ELSEVIER

Endopolygalacturonase secretion by Kluyveromyces marxianus and other cocoa pulp-degrading yeasts

Rosane F. Schwan,*?+ Richard M. Cooper,* and Alan E. Wheals*
*School of Biology and Biochemistry, University of Bath, de Pesquisas do Cacau/SETEA, Itabuna, Bahia, Brazil Bath, United Kingdom CEPLAUCentro

Among 12 yeast strains isolated from cocoa fermentations, only four showed extracellular pectinase activity. Kluyveromyces marxianus was the most pectinolytic with 85% of total secreted protein consisting of a constitutive endopolygalacturonase (PG). No pectic lyases or methylesterases were produced. The pH and temperature optima for PG activiv were 5.0 and 40C respectively. Purified PG comprised four proteins of M,. 47, 41, 35, and 33 kDa based on gel filtration and 45, 42, 39, and 36 according to SDS-PAGE. Activity-stained, isoelectric focusing gels showed three major bands (pls 5.9, 5.6, and 5.3) and up to six minor bands from pl 6.4-5.0. PG had a typical random mode of action, very high macerating activity on plant tissues, and reduced the viscosity of cocoa pulp. PG secretion started in early exponential phase and was completed after 24 h. Only five out of 138 mutants with altered PG levels produced after nitrosoguanidine mutagenesis showed modest (up to 25%) increase in PG production. Most mutants were underproducers of the full complement of PG isoforms including five which had high intracellular PG located in low-densi@ vesicles, vacuoles, and ER fractions. In most mutants, there was a clear correlation between PG and inulinase activity secreted from cells. The implications for both cocoa and enzyme production are discussed. 0 1997 Elsevier Science Inc.

Keywords: Kluyveromyces marxianus; Kluyveromyces thetmotolerans; Saccharomyces cerevisiae var. chevalieri; Candida rugopelliculosa; endopolygalacturonase;inulinase; cocoa fermentation; yeasts

Introduction
Cocoa pulp is a rich medium for microbial growth. Yeasts tend to dominate the initial phase of the natural fermentation. The primary activity of yeasts is the conversion of sucrose, glucose, and fructose to ethanol and carbon dioxide. In addition, some yeast species are capable of secreting pectinolytic enzymes, 2,3 these enzymes break down the walls of pulp cells resulting in loss of fluids. An isolate of Kluyveromyces marxianus isolated from cocoa fermentations was found to be the most active in this respect. K. murxianus is a diploid yeast which has been widely used for the production of lactase and pectinases; these enzymes are deployed for whey utilization and apple juice

clarification,

Address reprint requests to Dr. A. E. Wheals, School of Biology and

Biochemistry, University The present- address oi Universidade Federal de Brazil Received 24 April 1996; ber 1996 of Bath, Bath, BA2 7AY, United Kingdom Dr. Schwan is the Departamento de Biologia. Lavras, Cx Postal 37, 37200-000, Lavras, MC, revised 18 November 1996; accepted 27 Novem-

respectively. Pectinolytic enzymes secreted by have been partially purified and characterized4- but the results obtained were very variable and partially contradictory warranting a study of the biochemical properties of the enzyme of K. marxianus CCT 3172. Furthermore, little attention has been paid to PG secretion by K. marxianus or to the inUacellular localization of secreted proteins. Microbial synthesis of enzymes at the industrial level requires highly productive strains to reduce costs but regulatory mutants have only occasionally been exploited. Most pectinases are induced by pectins and subject to catabolite repression; however, the PG of K. marxianus is very unusual because production is constitutive and not repressed by carbohydrates. Maximal production occurs in 10% (w/v> glucose under self-induced anaerobic conditions. The use of polygalacturonase from K. marxianus has attracted considerable interest;-* however, no attempts to enhance polygalacturonase production of K. marxianus have been reported. The goals of this study were to investigate pectinase production by strains of yeasts isolated from cocoa fermenK. marxianus

Enzyme and Microbial Technology 21:234-244, 1997 0 1997 Elsevier Science Inc. All rights reserved. 655 Avenue of the Americas, New York, NY 10010

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fndopolygalacturonase
tation and determine the nature and properties of the pectinase activity which is elaborated by the most pectinolytic of these yeasts. We describe the isolation and characterization of K. marxianus secreting extracellular, constitutive endopolygalacturonases with high activity against native pectins and the isolation of mutants with increased activity.

from Kluyveromyces

marxianus:

R. Schwan et al.

Materials and methods Strains, culture media, and growth conditions

The yeast strains used were K. marxianus NCYC 587; K. marxianus CCT 3 172; K. thermototerans CCT 1701; Candida rugopelliculosa CCT 1702; Candida pelliculosa CCT 1696; Candida rugosa CCT 3 176; Candida bombi CCT 1689; Torulaspora pretoriensis CCT 1688; Lodderomyces elongiosporus CCT 1690; Saccharomyces cerevisiae var. chevalieri CCT 1698; Saccharomyces cerevisiae CCT 3174; Pichia fermentans CCT 3175, and Kloeckera apiculata CCT 3 199. These strains, except NCYC 587, were isolated during cocoa fermentations in Bahia, Brazil and have been identified in conjunction with, and deposited in, the culture collection of the FundaGao Tropical de Pesquisas e Tecnologia Andre Tosello (CCT), Campinas, Sao Paulo, Brazil. All strains were maintained at 4C on agar slants containing (I -I): yeast extract (Lab M), 3.0 g; peptone (Lab M), 5.0 g; glucose, 10.0 g: malt extract (Lab M), 3.0 g; and agar, 20.0 g.

cells and in the washed cell supernatant (cell wall enzyme fraction). Cell-bound enzymes were obtained from washed cells (100 mg dry wt) suspended in 4 ml of either 0.05 M potassium phosphate pH 5.0 or 0.1 M acetate buffer pH 5.0. The suspension was transferred to a Braun bottle containing 35 g of 0.45-0.50 mm diameter glass beads and shaken for six periods each of 15 s in a Braun homogenizer, and then cooled with expanding CO,. The beads were allowed to settle and the supematant removed. All fluids were dialyzed versus distilled water at 4C for 18 h before enzyme assay. Polygalacturonase (PG). Two methods were used to measure PG activity (versus polygalacturonic acid Na-salt) in culture filtrates; release of reducing groupsI or decrease in viscosity of substrate.h PG activity (reducing groups) is expressed as pmol galacturonic acid equivalents released pgg protein mini . PG activity (viscometry) is expressed as relative viscometric units (RVU) pg- protein ml- RVU is defined as 10 times the reciprocal of time (min) for a 50% decrease in relative viscosity. Macerating activity was determined with 8 X 2 mm discs used at 5 per ml cut from potato and cucumber tissue and submerged in an enzyme solution in Petri dishes and incubated at 20C for up to 2 h. Macerating activity was estimated from the loss of coherence of tissue. Pectin methylesterase (PME). Pectin methylesterase was assayed by continuous titrimetric determination of the carboxyl groups liberated from methylester bonds. PME activity was expressed as microequivalents of polygalacturonic acid produced ml- h- . Pectin lyase (PL) and pectate lyase (PGL). Pectin lyase and pectate lyase activities were determined by standard methods.s One activity unit was defined as the amount of enzyme which released 1 pmol of unsaturated product min -. Inulinase. lnulinase was measured by determining the rate of appearance of fructose and glucose with a Boehringer glucose/ fructose test combination. The reaction mixture consisted of 2% (w/v) sucrose or 2% (w/v) inuhn in a 0.1 M sodium acetate buffer pH 4.5 at 40C.4 One unit of inulinase activity was defined as the amount of enzyme catalyzing the liberation of I p.mol fructose min-.

Screening for pectinase

activity

Screening of cells for pectinase activity was done using basic medium (MP-5).13 It contained 500 ml of mineral salt solution [(NH&SO,, 2 g; KH,PO,, 4 g; Na,HPO,, 6 g; FeSo, - 7H,O. 0.2 g; CaCI,, 1 mg; H,BO,, 10 pg; MnSO,. 10 pg; ZnSO,, 70 pg; CuSO,, 50 kg: MOO,, 10 pg, pH 5.0; I-)]; yeast extract, 1 g; polygalacturonic acid, 5 g; D-glucose, 5 g; agar, 15 g; and 500 ml of distilled water. Petri dishes were incubated for 48 h and then flooded with a I % (w/v) aqueous solution of hexadecyltrimethylammonium bromide (Cetrimide, Sigma, St. Louis, MO). This reagent precipitates intact polygalacturonic acid (5 g 1-l) in the medium and thus clear zones around a colony in an otherwise opaque medium indicate degradation of the polygalacturonic acid.x Pectinase-producing yeasts were grown in a medium which contained (g I_ ): glucose, 10.0 (sterilized separately); (NH&S04, 3.0; KH,pO,, 4.5; yeast extract (Lab M), 1.0; MgSO, +7H,O, 0.25: and CaCl, * 2H,O, 0.25; adjusted to pH 5.0 with HCI. The medium (I 1) was dispensed in 2-l round flat-bottomed flasks fitted with fermentation locks. Flasks were inoculated with 1.0 mg dry wt equivalent of organisms from a 24-h starter culture. Cultures were incubated at 30C under self-induced anaerobic conditions, stirred continuously, sampled at intervals and growth (dry wt ml-) was determined from optical density (OD,,e) measurements against an approprrate calibration curve.

Enzymatic

characterization

of subcellular

organelles

Enzymatic activities of plasma membrane vanadate-sensitive Mg2+ -ATPase, alkaline phosphatase, and NADPH-cytochrome c oxidoreductase were measured according to published methodstY with minor modifications. Plasma membrane ATPase. Plasma membrane vanadate-sensitive MgZf -ATPase activity was assayed by following release of phosphate, Pi, from ATP. * The reaction mixture consisted of I ml of 100 mM MES-tris buffer pH 6.5 containing 80 ITiM KCI, 6 ITIM MgCt,, 6 ITIM Na-ATP, and enzyme preparation containing 25-50 pg protein. Sodium orthovanadate (100 PM) was included in control mixtures to establish that the enzyme being assayed was inhibited by this compound. The reaction was started with addition of Na-ATP, the mixture was incubated for lo-40 min at 3OC, and the amount of Pi liberated was measured. The reaction was stopped by addition of 2 ml acidified molybdate solution (2.0% (v/v), cone, H,SO, containing 0.5% (w/v) ammonium molybdate and 0.5% (w/v) sodium dodecyl sulfate). Ascorbic acid [0.02 ml, 10% (w/v)] was added and the color was allowed to develop at 30C for 5 min. Absorbance of the solution was measured at 750 nm and Pi concentration determined by comparison with a stan-

Enzyme assays
Samples of cultures containing approximately 100 mg dry weight yeast cells ml- were harvested by cenhifugation at 4,000 g for 4 min at 4C. This culture medium supematant was used as a source of extracellular enzymes. For the cell wall-bound enzyme fraction, cells (100 mg dry wt) were suspended in 10 ml of enzyme release buffer (50 mu potassium phosphate pH 7.0 containing 10 mM 2-mercaptoethanol, 10 mM dithiothreitol, and 2 mM MgS0,.14 The suspension was incubated for 1 h at 30C centrifuged as before, and then rewashed with similar buffer. Enzyme activities were analyzed in the growth medium supematant after incubation of the

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dard curve. ATPase activity was expressed mg- protein min- . as pm01 Pi released

Preparation

and fractionation

of spheroplasts

Alkaline phosphatase. Alkaline phosphatase was assayed by following release of p-nitrophenol (NP) from p-nitrophenol phosphate (PNPP). The reaction mixture contained 100 mu Tris-HCl pH 8.9, 5 mM MgCl,, 10 mM PNPP, and 50-100 kg protein in a final volume of 2 ml. After 30 min at 3OC, the reaction was stopped by addition of 1.O ml of 1 M NaOH and the absorbance of the mixture was measured at 410 nm.** Alkaline phosphatase activity was expressed as pmol NP formed mg- protein min- . NADPH-cytochrome c oxidoreductase. This enzyme was assayed 23 by following the increase in absorbance at 550 nm as cytochrome c was reduced in the presence of NADPH. The reaction mixture contained in a final volume of 2 ml; 50 mM Tris-HCl pH 7.7,0.1 mM EDTA, 2.2 mM KCN, 0.1 ItIM NADPH, 0.05 DIM cytochrome c, and 0.1 ml enzyme preparation containing 40 pg protein. The reaction was started by addition of the protein and the absorbance of the reaction mixture was measured for 3 min at room temperature. Enzyme activity was calculated using an absorption coefficient of 21 Fmol ml- cm- for cytochrome c,,~-c Activity was expressed as nmol cytochrome c reduced mg -oXprotein min- . Protein assay. Protein assay kit.24 was measured using a Bio-Rad protein

Characterization

of polygalacturonase

Determination of relative molecular mass by gel fdtration. A sephacryl SlOO HR (Pharrnacia, Uppsala, Sweden) column (90 cm X 1.6 cm) was equilibrated and eluted with 0. I M acetate buffer pH 5.0 at a flow rate of 1 ml min-. After checking the packing with blue dextran, the column was calibrated with cytochrome c (Mr. 14,200), soya bean trypsin inhibitor (Mr. 21,500), carbonic anhydrase (Mr. 29,000), egg albumin (Mr. 45,000), and bovine serum albumin (Mr. 66,000). All standards and samples were passed through the column at least twice, and a mean of their elution volumes was taken. Fractions (2.7 ml) were collected and assayed for PG activity and A,,,. SDS-PAGE. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) was performed as described by Laemmli. 25 Acrylamide gels (12% w/v) were calibrated with marker kit SDS-7 (Sigma). Freeze-dried samples were dissolved in 10% (v/v) trichloroacetic acid and left for 30 min in ice-cold water. After centrifugation (6,000 g for 15 min), the pellet was washed three times with 50% (v/v) ethanol ether. Samples were freezedried to remove the remaining ethanol ether, mixed in a 1: 1 ratio (v/v) with a loading buffer, and heated at 100C for 10 min before loading onto gels. Electrophoresis was performed at 200 mV constant current until the bromophenol blue marker ran off the bottom of the gel. Gels were stained with Coomassie brilliant blue. Isoelectric focusing. Isoelectric focusing was performed using a LKB flat bed gel electrophoresis kit at 4C for 90 min. The gels used were ready-prepared 1 mm polyacrylamide (PAG plates) which contained Pharmalyte carrier ampholytes to give a pH range of 3-10. The manufacturers instructions for PAG plates were followed. Separated PG isozymes were visualized by a pectateagarose overlay ge1.26 Following staining with ruthenium red (Sigma), clear bands in a red background revealed the position of PC isozymes. The PAG plates were then stained with Coomassie Brilliant Blue (30 min) and destained overnight in a solution containing water:methanol:acetic acid (6:3:1).

Spheroplasts were prepared by minor modifications of previous methods.27 Spheroplast formation was followed by diluting 0.1 ml of the suspension into 2.9 ml water, measuring the optical density at 600 nm, and assessing whole cell counts by microscopic examination with a hemocytometer. Spheroplast formation was also monitored by scanning electron microscopy (SEM) following dehydration by critical point drying (Polaron Equipment Ltd., Watford, UK). Cell breakage was effected by osmotic lysis through formation and disruption of spheroplasts. Cultures grown in a medium containing 50 g 1_ glucose under self-induced anaerobic conditions for different periods of time were harvested and converted into spheroplasts. All subsequent operations were performed in an ice-water mixture or a centrifuge at 4C. Spheroplasts from 100 mg dry wt were washed gently three times with buffered sorbitol and suspended in 5 ml buffered mannitol(0.3 M, containing 50 mM Tris, adjusted to pH 7.2 with HCl). Lysis of spheroplasts were effected by a combination of osmotic lysis and gentle mechanical disruption by incubating the spheroplast suspension for at least 20 min in an ice-water mixture and then subjecting the suspension to ten strokes of a tenon-glass hand homogenizer.28 Discontinuous sucrose-density gradients consisting of 3 ml each of 60,50,45,40, 35,30,25,20, 15, and 10 (w/v) sucrose in buffered mannitol were constructed and 5 ml of the spheroplast lysate applied to the top of each gradient. The gradients were centrifuged at 50,000 g for 120 min at 4C in an Sorvall ultracentrifuge fitted with a 6 X 35 ml swing-out rotor. Fractions (1 ml) were collected using a peristaltic pump. The sucrose density in the fractions was measured using an Atago Hand Refractometer (Type 500; Atago Ltd., Tokyo, Japan). Each fraction was then assayed for protein, PG, and marker enzymes for different organelles.

Mutagenesis
A 5-10% survival rate after N-methyl-N-nitro-N-nitrosoguanidine mutagenesis was used. 29 Strains selected on the basis of halos in solid pectic medium were further tested in liquid media.

Chemicals
All chemicals used were AnalaR grade or of the highest purity available commercially. Polygalacturonic acid Na-salt (PGA), dinitrosalicylic acid (DNS), N-methyl-N-nitro-N-nitrosoguanidine and hexadecyltrimethylammonium bromide were purchased from Sigma Chemical Co.

Results
Screening for pectinolytic activity
Most of the yeast strains studied were found during the first

36 h of cocoa fermentation by which time the pulp was degraded. The exception was for K. thermotolerans which only appeared at the end of the fermentation. Pectinolytic activity was determined for all 12 yeast species. K. marxianus showed a halo diameter of 30 - 2 mm, S. cerevisiae var. chevalieri a diameter of 7 2 3 mm; however, a halo was barely detectable with C. rugopelliculosa and K. thermotolerans. The remaining strains tested showed no extracellular pectinase activity.

236

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Endopolygalacturonase
Table 1 Extracellular cocoa fermentation PG activity of four yeasts isolated from

from Kluyveromyces

marxianus:

R. Schwan et al.

Yeast K. K. S. C.

PG activity (kmol galacturonic acid (pg- protein min-) 21.69 12.03 7.16 4.05

marxianus (CCT 3172) thermotolerans (CCT 1701) cerevisiae var. chevalier; (CCT 1698)
rugopelliculosa (CCT 1702)

Cultures were grown in a medium containing 1% (w/v) glucose + 1% (w/v) pectin under self-induced anaerobic conditions for 5 days

Production of pectinolytic enzymes by yeasts

The ability of K. marxianus, 5. cerevisiae var. chevalieri, C. rugopelliculosa, and K. thermotolerans to secrete pectinolytic enzymes in liquid medium was determined. Cells were grown in a basal medium containing both 1% (w/v) glucose and 1% (w/v) pectin at 30C for 5 days. Pectinolytic enzymes were produced only during exponential growth; therefore, the highest yields were obtained from lower inoculum levels which resulted in extended periods of exponential growth. All samples were assayed for pectinolytic activity including pectinmethylesterase (PME), pectin lyase (PL), pectate lyase (PGL), and polygalacturonase (PG). PG activity was measured by viscometry and the increase in reducing groups. All four yeast strains showed extracellular PG activity measured by the increase in reducing groups; however, the amount of enzyme secreted varied considerably between strains (Table 1) with K. marxianus as the most active secretor. Neither PME nor pectic lyases were detected in culture filtrates. When 1% (w/v) pectate was used as enzyme substrate, activity measured by reducing groups assay was threefold higher than when 1% (w/v) pectin was used as substrate. This suggested that pectate was the preferred substrate of these enzymes. Since lyase activity was not detected under these conditions, it can be concluded that these enzymes were polygalacturonases.
2 3 4 5

Effect of addition of pectic substances on growth and PG secretion

Including pectin or PGA in media containing 1.0 or 2.0% (w/v> glucose had no significant effect on growth or PG production by any of the yeasts when compared with that in media lacking these pectic compounds. Growth and PG secretion occurred only when glucose or another growth substrate was added to the media; thus, these yeasts were able to break down pectin, but were unable to utilize pectin or its monomer, galacturonic acid.

Figure 1 Effect of pH on PG activity. PG activity was measured at different pH values using a range of buffers: Citrate buffer (0) for pH 3.0-5.5, acetate buffer (A) for pH 3.5-6.0, and phosphate buffer (0) for pH 4.5-7.0. K. marxianus (A) and S. cerevisiae var. chevalieri (B)

Properties of polygalacturonase secreted by K. marxianus and S. cerevisiae var. chevalieri

The enzymes
cerevisiae

tested were produced by K. marxianus and S. grown in a medium containing 1% (w/v) glucose in l-l self-induced anaerobic fermentation for 5 days. PG activity was assayed by the increase in reducing sugars. Maximum enzyme activity was obtained at pH 5.0
var. chevalieri

for PG secreted by K. marxianus (Figure la) and pH 4.5 for PG secreted by S. cerevisiae var. chevalieri (Figure lb). The effect of pH on the stability of the enzyme was investigated by measuring the remaining activity after incubation at 40C in buffers giving pH values of 3.0-8.0. The enzyme secreted by K. marxianus was stable in the pH range from 4 to 6 but PG from S. cerevisiae var. chevalieri showed decreased activity at all pH values tested (data not shown). Tbe effect of incubation temperature on PG activity was tested from 20-70C and gave maximum reaction rates of PG from K. marxianus and S, cerevisiae var. chevalieri at 40C and 3OC, respectively (Figure 2). Beyond the optimum temperature of 40C for K. marxianus PG, activity was lost rapidly. The enzyme secreted by S. cerevisiae var. 1997, vol. 21, September 237

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Temperature

(C)

Figure 2 Effect of temperature on PG activity. Reactions used 0.4% (w/v) polygalacturonic acid in 0.1 M citrate buffer pH 5.0 for 1 h. PG activity was expressed as pmol galacturonic acid released pg- protein min-. K. marxianus (0) and S. cerevisiae var. chevalieri (0)

10

20

30

40

50

60

Time (min)
Figure 3 Decrease in cocoa pulp viscosity secreted from K. marxianus produced by PG

chevalieri was more sensitive. It was rapidly denatured at temperatures above 35C after 5 min of incubation. PG secreted by K. murxianus was stable in 0.1 M acetate buffer pH 5.0 for 10 weeks at 4C and was stable at -20C. It lost less than 10% activity after 4 months while PG activity from S. cerevisiae var. chevulieri was lost completely after a few days at either 4C or at -20C.

Enzymatic characterization

of yeasts

Hydrolysis

of native pectin in plant tissue by secreted by K.

polygalacturonase

marxianus

Polygalacturonase secreted by K. marxianus was tested against native pectin in plant tissues. Its effect on cocoa pulp was measured by the decrease in viscosity and against native pectin present in vegetables such as potato and cucumber by loss of coherence of tissue (maceration activity). Cocoa pulp was removed from the seeds using a depulping machine. Pulp was centrifuged (6,000 g for 20 min) to remove solid material. The supematant was then used as a substrate for the viscometric method. The reaction mixture containing 8 ml of cocoa pulp and 2 ml of enzyme (680 RVU) was incubated at 25C. Reduction in viscosity was recorded at l-2 min intervals. A sharp decrease in viscosity was observed after a 2-min incubation and a 50% decrease in viscosity was achieved within 18 min (Figure 3). PG produced by K. marxianus had very strong maceration activity on potato and cucumber discs (data not shown). Maceration of tissue from both species was extremely rapid with softening apparent even after only 5 min. Complete cell separation occurred between 40 (2,000 RVU preparation) and 60 (1,000 RVU) min for potato and 30-60 min with cucumber. 238 Enzyme Microb. Technol.,

Enzymatic characterization of the four pectinolytic yeasts was assessed using a semi-quantitative micromethod (APIZYM) which gives a calorimetric visualization of the hydrolysis rate of p-nitrophenol glycosides. All four yeast strains tested showed similar extracellular activity of nonspecific esterases, esterase lipase, lipases, phosphoamidase, and acid phosphatase, and K. marxianus alone showed slight alkaline phosphatase activity. Determination of relative molecular K. marxianus endopolygalacturonase mass of

From concentrated (versus polyethylene glycol Mr 20 kDa) culture supematant of K. marxianus, gel permeation resolved four peaks containing PG activities (Figure 4); molecular masses were calculated using a calibration curve constructed by plotting the log (Mr) against the elution volume minus void volume (Ve-Vo). The four PG forms had apparent Mr of 47, 41, 35, and 33 kDa. SDS gel electrophoresis separation in total denaturing conditions was performed on the protein of K. marxianus culture filtrates. The molecular masses of the proteins were calculated by comparing the relative mobilities of this enzyme with those of the standards. The four bands (Figure 5) between 45 and 36 kDa were assumed to be PG based on the gel filtration results for PG activity and total protein (Figure 4). The relative molecular mass of the four protein bands were estimated as 45,42,39, and 36 kDa (Figure 5). According to analysis of all bands by laser densitometry,

1997, vol. 21, September

Endopolygalacturonase

from Kluyveromyces

marxianus:

R. Schwan et al.

IV

,-

I-

40 Fraction Numher

Figure 4 Separation profile of extracellular PG applied to a Sephacryl SlOO column. One ml of 20X concentrated dialyzed culture supernatant was applied to the column and 2.7 ml fractions were collected and analyzed for protein (0) and reducing sugars released (a). Peaks I, II, Ill, and IV correspond to PG activity measured as pmol of galacturonic acid released min-. The elution points of standard relative molecular mass markers applied to the column are indicated by the arrows with Mr in kDa

about 85% of total protein secreted into the culture medium by K. marxianus consisted of PG.

Mechanism of enzyme action

Viscometric assay and identification of hydrolysis products were used to determine the mechanism of action of PG. An endoPG is characterized by a strong reduction in viscosity (e.g., 50%) with a concomitantly low release of reducing groups (e.g., l-3%) whereas an exoPG has to hydrolyze greater than 20% of the glycosidic linkages to obtain an equivalent viscosity reduction.25 The time required for a 50% decrease in viscosity of a 3.0% (w/v) polygalacturonic acid solution at 25C was approximately 18 min at which time about 1.5% of the total galacturonide bonds had been hydrolyzed (data not shown). These results revealed a random mechanism of hydrolysis and the enzyme was therefore a poly 01 (1,4)-D-galacturonide glycanohydrolase (EC 3.2.1.15) or endoPG.

three, closely associated bands (I-IV) found at sucrose densities between 1.16 g ml-i and 1.20 g ml-. The identification of the bands was achieved using marker enzymes and transmission electron microscopy. Band I included many vesicles and a peak of alkaline phosphatase activity (a vacuolar marker in yeasts). It was located at a density of 1.05 g ml-. The NADPH cytochrome c oxidoreductase activity, an endoplasmic reticulum marker, had a peak at a density of 1.11 g ml-. Transmission electron microscopy (TEM) revealed the presence of lipid vesicles

Mr
(X lo31

Location of endopolygalacturonase in batch cultures

The distribution of PG as culture medium supematant, cell wall-associated, and cell-bound enzyme was determined in K. marxianus during the time course of growth in 5% (w/v) glucose medium. PG secretion started between 8 and 12 h after inoculation. Approximately 90% of total PG was secreted in the early stationary phase. The subcellular location of PG was studied in cells disrupted by osmotic lysis through formation and disruption of spheroplasts. Although young cells were very (95%) susceptible to treatment with Zymolyase, they became more resistant to attack after 14 h of growth. To overcome this problem, cells were pretreated with 0.1 M mercaptoethanol for 30 min at 30C. The sulfhydryl agent reduces disulfide bridges in the outer mannan-protein layer. This aids formation of spheroplasts and induces enzyme release.30 Spheroplast lysates from self-induced anaerobically grown cells produced two distinct regions of turbidity in a discontinuous sucrosedensity gradient. There was a single, milky-white band at the top of the gradient ( 1.04 g ml- sucrose) and a group of

Figure 5 SDS-polyacrylamide gel electrophoresis of culture supernatant of K. marxianus CCT 3172. Freeze-dried samples were resuspended in distilled water, mixed with an equal volume of sampling buffer, and heated at 100C for 5 min. Aliquots of 10 ~1 were applied to a 12% (w/v) discontinuous SDS-polyacrylamide gel system. Molecular masses of standard proteins (Sigma): Bovine serum albumin (66 kDa), egg albumin (45 kDa), glyceraldehyde-3-phosphate dehydrogenase (36 kDa), carbonic anhydrase (29 kDa), trypsinogen (24 kDa), and alactalbumin (14.2 kDa). Lanes 2,3,4, and 1 are increasing diluted culture supernatants of K. marxianus, respectively

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Table 2 Distribution of PG in spheroplasts and subcellular fractions of wild type and mutant K. marxianus PG activity= Wild type Time (hjb Cellular fraction Spheroplasts Low-density vesicles Vacuoles ER Plasma membraneC 8 673 350 84 160 59 10 715 314 115 50 246 12 516 210 88 48 170 14 356 81 32 38 205 16 212 57 0 0 148 24 0 0 0 0 0 8 275 150 55 70 0 Mutant 3 120 255 215 25 15 0 cells

aExpressed as RVU bHours after inoculation of self-induced anaerobic culture Organelle identified by peak of marker enzyme activity

which were identified by their affinity for osmic acid3 at densities 1.08 and 1.14 g ml-. The peaks for protein content corresponded to densities of 1.045 g ml- and Mg++ 1.20 g ml-. Over 85% of the vanadate-sensitive ATPase activity, marking yeast plasma membranes, was detected at densities 1.16-1.20 g ml-. When the bands II, III, and IV were examined by TEM, they were found to contain plasma membrane. This confirmed the results obtained using ATPase as a marker enzyme for this organelle. Vesicles were also detected in these bands but they appeared to be part of the plasma membrane and had not resulted from fraction contamination. Spheroplast lysates fractionated on sucrose-density gradients showed sequential movement of PG from the ER via low density vesicles to the plasma membrane (Table 2). PG activity was not found in fractions of cells harvested after 24 h.

Isolation and enzymatic characterization K. marxianus mutants

of

Nitrosoguanidine was used to produce mutants in an attempt to isolate strains with enhanced PG production. About 18,000 colonies were initially screened and approximately 0.1% of survivors had altered PG levels based on the size of halos. Mutants (138) produced clearing zones of repeatable and different diameter compared to the parent strain (diameter of 30 2 2 mm). PG activity of each mutant was then measured in the supernatant from liquid cultures. Only five mutants showed increased extracellular PG activity compared with the parental strain. The highest activity was 23.4 PG units which is 25% above the wild-type level. The great majority of mutants (107) showed reduced levels of PG activity ranging from O-15 PG units. Some underproducers had lower growth rates than the wild type. Mutants with reduced PG levels could have resulted from impaired secretion and/or synthesis. Mutants (130) showed similar or lower intracellular PG levels compared to the wild type. These comprised both over- and underproducers. Three mutants with higher intracellular activity secreted the enzyme later than the wild type. Five mutants with reduced levels of secretion had very high intracellular PG activity. This possibly indicated a defective secretory 240 Enzyme Microb. Technol.,

pathway; however, total PG activity remained below wildtype levels. Subcellular fractionation was performed in order to investigate the intracellular location of accumulated PG in those mutants. Spheroplast lysates of mutants 3,20,44, 103, and 110 were separated on a discontinuous sucrose-density gradient. The same numbers of bands were observed as in the parental strain; however, the milky-white band at the top of the gradient was larger and more dense in all five mutants than that found in the wild type (data not shown). A greater number of vesicles in comparison with the wild type was observed in the top band from mutants 3, 20, and 103. The distribution of PG activity in subcellular organelles was broadly similar in these five strains and is shown for mutant 3 (Table 2). PG activity was detected in low-density vesicles, vacuoles, and ER fractions in samples harvested during the early exponential phase of growth; however, PG levels were always lower (at least 1.5-fold) than those found in the wild type. Cells of the mutants harvested during the stationary phase of growth showed that 84% of total intracellular PG activity was located at sucrose densities 1.04-1.06 g ml- which corresponded to the vesicle fraction (Table 2).

Isoenzyme profile of polygalacturonase and mutant strains

in wild-type

Isoelectric focusing (pH 3-10) of dialyzed, concentrated culture supematant from wild type grown on glucose medium under self-induced anaerobic conditions showed up to nine grouped apparent PG isoenzymes. Three major bands (estimated pIs 5.9, 5.6, and 5.3) and six minor bands of apparent isoforms range from 6.4 to 5.0 (Figure 6). The time course of growth showed that all nine isoforms were present in the culture filtrate after only 24 h of incubation. The apparent absence of some bands before this time may reflect relative amounts and sensitivity of the overlay technique. Eighteen mutant isolates showing a wide variation in PG activity were selected as representative samples for the study of isoenzymes. Extracellular PG activities of these mutants grown as above for the wild type but for 48 h are given in Figure 8.

1997, vol. 21, September

fndopolygalacturonase

from Kluyveromyces

marxianus:

R. Schwan et al.

P
9.3 -

6.85

5.20-

5.85

5.20

Figure 7 lsoenzyme profiles of K. marxjanus mutants. WT indicates wild-type strain and other numbers indicate mutants with varied PG activities

Discussion 3.50 Of 12 yeast species isolated from cocoa fermentations, K. marxianus, S. cerevisiae var. chevalieri, C. rugopelliculosa, and K. thermotolerans produced extracellular, constitutive PG but no PME, PL, or PGL although some strains of S. cerevisiae are known to produce several pectinolytic enzymes.3 The amounts and properties of each PG differed but all were relatively unstable compared with that of K. marxianus which was also produced at the highest levels. It follows that K. marxianus is likely to be the key yeast for the hydrolysis of cocoa pulp pectin in initial fermentations. PG secreted by K. marxianus CCT 3 172 had a strong activity in reducing the viscosity of cocoa pulp. This strain was found during the first 36 h of cocoa fermentation when the pulp is degraded. PG secreted by K. marxianus CCT 3 172 showed activity from pH 4-6 with an optimum at pH 5 typical of PG secreted by yeasts*7.,3J and very similar to that obtained previously for Saccharomyces ,fragilis.35 Unlike some pectinases. K. marxianus PG activity was not affected by buffers used across the pH range studied. The effect of temperature on PG activity from K. marxianus was similar to that reported for PGs from yeasts.3.7,7h Although the reason for the lack of PG stability from other yeasts is not known, the biological significance is clear. Only PG from K. marxianus is produced in sufficient amounts at the right time with the correct optimum pH and the right stability to reduce viscosity in cocoa fermentations. Gel filtration indicated that four proteins were present even though the size separation between them was minimal. The relative molecular masses were in close agreement with previous estimates. 6~37 PG secreted from strains of S. cerevisiae had similar Mr values around 40 kDa.* (Also A. Belarbi, personal communication.) Activity-stained IEF gels revealed the presence of nine multiple forms that could be divided into two acidic groups 1997, vol. 21, September 241

Figure 6 lsoenzyme profiles of K. marxianus. Detection of PG activity was on polygalacturonic acid-agarose overlay gel after resolution by ultra thin-layer isoelectric focusing (PAG plates). The overlay gel was then stained in 0.02% (w/v) ruthenium red. Samples l-5 were dialyzed culture filtrates from 8, 12, 14, 16, and 24 h growth after inoculation, respectively

Mutants and wild type showed a common pattern of at least three major isoenzymes (Figure 7) and six minor apparent isoforms. Profiles of PG from wild type and the very low underproducers (mutants secreting less than 1% of the normal PG) were equivalent in band number (nine), but the intensity of all bands from the mutants was generally weaker; however, some mutants which were also underproducers (2, 3, and 4) had almost identical profiles to the wild

type.

Inulinase

A comparison was made with inulinase, another secreted enzyme. Inulinase levels of K. marxianus mutants in shake flasks cultures grown on inulin were determined in the culture supernatant and in both cell wall-associated and ground-cell portions. There was considerable variation in the amount of enzyme partitioned between cell wall and cytoplasm, but with few exceptions, the proportion of total activity found in the supernatant was approximately 70% (data not shown). There was a clear relationship between extracellular inulinase and PG production (Figure 8).

Enzyme Microb. Technol.,

Papers

2oc
0 0

5 150 T )1 .= > .P 8 2 .2 50
0

100
0
0

l
0

50

100

150

PG activity (%)
Figure 8 lnulinase and endopolygalacturonase production by wild type and mutant strains of K. marxianus. Cell were grown in flasks at 30C for 48 h under self-induced anaerobic conditions with glucose at 50 g I-. PG activity and inulinase activities for each strain are expressed as percentages of wild-type values which were 18.6 wmol galacturonic acid hydrolyzed kg- protein min- and 51.0 pmol fructose hydrolyzed mg- dry weight min-, respectively

in relation to the intensity of bands. The first one consisted of three major bands (~1s estimated 5.9,5.6, and 5.6) and the second with five or six minor isoforms showed weaker band intensities. The p1 values in this study are in agreement with previous estimates. 37 The numbers of apparent isoenzymes are sometimes caused by variations in glycosylation, glycolytic, or proteolytic degradation (especially apparent in older cultures), or artifacts from IEF, however, the presence of all nine PG isoforms in growth medium from 24 h culture suggests that they are not artifacts. Aspergillus niger has at least three PG genes 39 but the number of observed isoforms can greatly exceed this value. None of the underproducer mutants had lost a single isoenzyme indicating that none of the mutations were in structural genes. Those underproducer mutants showing nine isoenzymes with similar intensities of each band (strains 6, 7, 30, and 31 in Figure 8) seem to have had a mutation in the regulatory genes affecting a subset of the isoenzymes. It has been suggested previously that the weak intensity of bands in activity-stained gels indicates an overall reduction in the release of the isoenzymes.26 Differences were found in isoenzyme profile, growth rate, and total excreted proteins in mutants which showed about 20% extracellular PG activity of wild-type levels, a phenotypitally pleiotropic effect in which various enzyme systems were modified (unpublished data). An attempt was made to reveal similar profiles from the other three pectinolytic yeasts. Neither K. thermotoleruns nor C. rugopelliculosa produced sufficient stable enzyme for the analysis. S. cerevisiue var. chevalieri had a profile similar to K. marxianus (data not shown). The lower 242 Enzyme Microb. Technol.,

amounts and stabilities of endoPG from the other three yeasts indicated that their contribution to pectin degradation in situ must be less than that of K. marxianus. The endoaction of the K. marxianus PG was reflected in an extremely rapid attack on plant tissue. This activity appears to be at least equivalent to that of several commercial pectinase preparations used for separating plant cells for protoplast preparation (RMC, unpublished data). Most of the endoPGs produced by plant pathogens and saprophytes have so far been reported to possess macerating activity. The strong activity of PG in reducing the viscosity of cocoa pulp reveals the key role of polygalacturonan in the physical properties of this mucilage. Pectin makes up about l-1.5% (w/w) of cocoa pulp and is 68% esterified. This demonstration provides a rare example of enzymatic degradation of a native, nonextracted, complex polymer. It is tenable that PG obtained from K. marxianus may be utilized directly on cocoa beans to speed up the fermentation process and also to obtain higher quality fermented beans. Most studies on PG from K. marxianus show that it is almost entirely extracellular in contrast to lactase and inulinase which can be found both intra- and extracellularly. The distribution of PG activity produced by K. marxianus among supematant, cell-wall and cell-bound fractions changed throughout the time course of growth. It is likely that PG activity found in cell-wall fractions was the enzyme in the process of being released rather than located in the periplasmic space. Studies on the subcellular location of PG using sucrose density gradient showed that during the early exponential phase of growth, PG was distributed in fractions containing low-density vesicles, high alkaline phosphatase, NADPH-cytochrome c oxidoreductase, and vanadate-sensitive ATPase activities; however, the proportion of PG in these fractions varied during batch growth which suggested that PG was synthesized and secreted by the classic yeast secretory pathway. This conclusion is consistent with the results from the five mutants which synthesized PG but were unable to release it into the medium. Subcellular fractionation revealed that 90% of intracellular PG activity was located at the vesicle fraction. This indicated a specific transfer block at the stage from vesicles to plasma membrane. Vesicles are involved in transport of enzymes from their site of synthesis to their site of accumulation. This is the first study of subcellular location in K. marxianus. Sucrose-density gradients were used to fractionate spheroplast lysates of K, marxianus. Electron microscopy confirmed the presence of many low-density vesicles in the top (lowest density) band. Vesicles and vacuoles from S. cerevisiae have been isolated by various authors.4 Vacuoles were not found in the vesicle fraction examined by electron microscopy although the peak of alkaline phosphatase (a vacuolar marker enzyme) was located close to the top band. Identification of vacuoles has caused confusion because of their contamination by various types of vesicles; for example, lipid vesicles, lipid-rich enzyme-containing vesicles, or plasma membrane vesicles. The plasma membrane, identified by transmission electron microscopy, and plasma membrane ATPase marker enzyme was separated into two or three fractions. The plasma membrane of K. marxianus was found between densities 1.16-l .20 g ml- sucrose. Some vesicles were found associated with it.

1997, vol. 21, September

Endopolygalacturonase The screening method based on the diameter of halos yielded only five strains with at least 20% more PG than the wild type. Most attempts to enhance pectinase production have been made in filamentous fungi in which enzyme production is regulated by induction and catabolite repression mechanisms.42 By contrast, PG produced by K. marxianus is constitutive and not subject to catabolite repression. PC was already found to be the most prolifically secreted protein of K. marxianus and failure to find greatly enhanced secretion levels is consistent with the idea of a constitutive gene. To obtain substantial overexpression, strains will probably require cloning of the PG gene and subsequent genetic modification of wild isolates.4 Inulinases (EC 3.2. I .7) from yeasts in particular are able to hydrolyze both inulin and sucrose very efficiently. Inulinase in K. marxianus was efficiently secreted into the culture medium.44 Although inulinase is regulated by both induction and repression, reasonable enzyme levels were produced in the absence of inducer with cultures grown on glucose as the sole carbon source. Figure 8 shows that all mutants of K. marxianus that produced high PC activity showed high total inulinase activity and PC underproducers also showed low levels of inulinase. This suggests that these mutations were affecting the capacity of the cell to export extracellular enzymes and that PC and inulinase rely on common mechanisms of secretion. Some mutants were inefficient in secreting one or the other of the two enzymes and are likely to be caused by mutations affecting the structure or regulation of only one of the two enzymes. A NTG-induced K. marxianus mutant (KF28) selected as an inulinase overproducer was also a pectinase overproducer.4s No other pectinolytic enzymes, apart from PC, were secreted by K. marxianus into the culture media. This should facilitate the process of producing a pure enzyme which could be used directly on cocoa beans for the extraction of cocoa juice. The pulp obtained with the aid of pectinolytic enzymes has a lower viscosity. This aids processing of pulp for pasteurized juice and soft drinks from cacao. A more long-term goal would be to construct overproducing strains of K. marxianus which could be used as starter cultures for cocoa fermentation. It has already been demonstrated that K. marxianus has considerable economic advantages over Aspergillus as a source of endoPG even without genetic improvement of the strain. Cloning of the gene for PC would be highly advantageous for the wine, cider, and fruit juice industries. Other potential industrial applications are in softening of vegetables for preparation of baby foods and manufacture of fruit nectars. At present, large quantities of PG are added to batch fermentations to clear haze from fruit juices and eiders. The current source of the enzyme is the filamentous fungus Aspergillus niger. There are four actual or potential problems with this source. First. many commercial pectinases are low in PG activity. Secondly, A. niger also secretes pectinmethylesterase which, if left in the pectinolytic digest, produces the toxic alcohol methanol. Thirdly, production costs of this enzyme are high if pure enzyme is needed since separation from other enzymes is required. Lastly, A. niger also secretes a variety of other enzymes. Some possess oxidizing activities that are not desirable in the production of wine and fruit juices, and others (for

from Kluyveromyces

marxianus:

R. S&wan

et al.

example, arabinofuronosidase) can cause the formation of haze. Furthermore, many commercial pectinases contain beta-glycosidases which can hydrolyze cyanogenic glycosides which may be present in indigenous fruits, thereby releasing cyanide. 46 With K. ma&anus additionally achieving the status of being designated GRAS (Generally Regarded As Safe), it presents excellent opportunities for further development.

Acknowledgments
R. F. Schwan was supported by the Brazilian Council (CNPq) under grant no. 200.122/91.6. Research

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