medium used was t.he standard synthetic Neurospora mini- mal (15), but contained 0.5 per cent glucose and 1 per cent pectin N. F. or pectic acid3 N. F. as carbon sources. 500 ml. Erlenmeyer flasks con- taining 250 ml. of this medium were inoculated and incubated with constant agitation at 30” for at least 8 days.

The activities of enzyme preparations were assayed by incubating the enzyme with 1 per cent pectic acid N. F. in a 0.1 M solution of sodium chloride at a temperature of 26.5”, unless otherwise indicated. Progress of the enzymatic reaction was followed by measuring the decrease in viscosity and the increase in aldose-reducing groups. In certain instances the rapid trichloroacetic acid method (16), which involves the volumetric estimation of precipitated residual pectic acid, was used.

Viscosimetric measurements were carried out in Ostwald viscosimeters. The enzyme action was expressed as per cent viscosity change. This is equivalent to the “Abbauzahl A” of Weber and Deuel (17) and was cal- culated as follows:

A _ vo - VI - ____ x 100

vo - v.

VO = flow time in seconds of pectic acid + inactivated enzyme; Vt = flow time in seconds at reaction time of pectic acid + active enzyme; V, = flow time in seconds of the solvent + inactivated enzyme.

The liberation of aldose groups as the result of enzymatic hydrolysis of the 1,4-glycosidic linkage was determined by the iodometric method of Willstatter and Schudel (18) as modified by Jansen and MacDonnell (11). The reducing values were calculated as galacturonic acid corrected for initial reducing value and expressed as per cent hydrolysis. In all cases in which iodometric determinations were made, the pectic acid was purified with 70 per cent ethanol (19) to remove arabogalactan, which would

2 In addition to the extracellular depolymerase, enzymes have been detected in homogenates of mycelium, which attack the polyuronide resulting from depoly- merase action, to yield free galacturonic acid.

3 Manufactured by California Fruit Growers Exchange, Ontario, California.

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E. ROBOZ, R. 1%‘. BARRATT, AND E. L. TATUM 461

otherwise give rise to reducing values not due to the polyuronide molecule proper.

Monogalacturonic acid was identified by the sensitive qualitative test of Ehrlich (20) with pure galacturonic acid as a standard.

Results

Relation of Enzyme Concentration and Reaction Rate-In order to com- pare enzymatic activities of various preparations and to follow the stages of purification, it became necessary to establish a common unit of activity. Viscosimetric assays were carried out on pectic acid as substrate with a series of enzyme concentrations. When enzyme concentration was plotted

3.0 4.0 5.0 UNITS DEPOLYMERASE (LOG SCALE)

FIG. 1. Effect of depolymerase concentration on viscosity change. Curve 0, 1 hour, 0,2 hours, l ,4 hour readings. 1 depolymerase unit is defined as the amount of enzyme necessary to produce a 50 per cent viscosity change of 500 mg. of pectic acid in 4 hours at 26.5”.

against per cent viscosity change, a non-linear relationship was obtained, even at very low enzyme concentrations and short time periods. However, when the logarithm of enzyme concentration was plotted against per cent viscosity change, as shown in Fig. 1, a linear relationship was found over the range from 20 to 80 per cent. Reid (7) also obtained a linear rela- tionship between per cent viscosity and the logarithm of enzyme concentra- tion at a given time for polygalacturonase. This relationship permits the establishment of a depolymerase unit. The unit was arbitrarily defined as the amount of enzyme necessary to produce a 50 per cent drop in vis- cosity of 500 mg. of pectic acid (1 per cent solution) in 4 hours at 26.5”. Similarly, 1 unit represents a viscosity change of 36 per cent in 2 hours or 20 per cent in 1 hour, as can be seen in Fig. 1. Unit activities of depolymer- ase preparations were determined by averaging the unit values from the 1, 2, and 4 hour readings, provided the viscosity change was between 20 and 80 per cent.

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462 PECTIN DEPOLYMERASE

Routinely, the activity of depolymerase was measured by viscosity change rather than by reducing values because the changes of the latter in the first hours of the reaction are somewhat low for accurate measure- ment. In order to study the relation between the changes measured by the two methods, the results of viscosimetric and iodometric determinations were compared. Zero values were determined with a mixture of pectic acid and inactivated enzyme. Data of a representative experiment are

TABLE I

Effect of Depolymerase on Changes in Viscosity and Reducing Value of Pectic Acid

Time of incubation

min.

15 30. 45 60 90 ha.

2 3 4 5 6 7 8

8% 9

11s dOYS

1 3 4

-

.-

-!-

Per cent viscosity change

18.4 0.48 35.8 0.77 47.2 2.13 56.0 2.52 67.0 4.55

73.4 80.7 84.2 86.2 87.5 86.4 86.1 86.1

6.0 8.9

11.8 13.2 15.5 17.1 16.8 18.5 18.9 19.2

I

.-

‘er cent hydrolysis calculated from reducing values

21.9 26.7 26.8

given in Table I, with enzyme Preparation III (to be described later). A viscosity change of 50 per cent was accompanied by only 2 per cent hy- drolysis, as measured by reducing values. When the viscosity change reached the maximal value, 87.5 per cent in this experiment,, only 15.5 per cent hydrolysis had taken place; when no further increase in reducing value could be obtained, hydrolysis was 27 per cent complete.

Production of Pectic Acid Depolymerase-The effects of different carbon sources on growth and enzyme production were investigated. Growth on pectin alone is very slow until about the 6th day. Of the sugars added to pectin, n-glucose was found most satisfactory in increasing both the weight of mycelium and the production of enzyme. Furthermore, more

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E. ROBOZ, R. W. BARRATT, AND E. L. TATUM 463

enzyme is produced with pectin than with pectic acid as a substrate. When tested under comparable conditions on pectic acid, filtrates from cultures grown on pectin gave 81 per cent change in viscosity, while only 33 per cent change in viscosity was obtained with filtrates from cultures grown on pectic acid. Under the conditions used, Neurospora will not grow on galacturonic acid as the sole carbon source.

Daily determinations of the enzymatic activity of filtrates from growing cultures were made for 28 days with the trichloroacetic acid precipitation test (16). Although active filtrates were obtained up to the 28th day of growth, harvesting on the 12th day gave the best preparations, since longer periods resulted in some autolysis of the mycelium which made purification of the culture filtrate more difficult.

Purijkation of Pectin Depolymerase-The mycelium was removed on a Biichner funnel with a Celite filter aid. The filtrate was concentrated to one-fourth of the original volume in vacua at temperatures under 30”, and refiltered. Dry preparations were made from this concentrate by three procedures: (a) 1 liter was treated with 4 volumes of cold 95 per cent ethanol with constant stirring. After standing at 4” overnight, the pre- cipitate was filtered, redissolved in water at 25”, purified by repeated pre- cipitation with ethanol, and &rally dried in vacua. (b) 1 liter of the concentrate was dialyzed for 48 hours at 4” against running distilled water. The dialysate was concentrated in vacua below 30” and precipitated with ethanol. After standing in the cold overnight, the precipitate was cen- trifuged, washed with cold ethanol, and dried in vacua. (c) 1 liter of the concentrate was passed through freshly regenerated IR-100 cation exchange and IR-4B anion exchange resins4 to remove the salts originally present in the nutrient solution. The solution was concentrated and the enzyme precipitated with ethanol. It was centrifuged, redissolved, and further purified as described for Preparation I. The ion exchange Procedure III (Table II) gave the best recovery (95.5 per cent), while the dialysis Proce- dure II gave the greatest enrichment (11-fold). The three different prep- arations, as well as the culture filtrate, when compared on pectic acid at equivalent enzyme activities, were qualitatively and quantitatively indis- tinguishable.

Properties of Pectin Depolymerase-The effects of temperature and hy- drogen ion concentration on the stability and reaction rate of depolymerase solutions were investigated. 120 mg. of the dry enzyme, Preparation I, were dissolved in 15.0 ml. of water, and 2.5 ml. aliquots were allowed to stand at six temperatures covering the range 26.5-90” for 1 hour. The residual activity was assayed by viscosimetry under standard conditions. The data reported are from the 2 hour viscosity readings. The activity

4 Manufactured by Rohm and Haas Company, Philadelphia, Pennsylvania.

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464 PECTIN DEPOLYMERASE

in units measured at 26.5’ was taken as 100 per cent, and the percentage of residual activities of the solutions exposed to other temperatures was calculated. As shown in Fig. 2, pectin depolymerase solutions were 50 per cent inactivated at 40” and completely inactivated at 70” in 1 hour.

TABLE II Depolymerase Activity of Diflerent Preparations

Activity in Relative Total Method of preparation units* per purifica- Yield units Per cent

WT. tion recovered recovery

gm.

Concentrate before treatment. . 0.04 31.0 1229 I. Ethanol Pptn.. 0.09 2.2 12.3 1078 87.6 II. Ion exchange.. . . . . 0.24 6.0 5.0 1172 95.5 III. Dialysis.. . . 0.46 11.7 1.3 590 48.0

* 1 unit is defined as the quantity of enzyme necessary for 50 per cent viscosity change of 500 mg. of pectic acid in 4 hours at 26.5”.

FIG. 2

k! O2 3 I 4 I 5 I 6 1 7 I

PH FIG. 3

FIG. 2. Effect of temperature on stability of depolymerase. Residual activity after 1 hour at indicated temperatures; results from 2 hour viscosimetric assay at 26.5”.

FIG. 3. Effect of pH on stability of depolymerase. Residual activity after 15 hours at indicated pH; results from 2 hour viscosimetric assay at 26.5’.

The effect of hydrogen ion concentration on the stability of depolymerase was determined as follows: To a series of six McIlvaine’s standard buffer solutions (2.5 ml. each) covering the pH range from 2.2 to 8.0, 20 mg. of dry enzyme Preparation I were added and allowed to stand for 15 hours at 25”. After readjustment to pH 6, the residual activity was assayed and calculated in the manner previously described. The data in Fig. 3, taken from the 2 hour readings, show that stability is greatest at pH 6.0.

The effect of temperature on the reaction rate of depolymerase (Prep-

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E. ROBOZ, R. W. BARRATT, AND E. L. TATUM 465

aration I) was determined by following the viscosity change at four tem- peratures; 26.5”, 34.0”, 37.5”, and 40.5”. The reaction rates at the four temperatures show the usual temperature coefficient over the range inves- tigated, with the optimum above 40.5”.

The influence of pH on the reaction rate was determined by adding enzyme Preparation I to a series of fourteen buffered pectic acid solutions covering the pH range 2.5 to 7.5. The results of the 2 hour assay are presented in Fig. 4 and show an optimal rate of reaction from pH 5.5 to 6.0.

The action of depolymerase on purified pectin and pectic acid was com- pared by using Preparation III. Pectin depolymerase reduces the viscos- ity of pectin at least as rapidly as that of pectic acid. The final reducing

,100 z? 2 80

& 60- 0

ts 5 40-

l-

5 2 20-

it

‘2 3 4 5 6 7

PH

FIG. 4. Influence of pH on reaction rate of depolymerase. viscosimetric assay at 26.5”.

Results from 2 hour

values of the two hydrolysates were almost identical, 1.245 and 1.241 mM aldehyde per gm. of substrate respectively.

Comparison of Depolymerase with Polygalacluronase-Since pectin de- polymerase does not hydrolyze pectic acid completely, further studies were undertaken to characterize the reaction. The action of depolymerase (Preparation III) was compared with that of Pectin01 (polygalacturonase) on a solution of purified pectic acid containing 93.2 per cent galacturonic acid.5 The solution used contained 780 mg. of pectic acid per 100 ml. and was adjusted to the optimal pH values for the two enzymes, 5.5 for depolymerase and 3.5 for polygalacturonase (21). The hydrolyses were carried out at 30” under toluene and were considered to be complete when, after several days, the further addition of enzyme did not increase the reducing value. The final reducing values expressed as galacturonic acid

6 Water 3.6 per cent, ash 2.0 per cent, methoxyl 0.35 per cent, uranic acid anhy- dride corrected 90.1 per cent. Pectic acid and analysis generously provided by Dr. E. F. Jansen.

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466 PECTIN DEPOLYMERASE

and the calculated per cent hydrolysis are given in Table III. To compare the two enzymes further, polygalacturonase was added to the depolymerase hydrolysate, and the hydrolysis increased from 33 to 97.5 per cent (Table III). These experiments show that the two enzymes are different in action.

Isolation and Partial Characterization of Low Molecular Weight Poly- uronide from Pectic Acid-2 gm. of purified pectic acid were dissolved in 200 ml. of distilled water, and the pH adjusted to 5.5 with NaOH. An excess of depolymerase (50 mg. of Preparation III) was added and the mixture incubated at 30” for 5 days under toluene. No further increase in reducing values was detectable after 3 days. The hydrolysate was cen- trifuged and treated with 4 volumes of ethanol. After standing overnight at 4”, the precipitate was centrifuged and purified by repeated precipitation with ethanol. An aqueous solution of the precipitate was passed through a

TABLE III

Comparison of Reducing Values Obtained with Depolymerase and Polygalacturonase on Pectic Acid

Enzyme added Reducing values1 as

mg. galacturon1c acid anhydride

Per cent hydrolysis

Pectin depolymerase.. . . 240

~ I

33.0 Polygalacturonase* . 654 90.0 Depolymerase followed by polygalacturonaset 709 97.5

* Pectin01 extracted with 80 per cent ethanol at room temperature to remove reducing sugars.

t pH adjusted to 3.5 before adding polygalacturonase.

column of IR-100 cation exchange resin, the effluent concentrated and treated with ethanol. The white flocculent precipitate was washed with ethanol and with ether and dried in vacua. From 2.00 gm. of pectic acid, 1.13 gm. of low molecular weight polyuronic acid were obtained (56 per cent). In other experiments, yields ranging from 50 to 56 per cent were obtained.

The low molecular weight polyuronic acid is readily soluble in cold water. A solution does not exhibit the colloidal properties of pectic acid, namely gelling on addition of ethanol or 10 per cent calcium chloride; nor is it precipitated by trichloroacetic acid. 1 gm. of the polyuronic acid dissolved in 100 ml. of water has a pH of 2.8, and, titrated with 0.1 N NaOH to pH 6.8, gave a neutralization equivalent of 187.

The compound was hydrolyzed at pH 3.5 with ethanol-extracted Pec- tinol. After hydrolysis, a reducing value equivalent to 97.5 per cent galacturonic acid anhydride was obtained. Analysis, according to the modified Lef&vre and Tollens method (22), gave a uranic acid anhydride value of 98.1 per cent.

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E. ROBOZ, R. W. BARRATT, AND E. L. TATUM 467

The optical rotation of the low molecular weight polyuronide was de- termined in water and compared with the values found for pectic acid and galacturonic acid (Table IV).

The purity of the polyuronide was determined by electrophoresis in a Tiselius cell by the schlieren scanning photographic method (23, 24). A 0.5 per cent solution of polyuronide was prepared in acetate buffer at pH 4.98 and the mobility measured with a field strength of 5.07 volts per cm. at 0.6”. The main component had uncorrected mobilities of - 10.6 X 10ds and - 10.9 X 1O-6 cm. per second per volt per cm. for the descending and ascending boundaries respectively. The electrophoretic diagram indicated that the uronide was approximately 90 per cent homogeneous.

The molecular weight was determined from the diffusion and sedimenta-

TABLE IV Action 0-f Depolymerase (Preparation III) on Peck Acid and on Methyl Glycoside

of Potygalacturonic Acid (MP)

Pectic acid .................................. Depolymerase hydrolysate of pectic acid ..... Low polyuronide from pectic acid. .......... MP ......................................... Depolymerase hydrolysate of MP ............ Low polyuronide from MP. ................. a-u-Galacturonic acid. ......................

-

-_

Per cent yield

53

49

Reducing values, as per

cent uranic lcid anhydride

1.80 25.37 16.17

1.08 22.30 13.95 90.7

‘ptical rotation,

t&c

degrees +270

+14s

+146 +50.5

tion constants. The average value for DZO was 1.66 X 10m6 sq. cm. per second, and for SZO~ 1.16 X 10-13. Assuming a partial specific volume of 0.61 (25), the molecular weight was calculated to be approximately 4000. The molecular weight was also calculated from the end-group determination as used for degraded cellulose (26) by titrating the free aldose groups according to Willstatter. The results indicated a molecular weight of 1200 to 1300 or a multiple thereof. This would represent a minimal chain length of 7 to 8 uranic acid anhydride units.

Action of Depolymerase on Methyl Glycoside of Polygalacturonic Acid- Additional information would be derived from the use of methyl glycoside of polygalacturonic acid (MP) (27, 28) as a substrate for depolymerase. MP is prepared from pectic acid by methanolysis and consists of over 98 per cent uronide (29). 333 mg. of this compound were dissolved in water, and the pH was adjusted to 5.0, depolymerase Preparation III (the same as used for the preparation of the low polyuronide from pectic acid) was added, and the mixture incubated under toluene at 30”. After 1 week the

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468 PECTIN DEPOLYMERASE

characteristic colloidal properties had disappeared. The reducing value was determined and, to make certain that the reaction had gone to com- pletion, more enzyme was added. After incubation for 3 additional days, the reducing value had not increased. The hydrolysate was filtered and 41 ml. (equivalent to 264 mg. of the original compound) were treated with 160 ml. of cold ethanol. The white amorphous precipitate was purified further by the procedure given for the preparation of the low molecular weight polyuronide from pectic acid. 160 mg. of dry material were ob- tained, a yield of 49.2 per cent after correcting for the enzyme added. The reducing value and optical rotation of this preparation were deter- mined. The properties of this preparation from MP are compared with those of the low molecular weight polyuronide from pectic acid in Table IV.

DISCUSSION

At the present state of purification of pectin depolymerase there can be no critical evidence that the same enzyme is responsible for the lowering of viscosity of pectic acid, and for the production of the polyuronide from pectic acid and from methyl glucoside of polygalacturonic acid. However, no separation of these properties has been observed during the various fractionations and divergent purification methods so far employed. Until evidence to the contrary is obtained, pectin depolymerase may be considered as a single enzyme.

Pectin depolymerase clearly differs from previously described pectic enzymes. It differs from polygalacturonase in the pH optimum, the pH stability, and the nature of the end-products. For polygalacturonase the pH optimum is 3.5 (21), with inactivation occurring rapidly above pH 6 (13), while for pectin depolymerase the pH optimum is 5.5 to 6.0, with the greatest stability at pH 6. Galacturonic acid is not a major end- product of depolymerase action, although it is essentially the sole end- product of polygalacturonase action. Although there is not pertinent evi- dence, it is possible that pectin depolymerase may be a component of polygalacturonase.

Several pectic enzymes have been reported to differ from polygalac- turonase, in that pectic acid is attacked without the liberation of galac- turonic acid. For the enzyme from Byssochlamys fulva (lo), the aberrant behavior appears attributable to a low specific polygalacturonase activity (30, 31). This seems not to be true for pectin depolymerase from Neuro- spora since highly active preparations, which give up to 90 per cent vis- cosity change in 1 hour, do not give more than 30 per cent of the theoretical maximal reducing value, even upon the addition of more enzyme and pro- longed incubation. Furthermore, purified low molecular weight polyuron- ide produced by depolymerase action is not attacked by any concentration

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I<. ROBOZ, R. WV. BrlRRATT, AND E. L. TATUM 469

of depolymerase, while it is rapidly hydrolyzed by polygalacturonase (Pec- tinol) to monogalacturonic acid. Pectin depolymerase also differs from the “pectolytic factor” from tomato (8) and a polygalacturonase-like enzyme from yeast (9) in pH optimum, stability, and in substrate attacked. The tomato enzyme has a pH optimum of 4.5, is only 85 per cent inactivated at 100” in 15 minutes, and is completely inactivated only after 2 hours (4). In contrast, pectin depolymerase has a pH optimum of 5.5 to 6.0 and is completely inactivated at 70” in 1 hour. The tomato enzyme also is almost inactive on pectin unless methyl esterase is present (3), while pectin depolymerase readily attacks even citrus pectin. The polygalacturonase- like enzyme from yeast (Saccharomyces fragilis) has a pH optimum of 3.5 to 4.0 and results in only 11 per cent of the theoretical maximal reducing value (32). These properties appear sufficient to distinguish it from the Neurospora enzyme.

The site of the hydrolytic attack of depolymerase cannot be definitely established since the exact structure of the pectin macromolecule is not yet clear. It has been shown that the purest pectin preparations contain about 10 per cent non-uronides, but there is not agreement as to whether or not these non-uronides are in the main pectin chain. If they are not, then pectin depolymerase necessarily attacks the uronide linkages. However, according to Lineweaver and Jansen (3) the occurrence of non-uronides in the main chain of pectin is indicated by x-ray data (33) and by the fairly homogeneous galacturonide of 32 units obtained by methanolysis of pectic acid. In this case pectin depolymerase might conceivably attack only non-uronide linkages. Nevertheless, pectin depolymerase has been shown to attack this methyl glycoside of polygalacturonic acid (MP). Since this compound is at least 98 per cent uronide, its hydrolysis by pectin depolymerase is strong evidence that uronide linkages per se are attacked by this enzyme. The possibility exists that in addition depolymerase attacks non-uronide linkages.

The finding that pectin depolymerase hydrolyzes MP and yields a lower polyuronide apparently identical with that produced from pectic acid has implications of considerable theoretical significance. In the first place it would strongly suggest that MP contains more than one type of glycosidic linkage. It would also imply that pectic acid contains a structural unit smaller than this compound, and contained therein. The evidence sup- ports the view that this basic unit is the polyuronide formed as the pri- mary end-product of pectic acid hydrolysis by pectin depolymerase from Neurospora.

The authors gratefully acknowledge their indebtedness to Dr. E. F. Jansen, Western Regional Research Laboratory, for supplying samples of

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470 PECTIN DEPOLYMERASE

purified pectic acid and of MP, and to Dr. N. J. Eldredge and Dr. L. Noda, Department of Chemistry, Stanford University, for carrying out and inter- preting the electrophoretic and ultracentrifuge studies.

SUMMARY

1. An extracellular enzyme produced by Neurospora crassa rapidly re- duces the viscosity of pectic acid and pectin and hydrolyzes uronide linkages.

2. This enzyme, pectin depolymerase, differs from polygalacturonase in pH optimum, pH stability, and the nature of the end-products.

3. More than 50 per cent of the pectic acid attacked by depolymerase is converted to a low molecular weight polyuronide.

4. The low molecular weight polyuronide has been isolated and found to be at least 90 per cent homogeneous by electrophoretic analysis. The molecular weight calculated from electrophoretic data is about 4000, while that from end-group determinations is about 1300 or a multiple thereof.

5. The evidence suggests that the polyuronide produced from pectic acid by depolymerase represents a basic unit of the pectin molecule.

BIBLIOGRAPHY

1. Phaff, H. J., and Joslyn, M. A., Wallerstein Lab. Communicat., 10, 133 (1947). 2. Kertesz, Z. I., and McColloch, R. T., Advances in Carbohydrate Chem., 6, 79

(1950). 3. Lineweaver, H., and Jansen, E. F., Advances in Enzymol., 11, 267 (1951). 4. Kertesz, Z. I., in Sumner, J. B., and Myrback, K., The enzymes, New York,

745 (1951). 5. Lineweaver, H., Jang, R., and Jansen, E. F., Arch. Biochem., 20, 137 (1949). 6. Jermyn, M. A., and Tomkins, R. G., Biochem. J., 47, 437 (1950). 7. Reid, W. W., J. SC. Food and Agr., 1,234 (1950). 8. McColloch, R. J., and Kertesz, Z. I., Arch. Biochem., 17, 197 (1948). 9. Luh, B. S., and Phaff, H. J., Abstracts, American Chemical Society, Atlantic

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TatumElizabeth Roboz, R. W. Barratt and E. L.

FROM NEUROSPORASUBSTANCES BY A NEW ENZYME

BREAKDOWN OF PECTIC

1952, 195:459-471.J. Biol. Chem. 

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