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Enzyme and Microbial Technology 40 (2007) 524–532

Improved production by fed-batch cultivation and some properties ofCu/Zn-superoxide dismutase from the fungal strain Humicola lutea 103

E. Krumova a, P. Dolashka-Angelova b, S. Pashova a, L. Stefanova a,J. Van Beeumen c, S. Vassilev a, M. Angelova a,∗

a Institute of Microbiology, Bulgarian Academy of Sciences, Academician G. Bonchev 26, 1113 Sofia, Bulgariab Institute of Organic Chemistry, Bulgarian Academy of Sciences, Academician G. Bonchev 9, 1113 Sofia, Bulgaria

c University of Gent, Department of Biochemistry, Physiology and Microbiology Laboratory of Protein Biochemistry and Protein Engineering, K.L.Ledeganckstraat 35, B-9000 Gent, Belgium

Received 3 January 2006; received in revised form 5 May 2006; accepted 5 May 2006

bstract

Cu/Zn-containing superoxide dismutase (Cu/Zn-SOD) from the fungal strain Humicola lutea 103 is a naturally glycosylated antioxidant enzymehich has been demonstrated to have a protective effect against myeloid Graffi tumor in hamsters and experimental influenza virus infection

n mice. In this work, an improvement of enzyme production was achieved by using both an improved growing strategy and a more efficienturification protocol. The optimized fed-batch cultivation, with 7.5 mg/ml glucose fed daily after 24 h, resulted in prolonged growth and abundantycelium production, as well as in improvement in enzyme production. A 2-fold increase in the total SOD activity and a significant increase in

nzyme production were achieved (from 1.5- to 3.2-fold). Fed-batch technology contributed to a 24 h stabilization period of biosynthesis in whichvery stop of fermentation can be acceptable, thus making this fermentation a process of industrial interest. In addition, the improved purification

rocedure offers a reduction of purification steps and enhanced enzyme yield (1.6-fold).

The molecular mass was proven to be 15,940 Da for the subunit and the conformational dynamics of the protein in solution was studied bylectrospray ionization mass spectrometry (ESI-MS).

2006 Elsevier Inc. All rights reserved.

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eywords: Filamentous fungi; Fed-batch cultivation; Superoxide dismutase; Im

. Introduction

Filamentous fungi are important organisms in some biotech-ological industries where they provide a wide range of nativeroducts, mainly enzymes [1]. They have a number of advan-ages over traditional microbial cultures. Amongst others, fil-mentous fungi possess a wealth of enzymes that can modifyvariety of substrates [2]. Moreover, they demonstrate fast

rowth, an abundant mycelium, intensive respiration and a highevel of cyanide-resistant respiration [3]. Based on these physio-ogical peculiarities, an effective use of fungi for the production

f the intracellular antioxidant enzyme, superoxide dismutaseSOD), could be developed [4–6].

∗ Corresponding author. Tel.: +359 2 979 31 26; fax: +359 2 870 01 09.E-mail address: mariange@microbio.bas.bg (M. Angelova).

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141-0229/$ – see front matter © 2006 Elsevier Inc. All rights reserved.oi:10.1016/j.enzmictec.2006.05.008

ed production; Enzyme analysis; Electrospray ionization mass spectrometry

SOD, an enzyme naturally present in all aerobic cells, cat-lyzes the dismutation of the highly reactive superoxide radicalnion to hydrogen peroxide and molecular oxygen [7]. Theemoval of superoxide effectively blocks secondary reactionshat otherwise would lead to formation of the promiscuouslyeactive hydroxyl radical, which is highly damaging to all classesf biological macromolecules. The generation of ROS is annavoidable consequence of the oxidative metabolism that canause damages in all cellular constituents (DNA, lipids and pro-eins). It affects several cell functions, including replication,rowth, protein synthesis, and ion transport [8,9]. Additionally,any diseases are linked to damage from ROS as a result of an

mbalance between radical-generating and radical-scavengingystems in favour of the first—a condition called oxidative stress

10–13]. The involvement of oxidative stress in human diseasess the basis for antioxidant therapy [13,14]. The use of SODs a therapeutic agent has been proposed for several diseaseshere an important role by ROS has been suggested [15,16].

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E. Krumova et al. / Enzyme and M

imitations of SOD as a therapeutic agent are mainly due tots rapid clearance from blood by glomerular filtration [17,18].mong the strategies developed so far to improve the therapeutic

ction of SOD, its conjugation with polymers, its incorporationnto transport systems (liposomes), its chemical modificationnd the development of recombinant SOD should be mentioned19–21]. It would therefore be advantageous to provide SODith higher pharmacological activity. Our previous investiga-

ions have shown that the fungal strain Humicola lutea 103 ishigh producer of naturally glycosylated Cu/Zn-SOD [6,22],hich could be isolated in very few other cases. The secretory

etrameric extracellular mammalian SOD is the only glycosy-ated SOD, besides the H. lutea enzyme, described so far [23].lycoenzymes of this kind do not need additional processing for

onjugation and modification as do non-glycosylated enzymes.H. lutea Cu/Zn-SOD (HLSOD) was used in an in vivo model

o demonstrate its protective effect against myeloid Graffi tumorn hamsters [6]. Moreover, the fungal enzyme has been showno protect mice from mortality after experimental influenza/Aichi/2/68 (H3N2) virus infection. Using the glycosylatedLSOD, the survival rate is increased and the survival timerolonged, similar to the application of ribavarin, while non-lycosylated bovine SOD conferred lower protection [22].

The present study aims at the enhancement of the yield of nat-rally glycosylated Cu/Zn-SOD by using both fed-batch cultureechniques to increase enzyme production and a more efficienturification protocol. We also describe some properties of theurified enzyme.

. Materials and methods

.1. Microorganism

The fungal strain, H. lutea 103, from the Mycological Collection of thenstitute of Microbiology, Sofia, was used throughout and maintained at 4 ◦C oneer agar, pH 6.3.

.2. Cultivation, equipment and conditions

The compositions of the culture media (g/l) were as follows: (1) seededium—glucose, 40.0; NH4NO3, 3.0; KH2PO4, 1.0; MgSO4·7H2O, 0.5; KCl,

.5; FeSO4·7H2O, 0.001 and (2) production medium—glucose, 48.0; casein, 3.0;oybean flour, 4.0; MgSO4·7H2O, 0.5; CuSO4·5H2O, 0.0011; ZnSO4·7H2O,.0029; FeSO4·7H2O, 0.0043; MnSO4·7H2O, 0.0013.

The cultivation was performed in 500 ml Erlenmeyer flasks or in 3 or2 l bioreactor ABR-09 (working volume 2 and 7 l, respectively), developednd constructed by the former Central Laboratory for Bioinstrumentation andutomatisation (CLBA) of the Bulgarian Academy of Sciences. The bioreac-

or was equipped with pH monitoring, automatic DO monitoring and a controlystem.

For the inoculum, 80 ml of seed medium was inoculated with 5 ml of sporeuspension at a concentration of 2 × 108 spores/ml in 500 ml Erlenmeyer flasks.he cultivation was performed on a shaker (220 rpm) at 30 ◦C for 24 h. For shake-ask cultures, 6 ml of the seed culture were inoculated into 500 ml Erlenmeyerasks, containing 74 ml of the production medium. The cultures were grown at

0 ◦C for 120 h.

The bioreactors cultures were performed with 8% (v/v) 24-h-old shake-flasknoculum at 30 ◦C for 120 h. The fermentation parameters were: impeller speed,00 rpm, and air flow, 1 vvm (1 volume air per 1 volume liquid per min). Theesults obtained in this investigation were evaluated from repeated experimentssing three or five parallel runs.

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al Technology 40 (2007) 524–532 525

.3. Effect of fed-batch glucose addition

For shake-flask cultures, a sterile glucose solution (400 g/l) was added daily,tarting from 24 h of culture, to bring the final glucose concentration to 2.5,.0, 7.5 or 10.0 mg/ml, respectively. As batch variants, cultures without glucoseddition were used. For bioreactor cultures, feeding glucose solution was addedo bring the fermentation vessel up to concentration of 7.5 mg/ml according tohe same scheme.

.4. Analytical methods

The cell-free extract was prepared as described earlier [24]. Briefly,ycelium biomass was harvested by filtration, washed in distilled water and

hen in cold 50 mM potassium buffer (pH 7.8), and was resuspended in the sameuffer. The cell suspension was disrupted by homogenizer model ULTRA Turax25 IKA WERK. The temperature during treatment was maintained at 4–6 ◦C byhilling in an ice-salt bath and during the filtration through filter paper. Cell-freextracts were clarified at 13,000 × g for 20 min at 4 ◦C.

The SOD activity was measured by the nitro blue tetrazolium (NBT) reduc-ion method of Beauchamp and Fridovich [25]. One unit of SOD activity wasefined as the amount of SOD required to inhibit the reduction of NBT by0% of maximum (A560) and was expressed as units per mg protein [U/mg pro-ein]. Cyanide (2 mM) was used to distinguish between the cyanide-sensitivesoenzyme Cu/Zn-SOD and the cyanide-resistant Mn-SOD. The Cu/Zn-SODctivity was obtained as total activity minus the activity in the presence of 2 mMyanide. Protein was estimated by the Lowry procedure [26], using crystallineovine albumin as standard. Soluble reducing sugars were determined by theomogyi–Nelson method [27] and total nitrogen by the micro Kjeldal method28].

The dry weight determination was performed on samples of mycelia har-ested throughout the culture period. The culture fluid was filtered through ahatman (Clifton, USA) No. 4 filter. The separated mycelia were washed twiceith distilled water and dried to a constant weight at 105 ◦C.

The kinetic parameters were studied according to the procedures of Pirt [29].

.5. Purification of H. lutea Cu/Zn-SOD

Cell-free extract from H. lutea 103 obtained as described above was sat-rated to 30% with ammonium sulfate, placed at 4 ◦C for minimum 2 h, thenentrifuged for 30 min at 16,000 × g. The resulting supernatant was first appliedo: an Octyl-Sepharose CL-4B (Pharmacia, Fine Chemicals, Uppsala, Sweden)olumn (40 mm × 32 mm), equilibrated with 0.02 M Tris–HCl (pH 7.8) bufferaturated to 30% with ammonium sulfate and washed with the same buffer untilnbound substances, including SOD, eluted out from the column (absorbancet 276 nm dropped to about 0.08). The collected SOD containing fractions10–12 ml each) were then applied to a Phenyl-Sepharose CL-4B (Pharmacia,ine Chemicals, Uppsala, Sweden) column (62 mm × 35 mm), equilibrated with

he same buffer as used in the previous chromatographic step and eluted underhe same conditions. In the presence of 30% ammonium sulfate most of thempurities were not retained, while the SOD was adsorbed. The enzyme elutedith the same buffer containing 10% of ammonium sulfate as a narrow peak.oth steps column chromatographic steps were performed at a high flow rate of0–80 ml/h.

Fractions containing SOD were concentrated with Amicon (10 kDa cut-ff) and further purified by gel-filtration on a Sephadex G-100 column26 mm × 540 mm), eluted with 0.02 M potassium phosphate buffer, pH 7.8ontaining 0.02 M sodium chloride, at a flow rate of 12 ml/h. SOD-containingractions in all chromatography steps were detected by measuring enzyme activ-ty with NBT method [25].

.6. Polyacrylamide gel electrophoresis

The SOD isoenzyme profile was performed on polyacrylamide gels. Fortyg total protein was applied to 10% non-denaturing PAGE and was stained

or superoxide dismutase activity, as described by Beauchamp and Fridovich25]. Cu/Zn-SOD from bovine erythrocytes and Mn-SOD from Escherichia coliere used as standards. Purity control of the enzyme was performed by 10%

5 icrobial Technology 40 (2007) 524–532

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DS/PAGE, as described by Laemmli [30]. The standards used to make a plotf log molecular weight versus mobility of the protein band were: bovine serumlbumin (66 kDa), ovalbumin (44 kDa), soybean trypsin inhibitor (21.5 kDa) and-lactalbumin (14.4 kDa).

.7. Electrospray ionization mass spectrometry

All mass spectra were acquired on the electrospray ionization mass spec-rometrer (ESI-MS) Q-TOF equiped with a nanospray source. The equipmentas linked to an on-line micro-capillary LC-system (Ultimate, LC-Packings)

nd a Famos autosampler. Protein samples were prepared by diluting the proteintock solution in 10 mM ammonium acetate buffer of which the pH was adjustedith either NH4OH or HCO2H. The final protein concentration was 2 mM. All

olutions were kept at room temperature prior to analysis. ESI source settingsere kept constant throughout all measurements to avoid changes in the ion des-rption and transmission constants. The spectra were acquired at a rate of 5 s.o ensure a high signal-to-noise ratio, typically 180–280 scans were averaged

o record each spectrum.

. Results

.1. Growth and SOD production during batch cultivation

The batch cultivation of the fungal strain H. lutea 103 wasarried out in shake-flasks or stirred bioreactors first of 3, andater of 12 l total volume. Time courses of dry mycelium weightnd SOD activity in mycelia, as well as those of residual glu-ose and nitrogen concentrations in culture medium are shown inig. 1. The figure demonstrated typical fungal growth phases forach experiment. Glucose and nitrogen were quickly consumednd almost disappeared within 48 h. The time profile of totalOD production described a similar trend in flask and bioreac-

or cultures. Two maxima of enzyme activity were determined.he first maximum occurred in the earlier stationary phase ofrowth, and the second one at the end of the fermentation, whenhe nutrients had been depleted from the medium. Secondaryncrease in SOD activity during the late stationary phase haseen observed for SOD production by Candida maltosa [31]nd Cordyceps militaris [32]. It can be explained by an increasen the rate of ROS generation when the cells utilize endoge-ous sources of carbon and nitrogen (organic or amino acids)31].

An enhancement of biomass content and SOD productionas obtained by scaling up to a reaction volume to 12 l. This

llowed the formation of abundant mycelial growth, as well as a.0- and 1.5-fold higher SOD activity than in cultures in shake-ask or 3 l bioreactor, respectively.

.2. Effect of a glucose addition on biomass and SODroduction during fed-batch fermentation in shake-flask

In the batch fermentation, 4.5 g per 100 ml glucose initiallydded was totally consumed within 48 h of fermentation, butost of the SOD was produced after that. To see the effect

f glucose metabolism over the biosynthesis of the antioxidant

nzyme, fermentation was carried out in the fed-batch modehere glucose was added to the medium after 24 h (when 60%f initial glucose was consumed). Fed-batch fermentation wasarried out under conditions similar to batch fermentations.

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lucose (�) and nitrogen (�) concentrations in the fungal cultures during batchermentation in shake-flasks (A), 3 l bioreactor (B) and 12 l bioreactor (C) cul-ures.

For the shake-flask culture glucose was added at an intervalf 24 h, with the concentrations in the medium being 2.5, 5.0,.5 and 10.0 mg/ml, respectively (Fig. 2). The addition of glu-ose above 2.5 mg/ml had a pronounced effect on the growthf H. lutea strain. While the batch culture showed clear dis-inguishable growth phases (exponential, stationary and declinehases), fed-batch cultivation demonstrated a continuous growthp to the end of fermentation (120 h) (Fig. 2B–D), resulting in a.3–1.5-fold increase in biomass content. At the same time, theaximum level of dry weight showed little differences among

ariants with glucose addition of 5.5, 7.5 or 10 mg/ml, respec-ively.

Fig. 2 also shows the changes in SOD production during fed-atch experiments. Addition of glucose concentrations below.5 mg/ml did not result in any increase of enzyme level (35 and6 U/mg protein of the variants with 2.5 and 5.0 mg/ml glucose,espectively, versus 36 U/mg protein of the control) (Fig. 2A and). In the cultures with higher concentrations of glucose (7.5nd 10.0 mg/ml), enhanced enzyme production was establishedFig. 2C and D). Maximum specific SOD activity reached about

.5 times the level of the batch culture. On the other hand, timeourses of enzyme production were altered significantly uponrowth with glucose. The above-described two maxima of SODctivity during batch cultivation were not observed. The enzyme

E. Krumova et al. / Enzyme and Microbial Technology 40 (2007) 524–532 527

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uThe fungal strain produces two isoforms, namely Cu/Zn- andMn-containing SOD. After inhibition with 5 mm KCN theresidual activity was about 10% from the total one for the

ig. 2. Time courses of biomass production (�), SOD activity (©) and residual g.5 (A), 5.0 (B), 7.5 (C), and 10.0 mg ml−1 (D).

urve displayed a plateau for several hours. In the variants with.5 and 5.0 mg/ml glucose, this plateau was formed between 72nd 96 h. With using higher feeding levels (7.5 and 10.0 mg/mllucose), the period of equal SOD activity was obtained earlier,etween 48 and 72 h.

Despite the concentration of added glucose, the carbohydrateource was almost totally consumed by mycelium. The shake-ask cultures maintained constant glucose levels in the mediumfter 48th hour of fermentation.

.3. Fed-batch processes of SOD biosynthesis in bioreactor

The effect of the feeding mode on biomass and SOD produc-ion was evaluated also under scale up conditions in 3 and 12 lioreactors (Fig. 3). In those experiments glucose was addedaily at a concentration of 7.5 mg/ml 24 h after the start of fer-entation. Comparison of both types of cultivation performed in

ioreactors indicated that the switch from the batch to fed-batchode leads to approximately 30–44% enhancement of biomass

roduction. The profiles of mycelium dry weight were similaro those for fed-batch fermentation in shake-flasks. Fig. 3A and

show a prolonged exponential growth-phase and a lack oftationary phase up to 120 h.

Under fed-batch conditions, maximum specific enzymectivities of 64 and 96 U/mg protein were obtained in 3 and 12 lioreactors, respectively. The values are comparable to these of

he batch process in bioreactors, but they were achieved earlier.n addition, enzyme formation showed a similar trend as thatn the fed-batch cultures in shake-flasks (Figs. 2 and 3). Thehanges in specific activity over time showed a rapid rise in glu-

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e content (�) in shake-flask cultures during feeding by glucose in concentrations

ose feeding cultures, which reached a plateau between 36 and2 h.

The isoenzyme profiles of SOD produced by H. lutea 103sing batch and fed-batch cultivation, are illustrated in Fig. 4.

ig. 3. Biomass (�), SOD production (©) and residual glucose content (�)fter scale up of fed-batch cultivation in 3 (A) and 12 l (B) bioreactors.

528 E. Krumova et al. / Enzyme and Microbial Technology 40 (2007) 524–532

Fig. 4. Polyacrylamide gel electrophoresis (10% gel) of free-cell extracts fromfungal cultures stained for enzymatic activity; lane 1, Cu/Zn-SOD standard frombac

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ovine erythrocyte; lane 2, Mn-SOD standard from Escherichia coli; lanes 3nd 4, Mn- and Cu/Zn-SOD in the mycelium grown under batch and fed-batchultivation, respectively.

n-SOD enzyme (data not shown). Fed-batch cultivationaused an increase in Cu/Zn-SOD isoenzyme activity, whereashe activity of Mn-SOD did not change (Fig. 4, line 4).

.4. Comparison of the efficiency of SOD biosynthesis by. lutea grown under different conditions of fermentation

Comparison of kinetic relations (substrate consumption androduct formation parameters) for SOD production betweenhake-flask and bioreactor cultures as well as between batchnd fed-batch cultivation is given in Table 1. The results indi-ate that the scale up of batch cultivation from shake-flasks to 3nd 12 l bioreactor resulted in enhanced total SOD productionU/l, approximately 3.3- and 6.2-fold, respectively), and enzymeroductivity [Qp (U/l/h), approximately 5- and 9-fold, respec-ively]. Moreover, yield coefficients for biomass and productYx/c (g/g), Yp/c (U/g), and Yp/x (U/g)] in bioreactors was signif-cantly higher than that in flask cultures.

Fed-batch cultivation additionally improved the efficiency ofOD biosynthesis. Experiments with glucose feeding showedigher kinetic parameters for biomass [Yx/c (g/g)] and enzymeYp/c and Yp/x] production in comparison to batch fermentationTable 1). Although a lack of significant difference in specificnzyme activities between both types of cultivation was estab-ished, a higher total SOD activity was measured in the myceliumrown under fed-batch conditions. Because of enhanced levelsf biomass and intracellular protein, the glucose feeding ledo higher enzyme production compared to the batch fermen-ation. For shake-flask cultures, enzyme production increasedrom 21 × 103 U/l in batch cultivation to 40–50 × 103 U/l in fed-atch fermentation. The same trend was observed for bioreactorultures (from 70 to 90 × 103 and from 132 to 190 × 103 U/lor 3 and 12 l, respectively). The maximum yield of SODapproximately 190–195 × 103 U/l) was achieved during fed-atch cultivation in 12 l bioreactor. In this case, product yieldoefficients, Yp/c (5300–5900 U/g) and Yp/x (6000–7100 U/g)ere approximately 2-fold improved compared with 3 lioreactor.

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E. Krumova et al. / Enzyme and Microbial Technology 40 (2007) 524–532 529

Fig. 5. Hydrophobic chromatography of H. lutea SOD. (A) Octyl-SepharoseCL-4B column (40 mm × 32 mm); washed with 0.02 M Tris–HCl (pH 7.8) buffersaturated to 30% with ammonium sulfate until unbound substances, includ-i(a

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ng SOD (1) flow out from the column. (B) Phenyl-Sepharose CL-4B column62 mm × 35 mm), SOD (1) was eluted with the same buffer containing 10%mmonium sulfate.

.5. Improvement of purification protocol

The chromatographic elution profiles are shown inigs. 5 and 6 and the purification is summarized in Table 2.

The purification protocol consisted of two steps ofydrophobic interaction chromatography (HIC) before using theephadex G-100 column. The cell-free extract obtained after dis-

ntegration of fungal mycelium and centrifugation had a specific

pccm

able 2urification steps for Cu/Zn-SOD from H. lutea 103

urification steps Protein (mg) Total SOD activity (U) S

ell-free extract 166.00 10608ctyl-Sepharose 8.50 5660henyl-Sepharose 2.50 4025 1ephadex G-100 0.50 2050 4

n Sephadex G-100 column (26 mm × 540 mm), 0.02 M potassium phosphateuffer, pH 7.8 containing 0.02 M sodium chloride.

ctivity of 64 U/mg. After the first step on Octyl-Sepharose, a0.4-fold purification was achieved. The second step on Phenyl-epharose was an effective purification step, which yielded highpecific enzyme activity (1610 U/mg) at a high enzyme yield38%). The final chromatography on Sephadex G-100 resultedn an additional 2.5-fold increase in specific activity of Cu/Zn-OD (4100 U/mg), with an overall yield of 19.3% of the startingctivity.

Soluble protein fractions from crude extract and purifiedroduct were analyzed by SDS-PAGE (Fig. 7). After gel-hromatography step, a single protein band was observed, show-ng that Cu/Zn-SOD was homogenous.

.6. pH stability of H. lutea Cu/Zn-SOD

All mass spectrometric analysis were performed on a Q-TOFass spectrometer (Micromass, UK), interfaced to a chip-based

anoESI source (NanoMate100, Advion Biosciences, UK). Allass spectra were processed using MassLynx v3.1 Software

f Micromass. The ESI spectra were acquired using 10 mMmmonium acetate buffer at acid and native pH to calculate theolecular mass of the enzyme. The spectra collected at neutral

H contain only few protein-ion peaks, which form a narrowharge-state distribution. At pH 7.5 (Fig. 8A), the spectrumontained four peaks with a maximum intensity ion peak at/z = 2867. This result confirms that at this pH the native has

pecific SOD activity (U mg−1) Yield (%) Purification (fold)

64.0 100.0 1.0666.0 53.0 10.4610.0 38.0 25.2100.0 19.3 64.0

530 E. Krumova et al. / Enzyme and Microbial Technology 40 (2007) 524–532

Fig. 7. SDS-PAGE analysis (10% polyacrylamide gel) of soluble protein frac-tions from crude extract (lane 2) and purified product after final chromatog-rm

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Fig. 8. ESI spectra of H. lutea SOD in 10 mM ammonium acetate buffer. pHwwa

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aphy on Sephadex G-100 (lane 3). Lane 1 corresponds to molecular weightarkers.

molecular mass of 31,880 Da, which correlates to the masseasured by matrix assisted laser desorption mass spectrometry

MALDI).Science electrospray ionization mass spectrometry (ESI-MS)

s known to be a method that is also used to monitor protein con-ormational dynamics in solution, by following the changes inositive-ion charge-state distributions in response to changes ofmbient conditions (for example solution pH), are also mea-ure the spectra of SOD at lower pH values. When the pH ofhe protein solution is lowered below 6.0, higher charge-densitylower m/z) protein-ion peaks begin to appear in the spectra.he distribution still contains the peaks shown in Fig. 8A, but

wo maximum-intensity ion peaks are now observed (at m/z626.47 and 1957.61) (Fig. 8B). Such features suggest struc-ural changes within the native state of the protein moleculellustrating the formation of less compact structures leadingo ions with higher charge density. At pH values lower that, no peaks correlated with the native form of the moleculeere observed, confirming that only monomer form of SOD isresent. The ion signal at m/z 1957.61 of higher charge den-ity corresponding to one subunit of SOD and the unfoldedrotein with signal at m/z 1200.31 are represented in Fig. 8C.he protein was totally unfolded at a pH < 3. Only few peaksere observed at pH 2.5 with lower m/z and the maximal

on signal was at m/z 1200.31 (Fig. 8D). The molecular massf 15,940 Da, calculated from ESI-M spectrum correspondso the mass of one subunit calculated from the amino acidequence.

bofm

as adjusted with either NH4OH or HCO2H. The final protein concentrationas 2 mM and the samples were measured at different pH: A, 7.5; B, 6.4; C, 4.2

nd D, 2.7.

. Discussion

In this paper we concentrate on the effects of two approacheso improve the existing method for Cu/Zn-SOD production by H.utea 103: (a) glucose feeding that can improve the enzyme yieldf biosynthesis and (b) modification of the purification protocolhat improves the enzyme yield after isolation and purification.

Our results indicate that the fed-batch strategy can be useduccessfully to improve the Cu/Zn-SOD production by the fun-al strain. The enzyme is produced during the active growthhase of the fungus, implying that the volumetric enzyme pro-uction is dependent on the total biomass formation. Hence,onditions that provide enhanced mycelium content favour aigh SOD production. As it is known, fed-batch fermenta-ion technology allows obtaining larger biomass amounts andmproved enzyme production [33–35]. In our experiments, anttempt to increase the biomass production was made by the fed-

atch process using glucose as carbon source (Figs. 2 and 3). Theptimized fed-batch fermentation, when 7.5 mg/ml glucose wased daily after 24 h, resulted in prolonged growth and abundantycelium production.

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The enhanced biomass content by glucose feeding corre-ponds with an improvement in enzyme production (Table 1).he total SOD activity in the fed-batch stage was 1.3- to 2-

old higher than in batch fermentation. In addition, a significantncrease in enzyme productivity was achieved (from 1.5- to.2-fold). The possibility to improve the yield of biologicallyctive compounds by batch-type feeding of the carbohydratesuring the biosynthesis has been reported by several authors33,36–39]. A similar fermentation strategy for SOD produc-ion has been examined in a few previous studies concerning aecombinant human SOD synthesis by E. coli strains [40,41].

As was demonstrated during batch cultivation (Fig. 1), aftereaching a maximal SOD activity in the fungal cells, a drasticecrease in this activity occurs. This sharp drop impedes theetermination of the endpoint of the fermentation process. Theain finding of this study is that the fed-batch technology con-

ributes to the maintenance of maximum SOD activity for anxtended period of cultivation (Figs. 2 and 3). Daily additionf 7.5 mg ml−1 glucose leads to a plateau in the time course ofpecific activity that correlates with analogous trends in volumet-ic SOD activity and enzyme productivity (Table 1). A similarplateau phenomenon” allowed a 24 h stabilization period ofiosynthesis in which every stop of fermentation is acceptable,hus making this a process of industrial interest.

It should be noted that although the feeding process increasedlucose concentration in the medium, the data did not show anyepressive effect on SOD activity. These results confirmed ourrevious study that SOD activity in H. lutea is not subject toatabolite repression control even at relative high glucose con-entrations [24]. Similar data have been published for SOD syn-hesis in E. coli [42] and Hansenula polymorpha [43]. Accordingo Westerbeek-Marres et al. [44], in Saccharomyces cerevisiae,u/Zn-SOD is induced by catabolic repression, thus the increase

n the activity of this enzyme could also result from glucoseccumulation in the culture medium.

Advantages of the proposed purification procedure appar-ntly are both the reduction of purification steps and thenhanced enzyme yield. The high flow rate during two HIC stepseduce process time and allow fast purification of large volumes.he essence of improvement, which allows this fast and simpleurification, is the different behaviour of the enzyme at bothIC steps using the same chromatographic conditions. In theresence of 30% of ammonium sulfate, SOD was not bound onctyl-Sepharose and flowed out the column together with partf impurities. The opposite occurs on Phenyl-Sepharose wherehe enzyme is bound, and then eluted with buffer containing0% ammonium sulfate. The reason for this behaviour is veryikely the structure and amino acid composition of the protein. Itas found that H. lutea Cu/Zn-SOD contained 20 Phe residues,hich may explain a higher affinity to Phenyl-Sepharose [22].imultaneously, the enzyme yield from the new purification pro-edure was nearly 20%, which is 1.6-fold higher compared tour earlier report (12%) [6].

The ESI spectra of SOD were acquired over the pH range.5–7.5. Conformational dynamics of H. lutea Cu/Zn-SOD inolution using Electrospray ionization mass spectrometry (ESI-

S) showed that the native form of the enzyme is near neutral

[

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al Technology 40 (2007) 524–532 531

H 7.0 and that decreasing the pH below 4.0 reveals the presencef multiple protein conformers. The ion signals correspondingo different conformers and at pH values lower than 3.0 revealnly an unfolded subunit with molecular mass of 15,400 Da.

cknowledgments

This work was supported by grant no. LST.CLG980520/2003rom NATO and grant K-1302/03 from the NCSI of the Min-stry of Education and Science, Bulgaria, which are greatlycknowledged.

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