8/14/2019 r;l,l .'1.111,Y F_1 :1 0)tT Srpt r

1/6

1 1

0

r; l , l . '1 . 1 1 1 , Y f _ 1 :1 0)tTSrpt r : :!ler 196 7

C11arles C . PhoilGLl1Cl1SF: ISO: ;t :R11SE IN AR'rll'I'.ni:1CT1 :R F.JR 2453-2

isoni'rase act ;vity in resting cells were studied. Arthrobacter RJR 2453-2cells from fermentation batch No . 17 were thawed, resuspended in distilledwater, and the suspensions were standardized so that the cell concentrationwas 70 ng/ml (wet weii;ht) . Since this cell concentration is not obtainedduring the actual fcrnentations, it is superfluous to report the enzymeacti.vity as 11(~/ml . Therefore, all data are reported as 1.~ of activity

.1 1 ) , 1 9 6 7

In the prctvious report, an increasn in activity of glucose i .somcrasewas observed when the anionic detc.rf;et,t, sodium hcptadecyl sulfate (11llS),was rldded to the cells in the logarithmit or stationary phc!se of groiath ;and the cells were incubatr~d in the basal tnt-dium (XS-2) at 30 C. Inthsreport, the effccts of anionic and non-ionic surface active al;cnts on

d~cted at higher tc' l :,p e ratures, i . e . , 70 -CO C . , the results expressed asl:per mg of cells are only relative . When the assays %:ere performed at ~temperatures greater than 60 C, controls were always run to correct forthermal isomerization . The detergent concentration is reported as ug/n ~Jof cells (i;et ~;cight) ratcer than l,g/ml of culture fluid as used in theprcvious report .

Whenthe resting cell suspensionsat a pHof 6.8 =.2 wcre incuhatedat variousteiIperatllYCs tJoth In statiotiary flasns and during . ? c ,;iLationdefinte effectswore noted. The anonc detergen sodiumheptndecylsulfate (11DS) at conccntrations rc=n ;;i.ng from 0 to 16 11g/hb was evaluated.At 25 and 30 C, cl;z)'r.e

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c t c t i v i . t y b y ` ` a p p r o > : i m .ately 2 times. In these cells, t ; i e r e .lative,isotaerase

activity d,oeFnlt appear t o be dependent o n the IIDS concentratio n since

approxim~ataly the 'sar. t e ; ' r e s u l t s J.ere o btained at concentrations of 4 and30 of cells .

~'~Y On it~ChG coittrole . The additio n of, thv NUS dc[rrgent M .i~o incrcar. e d

t h a - e i i z l t n a :act'ivity at ; a l l , i o i - ' t p e r a t u r o i ;' but the two affects, vr,say

tGNpet :ttltra ;and . deter8ent coticentrnti6n, 4:cra no t a .tditive . In most

cases, tho ; .increase' in assay tetrperature incrcases tha rctlative enzyme

,

7 0 ~ t a . ;$0,C h4s : .si8ni[ic.ntlyi Cruat:er` than thof;a values obtainod at 60 C

aC 1 - C Is shown in FiEure 1, .Tita relative enzytna activity at3 hptire3 ,

:Sincu_the'cells' from batch No :` 17 exhibited a good initial enzymeactivity, it was desirable to substantiate these results with cells oflower activity . Therr.fore, cells frot a farctantation batch No . 22 weretreated with various concentrations of 11DS at 1 C for 30 hrs . 4: i.ththese cells, the relative enzyme activity was also significant1y increasedby ;the assay temperature and IiDS concentrations . These resrlts are givenin ;,the :followin8 table .

15

30

2

Relative E nzyme Activity

___ -- O /MAssay Temperzture60 C 70 c 75 C 80 c4 . 2

5 . 4

9 . 5

9 . 8

12 .1 13 .3 31 . 9

1 0 .6 12 . 7 10 . 9

19 . 7 25 .2 32 . 0

18 .6 23 . 5 29 . 9

Since'data was not collected at 11 DS concentrations o f 4 to 15 tl g/mg,,the optimum 11DS concentration cannot be determined in this experimrntalthough thesc data do show that the enzyme activity of initial lowactivity cells can be si .nificantly increased .

It was also desira"A .e to determine the effect of anothc-r anionicand other non-ionic surface active a-ents on their effect on Onzymeactivity in the resting cells . Therefore, another anionic detergent,sodium dodecyl sulfate (SDS) and the non-ionic surface active dete .r t;ents,T t.'een-60 (po1yoxyethy?.ene sorbit : ~ . ; 1 T ~ z :oster.rate) r:nrl Sp ; :n-60 (sorbitan

monoste ::rat.e) %,ere evaluated . Cells frora fcr ; c , ~ t c i t i c m b : a t c h : : o . 3 .7 ; .ere

standardized as above , cnd the deter8ents ucre :id(1e (t at concontr : : t ;nns

of 4 and 16 Ug/raF o f cells . The coll .s w ::re storc'd at l. C for 22 and4( hrs . All assays for enzyme activity wcore porforricd at 60 C . i 'he

greatest i ncrcase in enzycae activity i :as obtnined with SDS and i: DS a t a

coTtcent.ration of 4)1 , - /1 : 1 C ' of cell ; ; . Th re n ht i :frCtj to " b e no ii V ni r lctantdj*ffcYC' : 1 t.e bC'tKeL 'n tlli:se t1:O nln- . -A4 C l1 ;tl ' l ' 6iants,

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3

Twce,t60 and Shan-60, l ; : c V e r :liLhtl.y Lrv ; l I c t o : i t r . y l ; c l : : c :t ivttitcL t1Pii1 tlca

control but wera lee ls than those values obtnin4d with th e anionic dctc -tgonts .Other surface active cyents, such as sodium dioctyl sulphosuccinr+re, ananionic detergent, the cationic surface active agent, catyltritjethyl-a i ;leoniuin bromide (Cl'AR), aiid am, holytic tmrf .hcq ,,-tc tivo agent,, such as

dodecylglycine, are on order and will be ev,zluated as soor. as they

arrive.'ince the a bove data shot :P d that the sodium dodecyl sulfate andheptadecyl sulfate exhibited somewhat the saine stinul .htory action on theenzYme activity, the effects of these two detergents at Wder ranges of concentrations at different assay temperatures were evaluated . The con-centrations of detergents- ran from 0 to 230 Ug/mg of cells, and theenzymes were assayod at 60 and 75 C . The :;e cells were again obta3nedfrom batch No . 17, standardized, stored at 5 C, and assayed at 24 and .4 1 hrs . , Ar,ain, we vust mention that t he enzyme ac tivities (~,~u/mb)

reported at 75 C are only relative because of the definition of theterm enzyme activity . These results are shown in Figure 2 . It can bereadily seen that the relative enzyme activity 3s much greater when theassays were performed at 75 C than at 60 C . The SDS exhibit a greaterstimulation of enzyme activity than did the NDS. 7n these experiments,the results obtained with the IiDS detergent were much lower than hasbeeil obtained in previous exrer .iments ; various fresh solutions of IiDS

were prepared, and talese were evaluated . At i1DS concentrations of 6 and6 lig/mg, the enzyme activities obtained were 15 and 45 l-0/mg at 60 and

75 C, respectively . It can be seen that fresh detergent,solutions mustbe used in all these experiments . It can also be seen from Figure 2 thatthe sodium dodecyl sul fate has a r . ) a r k c:d opti.c?um corce;ltration for incroasin~

the enzyme activity . The addition of greater' than 1 . 8 l l g / ;ng of cel].s greatlyreduces the final enzyme activity in these cells . LIt should also be notedthat the reli?t:ive enzyme ac tivity 60 to 65 yU/i,1g is arproT : i i :ately 6 to

6-1/2 tiines ];reater tlirill norn. i a l l.y cypericaced in any fern . c :nt3t.io11 run .

These results refer to an cnr . y :;,e yield of approxicaately 4,000 pV/m1J The

effect s of sodium doclec.yl sulfate , nd other surface active ac-e nts on theenzyme aetivity as related to cell age, cell morphology, and the conditionsof growth are presently under investigation .The 4bility of baL : ~ s :,e stalks sld ;ic-c1 to the bac :aI m .:diura eonta.ininF

try,,tone, yeast extract, and salts to support cnzyl-e prodllction ac :d growthof A_rthrobacter_ RJR 2453-2 was evaluated . Al tllough the h : :ga sse couldserve as a carbon so ;lrce for this ort;anism r:ne] vould support adequategrowth, the onzyne activities %:ere all;ays less tllan 100 p0nl . Thedetert;,alts, SUS or }1DS, had no effe .t on incrc :?sing tl,is en~.yu?e activiLy .Al . t.11C)ULh tilC USCti at C6l?CC'lltl' :?i-10113 Va!'} . 1 7 1 c , l i l C L b : i _ i l 1 . : ? I l dchimical .nel ysis of tiie sc :peinatcilt flt :id, after

l '.oaciiliYatillil : : ! ' d l C:tCIi ;illetl ' b y the N i`lsOll ' s t.CL:t ofa xylo_e.0 6

a'ind the Eluco se cOilci'nt Yi3i:ioll w3s, as t:CtCl'i'I11 :'d by a6IUCoC;C U\1C1 : SC' t iat s0 . 26 mg/ml . Thcse r .:sul t s% :ere obtained af t e.r 60 hrs . at 30 t : . A l e.o, no

terapcrature - - f f c c t % : c> noted in ng sayii?g for ca, :ym :~ nctivity in

-

t l i ~ : . e

particulclr cells . cr .00~x