Stabilization of dextransucrase from Leuconostoc mesenteroides NRRL B-512F by nonionic detergents, poly(ethylene glycol) and high-molecular-weight dextran

Biochimica et BiophysicaActa, 785 (1984) 89-96

89

Elsevier

BBA 31847

STABILIZATION OF DEXTRANSUCRASE FROM LEUCONOSTOC M E S E N T E R O I D E S NRRL B-512F BY NONIONIC DETERGENTS, POLY(ETHYLENE GLYCOL) AND HIGH-MOLECULAR-WEIGHT DEXTRAN A R T H U R W. MILLER and JOHN F. R O B Y T *

Department of Biochemistry and Biophysics, Iowa State University, Ames, 1,4 50011 (U.S.A.) (Received August 2nd, 1983)

Key words: Dextran; Dextransucrase," Enzyme stabifity," Detergent," Poly(ethylene glycol)

Dextransucrase (sucrose: 1,6-a-D-glucan 6-a-D-glucosyltransferase, EC 2.4.1.5) (3 I U / m l culture supernatant) was obtained by a modification of the method of Robyt and Walseth (Robyt, J.F. and Walseth, T.F. (1979) Carbohydr. Res. 68, 95-111) from a nitrosoguanidine mutant of Leuconostoc mesenteroides NRRL B-512F selected for high dextransucrase production. Dialyzed, concentrated culture supernatant (crude enzyme) was treated with immobilized dextranase (EC 3.2.1.11) and chromatographed on a column of Bio-Gel A-5m. The resulting, purified enzyme lost activity rapidly at 25°C or on manipulation, as did the crude enzyme when diluted below 1 U / m l . Both enzyme preparations could be stabilized by low levels of high-molecular-weight dextran (2 pg/ml), poly(ethylene glycol) (e.g., 10 p g / m i PEG 20000), or nonionic detergents (e.g., 10 p g / m l Tween 80). The stabilizing capacity of poly(ethylene glycol) and of dextran increased with molecular weight. Calcium had no stabilizing action in the absence of other additions, but reduced the inactivation that occurred in the presence of 0.5% bovine serum albumin or high concentrations ( > 0.1%) of Triton X-100. In summary, dextransucrase could be stabilized against activity losses caused by heating or by dilution through the addition of low concentrations of nonionic polymers (dextran, PEG 20000, methyl cellulose) or of nonionic detergents at or slightly below their critical micelle concentrations.

Introduction Dextransucrase (sucrose: 1,6-a-D-glucan 6-a-oglucosyltransferase, EC 2.4.1.5) polymerizes the glucosyl moiety of sucrose to form dextran, an a-(1 ~ 6)-linked glucan with a-linked branches [1]. It is elaborated by Leuconostoc and Streptococcus species, and is primarily exocellular, although it is also found bound to the cell surface. The dextransucrase from Leuconostoc mesenteroides N R R L B512F, which synthesizes a dextran that has 95% a-(1 --, 6) and 5% a-(1 ---}3) branch linkages [2,3], is only produced when induced by sucrose [4]. Consequently, the crude enzyme contains a great * To whom correspondence should be addressed. 0167-4838/84/$03.00 © 1984 Elsevier Science Publishers B.V.

deal of dextran. Following a purification procedure that removed dextran (as well as many other impurities), Robyt and Walseth [5] found that the enzyme lost half its activity after storage for 2 days at 4 or - 15°C, irrespective of the presence of 25% (v/v) glycerol. This loss of activity could be prevented by adding back dextran. Subsequently, Miller and Robyt [6] found that dilute enzyme, whether crude or purified, lost activity rapidly at 25°C. This loss could also be prevented by the addition of dextran. In order to perform kinetic studies that would be handicapped by the presence of dextran, a search was conducted for alternative stabilizers. The results of this search have been previously reported in abstract form [6], and will be described here in detail.

90 Materials and Methods

Materials L. mesenteroides B-512F was obtained from the Northern Regional Research Center (formerly Northern Regional Research Laboratory, N R R L ) (Peoria, IL). Tween 80 and PEG 20000 were from Fisher Scientific (Pittsburgh, PA). Zwittergent 3-12 was from Calbiochem-Behring (La Jolla, CA). PEGs 400, 600, 1000 and 6000 were from J.T. Baker (Phillipsburg, N J). Bio-Gels P-2 and A-5m were from Bio-Rad Laboratories (Richmond, CA). Dextranase S was from Swiss Ferment (Basel, Switzerland). Octylglucoside, methyl cellulose (viscosity of 2% aqueous solution = 4000 cP), Triton X-100, Lubrol WX, Lubrol PX, sodium taurocholate (synthetic), mannan from Saccharomyces cerevisiae, bovine serum albumin (crystallized and lyophilized), Penicillium funiculosum dextranase (Grade I, chromatographically purified), and dextrans of various molecular weights were purchased from Sigma Chemical Co. (St. Louis, MO). Molecular weights (for Fig. 6) are weight-average molecular weights. Native dextran (industrial grade dextran from Sigma, M r (5-40)106 ) was precipitated twice from aqueous solution with 2 vol. ethanol. [U-14C]Sucrose was from New England Nuclear (Boston, MA) and ICN (Irvine, CA). [fructose-U-14C]Sucrose was from New England Nuclear. All other chemicals were of reagent grade. Mutation and culture L. mesenteroides N R R L B-512F was grown in sucrose medium [5], treated with N-methyl-N'nitro-N-nitrosoguanidine [7], and plated on agar medium containing sucrose. Colonies showing exceptional polysaccharide formation were selected. The dextransucrase activity in the resulting culture supernatant was 3 U / m l , compared to 0.02 U / m l for the parent organism. The dextrans produced by crude and purified dextransucrase derived from this culture supernatant by the procedure described in the next paragraph were identical to the dextran produced by the dextransucrase from the B-512F parent, as judged by 13C-NMR spectra and by the distribution of Penicillium funiculosum dextranase hydrolysis products (glucose, isomaltose, and branched tetra-, penta- and hexasac-

charides), which showed that the dextran was 4-5% branched.

Enzyme purification The culture supernatant was dialyzed and concentrated on a Bio-Fiber 80 miniplant (Bio-Rad). The concentrate was centrifuged at 12000 × g for 1 h to remove a particulate suspension. The centrifuged preparation (crude enzyme) had an activity of 90 U / m l and contained less than 0.02 U / m l of levansucrase, as judged by failure to incorporate label from [fructose-U-14C]sucrose into methanol-insoluble polymer. The specific activity of the crude dextransucrase was 98 U / m g protein (as bovine serum albumin), based on the protein determination of Bradford [8]. Dextranase S (an endodextranase) was immobilized to beads of ethylenediamine-modified Bio-Gel P-2 that had been treated with glutaraldehyde [9]. Crude dextransucrase was incubated with immobilized dextranase and passed over a 2.5 x 60 cm column of Bio-Gel A-5m. The pooled, active fractions constituted purified enzyme. Preincubations and assays Preincubations were performed in 0.05 M sodium acetate buffer (pH 5.0) at various temperatures in a circulating water bath. Dextransucrase assays were performed at the preincubation temperature by following the incorporation of label from [U-14C]sucrose into methanol-insoluble polymer [10]. One unit of enzyme is defined as the amount that will incorporate 1 /~mol of D-glucose into polysaccharide in 1 min at 25°C and pH 5. Results

Any manipulation of purified enzyme resulted in loss of activity. No loss of activity was observed from crude enzyme in the temperature range examined (4-30°C) unless it was diluted to less than 1 U / m l . The progressive inactivation that occurred then, when plotted semilogarithmically against time, was often biphasic or otherwise complex, but became more linear as enzyme concentration was reduced and was completely linear (showed simple exponential decay) at initial enzyme concentrations of 0.01 U / m l or less. Complex inactivation curves are well known for

91

enzymes in general [11], and have been observed previously for dextransucrase [12]. The rate of inactivation increased sharply at temperatures above 25°C, although it occurred even at 4°C. Inactivation occurred at equal rates in both glass (hydrophilic surface) and polystyrene (hydrophobic surface) vessels, making it unlikely that inactivation was caused primarily by the binding of the enzyme to the vessel walls. Bovine serum albumin, which is commonly added to enzyme preparations to stabilize them, unexpectedly increased the rate of inactivation of dextransucrase. Calcium, while by itself having no effect on the rate of inactivation, prevented the increase caused by bovine serum albumin. Only the increase was prevented: the inactivation in the presence of both 5 m g / m l bovine serum albumin and 5 mM added calcium occurred at virtually the same rate as in the absence of either. The inactivation could be stopped at any time by adding to the dilute enzyme solution a sufficiently high concentration of any of the stabilizers listed in Table I (see for example Fig. 1). Although the concentration range over which stabilization

developed was investigated with only one enzyme preparation (either crude or purified) for each stabilizer, all of the stabilizers were effective with both preparations. For most of the stabilizers, only a few concentrations (usually in the form of serial, 10-fold dilutions) were examined for their stabilizing effect. Therefore, the concentrations in Table I are given as 'lowest tested concentration' rather than as 'lowest concentration'. Anomalous results were obtained with Zwittergent 3-12 and Triton X-100. Increasing the concentration of these detergents only decreased the rate of enzyme inactivation up to a point. As the concentrations were increased above 100 /~g/ml for Zwittergent 3-12 or 50 # g / m l for Triton X-100 (at which concentrations almost complete stabilization was observed), the rate of inactivation began to increase again. For Triton X-100, the most likely cause of this effect was considered to be attack by the oxidizing impurity known to be present in commercial preparations of this detergent [13]. Curiously, while calcium did not prevent the loss of activity caused by high concentrations of Zwittergent 3-12, it did prevent the loss caused

TABLE I C O N C E N T R A T I O N R A N G E OVER W H I C H STABILIZATION DEVELOPS

Stabilizer

Enzyme preparation

Temperature of preincubation and assay (°C)

Lowest tested concentration giving any stabilization (/~g/ml)

Lowest tested concentration giving complete stabilization (/~g/ml)

Cricital micelle concentration in water (/,g/ml) a

Tween 80 Triton X-100 Lubrol WX Lubrol PX Zwittergent 3-12 Octylglucoside Sodium taurocholate Poly(ethylene glycol) 20000 Methylcellulose Yeast mannan High-molecularweight dextran

purified crude purified purified purified crude crude crude crude crude

27.5 27.5 27.5 27.5 27.5 30.0 30.0 30.0 30.0 30.0

2.5 5 1 > 1d > 10 d 280 260 5 - c 6

10 50 100 100 100 b 2 800 5 000 10 40 600

13 100-160 20-60 1 200 6 800 5 400-8100 -

crude

30.0

0.08

2

-

a From Refs. 32-35. Recalculated from molarity where necessary. b Complete stabilization was not observed. Stabilization was nearly complete at 100/~g/ml, but above this concentration, inactivation began to occur. c Lowest tested concentration (40 # g / m l ) gave complete stabilization. d No stabilization at this concentration.

92 !

i

|

i

!

!

220

2O0

~x

2

180 4O ..~ ,=c

°

160

a~

_= m

140

20

120 IO 100 I

I

I

I

I

I

1

2

3

4

5

Hours of enzyme incubation at 30°C before assay

Fig. 1. Stabilization of crude dextransucrase by Tween 80. After 2.5 h incubation, enzyme solution (0.15 U/ml before incubation) was diluted with an equal volume of water (O) or 1% Tween 80 (O). Activities plotted after this dilution were multiplied by 2 as a correction factor, zx, enzyme activity before dilution.

-i

I

I

I

0

I

2

Log {lJglml

I

3

Tween 801

Fig. 2. Activation of purified dextransucrase by Tween 80. Each type of symbol represents a different experiment.

100 80

by high concentrations of Triton X-100. For example, at 0.5% (w/v) Triton X-100, where inactivation occurred much more rapidly than in the absence of T r i t o n X-100, the addition of calcium to 0.2 m M resulted in complete stabilization of the enzyme. High dilution of crude or purified enzyme into buffers c o n t a i n i n g stabilizers gave an a p p a r e n t activation relative to controls n o t c o n t a i n i n g stabilizers (see, for example, Fig. 2). Some of this a p p a r e n t activation resulted from inactivation of the control enzyme before a n d during its initial assay. Because of the complex a n d somewhat unpredictable shape of the inactivation curves, the control velocities for Fig. 2 were not corrected for this inactivation. A b o v e 1 0 / ~ g / m l Tween 80, however, where complete stabilization occurred, the a d d i t i o n a l activation must be real. C a l c i u m a n d zinc ions affected enzyme stability. As noted above, the a d d i t i o n of calcium prevented increases in the inactivation rate caused by

60

._> "~

40

¢o

20

10 0

1

2

3

Hours of enzyme incubation at 30°C before assay

Fig. 3. Effect of EDTA on the stability of crude dextransucrase in the presence (closed symbols)and absence (open symbols) of 0.570 Tween 80. EDTA concentration (mM); O, O, 0; zx, -, 10. Initial enzyme activity was 0.15 U/ml. Calcium was present in all cases at 1.5 raM.

93 i

i

i

!

i

!

i

~

|

I

I

i

I

l

4

8

12

16

20

0.0 !

100; 80 60

"7'

O.

40

O. t..)

¢o

20

~

I0

o,

O.

8

0.5

0 2

4

6

Hours of enzyme incubation at 27.5°C before assay i

,

PEG Molecular Weight

8

i

i

0,0 .c:

(x lO-3)

Fig. 5. Effect of poly(ethylene glycol) molecular weight on the stability of crude dextransucrase. Dextransucrase (initial activity, 0.3 U / m l ) was incubated with 0.5% (w/v) poly(ethylene glycol) (PEG), with calcium present at 1.5 raM. Plots of In(activity) vs. time were linear for 1.5 h. Derivation of first-order decay constants is described in the legend to Fig. 4, The intercept on the ordinate axis is the decay constant in the absence of poly(ethylene glycol).

0.1 .

o

0,2

_

0.3

--

.

.

fF

.

-

0.0

t-

9

g

0.4

0. I

0.2

t.l_

0.5 0

I

I

I

I

i

2

4

6

8

i0

B

Zn +

""

0.3

~ ;7"

1).4

(raM) 0.5

Fig. 4. Effect of zinc(II) acetate on the stability of purified dextransucrase in the presence of 0.3% Tween 80 and 0.7 mM calcium. (A) Fraction of initial activity (0.14 U / m l in the absence of zinc) remaining with time. Zinc concentration (mM): (D, 0; o, 2.5; A, 5; a, 10. (B) Rate of inactivation as a function of zinc concentration. When the ordinate axis in part (A) is recalibrated in units of In(activity), the slopes of the inactivation curves drawn in the figure become equal to negative one times the the first-order decay constants for activity loss. Smaller decay constants therefore represent less rapid loss of activity, and a zero value represents complete stability.

0

I

I

40

80

I

120

I

160

Dextran /Wolecular Weight

I~

I

~-5000

(x lO-3)

Fig. 6. Effect of dextran molecular weight on the stability of crude dextransucrase. Dextransucrase (initial activity, 0.14 U / ml) was incubated with 0.5% (w/v) dextran, with calcium present at 1.5 raM. Plots of In(activity) vs. time were linear for 3 h. Derivation of first-order decay constants is described in the legend to Fig. 4. The intercept on the ordinate axis is the decay constant in the absence of added dextran.

94 high concentrations of bovine serum albumin or Triton X-100. Progressive inactivation was also caused by EDTA, and the addition of Tween 80 did not prevent this inactivation (Fig. 3). Likewise, zinc caused inactivation of the purified enzyme even in the presence of Tween 80 and a small amount of calcium (Fig. 4). These results were presumably due to the removal or displacement from the enzyme of stabilizing calcium. Both poly(ethylene glycol) and dextran (Figs. 5 and 6, respectively) stabilized the enzyme to an extent dependent on their molecular weight. A single polymer concentration (0.5% w / v ) was used for these experiments. Lower concentrations were tested only for the highest-molecular-weight polymers of each series (Table I). Discussion

Dextransucrase was inactivated at the low enzyme concentrations (under 1 U / m l ) required for accurate assays. Enzymes often undergo a time-dependent inactivation following dilution to low concentration [14,15], for reasons that include surface denaturation [14-16], binding of inactivators [17] and dissociation of active oligomers into inactive subunits [18]. One method commonly used to prevent this inactivation is to increase the total protein concentration by adding a large amount (1-2 m g / m l ) of an inert protein such as bovine serum albumin [14]. This was not effective in the present case (see Results). Another method is to add ligands (substrates, products, or effectors) of the enzyme to the enzyme solution [15,19]. While dextran was effective at low concentration (Table I), it was our original purpose to find an alternative stabilizer. Nonionic polymers other than dextran that stabilized the enzyme were methyl cellulose and poly(ethylene glycol) (Table I). At high concentration (0.5% w/v), both poly(ethylene glycol) and dextran showed a molecular weight dependence for the stabilization (Figs. 5 and 6, respectively), with the largest polymers being much better stabilizers than the smallest ones. At this relatively high concentration, one way the polymers might be stabilizing the enzyme would be by raising its effective concentration through steric exclusion [20]. However, both PEG 20 000 and high-molecu-

lar-weight dextran stabilized the enzyme at concentrations of only a few /~g/ml (see Table I), where steric exclusion could not be a significant factor. It is more likely, therefore, that both poly(ethylene glycol) and dextran stabilize the enzyme by binding to it. The stabilization given by a low concentration (10/~g/ml) of the largest polymers was greater than that given by a high concentration (0.5% w/v, or 5000 ~ g / m l ) of the smaller ones. In the case of dextran, this could be caused by structural differences. Low-molecularweight dextrans are produced by acid hydrolysis of high-molecular-weight dextran. Acid hydrolysis cleaves a-(1-3) linkages faster than et-(1-6) linkages [21], with the result that the low-molecular-weight dextrans are less branched. There is no corresponding possibility for poly(ethylene glycol), which is an unbranched polymer. The most interesting (because most unexpected) stabilizers were the detergents. All of the nonionic and mild, ionic detergents tested stabilized the enzyme. The concentrations at which they stabilized the enzyme (Table I) are so low as to suggest that stabilization may be mediated through detergent monomers rather than micelles. This is not certain, because critical micelle concentrations were not determined under the conditions of enzyme preincubation. Detergent effects have been described previously for streptococcal glucansucrases, but reports have not been consistent. For example, Figures and Edwards [22,23] found that Tween 80 reduced the aggregation state of glucansucrases from Streptococcus mutans 6715 and improved purification yields. By contrast, Luzio et al. [24] found that dextransucrase from Streptococcus sanguis ATCC 10558 was not disaggregated by nonionic detergents. However, in the presence of nonionic detergents at or below the critical micelle concentrations, their enzyme was disaggregated in active form by 0.005% SDS, which in the absence of nonionic detergents inactivated the enzyme. Similar results in reversing the effects of SDS were earlier observed by Russell [25] for the glucansucrases of S. mutans strain Ingbritt. Tween 80 was reported by Umesaki et al. [26] not to affect the stability or activity of glucansucrase from S. mutans OMZ 176, but Wittenberger et al. [27] found that it activated the enzyme from

95 Streptococcus salivarius up to 30%. Luzio et al. [24] f o u n d an initial activation b y n o n i o n i c detergents a n d p o l y ( e t h y l e n e glycol), b u t except for T r i t o n X-100, this was followed b y inactivation. T h e effects of detergents m a y be related to the m e m b r a n e origin of glucansucrases, as discussed b y Figures a n d E d w a r d s [22]. In this context, it was found b y H a r l a n d e r and Schachtele [28] that p h o s p h o g l y c e r i d e s s t i m u l a t e d the glucansucrases of S. mutans 6715. Interestingly, they also f o u n d that the activation b y p h o s p h o l i p i d s was independ e n t of (additive with) the activation b y dextran. This argues that the m e c h a n i s m s of stabilization b y d e x t r a n a n d detergents m a y also be different. C a l c i u m has n o t been r e p o r t e d to affect the activity or stability of steptococcal glucansucrases. However, it has been shown to affect b o t h of these p r o p e r t i e s for the d e x t r a n s u c r a s e s from L. mesenteroides N R R L B - 5 1 2 F [5,12,29] a n d L. mesenteroides I A M 1046 [30,31]. The studies cited d e m o n s t r a t e d that E D T A i n h i b i t e d the Leuconostoc enzyme, a n d that calcium best reversed the inhibition. F u r t h e r m o r e , the a m o u n t of activity that could be recovered after i n c u b a t i o n with E D T A decreased with the length of time that the e n z y m e was k e p t calcium-deficient. This was observed here (Fig. 3) even in the presence of an otherwise stabilizing c o n c e n t r a t i o n of Tween 80. Therefore, calcium a n d detergents have separate stabilizing roles. Z i n c is k n o w n to inhibit Leuconostoc d e x t r a n sucrases [5,31]. Fig. 4 shows that it also has a strong influence on d e x t r a n s u c r a s e stability. Even in the presence of Tween 80 a n d calcium, zinc inactivates the enzyme, most likely b y displacing the stabilizing calcium. Fig. 4b shows that the rate o f i n a c t i v a t i o n does n o t increase linearly with zinc c o n c e n t r a t i o n . M o r e t h a n one zinc-binding site m a y therefore be involved. In s u m m a r y , we find three classes of stabilizer for dextransucrase: nonionic ( a n d mild ionic) detergents, n o n i o n i c polymers, a n d calcium. T h e m e c h a n i s m s of stabilization, which m a y be different for the different classes of stabilizer, r e m a i n u n k n o w n , as d o the m e c h a n i s m s of inactivation.

Acknowledgement This w o r k was s u p p o r t e d b y G r a n t No. D E 03578 from the N a t i o n a l Institute of D e n t a l Re-

search, N a t i o n a l Institutes of Health, U.S. Public H e a l t h Service,

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