PROPUESTA ARQUITECTONICA

4.2.1. Factors influencing cell disruption

T he actual m echanism (s) by w hich solvents d estab ilise cells is u nclear, with a variety o f effects being o b served. T hese range from disruption of co-factor recycling [Hocknull and Lilly,(1988)] to exten siv e release o f in tracellu lar c o m p o n en ts [D eSm et et al. ( 1 9 7 8 )].

The action of the solvents studied here show some sim ilarities in their effects upon cell activity, despite the m arked differences in the hydrophobicities o f these solvents. E . c o l i JM 1 0 7 (p S S 2 ) and P . p u t i d a UV4 showed the most similarity in their patterns o f loss o f activity, with initial exposure p ro d u cin g a sm all bu t rapid declin e in activity in UV4 and JM 107(pSS 2) treated for up to 4 h o u rs in hexane and tetradecane satu rated b u ffers. T h is was followed by a slower rate of loss of activity in cells exposed for a g re a te r period of time. S im ilar p attern s o f activ ity loss were observed in A . s i m p l e x exposed to a variety of solvents [Hocknull, (1989)], and this may represent a general pattern of activity loss a m o n g st w hole cell biocatalysts. P . a e r u g i n o s a P A C lR ( p S S 2 ) d isplayed a unique pattern of activity reten tio n , w hich w ill be d iscu ssed later.

T h e c h an g e s in the n a p h th a le n e d io x y g e n a s e a c tiv itie s w ere observed to be dependent upon four main factors:

i) solvent presentation; ii) solvent hydrophobicity; iii) time of exposure;

iv) cell structure. S o lv e n t p resen ta tio n

T he severity of the action of solvents appears to be affected by the way in which the solvent is presented to the cell. Disruption due to dissolved levels of solvent, and that produced by exposure to the liq u id iliq u id in te rfac e, show m a rk e d d iffe re n c e s [Bar,

(1986)]. The ex ten t o f solvent d isru p tio n in tw o liq u id phase system s can be lim ited by im m o b ilisa tio n o f the b io ca ta ly st, th ere b y e ffe c tiv e ly re d u c in g in te rfa c ia l c o n ta c t [H o c k n u ll and L illy ,(1 9 9 0 )].

Cells exposed to dissolved levels of hexane retained activity, even a fter 21 hours o f treatm en t (Figs. 3.13.1, 3.16.1, and 3.19.1). How ever, when an hexane interface was introduced in tw o-liquid p h a se sy stem s, co m p lete in ac tiv a tio n o f all cell sam p les was observed (Figs 3.21., 3.22., and 3.23.).

A loss in the amount of cells recovered after exposure in the two- liquid phase system was noted, suggesting that cell lysis may have o c cu rred , or cells been reta in e d in the liq u id iliq u id in te rfac e form ed during the harvesting process. T herefore, not all o f the c e lls m ay h ave been r e c o v e re d a fte r the w a sh in g p ro c e ss , producing a lower activity than m ight have been possible. Sample m ea su re m e n ts carried out during b io cataly sis in the two liquid p h ase system s m ay give a truer rep re se n ta tio n o f the loss o f activity effected by interaction at the liquidiliquid interface.

S o lv e n t h v d ro p h o b icitv

L a a n e et a l (1 9 8 5 ) and B rin k and T r a m p e r ( 1 9 8 5 ) both d e m o n stra te d the relatio n sh ip betw een so lv en t w ater m iscib ility and activity retention of the biocatalyst. A lthough the param eters used to calculate the hydrophobic values of solvents by the two groups differed, both studies indicated that the higher the degree o f water m iscibility, the greater the loss in activity observed. This pred icted pattern of cell inactivation was seen for the solvents u sed in this study. The m o st hy d ro p h ilic so lv en t used in the satu rated b u ffer e x p o su re studies, p e n ta n o l (lo g P = 1 .3 3 ) caused total loss of activity in all cells, after ju st 30 minutes o f exposure in the saturated buffer. Cells treated in hexane and tetradecane saturated buffers retained activity, although the ex ten t o f activity loss and the rate of loss o f activity appeared to be d ependent u p o n the s o lv e n t h y d r o p h o b ic ity ; c e lls e x p o s e d to h e x a n e sa tu ra te d b u ffe r show ed a g re a te r fin al d e c re a se in a ctiv ity com pared with cells exposed to tetradecane saturated buffer (Fig 4.1. and Fig 4.2.) .

S o lv e n t h y d ro p h o b ic ity m ay be a sso c ia te d w ith the so lv e n t c o n c e n tr a tio n p r e s e n t in the b u ffe r, w ith m o re h y d ro p h ilic solvents expected to dissolve to a greater final concentration. This w ould resu lt in cells being challenged with a higher level of so lv e n t w h ich m ay d iffu se th ro u g h the m e m b ra n e , cau sin g a greater rate of cell inactivation.

T he loss o f p ig m en tatio n in U V 4 and P A C lR ( p S S 2 ), and the form ation o f a cloudy emulsion upon harvesting by all cells after e x p o s u r e to p e n ta n o l s a tu ra te d b u ffe r, in d ic a te s th a t o u ter m em brane disruption is likely to have occurred, solubilising lipids an d r e l e a s in g th e p e r ip la s m ic c o n te n ts ; s i m il a r lo s s e s o f cytochrom e C were observed when the outer m em branes o f cells were disrupted [Garrad,(1972)]. For this to occur, severe damage to the o u ter m em brane m u st be effected. E le ctro n m icro sco p y studies of P . o l e o v o r a n s exposed to 10% to 80% v/v solvents show ed loss o f lipids from both the cytoplasm ic m em brane and the outer m em brane, with the form ation o f protein free vesicles th o u g h t to be co m p o sed of e x tra cted lip o p o ly sa c c h a rid e s and p h o s p h o lip id s [W ith o lt et al.(1990)1. In o rder to explain these o b se rv a tio n s,W ith o lt pro p o sed two m ec h an ism s, in w h ich cells com ing into contact with solvent m olecules partitioned their lipids around a core of solvent, forming the vesicles seen. M ost damage was observed to occur in the cytoplasm ic m em brane, suggesting th a t in te rn a lisa tio n of the so lv e n t e n a b le d d is ru p tio n o f the m e m b r a n e .

Tim e of exposure

Studies into the action of solvent dam age on b io cataly sts have looked at reactions over a limited time span. W hilst this gives an in d icatio n of the activity reten tio n o v er th at specific reactio n p eriod, long term effects of solvent disruption on the biocatalyst are not considered. Microbial biocatalysts are likely to be subject to non-specific, cumulative solvent dam age, affecting a num ber of d if f e r e n t c e llu la r fu n ctio n s. T h e re fo re , in c re a s in g e x p o su re to solvents is likely to produce a continuous loss o f activity. The decrease in activity was observed as a two stage process in UV4 and JM 107; an initial rapid loss of activity over the first four

hours, follow ed by a slower rate of decrease over the rem aining period of exposure.

Cell structure

T he role of the outer m em brane structure in p ro tectin g cells against solvent disruption is seen by the greater levels of activity retained by P.putida UV4 compared with A. si m p l ex exposed to a ran g e o f solvents [H arrop et al.(1992)1. A m o n g s t the G ram - n e g a tiv e o r g a n is m s , c o m p o s itio n a l v a r i a ti o n s in th e o u te r m em brane are known to result in differences in the ability to take up a num ber of hydrophobic compounds. P , a e r u g i n o s a displays a h ig h to le ran c e to m any h y d ro p h o b ic a n tib io tic s , this p ro p erty being associated with the greater exclusion limits of its porins to these com pounds, or the greater num ber of porins found in the closed state. H ow ever, uptake of m any hyd ro p h o b ic antibiotics, such as quinolones, has been shown to occur either through the porins, or by diffusion through the lipid bilayer [C hapm an and G eo rg o p ap a d ak o u ,(1 9 8 8 ); M ic h e a -H a z e h p o u r et a l.(1991): H ooper et al.n989V. McCaffrey et al.(1992)1.

The com parative activity retentions of the cells (Figs 4.1, 4.2), sh o w s d if f e r e n c e s in the s o lv e n t t o le r a n c e s b e tw e e n th ese organisms. UV4 and JM107 show sim ilarities in their patterns of activity retention in the solvents tested, but differ in the rate of activity loss (after the initial 4 hour period o f exposure) and the final activity retained at the end of the 21 hour exposure period. JM 107 appears to be m ore susceptible to the effects o f solvents than UV4; exposure of UV4 in hexane saturated buffer resulted in an almost 55% final loss of activity (Fig 3.16.1.), w hilst activity of JM 107(pSS2) declined to alm ost 78% of the control sam ple (Fig 3 .1 3 .1 .). S im ila rly , e x p o su re to te tr a d e c a n e sa tu ra te d b u ffe r produced a decrease of approximately 13.4% for UV4 (Fig 3.17.1.) com pared with a 67.76% decrease for JM 107(pSS2) (Fig 3.14.1.), showing a greater long term solvent tolerance by UV 4 com pared with JM 107, in both hexane and tetradecane, when solvents were presented in a saturated buffer.

JM 1 0 7 (p S S 2 ) and UV 4 cells ex p o sed to betw een 0 .8 7 % and 1 0 % v /v e th a n o l a g a in r e s u lte d in v e ry d i f f e r e n t a c tiv ity retentions. The presence of ethanol appears to severely disru p t

the activity of JM 107(pSS2), as shown by the inability o f cells w hich had been preexposed to ethanol, to convert naphthalene to the d io l, and the d e crea se in a ctiv ity w ith the in c re a se in nap h th alen e solution, in cells th at had no t been pretreated this way. In contrast, preex p o su re to 10% ethanol was required to bring about a total loss of activity in UV4. These cells were able to to lerate p reex p o su re upto 2.5% v/v eth an o l w ith o u t any loss o f a c tiv ity .

In all cases, the level of activity was significantly greater than if c e lls had no t u n d e rg o n e any e th a n o l e x p o su re , e ith e r during pretreatm ent, or during biotransform ation. This m ay be due to the g reater surface area of the substrate p resented to the cell, the partial perm eabilisation of the cell wall allowing a greater rate of uptake of the substrate, or a combination of these effects.

The greater rate of activity loss observed in the E.coi strain was su g g ested earlier to be due to the g rea ter acc essib ilty of the so lv e n t in to the cell. T his m ay have o ccu rred both thro u g h m ovem ent via the porin channels, and diffusion through the outer m e m b r a n e .

Significant increases in activity were observed for P . a e r u g i n o s a P A C I R exposed for up to 4hours in both hexane and tetradecane saturated buffers (Figs 3.19.1., 3.20.1.). This is thought to be due to d isru p tio n of the highly o rd ered LPS array o f the o u ter m em brane by solvent, enabling an increased rate of diffusion of su b strate into the cell. Cells treated for 4h o u rs in tetradecane saturated buffer showed over 251% increase in activity com pared w ith the control sample. An equivalent exposure time in hexane saturated buffer only produced an increase in the activity level of j u s t under 157% of the control. The low er rate o f increase of a ctiv ity p ro d u ce d upon ex p o su re to h ex an e satu rated b u ffer, co m p ared with the tetradecane saturated buffer, m ay refle ct the greater dam aging potential of this solvent. H exane is thought to e ffe ct a greater disorganisation of the outer m em brane, allowing internalisation of the solvent to occur m ore readily, where it may destab ilise cellular functions.

Pro lo n g e d ex posure (up to 21 hours) results in a d ecrease in activ ity , as continued solvent dam age to c ellu la r fu n ctio n s d e­ stabilises the cell.

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Fig 4.1. Activity retention after exposure to hexane sa turated buffer Cumulative graph from Figs 3 .1 1 .1 ,3 .1 4 .1 , and 3.17.1.% activity as % o f untreated control. Dashed line indicates theoretical d eclin e in activity ..Cells preexposed to 50m l hexane saturated buffer atl50rpm .B iotransform ation carried out in single aqueous phase 60m l phosphate buffer at 750rpm ,28oC, air sparged at

l- 1 .3 3 v /v /m in .

• UV4; ^JM 107(pSS2); APAClR(pSS2)

26C 24C 22C 20C \ 18C 16C •'I' 14C ■g 12C Ï IOC 0 1 2 3 4 5 6 7 8 9 1 01 11 21 31 41 51 61 71 81 92 02 1 Period of exposure(hrs)

Fig 4.2. Activity retention after exposure to tetradecane saturated buffer

Cumulative graph from Figs 3 .1 2 .1 ,3 .1 5 .1 , and 3.18.1.% activity as % o f untreated control. Dashed line indicates theoretical decline in activity ..Cells preexposed to 50m l hexane saturated buffer at 150rpm .Biotransform ation carried out in single aqueous phase 60m l phosphate buffer at 750rpm ,28oC , air sparged at l-1 .3 3 v /v /m in .

It is p o stu lated th at the structure o f the o u ter m em b ran e o f P A C IR is thought to produce a very effective hydrophobic barrier, as o b se rv e d by the lo w e r ra te o f n a p h th a le n e c o n v e r s io n , compared with strain PAC610(pSS2) (see section 4.1.5.)-

T e tra d e c a n e and h e x an e w o u ld be e x p e c te d to be sim ila rly hindered in their ability to traverse this lipd bilayer. H ow ever, som e d iffu sio n o f these solvents is still likely to occur, with integration into the lipid bilayer, resulting in the disorganisation of this highly organised structure. Because the m em brane appears to be m ore resistan t to hydrophobic co m pounds co m p ared with