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Enzyme and Microbial Technology 40 (2007) 187–194

Rapid communication

Stimulation of novel thermostable extracellular lipolyticenzyme in cultures of Thermus sp.

Alberto Domınguez a, Pablo Fucinos b, M. Luisa Rua b, Lorenzo Pastrana b,Marıa A. Longo a, M. Angeles Sanroman a,∗

a Department of Chemical Engineering, University of Vigo, Lagoas-Marcosende, 36310 Vigo, Spainb Department of Biochemistry, Genetics and Immunology, University of Vigo, Spain

Received 29 September 2005; received in revised form 5 September 2006; accepted 12 September 2006

bstract

Selected organisms from the genus Thermus (T. aquaticus YT1, T. thermophilus HB8 and HB27) offer new opportunities for biocatalysis andiotransformations as a result of the extreme stability of their enzymes. In order to favour the secretion of extracellular lipolytic enzymes the effectf temperature and carbon source has been studied. All strains were able to grow within a wide temperature range (from 60 to 80 ◦C) with anptimum value of 70 ◦C for extracellular lipolytic activity.

On the other hand, several sugars were used as carbon source in the culture medium at different concentrations (from 0.5 to 14 g dm−3).upplementation of monosaccharides (glucose, fructose) and disaccharides (maltose, sucrose) was carried out in an attempt to increase biomass and

ipolytic enzyme production. The influence of carbon source was variable, depending on the strain. Nevertheless, the addition of low concentrationsf mono- or disaccharides helped to improve the productivity of the process in most cases. The most significant effects on extracellular lipolytic

ctivity were detected in T. thermophilus HB27 and T. aquaticus YT1 cultures, using sucrose and fructose as carbon source at an initial concentrationf 0.5 g dm−3. Moreover, the effect of addition of lipids (olive oil, coconut oil and tributyrin) was tested. In T. thermophilus HB27 cultures, theighest extracellular lipolytic activities were obtained when olive oil was added (70% higher than those obtained in control cultures).

2006 Elsevier Inc. All rights reserved.

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eywords: Thermostable lipolytic enzymes; Carbon source; Temperature; T. th

. Introduction

In recent years, there has been an increasing interest in thetudy of enzymes from extremophiles, since they are not onlyore thermostable but often more resistant to chemical agents

nd extreme pH values than their mesophilic homologues [1–3].Lipolytic enzymes catalyze a wide number of different reac-

ions, most of them of industrial application. In spite of theirotential interest, the available information about the productionf lipolytic enzymes by thermophilic microorganisms is scarce,nd it is mainly focused on the detection of this activity in someacteria isolated from hot springs, as well as the stability of the

nzymes at high temperatures [4,5].

The ability of T. thermophilus (HB8 and HB27) and T. aquati-us YT1 to produce significant lipolytic activity when cultivated

∗ Corresponding author. Tel.: +34 986 812383; fax: +34 986 812380.E-mail address: sanroman@uvigo.es (M.A. Sanroman).

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141-0229/$ – see front matter © 2006 Elsevier Inc. All rights reserved.oi:10.1016/j.enzmictec.2006.09.006

philus HB8; T. thermophilus HB27; T. aquaticus YT1

n a complex medium has been demonstrated in our laboratory.ll enzymes were stable at 80 ◦C over 30 min and showed a

emarkable activity on fatty acid esters with acyl chains longerhan 10 carbon atoms [6]. Two proteins with lipolytic activityere identified in intra- and extracellular extracts from the threehermus strains cultures by zymogram analysis, with moleculareights of 34 and 62 kDa [7,8]. The culture time-course in aasal medium has been described [8] and the influence of someariables (i.e. gas environment) in biomass and enzyme produc-ion has been investigated, both in Erlenmeyer flasks [8] and atirred tank bioreactor [9].

In previous papers [6–9], we have shown that enzyme produc-ion was not fully associated to growth rate, although absolutealues of total lipolytic activity and biomass were positivelyorrelated. However, cell growth was relatively low, and lipoly-

ic activity appeared to be largely retained within the biomass.herefore, it would be interesting to find culture conditions (i.e.edium composition, pH, temperature, aeration), allowing to

mprove growth and/or favour enzyme secretion.

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88 A. Domınguez et al. / Enzyme and M

In this work, optimisation of lipolytic enzyme production by. thermophilus HB8, HB27 and T. aquaticus YT1 has beenttempted. The influence of incubation temperature and theffect of the type and concentration of carbon source in the cul-ure medium have been studied.

. Materials and methods

.1. Microorganisms and culture conditions

T. thermophilus HB8, HB27 and T. aquaticus YT1 were kindly providedy Dr. J. Berenguer (Universidad Autonoma, Madrid, Spain).

Submerged cultures were carried out in 1000 cm3 Erlenmeyer flasksith 200 cm3 of medium (pH 7.5), prepared in distilled water and composedf 8 g dm−3 casein peptone, 4 g dm−3 yeast extract and 3 g dm−3 NaCl.hroughout this study, the basal medium was consecutively supplemented witheveral concentrations (0.5, 1.5, 7 and 14 g dm−3) of carbohydrates (glucose,ructose, maltose and sucrose). Also, some experiments were carried out inulture media supplemented with lipids, namely olive oil, coconut oil andributyrin (concentrations 1 and 10 g dm−3). Culture inoculation and samplereparation were realised according to Fucinos et al. [8]. Experiments wereone in duplicate and samples were analysed in triplicate. The values in thegures correspond to mean values with a standard deviation less than 15%.

.2. Analytical methods

.2.1. Cell growth determinationCell concentration was monitored spectrophotometrically at 600 nm and the

btained values were converted to g cell dry wt dm−3 using calibration curvesreviously determined.

.2.2. Lipolytic activity assayIt was determined using p-nitrophenyl laurate (p-NPL) as substrate following

he method described in Fucinos et al. [7]. One activity unit was defined as themount of enzyme that produced 1 �mol of p-nitrophenol per min under standardssay conditions. The activities were expressed in U dm−3.

.2.3. Protein concentrationIt was determined according to Lowry et al. [10], using bovine serum albumin

s standard.

.2.4. Total carbohydrates concentrationIt was determined by the phenol-sulphuric method of Dubois et al. [11],

sing d-glucose as standard.

. Results and discussion

In this work, some strategies to enhance lipolytic enzymeroduction by three Thermus strains have been investigated. Inprevious paper [6], the selected strains were reported to show

emarkable activity on fatty acid esters with acyl chains longerhan 10 carbon atoms, and the activity towards these substratesppeared to increase as substrate chain-length diminished.herefore, the denomination “lipolytic enzyme/activity” haseen utilised throughout this work, since incontrovertible attri-ution of true lipase activity would require further investigation.

Moreover, an operational classification of enzyme activitiess intra- or extracellular has been utilised. Thus, activity

etected in the culture medium after biomass separation byentrifugation was considered as extracellular, while that recov-red in solution after sonication of the buffer-resuspended cellsnd elimination of cell debris was considered as intracellular.

l

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ial Technology 40 (2007) 187–194

lthough the occurrence of cell-bound enzyme would beossible and is not regarded here, this categorization is based onsual industrial practice, and it is the most interesting from thetandpoint of industrial application of the described enzymes.

.1. Effect of temperature on growth and lipolytic activity

The three studied strains were grown in shake flasks,ithin a wide range of temperature (from 60 to 80 ◦C). The

ultures were stopped after 30 h of incubation, since previousxperiments indicated that, no significant increases in lipolyticnzyme activity were attained later in flask cultures. Besides,ell lysis was mostly negligible at this time. Moreover, theuthors indicated that no kinetic typification of the enzymes asrimary metabolites was possible for any of the Thermus strains,ecause of the lack of a good fitting of the experimental lipolyticctivity production rates to the Luedecking & Piret model6–8].

Time-course of cultures and final biomass, intra- andxtracellular lipolytic activity are shown in Fig. 1 and Table 1,espectively. In the three studied strains, the increase in temper-ture seemed to have a negative effect on biomass production:nal cell growth at 60 ◦C was almost two-fold higher than

hat at 80 ◦C. However, this effect was more remarkable in T.hermophilus HB8 cultures, while T. thermophilus HB27 and. aquaticus YT1 only showed a significant decrease in cellrowth at temperatures above 70 ◦C. These results are closeo those reported by Oshima and Imahori [12], who foundhat the optimal growth temperature for a locally isolated T.hermophilus strain was 70 ◦C. When biomass productionrofiles (Fig. 1) are considered, it can be seen that stationaryhase is approached rather rapidly at 80 ◦C. Similar finalell growth levels are attained at 60 and 70 ◦C, although aonger lag phase is generally observed at 60 ◦C, as it wasoreseeable.

More significant differences between strains were observedor lipolytic enzyme production. Maximum intracellularctivity was very similar in all T. aquaticus YT1 cultures, withn average value of 66 U dm−3, and production profiles wereairly close. On the other hand, the increase in temperature hadn acute effect on T. thermophilus HB8 and HB27 intracellularnzyme levels. Final activities of 125 and 93 U dm−3 werebtained, respectively, operating at 80 ◦C, about two-fold higherhan those attained at 60 ◦C, while intermediate values werebtained at 70 ◦C. Although the shape of the production patternsas somewhat similar to those of cell growth for each inde-endent temperature, enzyme production cannot be consideredtrictly growth associated. First, the highest activity levels wereound at 80 ◦C, which was the least favourable condition foriomass production. Secondly, at this temperature, attainmentf maximum enzyme levels was delayed with respect to those inell growth. Therefore, some additional feature must be involvedn the mechanism of lipolytic enzyme production at intracellular

evel.

T. thermophilus strains have been reported to producehaperonins, a class of proteins implicated in the folding ofther proteins. Taguchi et al. [13] recovered and purified two

A. Domınguez et al. / Enzyme and Microbial Technology 40 (2007) 187–194 189

at diff

iHtHdrcmrs

wdaocia

ihpcwt

oTt

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Fig. 1. Time course of cultures of Thermus strains, grown

ntracellular chaperonins from late log phase T. thermophilusB8 cultures performed at 75 ◦C, and demonstrated their ability

o promote refolding of several enzymes at high temperatures.owever, the effect of chaperonins on protein refolding isependent on the temperature: above 60 ◦C spontaneousefolding fails but if the native protein is sufficiently stable, thehaperonin induces productive refolding in an ATP-dependentanner. At temperatures below 60 ◦C (i.e. 50 ◦C) spontaneous

efolding of the proteins occurs, and the chaperonin arrests thispontaneous refolding in the absence of ATP.

It could be hypothesised that the occurrence of chaperoninsithin T. thermophilus cells might contribute to the phenomenaescribed in this work, by favouring lipolytic enzyme foldingnd therefore stability at high temperature. Since the mechanismf chaperonin action seems to be dependent of temperature, thisould account to a certain extent for the differences observed inntracellular lipolytic enzyme levels for T. thermophilus HB27nd HB8.

As for extracellular lipolytic activities, they were rather lown comparison with the intracellular enzymes. In all cases, theighest final values were obtained when operating at 70 ◦C. A

arabolic dependence of temperature was postulated for extra-ellular lipolytic activity. Experimental results for final activityere fitted to polynomic equations, and subsequently derivated

o obtain the theoretical optimum. According to this procedure,

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erent temperatures: (�) 60 ◦C, (©) 70 ◦C, and (�) 80 ◦C.

ptimal temperatures were found at 66.9, 70.6 and 70.5 ◦C for. thermophilus HB8 and HB27 and T. aquaticus YT1, respec-ively.

The lower extracellular enzyme activity at high tempera-ure could in part be due to the secretion of specific proteasesuring the last phase of the cultures. This fact was describedy Matsuzawa et al. [14] in previous studies carried out withther Thermus strains. Also, temperature-dependent differencesn extracellular enzyme activity could be related to the occur-ence of special enzyme secretion mechanisms in the studiedicroorganisms, a hypothesis that has already been proposed

y the authors in a previous work [8] to explain the seeminglyndependent dynamics of extracellular lipolytic activity andntracellular enzyme and biomass production. Several speciesf Thermus genus are able to form, under certain environmentalonditions, clusters of cells surrounded by a membrane formedy the fusion of the individual external membranes of each cell.hese formations are usually known as rotund bodies [15]. Itight be assumed that proteins could be firstly secreted by indi-

idual cells to this internal cavity and, from there poured intohe culture medium. Therefore, the integrity and permeability

f the external membrane of the rotund bodies might be impor-ant to control the liberation of enzymes to the culture medium,nd these properties could be influenced by the temperature.hus, the combined and opposed effects of increased release of

190 A. Domınguez et al. / Enzyme and Microb

Tabl

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tion

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ass

(gdm

−3)

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icen

zym

e(U

dm−3

)In

trac

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lar

lipol

ytic

enzy

me

(Udm

−3)

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27T.

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T1

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B27

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1

601.

051.

011.

053.

927.

6911

.57

51.1

258

.01

63.0

570

0.75

1.04

1.18

4.50

34.4

027

.25

72.8

81.7

968

.70

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640.

621.

6313

.16

14.6

112

4.78

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ial Technology 40 (2007) 187–194

nzymes to the culture medium and thermal or proteolytic deac-ivation at high temperatures could account for the results foundn the present work.

Although maximum intracellular lipolytic activity wasbtained at 80 ◦C in T. thermophilus HB8 cultures, the scarceeneficial effect in the other studied strains and the consequentncrease in production costs does not recommend operating athis temperature. Besides, one of the most interesting aspectsn this field is the enhancement of enzyme secretion, which inhis case appears to be favoured at 70 ◦C (especially for T. ther-ophilus HB27). Therefore, all subsequent experiments were

arried out employing 70 ◦C as culture temperature.

.2. Effect of the type and concentration of carbon source

A high degree of nutritional diversity has been detected inhermus strains (i.e. T. thermophilus HB8, T. aquaticus YT1) byegryse et al. [16], Alfredsson et al. [17] and Santos et al. [18].hese strains grew well on different carbon sources, although theresence of glucose, galactose, fructose, sucrose or maltose inhe culture medium seemed to have a particularly positive effect.

However, the above-mentioned studies were carried out byeans of agar plate cultures, and only provided qualitative

esults. Besides, they focused on the investigation of cellrowth, and no information was given on the production ofotentially interesting enzymes. In the present work T. ther-ophilus HB8, HB27 and T. aquaticus YT1 have been grown

n submerged cultures, using a complex medium supplementedith several concentrations (from 0.5 to 14 g dm−3) of mono-

nd disaccharides (glucose, fructose, maltose and sucrose). Thend-culture values of biomass, intra- and extracellular lipolyticctivity (after 30 h of incubation) for the three strains areompared in Figs. 2–4, respectively. The data are expressed asercentages referred to the results obtained in the basal mediumwith no additional carbon source) at 70 ◦C, shown in Table 1.

The effect of carbon source appeared to be somewhattrain-dependent and therefore difficult to generalise. However,ome common patterns could be established for the testedtrains. As it can be observed in Fig. 2, supplementation ofhe basal medium with low concentrations (up to 1.5 g dm−3)f mono- or disaccharides led, in most cases, to a moderatencrease (20–50%) in biomass production. Higher carbonource concentrations did not result in further improvements inell growth, with the exception of T. thermophilus HB8 cultures,n which biomass was increased two-fold and 2.5-fold whenhe basal medium was supplemented with sucrose (7 g dm−3)r maltose (14 g dm−3). Also, it is noteworthy that cell growthas significantly diminished when high levels of glucose were

dded to the medium, in all the studied strains.When intracellular lipolytic activity production was con-

idered (Fig. 3), the general behaviour of the microorganismsas similar to that described for cell growth. However, the

omparison of both sets of data indicated that intracellular

nzyme and biomass productions were not fully associated. Theddition of low concentrations (0.5–1.5 g dm−3) of mono- andisaccharides generally resulted in a noticeable ameliorationn enzyme production, the most promising results having been

A. Domınguez et al. / Enzyme and Microbial Technology 40 (2007) 187–194 191

Fig. 2. Influence of carbon source on biomass production by Thermus strains,aaT

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Fig. 3. Influence of carbon source on intracellular lipolytic activity productionby Thermus strains, after 30 h of culture time. Carbon source: (�) glucose, (©)fH6

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fter 30 h of culture time. Carbon source: (�) glucose, (©) fructose, (�) maltose,nd (�) sucrose (100% biomass production: T. thermophilus HB8, 0.75 g dm−3;. thermophilus HB27, 1.04 g dm−3; T. aquaticus YT1, 1.18 g dm−3).

btained with disaccharides, and for T. thermophilus HB8.arbon source supplementation above this level does not

eem to be advisable, since intracellular lipolytic activitiesemained mostly unaltered, or decreased. Only T. thermophilusB8 and HB27 showed any amelioration in intracellular

nzyme levels at high carbon source concentrations, withhree-fold and 1.6-fold rises in activity (referred to control) inhe presence of sucrose (14 g dm−3) and maltose (7 g dm−3),espectively.

Finally, extracellular lipolytic enzyme concentrations were

ssessed. No significant levels of extracellular activity wereetected for T. thermophilus HB8, in any case. As for the otherwo strains (Fig. 4), the general effect of carbon source supple-

entation was similar to that previously described for biomass

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ructose, (�) maltose, and (�) sucrose (100% lipolytic activity: T. thermophilusB8, 72.80 U dm−3; T. thermophilus HB27, 81.79 U dm−3; T. aquaticus YT1,8.70 U dm−3).

nd intracellular enzyme. A slight enhancement in extracellularnzyme production was obtained when low concentrations ofhe different carbon sources were added. The best results werebtained with sucrose for T. thermophilus HB27 and fructoseor T. aquaticus YT1 (both sugars at 0.5 g dm−3).

Total carbohydrates and protein consumption were evalu-ted in all the cultures. The assessed nutrients were not totallyepleted in any case, and most consumption occurred duringhe early stages of the cultures. Low nutrients consumption

n Thermus strains has already been mentioned in previousorks [6,19,20]. No relevant information could be inferred from

hese data, concerning the dynamics of cell growth and lipolyticnzyme production on different carbon sources.

192 A. Domınguez et al. / Enzyme and Microb

Fig. 4. Influence of carbon source on extracellular lipolytic activity productionbCl3

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ph

bbfeaais

madoibactTstictac(wp

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y T. thermophilus HB27 and T. aquaticus YT1, after 30 h of culture time.arbon source: (�) glucose, (©) fructose, (�) maltose, and (�) sucrose (100%

ipolytic activity: T. thermophilus HB8, 4.50 U dm−3; T. thermophilus HB27,4.40 U dm−3; T. aquaticus YT1, 27.25 U dm−3).

To our knowledge there are few reports about the produc-ion of thermostable lipases or esterases from thermophilicicroorganisms, and none on how it can be affected by the

hoice and concentration of carbon source. However, the influ-nce of this factor on lipolytic enzyme secretion by mesophilicicroorganisms has been investigated. The results are very dif-

erent depending on the microorganism and the experimentalonditions employed. Generally, monosaccharides have beeneported to favour lipase production by mesophilic microor-anisms. So, Benjamin and Pandey [21] found that glucosend fructose enhanced lipase activity in submerged cultures ofandida rugosa. Dalmau et al. [22] proposed a mixture of com-ounds as optimum carbon source for lipase production by theame microorganism. Polysaccharides (i.e. starch) and glycerolave been described as poor carbon sources for lipase productiony C. rugosa and Yarrowia lipolytica [21–23], although they ledo good results in the case of Penicillium citrinum and Rhizopuselemar [24,25]. Costa et al. [26] concluded that the ability of theeast Issatchenkia orientalis to secrete lipolytic activity in sub-erged culture was improved by using monosaccharides (glu-

ose, fructose), although di- and polysaccharides (sucrose, lac-

ose, maltose, starch) as well as glycerol were not recommended.

The results found in the present work seem to indicate that theresence of certain carbon sources in the culture medium mightave an influence on cell growth and lipolytic enzyme production

sape

ial Technology 40 (2007) 187–194

y Thermus strains. However, the observed effects appeared toe somehow less dramatic than those reported in some instancesor mesophilic microorganisms. This could be related to thextreme environments in which thermophilic microorganismsre usually found, generally characterised by low nutrients avail-bility. Therefore, the strains are naturally conditioned to surviven oligotrophic environments, and inhibition by excess of sub-trates is often encountered [27].

Supplementation of the medium with low concentrations ofono- and disaccharides generally resulted in improved biomass

nd lipolytic enzyme production. However, when high carbohy-rate concentrations were used, no further improvements werebserved, and in some cases (i.e. glucose) strong decreasesn biomass and enzymatic activity were detected. This coulde attributed to the occurrence of Maillard reactions betweenmino compounds and reducing sugars, promoted by the highulture temperatures utilised and favoured by values of pH inhe medium between 7.5 (initial) and 8.5 (at the end of culture).his kind of reaction is often found in cultures of thermophilictrains and may result in products that can be inhibitory tohe microorganisms [27]. In the present case, this potentiallynhibitory effect appears to be most remarkable with monosac-harides, and more precisely glucose. This could be due botho the higher reactivity of this sugar towards Maillard reaction,nd a possible stronger toxic effect of the formed products. Sus-eptibility of disaccharides to undergo the reaction is smalleri.e. maltose) or nonexistent (i.e. sucrose, non-reducing sugar),hich agrees with the lesser adverse effects associated to theirresence in the culture medium.

With respect to the mechanism by which disaccharides mighte metabolised by the studied strains, the results seem to indi-ate that they are not massively hydrolysed in the medium beforeeing incorporated to the cells. Extracellular hydrolysis of bothisaccharides would imply the generation of glucose, which washown to exert a strong inhibitory effect on both biomass andnzyme production, yet no drastic negative repercussions wereetected when high concentrations of maltose or sucrose wereonsidered. Therefore, either the disaccharides are hydrolysedt approximately the uptake rate by the cells, thus maintain-ng a concentration below the critical one for inhibition, orhey are directly up taken by the cells. The latter hypothesiss supported by the recently ascertained genome sequence of T.hermophilus, in which maltose/trehalose permease and ABCransporter encoding genes were found [28]. Besides, the mal-ose/trehalose ABC transporter was recently reported to alsoecognize sucrose [29]. The genes encoding two �-glucosidades,�-glucosidase and a maltodextrin glucosidase were also found,ut there are no indications that these enzymes could be secretedy the microorganisms to the culture medium.

Finally, the effect of the addition of lipidic compounds to theulture medium in biomass and lipolytic enzyme production haseen assessed in T. thermophilus HB27 cultures. This strain waselected for this preliminary study, due to the higher enzyme

ecretion levels detected in previous cultures, and the morebundant information available on its lipolytic enzymes [7]. Theresence of lipids has been reported to be crucial for lipases andsterases production by a number of microorganisms [30]. Thus,

A. Domınguez et al. / Enzyme and Microb

Fig. 5. Intra- and extracellular lipolytic activity production by T. thermophilusHct

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aa

R

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B27 cultures in presence of several lipidic compounds with different con-entrations (1 and 10 g dm−3): B, control; OO, olive oil; CO, coconut oil; TB,ributyrin.

he basal medium was supplemented with different concentra-ions (1 and 10 g dm−3) of triglycerides (olive oil, coconut oilnd tributyrin). Intra- and extracellular enzyme activities after0 h culture time are presented in Fig. 5. Tributyrin appearedo have a strong inhibitory effect in the microorganism, whileell growth did not undergo dramatic changes with the additionf olive or coconut oil. On the other hand, the presence of oliveil helped to increase lipolytic enzyme production levels, bothntra and extracellular up to 33% and 70%, respectively. As itccurred with carbohydrates, the best results were obtained withow concentrations of added carbon source. Therefore, it wouldppear that the presence of lipids could trigger to some extent theroduction of lipolytic enzymes. The occurrence of a biphasicedium could lead to hydrophobic interactions that might alter

he permeability of the membranes surrounding the alreadyentioned rotund bodies, therefore accounting for the observed

ariations in enzyme secretion levels. However, further inves-igation is required on this topic before attempting any definiteonclusion.

. Conclusions

According to the results obtained, it can be concluded thathe progressive increase in incubation temperature appeared toave a negative effect on cell growth, and 70 ◦C was selecteds an optimum for this variable, a compromise in terms of

[

ial Technology 40 (2007) 187–194 193

iomass, intra- and extracellular enzyme production, and costptimisation.

Disaccharides (i.e. sucrose and maltose) have been estab-ished as good carbon sources allowing to improve growth and/oravour enzyme secretion by these microorganisms, although theost adequate concentration seems to depend on the strain.n the other hand, the addition of high levels of glucose or

ructose to submerged cultures of Thermus sp. did not bringbout significant ameliorations in lipolytic activity. Moreover,he addition of some lipidic compounds (olive and coconut oils)o the culture medium increased total lipolytic activity levels upo two-fold with respect to the control culture. These promisingesults encourage to realise more studies in which surfactantsr other lipidic compounds would be added to culture medium.hese compounds could help to increase the secretion of lipoly-

ic enzymes due to changes in cell wall permeability or surfactantffects on cell bound enzyme.

cknowledgements

This work was financed by the Spanish Ministry of Sciencend Technology and European FEDER (Project PPQ2001-3361)nd XUNTA de Galicia (Project PGIDT03PXIB30103PR).

eferences

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