World
Journal
of Microbiology
& Biotechnology
10, 657-663
Effects of the detergent Tween 80 on Thermomonospora curvata E. Thies,
T. Jenkins
and F. Stutzenberger*
Tween 80 (O.l%, v/v) added to T’hermomonospora curoutu growing in minimal medium caused a transient lowering of the dry cell mass, decreased the optimal growth temperature of the thermophile from 62 to 54”C, and increased extracellular esterase activity. Cells grown in the presence of Tween 80 had decreased concentrations of branched chain fatty acids and increased concentrations of oleic acid. The detergent removed surface protuberances from mycelia and increased the liberation of enzymes active against crystalline cellulose, but did not stimulate liberation of enzymes active against carboxymethylcellulose, starch or pectin. Key words: Actinomycete,
amylase, cellulase, detergent, fatty acids, pectinase, Thermomonospora,
Detergents have been frequently used to dissociate cell membrane components in a variety of biological systems (Neugebauer 1992). Some of the non-ionic detergents such as the polyoxyethylene octyl phenols (Triton X series) can solubilize membrane lipid bilayers and release the associated enzymes (Scopes 1987). In contrast, the polyoxyethylene sorbitol esters (Tween 20 to 80 series) are ineffective in solubilizing membrane lipids (Womack et al. 1983), yet are widely successful in improving exoenzyme secretion from growing bacterial and fungal cultures (Jacques et al. 1985; Asther & Corrieu 1987; Stutzenberger 1987; Hung et al. 1988; Bertrand et al. 1989; Gillespie et al. 1990; Capalash ef al. 1991). However, the effects of the Tweens on the cells themselves, as well as the mechanism by which they enhance enzyme secretion, have not been determined. Studies to date on the effects of Tween detergents in microbial systems have been confined to mesophilic species. It was of interest to test the effects of detergents such as Tween 80 (which causes changes in fatty acid composition) on a thermophilic species, since thermophilic bacteria have very low ratios of unsaturated/saturated fatty acids compared with mesophiles (Amelunxen & Murdock 1978). Here we report some effects of Tween 80 on the growth, fatty acid composition, soluble enzyme levels and surface topology
E. Thies and T. Jenkins are with the Department of Animal, Dairy Veterinary Sciences and F. Stutzenberger is with the Department Microbiology, Clemson University, Clemson, SC 29634-1909, USA; 603 65Gll27. ‘Corresponding author. @ 1994
Rapid
Communications
of Oxford
and of fax:
Tween
80.
of Thermomonosporn curvafa, a thermophilic which secretes a variety of depolymerizing (Bemier et al. 1988).
Materials
actinomycete exoenzymes
and Methods
Strain and Growfh
Conditions
The strain of T. curvata used was isolated from municipal solidwaste compost at the US Public Health Service/Tennessee Valley Authority Joint Composting Project at Johnson City, TN, USA (Stutzenberger et al. 1970). Stock cultures were stored in 50% (v/v) glycerol at - 86°C. Cultures (100 ml in 250-m] baffled Erlenmeyer flasks inclined at 30” from vertical in a rotary waterbath and shaken at 140 rev/min) were grown in triplicate at 53 to 55°C in mineral salts/minimal medium (Stutzenberger 1972) containing 0.5% cellobiose as sole carbon source. In some experiments, cultures were grown on 0.5% cellobiose octaacetate or native cotton fibres to induce cellulase production. Pectinase and amylase were induced by 0.5% apple pectin or soluble starch, respectively. Detergents of the polyoxyethylene sorbitan series, monolaurate (Tween 20), monopalmitate (Tween 40), monostearate (Tween 60) and monooleate (Tween 80), were prepared as stock solutions (IO%, v/v) in distilled water and sterilized either by autoclaving or by filtration through o.@prn pore membranes. Detergent solutions were added aseptically in various concentrations up to I% (v/v) to early-exponential-phase cultures. Comparisons of growth in the presence and absence of detergents were based on the increase in washed cell absorbance (1.0 mg dry cell wt/ml giving A,,, of 1.7). Specific growth rate constants (k) were calculated by the forrnula: In X - In X0 = kt
Ltd World
Jound
of Mtcroblology
6 Biotechnology,
Vol 10. 1994
657
E. Thies, T. Jenkins and F. Stutzenberger where X is cell mass concentration and t is time in h. Due to the particulate nature of actinomycete growth in liquid media and the subsequent large variation (average standard deviation of 14.2%) in dry cell mass determinations, A,,, were determined in triplicate for each sample from triplicate flasks and averaged. Cu2ture Sample Prepurution Samples (20 ml) of culture fluids were clarified by centrifugation (12,000 x g, 10 min, 22°C). Cells were washed twice with 40 vol (relative to packed cell volume) of mineral salts/minimal medium to remove free Tween SO. In preliminary experiments, additional washings did not significantly alter the detectable fatty acid composition. Freeze-dried residues from culture samples were weighed into 15 x 125 mm screw-capped culture tubes with Teflon liners prior to fatty acid analysis. Fatty Acid Analysis Fatty acids were methylated with 3 ml 5% HCI in absolute methanol according to Browse et al. (1986) using heptadecanoic acid (O.l%, w/v) in benzene as internal standard. Methyl esters were analysed by GC with a 30 m x 0.25 mm int diam fused silica capillary column (helium carrier gas) and a flame ionization detector. Fatty acids were identified by retention time comparisons with bacterial fatty acid methyl ester standards and verified by GC-MS spectral comparisons. Data were statistically evaluated by analysis of variance using the GLM procedure of the Statistical Analysis System (Anon. 1985).
7 kP N,. Stirred retentates were washed repeatedly with equal volumes of 0.1 M phosphate buffer, pH 7.2, to remove small mol wt comoonents. The retained nroteins from each set of culture fluids where pooled and fractionated by size-exclusion chromatography at an eluant flow rate of 1.0 ml/min (0.1 M phosphate buffer, pH 7.2 with 0.15 M NaCI) through a Phenomenex BioSep S2000 size-exclusion chromatography (200,000 mol wt exclusion) analytical column (300 x 7.5 mm) in an HPLC system with detector set at 280 nm. Protein concentrations were determined by the method of Bradford (1976). Scanning Electron A4icroscopy (SEM) Cell surface topology of T. curvutu mycelia was visualized under SEM by a modified method of Lamed et uI. (1987). One-ml cell samples were centrifuged for 1 min at 13,600 x g, 22°C. washed twice and resuspended in 1 ml 0.15 M saline. Supematant fluid from 0.2 ml washed cell suspension was removed by filtration through O.&pm pore membranes, then washed with 1 ml saline. Washed cells were labelled with horse spleen ferritin coupled with N,iV-dimethyl-x,3-propanediamine (Danon et al. 1972). applied as a solution (0.2 ml, 1 mg/ml) to each filter for 10 min. Filters were washed with two 5-ml volumes of saline, then fixed in 3.5% glutaraldehyde, pH 7.3, overnight. Specimens were dried in an ethanol series (50% to lOO%), dried at the critical point with liquid CO,, and coated with gold before viewing.
Results Enzyme
Assays
cc-Arnylase (EC 3.2.1.1) activity was measured in 2 ml 2% soluble starch buffered with 0.1 M MES, and 0.2 ml of enzyme for 30 min at 65°C. Extracellular esterase (EC 3.1.1.3) was estimated in cell free culture fluid samples by the rate of hydrolysis of p-nitrophenylpalmitate (Winkler & Stuckmann 1979) adjusted to pH 8 and 54°C to approximate culture fluid conditions. The substrate for measurement of total cellulase activity against crystalline cellulose was 3% (w/v) Red Cross, surgical-grade, absorbent cotton fibres in 2 ml reaction mixture buffered to pH 6.1 with 0.1 M MES. Cotton fibre (CF) activity was determined after incubation for I h at 65°C with 0.5 ml enzyme. For specific measurement of cellulase as endoglucanase (EC 3.2.1.4) activity, 100 ~1 of appropriately diluted enzyme was incubated for 10 min at 65°C in 2 ml reaction mixture (2.5% carboxymethylcellulose (CMC; Hercules type 7L) in 0.1 M MES buffer, pH 6.11. Product accumulation for amylase and cellulase was quantified as liberation of soluble reducing sugars (expressed as glucose equivalents) determined by the method of Bemfeld (1955). Pectinase as polygalacturonate lyase (PL; EC 4.2.2.2) activity was measured spectrophotometrically by increase in A,,, (Albersheim & Killias 1962) at room temperature in reaction mixtures containing 0.25 ml 0.2 M 3-(cyclohexylamino)-I-propanesulfonic acid (CAPS buffer), pH 10.4, with 0.8 mM CaCl,, 0.25 ml 0.6% polygalacturonic acid (PGA; Sigma) and 0.1 ml appropriately diluted enzyme. Enzyme units were calculated as pmol product per min (esterase units in nmol/min). The average standard deviations for these assays, based on sets of six replicates, were as follows: amylase, 6.6%; general esterase activity, 8.8%; CF activity, 10.1%; endoglucanase, 5.6%; pectinase, 4.2%; protein, 3.7%; and reducing sugar, 1.3%. Emprotein Fractionation Concentrates (lo- to x2-fold) of exouroteins from cell-free fluid from duplicate cellulose-grown cultures grown either in the presence or absence of Tween 80 were obtained by ultrafiltration on an Amicon YMlO (10,000 mol wt exclusion) membrane under
Effect of Tween Defergenfs on Growth Addition of Tween detergents at 0.1% (v/v) to unadapted cells of T. cttrvata growing exponentially in cellobiose/minera1 salts medium caused a rapid decline in apparent dry cell mass (Figure I). This decline was accompanied by a transient doubling in soluble protein [from about 60 to 110 pug/ml in mid-exponential-phase cultures (data not shown)], indicating cytolysis of some cells by the detergent. This was probably attributable to the fatty acids released from the Tweens, since addition of palmitic acid or oleic acid equivalent to their corresponding concentrations in 0.1% Tween 40 or Tween 80, respectively, caused similar declines. Growth resumed after the decline and reached a stationary-phase dry cell weight comparable with that observed in the absence of the detergents. However, cultures which received Tweens sterilized by autoclaving rather than filtration had a 12- to 20-h lag period before resumption of growth (data not shown); filtered stock solutions were employed in all subsequent experiments. Tween 20, 40 and 60, whether autoclaved or filtered, were inhibitory to growth at concentrations higher than O.l%, whereas Tween 80 caused no inhibition to adapted T. ctlrvata cells grown with concentrations up to 1.0%. Therefore, the remainder of the study used Tween 80. The fatty acid composition of Tween 80 consisted of cis18:l (77%), truns-l&l (lo%), 16:0 (7%), l&O (I%), anfeiso17:O (2%). iso-Id:0 (1%) and other fatty acids (< 1% each). The major fatty acid, cis-l&l (oleic acid), provided an indicator by which to monitor disappearance of the deter-
Tween 80 effects on T. curvata
IO
20
30
50
40
60
Time (h) Time (h) Figure 1. Decreases in apparent dry cell wt (compared with control, 0) after addition of Tween 40 (0) or Tween 80 (A) to mid-exponential-phase 7. curvata cultures in cellobiose/minimal medium. l-time of addition.
=E 0.16 t
Figure 3. Increase in dry cell wt (0) and extracellular esterase activity (0) in T. curvata adapted to grow in Tween/cellobiose/ minimal medium. One unit of esterase activity was defined as 1 nmol of p-nitrophenol liberated per min. Table 1. Composition cells gown at 55°C.
of long-chain
fatty
acids
of T. curvata
Fatty acid
0.04 0.02 0.02 0
O\-0
-0 I 20
I 10
J
Time (h) Figure 2. Decrease of cis-18: 1 (oleic acid) in cell-free fluid addition of Tween 80 to 7. curvata mid-exponential-phase culture.
% of total
Branched-chain iso-15:o anteiso-15: 0 iso-16:0 iso-17:0 anteiso-17 :0 iso-18:0 Total
30
after
gent after addition to T. curvufa cultures. When Tween 80 (initially at 0.1%) was added at early exponential phase, the concentration of cis-18:l in culture fluid decreased markedly (Figure 2). Other fatty acids (tram-Id:1 and 16:0), which contributed significantly to Tween 80 composition, decreased to a comparable extent (57% and 59%, respectively). The rapid decline of Tween 80 in T. curvufu culture fluid prompted the estimation of extracellular esterase, the activity of which could account for the detergent’s disappearance (Asther & Corrieu 1987). While hydrolysis of p-nitrophenylpalmitate cannot invariably be construed to indicate total lipase activity, the usefulness of the substrate as a general esterase indicator in a variety of bacteria has been confirmed (Fay et al. 1990). The addition of Tween 80 (to give 100 pg/ml) to a T. curvafa culture tripled esterase secretion
dry
1 2 38 12 24 12 89
Straight-chain, 15:o 16:0 1810 19:o Total
saturated
Straight-chain, cis-18: 1 trans-18 : 1 18:2 Total
unsaturated
Cl 3 5 Cl 8
Cl 2
from about 2.6 to 7.7 U/mg dry cells within two generation times. In a Tween-adapted culture, esterase accumulated in cell-free fluid in proportion to cell mass (Figure 3). The presence of Tween 80 influenced the temperaturedependence profile for growth of the actinomycete. 7. curvufu is a facultative thermophile able to grow in minimal medium from 35 to 65°C; however, cultures grown in the presence of 0.1% detergent exhibited a narrowed temperature range with a shifted optimum (Figure 4). Changes in bacterial growth temperature requirements often coincide with changes in the cellular fatty acids, particularly in
World]oumal
of Minobmlogy
b Biotechnology.
Vol IO. 1994
659
E. Thies, T. Jenkins and F. Stutzenberger 0.5 ,
1
h
50
60
Temperature (“C) Figure
4. Influence
of Tween
80 on the specific growth rate of T. medium with (A) and without (0)
curvata in cellobiose/minimal 0.1%
Tween. 12
0 U.0
I
Growth temperature (“C) . I . I . I .
0.2
0.4
0.6
0.8
1.0
Initial Tween 80 concentration (g/l 00 ml) Flgure 5. Influence of Tween 80 on cis-18:1 concentration in long-chain fatty acids (LCFA) of T. curvafa grown at 54°C. Inset shows growth temperature dependence of cis-18: 1 contribution to LCFA in the presence of the detergent.
0 B 5
I
0.2
0.4
5
m’
Initial Tween 80 concentration (g/l00 ml)
Figure grown
660
I
70’ 0.0
6. Decrease in branched-chain at 54C in the presence of Tween
WorldJournal
of Minobiology
fatty 80.
& Biotechnology.
acids
0.6 in T. curvata
Vol ~0, 1994
membrane composition (Brock & Mad&an 1991). Since almost all lipid ( > 90%) in Gram-positive bacteria is cytoplasmic membrane-associated (Lennarz 1966), whole cell extracts were used in the comparison of cells grown in the presence and absence of Tween 80. The presence of Tween 80 during growth caused little detectable difference in the total long-chain fatty acid content (3.4%, 3.6%, 3.5% and 3.6% of the dry wt of cells grown in 0, 0.05, 0.1, 0.2 and 0.4% Tween, respectively). Although the relative proportions remained similar, the amounts of individual fatty acids changed during growth in the presence of the detergent. A typical long-chain fatty acid composition of dry cells grown in the absence of Tween 80 is shown in Table 1. The greatest change from control values was a Is-fold increase in the cis-18:l content of the cells; the magnitude of this change was strongly temperature dependent (Figure 5). Other changes in straight-chain fatty acid composition included decreases of about 20% in 16:O and l&O. The presence of the detergent also had an effect on the total branched chain fatty acids content of T. curvata (Figure 6). This effect was largely attributable to cumulative decreases in anfeiso-15:O and anteiso-17:O. Tween 80 also influenced the T. curvata cell-surface topology. During growth on cellobiose octacetate or cellulose, mycelia were covered with multiple surface protuberances (Figure 7a). These protuberances are similar to the cellulosomes of the thermophile, Clostriditrm fhermocellum, which contain both cellulolytic enzymes and substrate attachment sites (Bayer & Lamed 1988). The addition of Tween 80 to T. curvafa mycelia resulted in the loss of most of these surface structures, leaving a stripped, ragged appearance with few vestiges of their cellulosomal structure (Figure 7b). The presence of the detergent also caused an increase in soluble enzymes active against crystalline cellulose, but not in endoglucanases active against CMC (Figure 8). This increase of enzymatic activity against crystalline cellulose was apparently not the result of cytolysis since sonic breakage of T. curvafa cells did not release detectable cellulolytic enzymes. Although cell-free-fluid endoglucanase remained quantitatively unchanged, cultures grown in Tween 80 produced exoprotein and endoglucanase patterns chromatographically different from those of the controls (Figure 9). In addition to cellulases, T. ctrrvafa also secretes exoenzymes which act on a variety of other polysaccharides (Bemier et al. 1988). It was therefore of interest to determine whether the presence of Tween 80 stimulated the release of other extracellular enzymes from the actinomycete. When T. curvafa was grown on starch or pectin in the absence of Tween 80, amylase and pectinase activities in culture fluids reached peak levels of 16.6 and 4.6 U/ml, respectively. Slightly lower peak levels (15.1 and 4.0 U/ml) were observed during growth of T. curvafa with 0.1% detergent in the medium.
Tween 80 effects on T. curvata
a
b
Figure 1. Scanning electron microscopical comparison of cell surface topologies of T. curvata mycelia grown on cellobiose/ octaacetate/minimal medium in the absence (a) and presence (b) of 0.1% Tween 80. Bar 1/~m. 200
gE E~ E
m E
>.'Z'--
O :3
.->
100
0----
0
1
2
'
I
3
4
,
I
5
,
I
6
i
7
T i m e (days) Figure 8, Cotton fibre (CF) activity (O, @) and endoglucanase activity (A, A) in cell-free fluid from cultures with (@, A) and without (O, A) Tween 80.
Discussion The lyric effect of Tween detergents on T. curvata appears to be due to their long-chain fatty acid content, since the effect could be duplicated by using an equivalent amount of the corresponding acids. This conclusion is supported by earlier reports on inhibitory effects of long-chain fatty acids in other bacteria (Galbraith et al. 1971; Maczulak et aI. I981). The ability of T. curvata to resume growth after prolonged Tween 80 exposure would be due, at least in part, to its relatively high extracellular esterase activity [compared with a wide variety of other bacteria, (Winkler & Stuckmann 1979; Fay e~ al. I990)] which enables the actinomycete to initiate degradation of the detergent. The continual disappearance of oleic acid from culture fluid after Tween 80 addition, together with the slight stimulation of growth in detergent-adapted cells at the adapted tempera-
ture optimum of 54°C, is consistent with the view that the actinornycete utilizes the liberated fatty acid after de-esterification of the detergent. The presence of Tween 80 caused a decrease in the optimal growth temperature of the facultative thermophile from 62 to 54°C. This effect may be related to changes in fatty acid composition. T. curvata long-chain fatty acids consisted mainly (almost 90%) of iso- and anteiso-branchedchain fatty acids from C1~ to CI8, a composition comparable with that observed in other thermophilic bacteria (Langworthy & Pond 1986). A high concentration of these saturated, branched-chain fatty acids confers membrane stability at elevated temperatures (Brock & Madigan 199I). Fatty acid moieties of Tween detergents are incorporated into both bacterial and fungaI membranes (Jacques et al. 1985; Asther & Corrieu I987). This incorporation causes increased degrees of unsaturation in membrane fatty acids and subsequently enhances membrane fluidity. Increased membrane fluidity has been suggested (Wittenberger et al. 1978) as a possible mechanism to explain the enhanced exoenzyme secretion rates in the presence of Tween detergents. However, in Streptococcus sanguis (Jacques et al. 1985) and Trichoderma reesei (Panda et al. I987), stimulation of protein secretion is not totally attributable to changes in membrane lipid composition. In T. curvata, it is likely that the enzyme-selective nature of Tween 80 stimulation (increased cotton fibre cellulase activity with unchanged levels of amylase, endoglucanase or pectinase) results from the solubilization of enzymes already secreted and bound to the cell surface in cellulosomes rather than stimulation of enzyme biosynthesis or secretion. This view is supported by the observation that cellulases remain bound to the T. curvata mycelia, while mycelial surfaces are devoid of detectable amylase or pectinase activities (Hostalka et al. 1992).
World Journal of Microbiology & Biotechnology, Vol I0, 1994
6 61
E. This, T. Jenkins and F. Stutzenberger
0
0 5
6
7
8
9
10
11
5
Figure 9. Alterations in elution patterns, determined by size-exclusion HPLC, ultrafiltration concentrates from cell-free fluids from cultures grown in the absence
Acknowledgements We thank J. 0. Eubanks,J. E. Mann and E. G. Porter for excellenttechnicalsupport.
References Albersheim, P. & Killias, U. 1962 Studies relating to the purification and properties of pectin transeliminase. Archives of Biochew&y and Biophysics 97,107-115. Amelunxen, R. E. & Murdock, A. L. 1978 Microbial life at high temperatures: mechanisms and molecular aspects. In Microbial Life in Extreme Environments, ed Kushner, D. J. pp. 217-277, London: Academic Press. Anon. 1985 Statistical Analysis System Users’ Guide. Version 5 Edition. Cary, NC: SAS Institute. Asther, M. & Corrieu, G. 1987 Effect of Tween 80 and oleic acid on ligninase production by Phunerochuefe chrysosporium INA-12. Enzyme and Microbial Technology 9,245-249. Bayer, E. A. & Lamed, R. 1988 Ultrastructure of the cell surface cellulosome of Closfridium fhermocellum and its interaction with cellulose. Journu~ of Bacteriology 167, 828-836. Bemfeld, P. 1955 Amylases, alpha and beta. Methods in Enzymology
1,149-154. Bemier, R., Kopp, M., Trakas, B. & Stutzenberger, F. 1988 Production of extracellular enzymes by Ihermomonosporu curoufu during growth on protein-extracted luceme fibres. Journal of Applied Bacteriology 65, 411-418. Bertrand, J.-L., Morosoli, R., Shareck, F. & Kluepfel, D. 1989 Expression of the xylanase gene of Sfreptomyces lividuns and production of the enzyme on natural substrates. Biotechnology and Bioengineering 33, 791-794. Bradford, M. M. 1976 A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72, 248-254. Brock, T. D. & Madigan, M. T. 1991 Biology of Microorganisms, 6th edn. Englewood Cliffs, NJ: Prentice Hall. Browse, J., McCourt, F. J. & Somerville, C. R. 1986 Fatty acid composition of leaf lipids determined after combined digestion and fatty acid methyl ester formation from fresh tissue. Analyficul Biochemistry 152, 141-145.
662
World Jourtd
of Microbiology
6 Biotechnology,
Vool IO, 1994
6
7
8
9
10
1
Eluted volume (ml)
Eluted volume (ml)
of total exoprotein (0) and presence
(a) and endoglucanase (0) of Tween 80.
(b) in
Capalash, N., Gupta, K. G. & Sharma, P. 1991 Effects of additives on the activity of Bacillus sp. /I-xylanase. Letters in Applied Microbiology 12,31-33. Danon, D., Goldstein, L., Marikovsky, Y. & Skutelsky, E. 1972 Use of cationized ferritin as a label of negative charges on cell surfaces. Journal of Ultrastructure Research 38, 50&510. Fay, J. P., Jakober, K. D. & Cheng, K.-J. 1990 Esterase activity of pure cultures of rumen bacteria as expressed by the hydrolysis of p-nitrophenylpalmitate. Canadian Journul of Microbiology 36, 585-589. Galbraith, H., Miller, T. B., Paton, A. M. & Thompson, J. K. 1971 Antibacterial activity of long chain fatty acids and the reversal with calcium, magnesium, ergocalciferol and cholesterol. Journal of Applied Bacteriology 34, 803-813. Gillespie, A. M., Keane, D., Griffin, T. O., Tuohy, M. G., Donaghy, J., Haylock, R. W. & Coughlan, M. P. 1990 The application of fungal enzymes in flax retting and properties of an extracellular polygalaturonase from Penicillitrm cupsulufum. In Biotechnology in Pulp and Paper Munufacfure: Applications and Fundamenful Invesfigufions, eds Kirk, T. K. & Chang, H. M. pp. 211-219, Boston: Butterworth-Heinemann Hostalka, F., Moultrie, A. & Stutzenberger, F. 1992 Influence of carbon source on cell surface topology of Thermomonosporu curvufu. Journul of Bacteriology 174, 7048-7052. Hung, B. R., Lara, L., Patron, M. A., Ugarova, N. N., Bechstedt, W. & Clappes, S. 1988 Tween 80 and proteose peptone effect on cellulase production. Acfu Biofechnologia 8, 461464. Jacques, N. A., Jacques, V. L., Wolf, A. C. & Wittenberger, C. L. 1985. Does an increase in membrane unsaturated fatty acids account for Tween 80 stimulation of glucosyltransferase secretion by Sfrepfococcw salivarid Journal of General Microbiology
131,67-72. Lamed, ized
R., Naimark, J., Morgenstem, E. & Bayer, E. 1987 Specialcell surface structures in cellulolytic bacteria. Jownal of Bacteriology 169,3792-3800. Langworthy, T. A. & Pond, J. L. 1986 Membranes and lipids of thermophiles. In Thermophiles: General, Molecular and Applied Aspects, ed Brock, T. D. pp. 107-135. New York: John Wiley and Sons. Lennarz, W. J. 1966 Lipid metabolism in bacteria. Advances in Lipid Research 4,175-221. Maczulak, A. E., Dehority, B. A. & Palmquist, D. L. 1981 Effects
Tween of long-chain fatty acids on growth of rumen bacteria. Applied and Environmental Microbiology 42, 856862. Neugebauer, J. 1992 A Guide to the Properties and Uses of Defergents in Biology and Biochemistry. La Jolla, CA: Calbiochem-Novabiothem Corp. Panda, T., Gruber, H. & Kubicek, C. P. 1987 Stimulation of protein secretion in Trichoderma reesei by Tween surfactants is not correlated with changes in enzyme localization or membrane fatty acid composition. FEMS Microbiology Letters 41, 85-90. Scopes, R. K. 1987 Protein Purification: Principles and Practice. New York: Springer-Verlag. Stutzenberger, F. J. 1972 Cellulolytic activity of Themomonospora mrvafu: nutritional requirements for cellulase production. Applied Microbiology 24, 77-82. Stutzenberger, F. J. 1987 Component-specific stimulation of cellulase secretion in Thermomonospora curvata by the surfactant Tween 80. Journal of Applied Bacteriology 63, 239-244.
80 effects on T. curvata
Stutzenberger, F. J., Kaufman, A. J. & Lossin, R. D. 1970 Cellulolytic activity in municipal solid waste compost. Canadian Journal ofMicrobiology 16, 553-560. Winkler, U. K. & Stuckmann, M. 1979 Glycogen, hyaluronate and some other polysaccharides greatly enhance the formation of exolipase by Serrafia marcescens. Journal of Bucferiology 138, 663-670. Wittenberger, C. L., Beaman, A. J. & Lee, L. N. 1978 Tween 80 effect on glucosyltransferase production in Sfrepfococcus mutans. Applied and Environmental Microbiology 34, 23 I-239. Womack, M. D., Kendall, D. A. & MacDonald, R. C. 1983 Detergent effects on enzyme activity and solubilization of lipid bilayer membranes. Biochimicu ef Biophysics Acta 733, 210215.
(Received
in revised
World
Journal
form
I July
of Mmbiology
1994; accepted
6 Biotechnology.
3 ]uly
Vol IO, 1994
1994)
663
Journal
of Microbiology
& Biotechnology
10, 657-663
Effects of the detergent Tween 80 on Thermomonospora curvata E. Thies,
T. Jenkins
and F. Stutzenberger*
Tween 80 (O.l%, v/v) added to T’hermomonospora curoutu growing in minimal medium caused a transient lowering of the dry cell mass, decreased the optimal growth temperature of the thermophile from 62 to 54”C, and increased extracellular esterase activity. Cells grown in the presence of Tween 80 had decreased concentrations of branched chain fatty acids and increased concentrations of oleic acid. The detergent removed surface protuberances from mycelia and increased the liberation of enzymes active against crystalline cellulose, but did not stimulate liberation of enzymes active against carboxymethylcellulose, starch or pectin. Key words: Actinomycete,
amylase, cellulase, detergent, fatty acids, pectinase, Thermomonospora,
Detergents have been frequently used to dissociate cell membrane components in a variety of biological systems (Neugebauer 1992). Some of the non-ionic detergents such as the polyoxyethylene octyl phenols (Triton X series) can solubilize membrane lipid bilayers and release the associated enzymes (Scopes 1987). In contrast, the polyoxyethylene sorbitol esters (Tween 20 to 80 series) are ineffective in solubilizing membrane lipids (Womack et al. 1983), yet are widely successful in improving exoenzyme secretion from growing bacterial and fungal cultures (Jacques et al. 1985; Asther & Corrieu 1987; Stutzenberger 1987; Hung et al. 1988; Bertrand et al. 1989; Gillespie et al. 1990; Capalash ef al. 1991). However, the effects of the Tweens on the cells themselves, as well as the mechanism by which they enhance enzyme secretion, have not been determined. Studies to date on the effects of Tween detergents in microbial systems have been confined to mesophilic species. It was of interest to test the effects of detergents such as Tween 80 (which causes changes in fatty acid composition) on a thermophilic species, since thermophilic bacteria have very low ratios of unsaturated/saturated fatty acids compared with mesophiles (Amelunxen & Murdock 1978). Here we report some effects of Tween 80 on the growth, fatty acid composition, soluble enzyme levels and surface topology
E. Thies and T. Jenkins are with the Department of Animal, Dairy Veterinary Sciences and F. Stutzenberger is with the Department Microbiology, Clemson University, Clemson, SC 29634-1909, USA; 603 65Gll27. ‘Corresponding author. @ 1994
Rapid
Communications
of Oxford
and of fax:
Tween
80.
of Thermomonosporn curvafa, a thermophilic which secretes a variety of depolymerizing (Bemier et al. 1988).
Materials
actinomycete exoenzymes
and Methods
Strain and Growfh
Conditions
The strain of T. curvata used was isolated from municipal solidwaste compost at the US Public Health Service/Tennessee Valley Authority Joint Composting Project at Johnson City, TN, USA (Stutzenberger et al. 1970). Stock cultures were stored in 50% (v/v) glycerol at - 86°C. Cultures (100 ml in 250-m] baffled Erlenmeyer flasks inclined at 30” from vertical in a rotary waterbath and shaken at 140 rev/min) were grown in triplicate at 53 to 55°C in mineral salts/minimal medium (Stutzenberger 1972) containing 0.5% cellobiose as sole carbon source. In some experiments, cultures were grown on 0.5% cellobiose octaacetate or native cotton fibres to induce cellulase production. Pectinase and amylase were induced by 0.5% apple pectin or soluble starch, respectively. Detergents of the polyoxyethylene sorbitan series, monolaurate (Tween 20), monopalmitate (Tween 40), monostearate (Tween 60) and monooleate (Tween 80), were prepared as stock solutions (IO%, v/v) in distilled water and sterilized either by autoclaving or by filtration through o.@prn pore membranes. Detergent solutions were added aseptically in various concentrations up to I% (v/v) to early-exponential-phase cultures. Comparisons of growth in the presence and absence of detergents were based on the increase in washed cell absorbance (1.0 mg dry cell wt/ml giving A,,, of 1.7). Specific growth rate constants (k) were calculated by the forrnula: In X - In X0 = kt
Ltd World
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E. Thies, T. Jenkins and F. Stutzenberger where X is cell mass concentration and t is time in h. Due to the particulate nature of actinomycete growth in liquid media and the subsequent large variation (average standard deviation of 14.2%) in dry cell mass determinations, A,,, were determined in triplicate for each sample from triplicate flasks and averaged. Cu2ture Sample Prepurution Samples (20 ml) of culture fluids were clarified by centrifugation (12,000 x g, 10 min, 22°C). Cells were washed twice with 40 vol (relative to packed cell volume) of mineral salts/minimal medium to remove free Tween SO. In preliminary experiments, additional washings did not significantly alter the detectable fatty acid composition. Freeze-dried residues from culture samples were weighed into 15 x 125 mm screw-capped culture tubes with Teflon liners prior to fatty acid analysis. Fatty Acid Analysis Fatty acids were methylated with 3 ml 5% HCI in absolute methanol according to Browse et al. (1986) using heptadecanoic acid (O.l%, w/v) in benzene as internal standard. Methyl esters were analysed by GC with a 30 m x 0.25 mm int diam fused silica capillary column (helium carrier gas) and a flame ionization detector. Fatty acids were identified by retention time comparisons with bacterial fatty acid methyl ester standards and verified by GC-MS spectral comparisons. Data were statistically evaluated by analysis of variance using the GLM procedure of the Statistical Analysis System (Anon. 1985).
7 kP N,. Stirred retentates were washed repeatedly with equal volumes of 0.1 M phosphate buffer, pH 7.2, to remove small mol wt comoonents. The retained nroteins from each set of culture fluids where pooled and fractionated by size-exclusion chromatography at an eluant flow rate of 1.0 ml/min (0.1 M phosphate buffer, pH 7.2 with 0.15 M NaCI) through a Phenomenex BioSep S2000 size-exclusion chromatography (200,000 mol wt exclusion) analytical column (300 x 7.5 mm) in an HPLC system with detector set at 280 nm. Protein concentrations were determined by the method of Bradford (1976). Scanning Electron A4icroscopy (SEM) Cell surface topology of T. curvutu mycelia was visualized under SEM by a modified method of Lamed et uI. (1987). One-ml cell samples were centrifuged for 1 min at 13,600 x g, 22°C. washed twice and resuspended in 1 ml 0.15 M saline. Supematant fluid from 0.2 ml washed cell suspension was removed by filtration through O.&pm pore membranes, then washed with 1 ml saline. Washed cells were labelled with horse spleen ferritin coupled with N,iV-dimethyl-x,3-propanediamine (Danon et al. 1972). applied as a solution (0.2 ml, 1 mg/ml) to each filter for 10 min. Filters were washed with two 5-ml volumes of saline, then fixed in 3.5% glutaraldehyde, pH 7.3, overnight. Specimens were dried in an ethanol series (50% to lOO%), dried at the critical point with liquid CO,, and coated with gold before viewing.
Results Enzyme
Assays
cc-Arnylase (EC 3.2.1.1) activity was measured in 2 ml 2% soluble starch buffered with 0.1 M MES, and 0.2 ml of enzyme for 30 min at 65°C. Extracellular esterase (EC 3.1.1.3) was estimated in cell free culture fluid samples by the rate of hydrolysis of p-nitrophenylpalmitate (Winkler & Stuckmann 1979) adjusted to pH 8 and 54°C to approximate culture fluid conditions. The substrate for measurement of total cellulase activity against crystalline cellulose was 3% (w/v) Red Cross, surgical-grade, absorbent cotton fibres in 2 ml reaction mixture buffered to pH 6.1 with 0.1 M MES. Cotton fibre (CF) activity was determined after incubation for I h at 65°C with 0.5 ml enzyme. For specific measurement of cellulase as endoglucanase (EC 3.2.1.4) activity, 100 ~1 of appropriately diluted enzyme was incubated for 10 min at 65°C in 2 ml reaction mixture (2.5% carboxymethylcellulose (CMC; Hercules type 7L) in 0.1 M MES buffer, pH 6.11. Product accumulation for amylase and cellulase was quantified as liberation of soluble reducing sugars (expressed as glucose equivalents) determined by the method of Bemfeld (1955). Pectinase as polygalacturonate lyase (PL; EC 4.2.2.2) activity was measured spectrophotometrically by increase in A,,, (Albersheim & Killias 1962) at room temperature in reaction mixtures containing 0.25 ml 0.2 M 3-(cyclohexylamino)-I-propanesulfonic acid (CAPS buffer), pH 10.4, with 0.8 mM CaCl,, 0.25 ml 0.6% polygalacturonic acid (PGA; Sigma) and 0.1 ml appropriately diluted enzyme. Enzyme units were calculated as pmol product per min (esterase units in nmol/min). The average standard deviations for these assays, based on sets of six replicates, were as follows: amylase, 6.6%; general esterase activity, 8.8%; CF activity, 10.1%; endoglucanase, 5.6%; pectinase, 4.2%; protein, 3.7%; and reducing sugar, 1.3%. Emprotein Fractionation Concentrates (lo- to x2-fold) of exouroteins from cell-free fluid from duplicate cellulose-grown cultures grown either in the presence or absence of Tween 80 were obtained by ultrafiltration on an Amicon YMlO (10,000 mol wt exclusion) membrane under
Effect of Tween Defergenfs on Growth Addition of Tween detergents at 0.1% (v/v) to unadapted cells of T. cttrvata growing exponentially in cellobiose/minera1 salts medium caused a rapid decline in apparent dry cell mass (Figure I). This decline was accompanied by a transient doubling in soluble protein [from about 60 to 110 pug/ml in mid-exponential-phase cultures (data not shown)], indicating cytolysis of some cells by the detergent. This was probably attributable to the fatty acids released from the Tweens, since addition of palmitic acid or oleic acid equivalent to their corresponding concentrations in 0.1% Tween 40 or Tween 80, respectively, caused similar declines. Growth resumed after the decline and reached a stationary-phase dry cell weight comparable with that observed in the absence of the detergents. However, cultures which received Tweens sterilized by autoclaving rather than filtration had a 12- to 20-h lag period before resumption of growth (data not shown); filtered stock solutions were employed in all subsequent experiments. Tween 20, 40 and 60, whether autoclaved or filtered, were inhibitory to growth at concentrations higher than O.l%, whereas Tween 80 caused no inhibition to adapted T. ctlrvata cells grown with concentrations up to 1.0%. Therefore, the remainder of the study used Tween 80. The fatty acid composition of Tween 80 consisted of cis18:l (77%), truns-l&l (lo%), 16:0 (7%), l&O (I%), anfeiso17:O (2%). iso-Id:0 (1%) and other fatty acids (< 1% each). The major fatty acid, cis-l&l (oleic acid), provided an indicator by which to monitor disappearance of the deter-
Tween 80 effects on T. curvata
IO
20
30
50
40
60
Time (h) Time (h) Figure 1. Decreases in apparent dry cell wt (compared with control, 0) after addition of Tween 40 (0) or Tween 80 (A) to mid-exponential-phase 7. curvata cultures in cellobiose/minimal medium. l-time of addition.
=E 0.16 t
Figure 3. Increase in dry cell wt (0) and extracellular esterase activity (0) in T. curvata adapted to grow in Tween/cellobiose/ minimal medium. One unit of esterase activity was defined as 1 nmol of p-nitrophenol liberated per min. Table 1. Composition cells gown at 55°C.
of long-chain
fatty
acids
of T. curvata
Fatty acid
0.04 0.02 0.02 0
O\-0
-0 I 20
I 10
J
Time (h) Figure 2. Decrease of cis-18: 1 (oleic acid) in cell-free fluid addition of Tween 80 to 7. curvata mid-exponential-phase culture.
% of total
Branched-chain iso-15:o anteiso-15: 0 iso-16:0 iso-17:0 anteiso-17 :0 iso-18:0 Total
30
after
gent after addition to T. curvufa cultures. When Tween 80 (initially at 0.1%) was added at early exponential phase, the concentration of cis-18:l in culture fluid decreased markedly (Figure 2). Other fatty acids (tram-Id:1 and 16:0), which contributed significantly to Tween 80 composition, decreased to a comparable extent (57% and 59%, respectively). The rapid decline of Tween 80 in T. curvufu culture fluid prompted the estimation of extracellular esterase, the activity of which could account for the detergent’s disappearance (Asther & Corrieu 1987). While hydrolysis of p-nitrophenylpalmitate cannot invariably be construed to indicate total lipase activity, the usefulness of the substrate as a general esterase indicator in a variety of bacteria has been confirmed (Fay et al. 1990). The addition of Tween 80 (to give 100 pg/ml) to a T. curvafa culture tripled esterase secretion
dry
1 2 38 12 24 12 89
Straight-chain, 15:o 16:0 1810 19:o Total
saturated
Straight-chain, cis-18: 1 trans-18 : 1 18:2 Total
unsaturated
Cl 3 5 Cl 8
Cl 2
from about 2.6 to 7.7 U/mg dry cells within two generation times. In a Tween-adapted culture, esterase accumulated in cell-free fluid in proportion to cell mass (Figure 3). The presence of Tween 80 influenced the temperaturedependence profile for growth of the actinomycete. 7. curvufu is a facultative thermophile able to grow in minimal medium from 35 to 65°C; however, cultures grown in the presence of 0.1% detergent exhibited a narrowed temperature range with a shifted optimum (Figure 4). Changes in bacterial growth temperature requirements often coincide with changes in the cellular fatty acids, particularly in
World]oumal
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E. Thies, T. Jenkins and F. Stutzenberger 0.5 ,
1
h
50
60
Temperature (“C) Figure
4. Influence
of Tween
80 on the specific growth rate of T. medium with (A) and without (0)
curvata in cellobiose/minimal 0.1%
Tween. 12
0 U.0
I
Growth temperature (“C) . I . I . I .
0.2
0.4
0.6
0.8
1.0
Initial Tween 80 concentration (g/l 00 ml) Flgure 5. Influence of Tween 80 on cis-18:1 concentration in long-chain fatty acids (LCFA) of T. curvafa grown at 54°C. Inset shows growth temperature dependence of cis-18: 1 contribution to LCFA in the presence of the detergent.
0 B 5
I
0.2
0.4
5
m’
Initial Tween 80 concentration (g/l00 ml)
Figure grown
660
I
70’ 0.0
6. Decrease in branched-chain at 54C in the presence of Tween
WorldJournal
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fatty 80.
& Biotechnology.
acids
0.6 in T. curvata
Vol ~0, 1994
membrane composition (Brock & Mad&an 1991). Since almost all lipid ( > 90%) in Gram-positive bacteria is cytoplasmic membrane-associated (Lennarz 1966), whole cell extracts were used in the comparison of cells grown in the presence and absence of Tween 80. The presence of Tween 80 during growth caused little detectable difference in the total long-chain fatty acid content (3.4%, 3.6%, 3.5% and 3.6% of the dry wt of cells grown in 0, 0.05, 0.1, 0.2 and 0.4% Tween, respectively). Although the relative proportions remained similar, the amounts of individual fatty acids changed during growth in the presence of the detergent. A typical long-chain fatty acid composition of dry cells grown in the absence of Tween 80 is shown in Table 1. The greatest change from control values was a Is-fold increase in the cis-18:l content of the cells; the magnitude of this change was strongly temperature dependent (Figure 5). Other changes in straight-chain fatty acid composition included decreases of about 20% in 16:O and l&O. The presence of the detergent also had an effect on the total branched chain fatty acids content of T. curvata (Figure 6). This effect was largely attributable to cumulative decreases in anfeiso-15:O and anteiso-17:O. Tween 80 also influenced the T. curvata cell-surface topology. During growth on cellobiose octacetate or cellulose, mycelia were covered with multiple surface protuberances (Figure 7a). These protuberances are similar to the cellulosomes of the thermophile, Clostriditrm fhermocellum, which contain both cellulolytic enzymes and substrate attachment sites (Bayer & Lamed 1988). The addition of Tween 80 to T. curvafa mycelia resulted in the loss of most of these surface structures, leaving a stripped, ragged appearance with few vestiges of their cellulosomal structure (Figure 7b). The presence of the detergent also caused an increase in soluble enzymes active against crystalline cellulose, but not in endoglucanases active against CMC (Figure 8). This increase of enzymatic activity against crystalline cellulose was apparently not the result of cytolysis since sonic breakage of T. curvafa cells did not release detectable cellulolytic enzymes. Although cell-free-fluid endoglucanase remained quantitatively unchanged, cultures grown in Tween 80 produced exoprotein and endoglucanase patterns chromatographically different from those of the controls (Figure 9). In addition to cellulases, T. ctrrvafa also secretes exoenzymes which act on a variety of other polysaccharides (Bemier et al. 1988). It was therefore of interest to determine whether the presence of Tween 80 stimulated the release of other extracellular enzymes from the actinomycete. When T. curvafa was grown on starch or pectin in the absence of Tween 80, amylase and pectinase activities in culture fluids reached peak levels of 16.6 and 4.6 U/ml, respectively. Slightly lower peak levels (15.1 and 4.0 U/ml) were observed during growth of T. curvafa with 0.1% detergent in the medium.
Tween 80 effects on T. curvata
a
b
Figure 1. Scanning electron microscopical comparison of cell surface topologies of T. curvata mycelia grown on cellobiose/ octaacetate/minimal medium in the absence (a) and presence (b) of 0.1% Tween 80. Bar 1/~m. 200
gE E~ E
m E
>.'Z'--
O :3
.->
100
0----
0
1
2
'
I
3
4
,
I
5
,
I
6
i
7
T i m e (days) Figure 8, Cotton fibre (CF) activity (O, @) and endoglucanase activity (A, A) in cell-free fluid from cultures with (@, A) and without (O, A) Tween 80.
Discussion The lyric effect of Tween detergents on T. curvata appears to be due to their long-chain fatty acid content, since the effect could be duplicated by using an equivalent amount of the corresponding acids. This conclusion is supported by earlier reports on inhibitory effects of long-chain fatty acids in other bacteria (Galbraith et al. 1971; Maczulak et aI. I981). The ability of T. curvata to resume growth after prolonged Tween 80 exposure would be due, at least in part, to its relatively high extracellular esterase activity [compared with a wide variety of other bacteria, (Winkler & Stuckmann 1979; Fay e~ al. I990)] which enables the actinomycete to initiate degradation of the detergent. The continual disappearance of oleic acid from culture fluid after Tween 80 addition, together with the slight stimulation of growth in detergent-adapted cells at the adapted tempera-
ture optimum of 54°C, is consistent with the view that the actinornycete utilizes the liberated fatty acid after de-esterification of the detergent. The presence of Tween 80 caused a decrease in the optimal growth temperature of the facultative thermophile from 62 to 54°C. This effect may be related to changes in fatty acid composition. T. curvata long-chain fatty acids consisted mainly (almost 90%) of iso- and anteiso-branchedchain fatty acids from C1~ to CI8, a composition comparable with that observed in other thermophilic bacteria (Langworthy & Pond 1986). A high concentration of these saturated, branched-chain fatty acids confers membrane stability at elevated temperatures (Brock & Madigan 199I). Fatty acid moieties of Tween detergents are incorporated into both bacterial and fungaI membranes (Jacques et al. 1985; Asther & Corrieu I987). This incorporation causes increased degrees of unsaturation in membrane fatty acids and subsequently enhances membrane fluidity. Increased membrane fluidity has been suggested (Wittenberger et al. 1978) as a possible mechanism to explain the enhanced exoenzyme secretion rates in the presence of Tween detergents. However, in Streptococcus sanguis (Jacques et al. 1985) and Trichoderma reesei (Panda et al. I987), stimulation of protein secretion is not totally attributable to changes in membrane lipid composition. In T. curvata, it is likely that the enzyme-selective nature of Tween 80 stimulation (increased cotton fibre cellulase activity with unchanged levels of amylase, endoglucanase or pectinase) results from the solubilization of enzymes already secreted and bound to the cell surface in cellulosomes rather than stimulation of enzyme biosynthesis or secretion. This view is supported by the observation that cellulases remain bound to the T. curvata mycelia, while mycelial surfaces are devoid of detectable amylase or pectinase activities (Hostalka et al. 1992).
World Journal of Microbiology & Biotechnology, Vol I0, 1994
6 61
E. This, T. Jenkins and F. Stutzenberger
0
0 5
6
7
8
9
10
11
5
Figure 9. Alterations in elution patterns, determined by size-exclusion HPLC, ultrafiltration concentrates from cell-free fluids from cultures grown in the absence
Acknowledgements We thank J. 0. Eubanks,J. E. Mann and E. G. Porter for excellenttechnicalsupport.
References Albersheim, P. & Killias, U. 1962 Studies relating to the purification and properties of pectin transeliminase. Archives of Biochew&y and Biophysics 97,107-115. Amelunxen, R. E. & Murdock, A. L. 1978 Microbial life at high temperatures: mechanisms and molecular aspects. In Microbial Life in Extreme Environments, ed Kushner, D. J. pp. 217-277, London: Academic Press. Anon. 1985 Statistical Analysis System Users’ Guide. Version 5 Edition. Cary, NC: SAS Institute. Asther, M. & Corrieu, G. 1987 Effect of Tween 80 and oleic acid on ligninase production by Phunerochuefe chrysosporium INA-12. Enzyme and Microbial Technology 9,245-249. Bayer, E. A. & Lamed, R. 1988 Ultrastructure of the cell surface cellulosome of Closfridium fhermocellum and its interaction with cellulose. Journu~ of Bacteriology 167, 828-836. Bemfeld, P. 1955 Amylases, alpha and beta. Methods in Enzymology
1,149-154. Bemier, R., Kopp, M., Trakas, B. & Stutzenberger, F. 1988 Production of extracellular enzymes by Ihermomonosporu curoufu during growth on protein-extracted luceme fibres. Journal of Applied Bacteriology 65, 411-418. Bertrand, J.-L., Morosoli, R., Shareck, F. & Kluepfel, D. 1989 Expression of the xylanase gene of Sfreptomyces lividuns and production of the enzyme on natural substrates. Biotechnology and Bioengineering 33, 791-794. Bradford, M. M. 1976 A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72, 248-254. Brock, T. D. & Madigan, M. T. 1991 Biology of Microorganisms, 6th edn. Englewood Cliffs, NJ: Prentice Hall. Browse, J., McCourt, F. J. & Somerville, C. R. 1986 Fatty acid composition of leaf lipids determined after combined digestion and fatty acid methyl ester formation from fresh tissue. Analyficul Biochemistry 152, 141-145.
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Capalash, N., Gupta, K. G. & Sharma, P. 1991 Effects of additives on the activity of Bacillus sp. /I-xylanase. Letters in Applied Microbiology 12,31-33. Danon, D., Goldstein, L., Marikovsky, Y. & Skutelsky, E. 1972 Use of cationized ferritin as a label of negative charges on cell surfaces. Journal of Ultrastructure Research 38, 50&510. Fay, J. P., Jakober, K. D. & Cheng, K.-J. 1990 Esterase activity of pure cultures of rumen bacteria as expressed by the hydrolysis of p-nitrophenylpalmitate. Canadian Journul of Microbiology 36, 585-589. Galbraith, H., Miller, T. B., Paton, A. M. & Thompson, J. K. 1971 Antibacterial activity of long chain fatty acids and the reversal with calcium, magnesium, ergocalciferol and cholesterol. Journal of Applied Bacteriology 34, 803-813. Gillespie, A. M., Keane, D., Griffin, T. O., Tuohy, M. G., Donaghy, J., Haylock, R. W. & Coughlan, M. P. 1990 The application of fungal enzymes in flax retting and properties of an extracellular polygalaturonase from Penicillitrm cupsulufum. In Biotechnology in Pulp and Paper Munufacfure: Applications and Fundamenful Invesfigufions, eds Kirk, T. K. & Chang, H. M. pp. 211-219, Boston: Butterworth-Heinemann Hostalka, F., Moultrie, A. & Stutzenberger, F. 1992 Influence of carbon source on cell surface topology of Thermomonosporu curvufu. Journul of Bacteriology 174, 7048-7052. Hung, B. R., Lara, L., Patron, M. A., Ugarova, N. N., Bechstedt, W. & Clappes, S. 1988 Tween 80 and proteose peptone effect on cellulase production. Acfu Biofechnologia 8, 461464. Jacques, N. A., Jacques, V. L., Wolf, A. C. & Wittenberger, C. L. 1985. Does an increase in membrane unsaturated fatty acids account for Tween 80 stimulation of glucosyltransferase secretion by Sfrepfococcw salivarid Journal of General Microbiology
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R., Naimark, J., Morgenstem, E. & Bayer, E. 1987 Specialcell surface structures in cellulolytic bacteria. Jownal of Bacteriology 169,3792-3800. Langworthy, T. A. & Pond, J. L. 1986 Membranes and lipids of thermophiles. In Thermophiles: General, Molecular and Applied Aspects, ed Brock, T. D. pp. 107-135. New York: John Wiley and Sons. Lennarz, W. J. 1966 Lipid metabolism in bacteria. Advances in Lipid Research 4,175-221. Maczulak, A. E., Dehority, B. A. & Palmquist, D. L. 1981 Effects
Tween of long-chain fatty acids on growth of rumen bacteria. Applied and Environmental Microbiology 42, 856862. Neugebauer, J. 1992 A Guide to the Properties and Uses of Defergents in Biology and Biochemistry. La Jolla, CA: Calbiochem-Novabiothem Corp. Panda, T., Gruber, H. & Kubicek, C. P. 1987 Stimulation of protein secretion in Trichoderma reesei by Tween surfactants is not correlated with changes in enzyme localization or membrane fatty acid composition. FEMS Microbiology Letters 41, 85-90. Scopes, R. K. 1987 Protein Purification: Principles and Practice. New York: Springer-Verlag. Stutzenberger, F. J. 1972 Cellulolytic activity of Themomonospora mrvafu: nutritional requirements for cellulase production. Applied Microbiology 24, 77-82. Stutzenberger, F. J. 1987 Component-specific stimulation of cellulase secretion in Thermomonospora curvata by the surfactant Tween 80. Journal of Applied Bacteriology 63, 239-244.
80 effects on T. curvata
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(Received
in revised
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1994; accepted
6 Biotechnology.
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Vol IO, 1994
1994)
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