kappa-Carrageenase from Pseudomonas carrageenovora.

Eur. J. Biochem. 93, 553-558 (1979)

x-Carrageenase from Pseudomonas carrageenovora Maitland W. McLEAN and Frank B. WILLIAMSON Department of Biochemistry, University of Aberdeen, Marischal College (Received August 30, 1978)

A x-carrageenase was isolated from the cell-free medium of cultured Pseudomonas carrageenovora. From dodecylsulphate/polyacrylamide gel electrophoresis, a single protein (identified as the x-carrageenase) was detected in the medium. Activity against nominal carrageenan types and inspection of the products indicate the enzyme to be a x-carrageenase. Purification is described here by ammonium sulphate precipitation and subsequent CM-Sepharose C L d B ion-exchange chromatography. Molecular weight was estimated as 35 000 by dodecylsulphate/polyacrylamide gel electrophoresis. Products of degradation were analysed by gel filtration, spectrophotometric assays and 13Cnuclear magnetic resonance. These results are consistent with the product of limit digest being neocarrabiose 4-O-sulphate.

Carrageenans are sulphated galactans extracted from certain marine red algae and comprise an structure [l]. Carrageenanalternating c( 1 -3J1-4 degrading enzymes have been reported from bacteria [2,3] and more recently from sea-urchin gut [4]. Weigl and Yaphe [2] described the presence of both kappa and lambda carrageenase activities in the cellfree medium of Pseudomonas carrageenovora from which they purified a x-carrageenase. Johnston and McCandless [3] reported further on A-carrageenase from the same bacterium. Nuclear magnetic resonance (NMR) has recently been employed in studies of carrageenans [5,6]. In the latter paper [6], enzymic degradation products were used as structural markers. Our research has necessitated a 3c-carrageenase and a study of the cell-free culture medium of Pseudomonas carrageenovora was undertaken. A method is described for the rapid purification of a x-carrageenase from Pseudomonas carrageenovora involving two steps to an electrophoretically pure enzyme. Dodecylsulphate/polyacrylamide gel electrophoresis indicated a single protein species in the cellfree medium. This was identified as the x-carrageenase. The purified enzyme exhibited greatest activity against substrates comprising wholly or partly x-carrageenan although significant reducing power increments and viscometric decrements were detected in substrates described as z-carrageenan and A-carrageenan. Abbreviations. CM-Sepharose, carboxymethyl-Sepharose; NMR, nuclear magnetic resonance. Enzyme. x-Carrageenanase (EC 3.2.1.83).

MATERIALS AND METHODS Materials

Reagents were products of BDH Chemicals Ltd (Poole, England). Sephadex and CM-Sepharose CL-6B were supplied by Pharmacia Ltd (Uppsala, Sweden). Deuterium oxide (> 99.8 % 'H) was obtained through Nuclear Magnetic Resonance Ltd (High Wycombe, England). Organism Pseudomonas carrageenovora (NCMB no. 302) was received as a lyophilized culture from the National Collection of Marine Bacteria (Torry Research Station, Aberdeen, Scotland). Culture Media

Liquid medium comprising (dm-3): NaCl 25 g, MgS04 . 7 H 2 0 5.0 g, CaClz . 2 H 2 00.2 g, KClO.1 g, FeS04 . 7 H20 0.02 g, casein hydrolysate 2.5 g, commercial carrageenan (from Chondrus crispus, through the Sigma Chemical Company) 2.5 g, Na2HP04 1.5 g, Na N 0 3 2.0 g was used for bulk culture in shake flasks, and with agar (1.5 w/vj for slopes and plates. For enzyme preparations, shake flasks (2-dm3 nominal capacity) containing liquid medium (350 cm3) were inoculated with aliquots (0.5 cm3) of vigorous culture. Incubation was carried out in an orbital incubator (25 "C, 100 rev. min-', 60- 80 h) before removing the cells and processing the medium. Growth rate was estimated by taking cell counts

554

x-Carrageenase from Pseudornonas carrageenovora

(Thoma grating) and enzyme activity by the reducing assay.

Protein estimation was by the method of Lowry et al. [ ll] .

Substrates

DodecylsulphatelPolyacrylamide Gel Electrophoresis and Molecular Weight Determination

Enzyme activity was routinely assayed by monitoring the increase in reducing power induced in standard carrageenan solutions. Substrates used were unfractionated carrageenan from Gigartina stellata (harvested locally, Cove Bay), or from Chondrus crispus (supplied by Sigma). Specific carrageenan types were REX 5400 (lambda) and REX 5401 (kappa) from Chondrus crispus through Marine Colloids (Rockland, Maine) and Auby Gel X52 (iota) batch L3940 from Eucheuma spinosum through Pierre Fitte Auby (Paris). Carrageenan was extracted from Gigartina stellata by a hot aqueous technique. Whole plant was washed (0.050 mol dm-3 sodium chloride) and then reduced by two passages in an X Press (Biotec, Stockholm) at - 20 "C. Carrageenan was extracted into hot aqueous solution (0.050 mol sodium chloride, 15 cm3 g-' wet plant, 85 "C, 2 h), the debris pelleted and the supernatant mixed with ethanol (3 vol.). The precipitated carrageenan was washed with ethanol and then dried under reduced pressure. Carrageenase Assays For the reducing power method, an aliquot of sample was incubated with carrageenan solution (0.2% w/v, 1 cm3, 0.050 rnol d mP3 sodium phosphate, pH 8.0,25 "C, 10 min). The equivalent of 200 pg of carrageenan was then assayed for reducing power as described below. The viscometric assay was routinely carried out in an OstwaId viscometer with 0.2 % carrageenan solution as substrate. Activity was examined by plotting specific viscosity against time after the addition of 5 mm3 of sample to 5.0 cm3 of carrageenan solution. Spectrophotometric Assays Total carbohydrate was estimated by the method of Dubois et al. [7] using galactose and methyl 3,6-anhydro-a-D-galactopyranoside as standards. 3,6-Anhydro sugars were estimated by the resorcinol method of Yaphe and Arsenault [8] using methyl 3,6-anhydro-a-~-galactopyranosideand fructose as standards. Reducing power was determined by the neocuproine method of Dygert et al. [9] which was found to be more precise at low values than the method of Somogyi [lo]. For carrageenans it was necessary to add ethanol (50% v/v) in the neocuproine method before measuring the absorbance to clear any opalescence. The final volume was 8 cm3 and the light path 1 cm.

The technique was based on that of Weber and Osborn [12]. Acrylamide concentration was 7.4 % (w/v) and urea (4.0mol dm-3) was incorporated into the gel mixture. Staining was with Coomassie brilliant blue solution. Molecular weight standards were bovine serum albumin (66 000), ovalbumin (45 000), trypsinogen (24 000) and j-lactoglobulin (18 400). Purification of x-Carrageenase Purification was carried out at 4°C. Culture (1 dm3) was centrifuged (14000 x g, 30 min), and

ammonium sulphate added to the supernatant (430 g dm-3). After standing (4 h), the precipitated protein was pelleted (14000 x g, 30 min) and taken up in sodium phosphate solution (50 cm3, 0.025 mol d m- 3 ,p H 6.5). Debris was pelleted (25000 x g , 10 min) and the solution column desalted with Sephadex G-25 (56 x 3.0 cm) equilibrated with sodium phosphate buffer (0.025 mol dm-3, pH 6.5). Fractions containing excluded material were pooled and applied to CM-Sepharose CL-6B (13.5 x 1.25 cm) equilibrated with the same eluent. After washing, a linear sodium chloride gradient (0-0.3 mol dm-3, 70 cm3) was superimposed on the starting buffer. Eluates were monitored continuously at 280 nm (LKB Uvicord 11) and fractions assayed for activity against carrageenan. Protein species in the fractions were detected by dodecylsulphate/polyacrylamidegel electrophoresis. Fractions containing the enzyme were pooled, filtered (0.45 pm Millipore) and stored at - 70 "C either in solution or lyophilized. Activity ProfiIes against p H and Temperature The profile of activity against pH was determined by plotting the reducing power increment induced in aliquots of carrageenan solution (0.2% w/v, 0.10 mol dm-3 sodium phosphate, pH range 4-10, 25°C). The temperature profile was determined at optimal pH as found above (temperature range 20 - 60 "C). Analysis of Degradation Products Molecular weight profiles of degradation products were examined by gel filtration on Sephadex G-25 fine (96 x 1.45 cm). The eluant was sodium chloride solution (0.10 mol dmW3)buffered with sodium acetate

M. W. McLean and F. B. Williamson

555

viscous for chromatography, it was dialysed against deionized water (20 vol., 4 " C , 18 h) and the dialysate concentrated. A suitable sample was applied to a column of Sephadex G-25 fine (125 x 3.0 cm), the fractions assayed, and peaks pooled and lyophilized for analysis by 13C-NMR.

(0.025 mol dm-3, pH 5.0). Analysis of the fractions was by one or more of the spectrophotometric assays. Large-scale fractionation was preceded by ethanol precipitation (60 % v/v) of high-molecular-weight material. In instances where the resulting sample was too

- 0.4

I3C-NMR Spectroscopy

-,.

Lyophilized samples were taken up in deuterium oxide and an aliquot (1.5 cm3) loaded into an NMR tube (803 x 10 mm) with a co-axial capillary of tetramethylsilane. Spectra were recorded on a Varian CFT-20 13Cpulse spectrometer at 20 MHz. Spectral width was 4000 Hz, acquisition time 0.65 s, pulse width 9 ps, and number of transients 13000 - 95 000. Samples were fully proton-decoupled and scanned at 34 "C.Sample concentration, as estimated by the phenol/sulphuric method of Dubois et al. [7], was 50-200 mg ~ m - ~ .

0.3

..-+" +

0.2

c a' a,

0.1

L m L

00-

Time (h)

Fig. 1. Curves for growth of Pseudomonas carrageenovora and production of x-carrageenuse. Cell number (A) and x-carrageenase activity (0)assayed by the reducing power method on 10 mm3 of cell-free medium

RESULTS Purification of x-Carrageenase

E 5 0.5 0.6

0.30

P .-

a35

0.25

0.4

m "

c m

2 0.2

0.15 P

m m

0.10

--_

__.____________-

0.20

0.3 m

0.1

0.05

0 0

Fig. 1 illustrates the growth of Pseudomonas carrageenovora and the associated rise in x-carrageenase activity. Dodecylsulphate/polyacrylamide gel electrophoresis of cell-free medium (100 mm3) indicated the presence of one protein species (from 25 h), which was identified as the x-carrageenase. After incubation (60 - 80 h), the cells were removed by centrifugation and the proteins precipitated by ammonium sulphate. The redissolved proteins were desalted into buffer for ion-exchange chromatography and applied to CM-Sepharose CL-6B (Fig. 2A). Ultra-

F r a c t i o n number

Fraction n u m b e r Fig. 2. Purijkation qf x-carrageenase: Chromatography on CM-Sepharose CL-6B. (A) Absorbance at 280 nm (----) and x-carrageenase activity (0)by the reducing assay. (B) Dodecylsulphate/polyacrylamidegel electrophoresis of column fractions. Aliquot applied to gel, 4 mm3

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x-carrageenase from Pseudomonas carrageenovora

Table 1. Purification of x-carrageenase from Pseudomonas carrageenovora One unit (U) of x-carrageenase activity is that amount which produces a change of 0.1 A450nmmin-' in the reducing assay described Step

Volume Protein x-Carrageenase Specific activity activity cm3

U mg protein-'

1940

491

220

Ammonium Sulphate

830

CM-Sepharose

930

Cell-free Medium

violet-absorbing material which failed to bind was eluted during loading and washing so that the absorbance base line was re-established before applying the gradient. Dodecylsulphate/polyacrylamide gel electrophoresis of fractions (Fig. 2B) consistently resolved one protein species during elution. This corresponded to the ultraviolet absorption and carrageenase activity peaks observed during the gradient. Recovery of activity during purification (Table 1) and the detection of only one protein species in the medium suggest that the high protein content for the first step is the result of non-specific material, for example, casein hydrolysate.

Molecular Weight Determination

0.6

The molecular weight as determined by dodecylsulphate/polyacrylamide gels was 35 000.

Activity Profiles against p H and Temperature Profiles of activity against both pH and temperature are depicted in Fig.3. From the pH experiment, pH 8.0 was taken as standard for assays and digests, and similarly from the temperature profile, 40 "C as the maximum workable temperature. Routine conditions were pH 8.0 and 25 "C.

0.2

0.1

2 DH

Temperature

("C)

Fig. 3 . Activity of purlfied x-carrageenase plotted against ( A ) p H and ( B ) temperature at p H 8.0 as estimated by the reducing assay. Reducing power at 5 min (A)and at 15 min (0)

Time

(min)

Analysis of Degrudution Products Fig. 4 illustrates degradation time courses of some carrageenans as measured by increase in reducing power and drop in specific viscosity. Degradation, as

Time (mln)

Fig. 4. Activity qf'pur$ed x-carragcenase on variou.s carrageenans. (A) Reducing assay. (B) Viscometric assay. Unfractionated carrageenan from Gigartina stellata (a), unfractionated carrageenan for Chondrus crispus (A), wcarrageenan from Chondrus crispus-REX 5401 (m), 2-carrageenan from Chondrus crispus-REX 5400 (O), and 1-carrageenan from Eucheuma spinosum-Auby Gel X52 (A)

M. W. McLean and F. B. Williamson

557

Zones 1.2

-E

10 .

0.8

0.6

i 0

-m

0.4

t 0

0.2

0 20

Fraction

v ,

35 number

105

100

80 6 (wm)

Fig.6. I3C-NMR spectra of material in zones 3 and 4 f r o m Sephadex G-25 gelfillration of x-carrageenan digests. (A) Zone 3. (B) Zone 4. Chemical shift, 6, were measured relative to tetramethylsilane

DISCUSSION

c, 20

40 45 35 Fraction number

Fig. 5. Gel ,fi'ltrution p r o f i k i ~ .o~/ clegradaticin products of' x-currageenan. (A) REX 5401 (20 mg) digested and applied to Sephadex (3-25 fine (96 x 1.45 cm). Total carbohydrate assayed by the phenol/ sulphuric acid method. (B) REX 5401 (5.0 8). Low-molecularweight material extracted from digest and fractionated on Sephadex G-25 fine (125 x 3.0 cm). Carbohydrate assayed by the phenol/sulphuric acid method (01, reducing power by the neocuproine method (A), and 3,6-anhydro sugars by the resorcjnol method (M)

monitored by the reducing assay, was allowed to progress to completion before fractionating the products. Gel filtration of whole digest through Sephadex G-25 resolved the material into four discrete zones as shown in Fig. 5 A.Zone 1 is fully excluded material, whereas zones 2, 3 and 4 contain material of discrete molecular size. In the large-scale preparation (Fig. 5 B), highmolecular-weight material has been largely removed by ethanol precipitation. In addition to the total carbohydrate profile, ones for reducing power and 3,6-anhydro sugars are included. Structural analysis of zones 3 and 4 was by 13C-NMR and deductions from the empirical assays. Typical spectra for these zones are depicted in Fig. 6.

A single protein isolated from the medium of Pseudomonas carrageenovora has been shown to be a x-carrageenase by virtue of its action on certain carrageenans. Attempts by us to bind this enzyme to DEAE-Sepharose CL-6B (pH 8.0) resulted in only partial adsorption, about 80 of the carrageenase eluting during loading. As the results indicate, the enzyme binds completely to CM-Sepharose (pH 6.5) thus affording a simple purification. Co-existence of a A-carrageenase activity has not been demonstrated in this study although the carrageenase assays were designed to measure this activity. This apparent inconsistency with previous studies [2,3] might be explained by a genetic change in the strain received by us, or by differences in the method of culture whereby a A-carrageenase is either not produced or is not similarly detectable. Validity of the reducing assays [9,10] is questionable in the monitoring of carrageenan degradation in view of the high initial values frequently encountered in time courses, and the disproportionately high reducing powers of the degradation products (Fig. 5 B). Indeed the reducing powers of zones 3 and 4 are respectively four and two times those consistent with a tetrasaccharide and disaccharide with single reducing ends. This result was experienced with both reducing assays used [9,10], which monitor the reduction of Cu(I1). An interpretation of this anomaly would be the sequential alkaline degradation, by the reagents of the reducing assay, of certain regions of the polymer and nascent regions exposed during enzymic digestion. The stoichiometric ratio of the 3,6-anhydro sugar content to that of total carbohydrate in the degradation products contained in zones 3 and 4 is 1 :2, and is therefore consistent with a basic repeating unit

558

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M. W. McLean and F. B. Williamson: x-Carrageenase from Pseudomonas carrageenovora

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