World

Journal

of Microbiology

and Biotechnology

9. 574470

Partial purification and properties lyase from Penicillium expansum

of pectin

D.O. Silva,* M.M. Attwood and D.W. Tempest A pectin lyase, poly(methoxygalacturonide) lyase, EC 4.2.2.10, from a culture filtrate of Penicilfium expansum was partially purified 33-fold with 7.3% yield. The enzyme was monomeric with a molecular mass of 36.5 kDa. The enzyme did not contain pectate lyase activity and degraded citrus and apple pectin best at pH 7.0 and 40 to 45’C. The K, for citrus pectin was 9 mg ml-‘. Key words: Partial purification,

pectin lyase, pectin transeliminase,

Poly(methoxygalacturonide) lyase, EC 4.2.2.10, (pectin lyase) removes 6-methyl-A-45 galacturonate residues from pectin. The enzyme was described originally as pectin transeliminase because the reaction released unsaturated gala&ironic acid groups from, in particular, the methyl esters of pectin (pectic acid) by means of transelimination (Albersheim et al. 1960). To date, it is the only known enzyme able to cleave, without prior action of other enzymes, the a-1-4 glycosidic bond of highly esterified pectins such as plant pectin (Fogarty & Kelly 1983; Pitt 1988). There are two closely related enzymes: pectin lyase, which is synthesized specifically by fungi, and pectate lyase (EC 4.2.2.1), mainly formed by bacteria, particularly those associated with phytopathological processes (Garibaldi & Bateman 1971). These enzymes, together with polygalacturonases, which catalyse a hydrolytic cleavage, are responsible for soft rot diseases of plant products after harvesting and during storage (Chesson 1980; Collimer & Keen 1986). Enzymes from non-pathogenic organisms catalyse the degradation of pectin polysaccharides during the decay of dead plant material and also have an important role in the food and textile industries. In the food-processing industry such enzymes are used in the extraction and clarification of fruit juices and grape musts and the maceration of vegetables and fruits (lshii & Yokotsuka 1971, D.O. Silva was and M.M. Attwood and D.W. Tempest are with the Department of Molecular Biology and Biotechnology, University of Sheffield, P.O. Box 594, Firth Court, Western Bank, Sheffield SlO 2UH, UK. D.O. Silva is now with the Departamento de Microbiologia, Universidade Federal de Vicosa, 36570-000 Vicosa, MG. Brazil: fax: (031) 899 2203 or (031) 891 3911. *Corresponding author. @I 1993 Rapid

574

Communications

World ]oumal

of Oxford

of Microbiology

Ltd

and Biotechnology, Vol 9, 1993

pectinase, Penicillittm expanstrm.

1972). In the textile industry the enzymes are used to separate cellulose fibres from cell walls (Tanabe & Kobayashi 1986); the production of pectinase and cellulase from F’enicillium expansum, which could be used in this process, has been investigated (Baracat et al. 1989). This paper describes the partial purification of pectin lyase from P. expansum and some of the enzyme’s properties.

Materials

and Methods

Microorganism

The strain of P. q~~stlrn used in this study was isolated in Brazil and kindly provided by JJ. Muchovej (Universidade Federal de Vicosa, Brazil). It was maintained by incubation on potato/ glucose/agar for 4 days at 25 “C and stored at 4 “C. Growth Conditions Cultures

were

grown

in shaking

(150 rev/min)

Erlenmeyer flasks phosphate buffer, (NH,),SO,, 10 pg pectin (Sigma). The pectin was not sterilized but added after the sterilization of the medium. To each flask of spore suspension, 2 x lo6 spores were added as the inoculum and incubated 120 h at 25°C. Contamination of the flasks was negligible; any contaminated flasks were removed.

(250 ml) with 100 ml of 0.05 M potassium pH 7.0 (Buffer A), containing 1% (w/v) chloramphenicol ml-’ and 0.3% (w/v) citrus

Enzyme Assay Pectin lyase activity was determined spectrophotometricaly by measuring the increase in absorbance at 235 nm as previously described (Albersheim & Killias 1962). The reaction mixture (2.5 ml) containing 0.05 M phosphate buffer, pH 6.7 (Buffer B), 0.4% (w/v) citrus pectin and enzyme was incubated at 40T for 20 min. The reaction was stopped by adding a sample (0.5 ml) of the mixture to 4.5 ml of 0.01 M HCl. One unit of activity was defined

Pectin lyase frum

Penicillium

expansum

as the amount of enzyme which produces an increase of one unit at 23.5 nm per min. Pectate Iyase activity was assayed by the same method using polygalacturonate as substrate. Absorption

Specfrum

-7 z

The absorption spectrum was recorded at room temperature against a buffer blank in !%'I800 Pye-Unicam Spectrophotometer with cuvettes of I cm path length.

3-

-1.2

vL

v) .z 5

Enzyme Ptlrificution The mycelia were removed from 150 Erlenmeyer flasks (15 1) by filtration, and chloroform (1 ml) added to the culture filtrate before it was concentrated to 10 ml by dry dialysis with Aquacide II (Calbiochem) at 4°C. The concentrated culture filtrate was centrifuged at 8000 x g for 20 min and the clear supematant used as the crude enzyme extract. All steps were at 4’C.

9

x

2-

‘5 2 3 p

l-

.c 5 a

Sephader G-75 Filtration. The crude enzyme was applied to a Sephadex G-75 (Pharmacia) column (1.6 x 40 cm), equilibrated and eluted with Buffer B at a flow rate of 1.33 ml h-‘. The active fractions were pooled and concentrated with Aquacide II.

I

0 I

I

4

5

6

I r

6

1

growth

medium

pH on pectin

PH DEAE-cellulose Chromatography. The concentrated active fractions were loaded onto a DE-52 column (1.0 x 40 cm) equilibrated and eluted with Buffer B at a flow rate of 0.6 ml h-‘. The pectin lyase activity was not retained on the column and was recovered in the eluate before a NaCl gradient (0 to 1 M) in Buffer B was applied.

The active fractions were pooled and concentrated as before. S-Sepharose Fast Flow Chromatography. The active fractions were applied to S-S ep h arose (Pharmacia) (4 x 8.5 cm) column equilibrated and eluted with Buffer B at a flow rate of 2.6 ml hK’. The active fractions were pooled, concentrated as before and applied to a second Sephadex G-75 column and eluted at a flow rate of 2.7 ml h-‘. The active fractions were pooled and concentrated before storage at -220°C. Gel Filtration for Molecdur Muss Determination Sephadex G-200 (Phannacia) column (2.5 x 82 cm) was prepared and equilibrated with Buffer B. The column was calibrated using

fl-galactosidase (510 to 530 kDa), catalase (230 to 250 kDa), lactate dehydrogenase (130 to 140 kDa), bovine serum albumin (65 to 70 kDa) and cytochrome G (12.5 kDa) (Andrews 1964). The partially purified enzyme was applied to the column and eluated with Buffer B at the flow rate used to calibrate the column. The elution volume containing the pectin lyase was used to determine the native molecular mass of the enzyme (Andrews 1964). Deterrninufion of Protein Concenfrution Protein content in the cell-free broth was determined by method of Lowry, with bovine serum albumin as standard. Cell Dry Weight Growth was measured

Results

by the method

of Calam

the

(1969).

and Discussion

Production of Pectin Lyase For optimum pectin lyase synthesis, the culture media had to be carefully buffered. If the potassium phosphate concentration in the growth medium was less than 0.05 M the pH of the medium at the end of the growth period

Figure

1. The effect

activity Standard

(0) and growth

of initial

mycelial dry wt (0) of Penicillium media were used throughout.

lyase

expansum.

could be as low as 3.0 and, although significant amounts of mycelia were formed, the pectin lyase activity was then low (Figure I). This requirement for growth medium of neutral or alkaline pH for the production of an active enzyme has been reported in other microorganisms (Fogarty & Ward 1974; Silley 1986; Manachini et al. 1988). Since the mycelial mass (dry wt) of I? erpansum was constant over the pH range 3.0 to 7.5 (see Figure l), other polygalacturonases must be present in the media to break down the substrate, pectin, used in the production of biomass. Indeed, the expression of polygalacturonase activity in I? expansum is higher when the growth medium has a pH value below 6.0 (data not shown). Pwificatiofl

Table 1 summarizes a typical purification from 15 1 of culture filtrate. The pectin lyase was purified 33-fold, with a 7.3% yield and a specific activity of 89.7 units mg protein-’ at 40°C. This level of purification and yield were similar to that reported for other pectin lyases purified from Penicillium spp. and the specific activity was within the range reported for

other

fungal

pectin

lyases

(Bush

& Codner

1968,

1970).

Throughout the purification procedure, one peak of activity was demonstrated (Figure 2A to D), indicating P. expansum produces just one pectin lyase. Other Penicillium spp. also produce single pectin lyases (Wijesundera et al. 1984) but other microorganisms produce more than one each (Blieva & Rodionova 1987; Manachini et al. 1988; Parini et al. 1988; Pitt 1988; Baldwin & Pressey 1989). The enzyme was not purified to homogeneity as judged by SDS polyacrylamide gel electrophoresis (data not shown). However, enzyme

World ]oumal of Mtcrobiofogy

and Bmtechnology, Vol 9, 1993

575

Table

1. Purifkatlon

Purifkatlon

of pectin

lyase

from

step

Concentrated Sephadex-Gi’5 DEAE-cellulose S-Sepharose Sephadex-G75

culture

flltrates. Yield

TOtA

Volume (ml) 1. 2. 3. 4. 5.

P. expansum

supernatant

(fast flow)

Protein OW

10 1 6.5 1.3 0.45

265 45 14.2 3.0 0.59

analysis indicated that the partially purified enzyme did not contain pectate lyase activity. Recently, the pectin lyase from P. italicurn has been purified to homogeneity (Alana et

AcNvity (units)

(units

719 419 262 131 53

mg protein

-3

Purification (fold)

v4 f

2.7 9.2 18.4 43.1 89.7

100 58.2 36.4 18.2 7.3

1 3.4 6.8 16 33

(31 to 35 kDa) reported for pectin lyases from microorganisms which produce only one pectin lyase each (Alana et al. 1991).

al. 1991).

The molecular mass of pectin lyase from P. ezpa~~~ was estimated, with a calibrated Sephadex G-200 gel filtration column, to be of 36.5 kDa. This value is similar to the values

Enzyme Fyffper~ies

The absorption spectrum of the partially purified enzyme shows a typical spectrum for a protein with no chromogenic prosthetic groups (i.e. no absorption between 320 and 600 nm and an E,,,/E,, of 0.83). This was not dissimilar to the spectra reported for the pectin lyases from ~erg~~f~ japonictrs(Ishii & Yokotsuka 1975) and P. if&urn (Alana et al. 1991).

Pectin lyase from P. expansum catalysed the cleavage of pectin from different sourcesbut was more active with citrus pectin than apple pectin and had little or no activity with polygalacturonic acid (Table 2). This variation has been reported with other pectin lyase preparations (Ishii & Yokotsuka 1975: Alana et al. 1991). Further work is required to understand the ways in which the degree of ester&cation of the substrate may interfere with the specificity of the enzyme. The effect of temperature on the activity of pectin lyase from P. e~~~u~ was similar to that reported for other fungal and bacterial pectin lyases. Activity was measured over a wide range of temperatures (5 to 70 “C) with an optimum range of 40 to 45 “C (Figure 3). Like many fungal pectin lyases, the enzyme was fully thermostable up to 45°C. Above this temperature, activity was lost rapidly and the enzyme was completely inactivated at 60 “C and 70 “C after 8 and 6 min, respectively. -1

4

I

b\

Table

ol---jo 2

Substrate

?a

IO

2. Substrate

specifkity

of pectin

at 1% (w/v)

lyase

from

I? ex~~uurn. Relative

Praction2&mber Figure 2. Partial purification of pectin lyase exp~ffsum by: (A) Sephadex G-75 gel filtration, (B) DEAE-cellulose chromatography, 2.4ml Sepharose fast flow chromatography, 2.7-ml a second Sephadex G-75 gel filtration, 2.7-ml the level of enzyme activity (a), absorbance O), and the NaCl gradient (----).

activityt W)

from Penicii/iu~ 2.4ml fraction; fractions; (C) Sfractions; and (D) fractions, showing at 280 nm (A,,,;

Pectin (citrus) Pectin (apple) Polygalacturonic

100 43 4

acid

* In 0.05 M potassium phosphate t Activity was measured using 100% activity = 87 units ml-‘.

buffer, pH 6.7. the standard assay

procedure.

Pecfin lyase from Penicillium

Temperature

(“C)

Figure 3. Effect of temperature on pectin lyase activity and thermostability. Enzyme activity was measured with the standard assay procedure either at different temperatures (0) or at the optimum temperature after 40 min preincubation at the given temperature (Cl).

The enzyme showed activity from pH 3.8 to 8.6, with an optimum at 7.0. Unlike some pectin lyases, the activity of the partially purified enzyme was not affected by the buffers used across the pH range studied (see Figure 4). The enzyme from I? expff~s~~ was not as stable at basic pH values as some fungal pectin Iyases (Alana et al. 19%) but was active over a wider range of pH values (Ishii & Yokotsuka 1975; Parini et al. 1988; Baldwin & Pressey 1989). The K,,, for pectin was calculated by standard procedures from initial enzyme rates over a range of substrate concentrations, using the assay procedure described. Double reciprocal plots were used to calculate the K,,, value; Michaelis-Menten kinetics was observed and the correlation coefficient was 98%. The K, for pectin was 9 mg ml-’ with a V,,, of 105 units ml-‘. The high viscosity of pectin makes it difficult to work with concentrations above 0.4% (w/v) in the reaction mixture. Therefore all enzyme assays were performed for 20 min since activity varied linearly with respect to the amount of the enzyme used. Due to the lack of a standard substrate and the different pH values used in their estimation, it is difficult to compare K, values for pectin lyases from different sources. However, the value for pectin lyase from I? e~~~s~~ is not si~i~cantly different from those reported for pectin lyase from P. paxilli (Szajer & Szajer 1982) (2.5 mg ml-‘) and P. ifahm (Alana et al. 1991) (15.0 mg ml-‘). Like pectin lyases from other fungi, the activity of the I? expansum enzyme was not inhibited by chelating agents such as EDTA or by metal ions (Parini et al. 1988). However, unlike some pectin lyases (Ishii & Yokotsuka 1975)‘ the partially purified enzyme was not stimulated by the presence of Ca” + (Table 3), perhaps because the citrus pectin used

Table lyase

3. Effect actlvity.

of EDTA

Compound

and metal

I

I

I

6

I

I

i

8

PH Figure 4. The pH dependence of activity of pectin lyase from P. expansum measured by the standard assay. The buffers used were citrate/acetate (0). phosphate (0). and TrislHCl (A).

2 2 10 50 100 2 2 2 2 2 2

cuso, ZnSO, MgSO, MnCI, coso, Mix* * Each metal ion at 2mu. t 100% activity = 92 units

ions

Final concentration (mMn)

None EDTA CaCI,

4

expansum

ml-‘.

on P. expansum

pectin

Relative activityt (%I loo 108 100 100 78 48 78 113 100 108 100 96

D.O. Silva, M.M.

Ahood

and D. W. Tempest

contained bivalent cations. However, high concentrations of Ca2 + did inhibit growth (data not shown). The enzyme has a potential use in studies on plant cell wall degradation during plant pathogenesis, in the isolation of plant cell protoplasts and in our main interest, the maceration of plant tissues. Additional studies are therefore required to fully characterize the pectin lyase produced by l? ~pa~rn.

Acknowledgements DOS was the recipient of a scholarship from CNPq and the research work was part of a project supported by FINEP at the Universidade Federal de Vicosa, Brazil.

Calam, C.T. 1969 The evaluation of mycelial growth. Mefho~ in Mi~obiolo~ 1. 567491. Chesson, A. 1980 Maceration in relation to the post-harvest handling and processing of plant material. Journal of Applied Bacteriology

48,

1-45.

Collimer, A. & Keen, N.T. 1986 The role of pectic enzymes in plant pathogenesis. Annual Review of Phytopathology 24, 383-409.

Fogarty, W.M. & KeIly, CT. 1983 Pectin enzymes. In ~i~obia~ Enzymes and Biotechnology, ed Fogarty, W.M. pp. 131-182. London: Applied Science. Fogarty, W.M. & Ward, O.P. 1974 Pectinases and pectic polysaccharides. Progress in Industrial Microbiology 1.3, 59-119. Garibaldi, A. & Bateman, D.F. 1971 Pectic enzymes produced by Erwiniu ch~sanfhemi and their effects on plant tissue. Physiology and Plant Pathology 1, 25-40. Ishii, S. & Yokotsuka, T. 1971 Pectin trans-eliminase with fruit juice clarifying activity. ]ourd of Agricuifwe and Food Chemistq 19, 958-961.

Ishii, S. & Yokotsuka, T. 1972 Clarification of fruit juice by pectin trans-eliminase. Journal of Agriculture and Food Chemistry 20,

References Alana, A., Llama, M.J. & Serra, J.L. 1991 Puri~cation and some properties of the pectin lyase from PenicilIium ifuIicum, Federation of European Biochemical Sociefies Letfers 2, 335-340. Albersheim, P. & Killias, U. 1962 Studies relating to the purification and properties of pectin transeliminase. Archives of Biochemistry and Biophysics 97, 107-115. Albersheim, P., Neukom, H. & Deuel, H. 1960 iiber die bildung von unges~~igten abbauprod~ten durch ein pektinabbauendes enzym. Helvetica Chimica Acfa 43, 1422-1426. Andrews, P. 1964 Estimation of the molecular weights of proteins by Sephadex gel-filtration. Biochemical ]ournal 91, 222-233. Baldwin, E.A. & Pressey, R. 1989 Pectic enzymes in Pectolyase: separation, characterization, and induction of ethylene in fruits. Phmt Physiology 90, 191-196. Baracat, MC., Valentim, C, Muchovej, J.J. & Silva, D.O. 1989 Selection of pectolytic fungi for degumming of natural fibers. Biofechnoiogy Letters 11, 899-902. Blieva, R.K. & Rodionova, N.A. 1987. Fractionation and purification of pectin-degrading enzymes produced by immobilized cells of Aspergillus azuumori. Prikladnaia Biokhimiia i Microbiologiia 23, 561-567. Bush, D.A. 8r Codner, R.C. 1968 The nature of macerating factor of Penicilliu~ d~ifa~u~ Saccardo. Phyfuch~~~ 7, 863-869. Bush, D.A. & Codner, R.C. 1970 Comparison of the properties of the pectin transeliminases of Penicillium digitafum and Penicillium italicum. Phytochemisty 9, 87-97.

787-791.

Ishii, S. & Yokotsuka, T. 1975 Purification and properties of pectin lyase from ~ffg~~~~ japoni~. ~~cu~~ral and biological Chemistry 39, 313-321. Manachini, P.L., Parini, C. & Fortina, M.G. 1988 Pectic enzymes from Aureobasidium pullulans LV 10. Enzyme and Microbial Technohgy 10, 682485. Parini, C., Fortina, M.G. & Manachini, P.L.J. 1988 Properties of two pectin lyases produced by Aureob~idium pullulans LV 10. ~0~~1

of Applied

~eriolo~

65, 477-481.

Pitt, D. 1988 Pectin lyase from Phoma medicaginis

var. pinode~~. in Enzymology 161, 3!%--354. Silley, P. 1986 The production and properties of a crude pectin lyase from Lachnospira multipams. Letters in Applied Microbiology 2, 29-31. Szajer, I. & Szajer, C. 1982 Pectin jyase of Penicikm pa&h. Biofechno~gy Letters 4, 549-552. Mefhods

Tanabe, H. & Kobayashi, Y. 1986 Enzymatic maceration mechanism in biochemical pulping of Mitsumata (Edgeworfhia papyrifera Sieb. et Zucc.) Bast. Agricultural and Biological Chemistry

50. 2779-2784.

Wijesundera, R.L.C., Bailey, J.A. & Byrde, R.J.W. 1984 Production of pectin lyase by Colletotrichum lindemufhiunum in culture and in infected bean tissue. fournal of General Microbiology 130, 285-290.

(Received

in revised

form

I

April 1993; accepted

7 April 1993)