EXPERIMENTS

Page. . 34

3.1 Grawth and cyclosporine A production under shake flask

conditions using Tolypocladium sp.

3.1.1 Effect of media constituents on cyclosporine A yield.

A wide range of carbon and nitrogen sources were found to s u m r t

the growth and cyc A production. Agathos -- et al. (1986) have reported

that 3% sorbose (w/v) based semisynthetic medium resulted in highest 0

cyc A production after 10 days of fermentation at 27 C usinq

Tolypocladium inflatum. Agathos &. ( 1987) have also reported

that addition of maltose as a sequential carbon source resulted in an

increased production of cyc A irrespective of the initial carbon

source.

Kobe1 and Traber (1982) have reported that the specific

production of a particular cylosporine type could be influenced by

the specific incorporation of an amino acid in excess in tha medim.

In the present study, the effect of different media on the

production of cyc A by the VCRC isolate of Tolypocladium sp. F-21 is

studied.

Page.. 35

Materials and methods

Organism used:

The fungal strain used in the present study was Tolypocladium SD.

F-21 obtained from the culture collection of Vector Control Research

Centre, Pondicherrv, India.

Submerged cultivation of the organism:

The Talypocladium so. was cultivated in 500 ml Erlenmeyer flasks

containinq 100 ml of the different media (1, 21 3 and 4). Cultures 0

were maintalned in a rotary shaker at 150 run at 25 C. Samples

were taken at different time intervals and analysed for biomass, DHI

resldual glucose, total carbohydrate and amino nitrogen levels and

cyc A yield. Biomass was worked out bv centrifuqing the culture at

5000 rpn for 15 min and expressed as wet weight w r litre of the

culture. pH of the culture was measured using an Elico model (India)

digital pH meter. Glucose was estimated using ortho toluidine

reagent by the method of Sasaki and Matsui (1972). The culture

supernatant (0.1 ml) was boiled for 8 min with 5 ml of ortho

toluidine reagent (6% ortho toluidine in qlacial acetic acid

containing 0.25% of thio urea) and the colour d e v e l W was measured

using a swctrophotometer at 660 run. The concentration of glucose

was calculated from the standard graDh lotted using different

concentrations of glucose.

Total carbohydrate *as estimated using anthrone reaqent by the method

of Morales g. ( 1975). The culture supernatant (0.1 m l ) was

boiled with 5 rnl of anthrone reagent (0.2% anthrone in conc.

H SO ) for 10 min and the colour developed was measured at 660 run 2 4

using a smctrophotaneter. The concentration of carbohydrate was

calculated from the standard graph olotted using different

concentrations of glucose.

Amlno nitrogen was estimated by Sorenson's formal titration (Lew,

1957) .

Cyc A was monitored by a modifled HPLC method of Kreuziq (1984) using

a Sl-lmadzu LC 6 A HPLC (Japan). Samples were inlected on to a 0

reversed ohase C18 bonded silica column which was mitained at 60 C

by means of an column oven (CIY) 6 A , Shimdzu, Japan) and

acetonitri1e:water containing 0.1% trifluoro acetic acid (80:20) at a

flow rate of 2 ml/min was used as the mobile phase. Cyc A was

identified and quantified based on the similarity of retention time

wrth that of standard cyc A which was used for the calibration of the

system.

Page.. 37

Canposition of the different media used:

Medium 1

Sucrose 30.OOg/1 Amnonium sulphate lO.OCQ/l Potassium dihvdro- 0.75q/l gen ohosphate * R a c e metal solution 1.00ml pH 5.4

Medium 2

Sucrose 3O.Kkg/1 Amnonium sulohate lO.C%g/l Potassium dihydro- 0.75q/l gen phosphate * Trace metal solution 1.01111 Amino tjutyric acid 0.5% DL-valine 0.5% pH 5.4

Medium 3

Glucose lO.Kkg/l Ma1 tose lO.Kkg/l Casein acid hydrolysate lO.OOg/l Yeast extract 5 .cOq/l Sodium acetate 1 .CCq/l Amino butyric acid 0.5% DL-val ine 0.5% pH 5.4

Page.. 38

Medium 4

Glucose Maltose Pept one Yeast extract Sodium acetate Amino butyric acid DL-val ine pH

* Comwsition of trace metal solution.

l O O X sa l t solution.

Zinc sulphate 0.4%. Manganous chloride 0.189. Amnonium mlybdate 0.0029. Copper sulphate 0.008g. Ferrous sulphate 0.5g. Sul~huric acid 0 . h l .

Volume made u ~ t o IOCml with d i s t i l l ed water.

Page.. 39

Results

C

The results of the growth of Tolypocladium s ~ . and cvc A yield in

different media is given in Fig. 2 & 3 and oH changes and

carbohydrate utilization during their growth is given in Table 2.

Amino nitrogen levels during their growth in the different media is

given in Fig. 4. In medium I, the bufferinq cawcitv was less as

evidenced by the decrease in the pH during the initial qrowth of the

fungus which did not rise thereafter. However in medium 2, the

addition of amino acids aminobutyric acid and valine at 0.5% level

led to the maintanance of pH in the desired range. In media 3 & 4,

the pH was maintained in the acceptable range, which may be due to

the presence of complex media c-nents which mav have acted as

buffers.

Between media 1 6, 2, there was no significant differences in the

biomass but the level of cyc A was significantly higher in medium 2

which indicates that addition of the amino acids, aminobutyric acid

and valine had a m influence on the yield.

Among media 3 6 4, the latter sumrted goad growth of the

mycelium. Maximum biomass of 503 g/l was attained on day 6 in medium

4 as c m r e d to the biomass level of 242 qll attained on dav 4 in

medium 3. However, the yield of cvc A was maxim in lnedium 3 (203

Page., 40

Fig. 2 . Growth of To1 ypocladium sp. in different media.

V - Medium 1 0 - Medium 2 0 - Medium 3 0 - Medium 4

For composition of Yedia, refer Materials L Methods.

Page.. 41

0 2 4 6 8 10 12 14

Time of Fermentation in daysC

Fiz. 3. Cyclosporine A yield during fermentation of Tolypocledium sp. in different media.

V - Medium 1 0 - Medium 2 0 - Medium 3 a - Medium 4

Table: 3

Carbohydrate utilization during the growth of Tolv~ocladium sp.

in different media:

Medium Parameters Days

fl 5.40 4.00 4.10 5.00 6.10 6.50 6.80

2 Sucrose (g/l) 28.50 25.50 23.50 21.85 19.85 19.00 20.20

PH 5.40 4.75 6.50 7.00 7.50 7.80 8.60 * 3 Glucose(g/l) 10.00 5.00 3.33 2.00 1.10 0.00 0.00

Total CHO (g/l) 20.00 14.70 10.20 5.70 1.80 1.30 1.10

4 Glucose (g/l) * 10.00 7.50 3.33 2.10 0.00 0.00 0.00

Total CHO (g/l) 19.90 14.60 10.28 5.70 1.60 1.40 1.10

* All values exoressed as mean of triplicates.

@ For comoosition of the 4 media, refer materials b methods sectim.

Page. . 43

Time of Fermentation in days

Fig. 4 . Ut i l i za t ion of aminoacids during growth of Tolypocladium sp. in media 3 and 4 .

V- Medium 3 0 - Medium 4

C

For composition of Media 3 & 4 , refer Materials and Methods.

Page. . 44

q/l) on day 10 as canpared to the yield of 110 mq/1 in d i m 4 on

day 14.

In all the 4 media tested, the synthesis of cvc A was found to

increase onlv when the maximum qrowth of the mvcelim was attained.

The level was higher when the pH of the culture was above 7.0.

In media 1 6 2, the utilization of the carbon source, surrose~

was very less (about 25 % utilization). Howeverr in media.3 & 41

near com~lete utilization (99%) of the added carbon sources, glucose

and maltose was observed (Table 3 ) . Also fran the table, it is

evident that the readily available carbohydrate source, qlucose, wa?

utilized durinq the early growth phase of the fungus (complete

utilization by day 8) followed by maltose during the late growth and

stationarv phases.

Discussion

The fungus, Tolywcladium sp. was grown in four different media

and the yield of cvc A was monitored for a wriod of 14 devs. Mediun

2 differed from medium 1 only in that it contained the amino acids

amino butyric acid and L-valine, each at a concentration of 0.5%. In

both these media, sucrose was the only carbohydrate source and

i amnonium sulphate the nitrogen source. Nediun 3 & 4 were c w l e x

media and medium 3 differed from medium 4 in that it contained casein

Wed hydrolyaate instead of peptone.

Page. . 45

In media 1 & 2, sucrose and ammniun sulohate van not utilized

completely and the biomass wan lees when compared to that obtained in

media 3 & 4. Also in medium 1, after the initial drop in the DH on

day 2, the pH did not rise indicating the poor buffering capacity of

the medium. However, in medium 2, after the initial drop in the DH

on day 2, the pH started increasinq and attained a DH of 6.8 on dav

14 indicating a better DH maintainance which may be due to the

addition of the amino acids. Aarino & Agathos (1990) have reported

that in Tolypocladium inflatum fermentation, OH plays an important

role in the final cyc A titre. Low final pH resulted in low cyc A

production whereas higher final pH was associated with higher titre.

The presence of precursor amino acids is k n m to increase the

specific production of cyc A (Lee & Agathost 1989) in Tolypocladium

inflatum. In the present investigation also, it was found that the

addition of the amino acids valine and aminobutyric acid increased

the production of cyc A by 18%. In media 3 6 4, the DH profile was

in the acceptable range indicating a better buffering capacity of the

complex media. The utilization of the carbon sources, glucose and

maltose and the nitrogen sources was also near camlete in these two

media. While medium 4 suwrted good biomass yield, cyc A yield was

better in medium 3.

From table 1, it is discernable that in media 3 & 4, the

available carbohydrate sources were utilized comletelv. Amonq the

Page.. 46

carbohvdrate sources, glucose was utilized dutinq the initiai b Browth phase to near ccmletion before switching w e r to l~ltO5e 1 &ring the late growth phase and the stationarv phase. Agathos

al. (1987) have reaorted that the carbohvdrate sources, sorbose~ *-

glucose, maltose, fructose and cellobiose favoured qood ,cvc A

production in 2.inflatum ATCC 34921. In shake flask cultivationr 5%

(w/v) maltose and glucose based semi synthetic medium yielded 50

q / l of cyc A on day 10. Also, they had recorted that, when maltose

was added as a sequential carbon source pulsetbthere was an increase

in cyc A production irrespective of the initial carbohydrate source.

The same authors had reported that 3 % (w/v) sorbose as the

carbohydrate source welded 105.5 mg/l of cvc A on day 10 and 2 %

(w/v) sorbose yielded a better cyc A titre (14.3 mg/g of biomass)

followed by 5 % mvoinositol (13.4 q / g of biomass 1. Margaritis and

Chahal (1989) have reported that in - T.inflatum ATCC 34921, the

highest levels of cyc A (170 mg/l) was obtained in a medium

containing 3 % fructose as the carbohydrate source with arrmbnium

hydrogen ~hosd?ate as the N and P source. In the Dresent study,

among media 1 & 2, there was an increase in the cvc A levels' in

medium 2 (18%) which indicates that the addition of amino butyric

acid and L-valine increases the yield of cvc A. Amorsg media 3 6 4,

the veld of cyc A was greater in medium 3 which contained casein

acid hydrolysate whereas in medium 4 which contained peptone there

was maximum bianass product ion.

Pran the above study, it is evident that among the 4 different

media studied^ medium 3 containing casein acid hvdrolvsate as the

nitroqen source sumrted maximum production of cvc A l while medium 4

which contained m t o n e as the nitrogen source suo#)rted maximum

bianass yield. The amino acids amino butyric acid and valine

increased the vield of cyc A when incornrated in the media.

Page.. 48

3.2 Studies on the imnobilization of TolvDocladiun so.

3.2.1 D i f f w i ~ ~ l and mechanical oraperties of calcim alginate gel

bQsas.

Natural and synthetic polymers are now being used as matrix in

many imnobilization techniques to entrao rotei ins^ enzymes, whole

microbial ~lant and animal cells. When an enzyme/microbial cell is

imrobilized in a carrier its orooert ies are altered considerably.

The w t imoortant of these effects is mass transmrt of substrate

and eroducts. Since the reaction in an imnobilized system takes

place in an heterogeneous Dhase, the substrate has to diffuse to the

catalyst surface for the reaction to take olace and the ~roduct to

diffuse back into the fluid ohase. Porous wrticles are generally

used for imnobilization because such materials can orwide large

surface area for imnobilization per gram of the sumo13 material. In

such cases, for the effective use of all the available internal L

surface area, the reactants must first dif- fran the fluid to the

outer surface of the pellet and then through the minute Wres of the

iwrfaco diffuse inside the pellet and reach the enzvne/cell for

biotranrformntion (Tanaka & g., 1983).

Ihc tmrp size of the gel which depends on the viscosity of the

c t m i ~ r and the method of immobilization can affect the diffusion of

Page.. 49

the wbstrates or the products and limit the rate of the raaction of

the mtramed cell or the enzymes. Hence, Dore size is a critical

parameter in ielecting a matrix for a particular process (Tanaka 5

al., 1983). -

While low molecular weight substrates and oroducts can easily

diffose in and out of the gel matrix, diffusional constraints are

encountered with hiqh molecular wight substrates and woducts.

Increasing the w r e size of the matrix mav lead to leakage of the

entraowd enzymes or cells. In addition to the pore size, the

hardness of the beads is also an imrtant character to be studied.

While too soft beads tend to break while beinq used in wcked and

continuous bed reactors, increasing the hardness of the beads bv

increasing the viscosity of the matrix tend to decrease the w r e size

of the surface and may lead to diffusional limitations.

m e diffusional characteristics of wlyacrvlamide gels have been

studied in detail by white g. (1960, 1961). The leakage of

enzymes and whole cells is minimal in wlvacrvlamide matrix because

of th'e amall pore size of the carrier but diffusional limitations of

substratrrs often result in low reaction rates. Havever, the average

pore size of calcium alginate gel is rewrted to be Larger,thhn that

of acrylamide gel and hence it is being widely used as a carrier for

imnobilization of whole cells and enzyms (Tanaka e., 1983).

In the ~teoent study, the diffusional and mechanical ~ m r t i e s

Page.. 50

of ~hdinat* bmda vrewred by using different concentrations of

aLgimLe and the cross linking agent calciun chloride were studied.

Materials and methods:

Sodiun alqinate was purchased f ran Robert Johnson Co. (India).

All other chemicals were of high qualitv analvtical qrade.

Effect of alginate concentration on the diffusional and mechanical

pro~cart ies of calciun alginate beads:

The Ca-alginate beads were oremred bv dro~oing alginate

solutions (50 ml) of different concentrations (1%, 2%, 3%# 4%) into

250 ml 0.5 M calcium chloride solution. Two hundred mg of bovine

8eW albumin (BSA) (M.W. 68#000) was added to 50ml of the alginate

solutions of different concentrations as molecular weight markers to

study its diffusional rates. The beads were Cured in calcium

chloride for 1 h after which they were washed with distilled water

an8 transferred to 250 ml conical flasks containing 50 ml distilled 0

watm and k w t on a rotary skaker (150 rpn 1 at 30 C. sMp1es were

taken at different time intervals and the level of a l b i n in the

water uu eat imatod by the method of LMy 2. (1951).

The mechanical strength of the beads wen exmessed in tenas of

hanineus of the beads to mild pressure amlied bv omsing them

Page.. 51

betwen the fingers. The diffusional wooerty of the be& were

expre6wd in relation to diffusion of altxanin.

Effect of calcium chloride on the mechanical and diffusional

prwrties of calciun alginate be*:

To study the effect of different strengths of calciun chloride on

the diffusional and mechanical prooerties of alqinate beads, 2%

alginate solutions containing 200 mg of BSA as molecular wight

marker was d r o m d in 250 ml of calcium chloride solutions of

different strenqths ( 5 0 mM - 500 mM) and the beads cured in it for 1

h. The diffusional and mechanical Drowrties of the different beads

were determined as described above. 61b.074

Diffusional orerties of 2% alqinate beads: SEE

The diffusional ~rowrties of 2% alginate beads were studied in

detail. The diffusion of BSA (M.W.66,000), cvanocobalamine (H.W.

1350) and qlomse (H.W. 180) from inside the beads into well stirred

solutions was studied by measuring the release of these markers frau

inside the beads into the solution.

Beads . containing these markers (200 mg of BSA, 50 mg of

Cyanombalamine and 200 mq of glucose) in 50 ml of 2$ alginate

solution was d r w in 0.5 H CaCl and wen, cund in it for 1 h. 2

After washim, they were transferred into 250 ml conical flaska

containing 50 ml of distilled water and their release into water was

measured at different time intervals. Albumin was measured by the

method of torrV g. (1957 1 , glucose by the method of Sasaki and

Hatsui (1972) using ortho toludine and cyanocobalmine by measuring

the abrbance at 540 nm.

m e diffusion of cyanocobalamine from the alqinate beads was

studied by incubatinq the beads in 1% cyanocobalarnine solution at 0

30 C I 150 rpn and measuring the absorbance in the solution at

different time intervals.

The rate of diffusion of cyanacobalamine from the beads and into

the beads was calculated from the formula given belov:

Rate of diffusion (D) = 0.D at "Owtime - 0.D at a given time X 100

0.D at "0" time

where D is the diffusional rate.

Results

Effect of alginate concentration on the diffusional and macianical

Fmwrties of the beads:

Tha diffusion of 0SA from beads ma& of different alginatk

Page. . 53

concentration over a period of time is sham in Piq.5. In be& slbde

of 1% alginate, the a l b i n concentration in the water increased

rapidly during the initial 30 min and gradually slowed vith time,

levelling off at about 60 min. In beads made of 2%, 3% and 43

alginate concentration, the albumin concentration in the water

increased gradually upto LOO min and levelled off between 100 - 120 min.

h e size of the beads are little affected bv the concentration af

alginate, but as the concentration of alqinate increases, the

hardness of the beads also increased £ran soft (1% alginate) to verv

hard (4% alqinate) (Table 4).

Effect of calcium chloride on the diffusional and mechanical

propert ies of the beads :

h e results on the effect of calcium chloride on the diffusional

and mechanical ~r-rties of alginate beads indicate that increasing

the stronqth of calciun chloride does not affect the diffusional or

mechanical prowrties of the alqinate beads (Table 5 ) .

Dif fuaional prapertiss of 2% alginate beads:

The diffusion of BSA, glucose and cvanocobalanine from 2%

alginate beads over a period of time is given in Fig 6 6 7. From the

Time of Incubation in minutes

Fig. 5. 1)iffusion of Albumin from alginate beads of different concentrations.

V - 1% alginate 0 - 2% alginate 0 - 3% alginate 0 - 4% alginate

Eftect of alginate cancentcatian on the Biffuaional and mcbnica l pa.apsrtie8:

cerrcentwtian Size of beads Hardnurs Diffusional of .Ig inate ( i n mn) ( @ I vrooert~

1 3-5 mn soft **** 2 3-5 mn mildly hard tt+

3 3-5 mn hard 4 3-5 mn very hard *

diffusional orowrtv based on the diffusion of albumin from inside the beads.

@ hardness of the beads based on the strenath of the beads t o light oressure awl i ed bv oressinq between the fingers.

Page.. 56

Table: 5

Effect of calcium chloride on the diffusional and mechanical properties

Strength of Size Hardness Diffusional calciun chloride (in mnl (@I ~roperty

X ) W 3-5 mn mildly hard **** 100 nFl 3-5 mn mildly hard **** 200mM 3-5 mn mildly hard **** 300W 3-5 mn mildly hard **** 4CQM4 3-5 mn mildly hard **** 500M 3-5 nm mildly hard ****

denotes the diffusional property based on the diffusion of albumin from inside the beads.

@ hardnea of the beada based on the strength of the be& to-light pressure amlied by pressing between the fingers.

All the beads were prepared using 2% alginate solution and cured in the remctive calcium chloride solutions for 1 h.

(0-4) - Glucose

in m

g.

(*-.4

) - Albumin

in m

g.

A

d

NC

-m

WO

N

00

00

0a

0

0 5 10 15 20 25 30

Time of Incubation in Minutes

F i g . 7 . Diffusion of Cyanocobalamine from 2% alginate beads.

Page.. 59

Time of Incubation in Minutes

F i g . 8 , Diffus ion nf Cyanocobalamine i n t o 2% a l g i n a t e beads.

Page.. 60

figurw, it can be seen that the albwnin concentration rises durinq

the initial 30 min levelling off betwen 60 and 100 min. In the case

of glucose and cvanocobalamine, maximum concentrat ion in the medium

was reached within 20 min. The results on the diffusion of

cyanocobalainin from the medium into the beads is given in Fig 8.

Pran the fiqure, it is clear that the diffusion into the beads

increases durinq the initial wriod and reaches a m a x i m at 30 min

after which it levels ott.

Discussion:

In these ex~eriments, molecular weights were used as an index of

the molecular size. The results oresented bravide quantititive and

systemic informtion on diffusion of many substrates into and from

alginate gel beads. The effect of alginate concentration and calcium

chloride strength on the dif fusimal and mechanical ~rowrties of the

beads were studied. These are imrtant characters which need to be

studied in detail before imnobilizing anv agent as they determine the

diffusion of substrates and oroducts throuqh the qel surface.

The hardness of the beads deoends on the wrcentaqe of the

alginate us& for making the beads. Tanaka 5 2. (1983) have reported that in the case of high molecular weight substrates such as

albumin, increasing the alginate concentration has more effect than

Paqe.. 61

increasing the CaCl strength. Beads made of 1% alginate were verv 2

soft and hence not suitable for use in wcked bed reactors and

cont inuow stirred tank reactors. Of the different concentrations of

alginate used, 2% alginate showed good diffusional and mechanical

properties and was hence studied in detail.

As rewrted earlier by Tanaka et al. (1983), the strength of - - CaCl did not influence either the mechanical or diffusional

2 properties of 2% alqinate beads. Diffusion of substrates fran 2%

alqinate beads was found to depend on the molecular weight of the

substrates. Of the three substrates used for the study, the

diffusion of albumin (M.W.66,OGO) levelled off at 60-100 min while it

was 30 min for cyanocobalamine (M.W.1350) and qlucose (M.W.180).

mese results suggests that low molecular weight substrates can

diffuse freely in and out of the alginate beads wh'ile in the case of

high molecular weight substrates, free movement is not wssible.

Diffusion of cvanocobalamine inside the beads fran the medium was

also found to be free and levelled off betveen 15-30 min.

Fran the above studv, it was found that 2% alqinate is ideal for

imnobilization of the whole myceliun since it msesses good

diffusional and mechanical ~ropert ies. Hence, 2% alginate d m w e d in

0.5 M CaCl and cured for 1 h was adooted as the standard ~thcid of 2

inmobilizat ion throughout the study.

3.2.2 Procbctiar of cvcloswrine A using the imnobilized

Toly~ocldlium sp.

Over the -st vears, there have been raoid develounents in the

use of enzymes as catalysts for industrial, analvticalt and medical

purposes. Enzvmes have been imnobilized in order to make their use

more convenientr in such a way that they resenble ordinary

wlid-ohase catalvsts used conventionally in the svnthetic chemical

reactions (Kennedv and Cabral, 1983).

In fermentative Drocess, many useful conpaunde are oroduced,

eswcially by the seauential action of several enzymes (mltienzm

systems) that are hiqhlv camartmentalized in the microorganism.

Individual enzvmes can be purified and co-imnobilized so as to

reconstitude multienzw ~athwavs & e. Hovever, the most

satisfactory way of utilizinq the multlenzvme reactions is bv

imnobilizinq the whole living cells as the enzyme extraction and

purification ~rocedures are tedious and time consuming.

The advantages of immobilization of whole cells are many. The

tedious and time consuminq procedures for enzvme extraction and

purification are eliminated, the cellular enzvmes are often already

organised into the requisite metabolic mthwavs, the problem

associatd with enzyme stability m y also eliminated. Many of the

Page. . 63

en- svstema require cofactors and coenzvmes fot their activity.

Them are e x m i v e and difficult to coirmnobilize. lhese are readily

present in the cell, eliminating the expensive method of

coinmobilization of the cofactors along with the enzymes. Thus,

imnobilizd livinq cells are oreferred to imnobilized enzymes for

degradative and synthetic pathways which require energy and exvensive

cofactors. Inmobilized cells are convenient to handle, appear to be

less susceotible to microbial contamination, and oennit easy

separation of the products. Also, nondividinq imnobilized cells

require onlv maintanence energy, and the vield will be qreater than

that obtained bv sutnnerqed fermentation methods. Imnobilization also

facilitates the use of dense cell woulation without any influence on

the rheological properties of the sospendinq medium.

In a recent report (Kobe1 G., 1989) have rewrted the

synthesis of cyc A bv imnobilization of the fungus on diataneceous

earth and have studied the effect of different orecursor amino acids

on the yield of cvc A. In the present studv an effort was made to

optimize the conditions for the o~timal oroduction of cyc A using

imnobilization technique.

Page. . 64

Materials and Methods.

Bi&ransfom~t ion media used:

Wivn 1 :L-qlvcine, L-valine, L-leucine, DL-alanine, aminobutyric

acid, all at 750 mq/l and glucose lO&q/l, di 7.4

Medim 2 : same as medium 1 exceot the concentration of L-valine

which was increased to 1.5 q/l.

Medim 3 : same as medium 1 excmt the concentration of L-valine

which was increased to 2 q/l.

Medim 4 : ssnw as medium 1 exceDt the concentration of L-leucine

ckrich was increased to 1.5 q/l.

McdiwlI 5 : same as medium 1 exceot the concentration of L-leucine

which was increased to 2 q/1.

M c d i u ~ 6 : s s l ~ as medium 1 e x c e the concentrations of L-valine and

L-feucine which were increased to 1.5 g/l .

Medium 7 : wne 8s medium 1 exceot the concentrations of L-valine and

L-leucine which were increased to 2 q/ l .

Grouth of the organism:

'Tolmladiwn SD. from the culture collection of Vector Control

R...srch Centre# Pondichem, was cultivated in 500 ml Erlenmeyer

flask@' containinq 100 m l of culture medim (dextrose 4%1 ~ e p t a r e l%#

glycerol 4 , malt extract 29, casein acid hvdrolvsate 3%,

DL-threanine 0.01% I DGvsline 0.01% I oH 5.2). Cultqreh were 0

maintained in rotary shaker at 150 run at 25 C for 10 days (late

growth ohasel after which the mycelium was harvested by

centrifugation at HXX) run for 20 min. 2he rnvceliun was extensivelv

washed with normal saline and used for imnobilization.

Itmobilization of the funqus:

Imnobiiization of the fungus was done in 2% calcium alqinate

accordinq to the method of Tanaka &. (1984). Mvcelium at 30% level (wet weiqht) was mixed with 2% sodium alqinate solution and

drowed in 0.5 H calcium chloride to make beads of 3-5 mn using the

model shown in Fiq.9 6 Plate 1. The beads were allwed to remain in

calciun chloride for 1 h after which they were washed rewatedlv with

sterile distilled water and used for the oroduction of cvc A in

-eked bed reactors under batch and continuous ~roduction mades.

Plate 2 shws the morphology of the imnobilized beads.

miobiliastian of the SpOrBd):

me fungus was cultivated in a medim containing 2% malt extract,

0.4% veast extract, pH 5.2 for 7 days which resulted in the

~roduction of 90-950 mores. 'Ihe SDores were harvested by

centrifugation at 6000 r m for 30 min and washed extensively with

normal saline. Spores at 0.1% level (wet weight were imnobilized in

2% alqinate and made into beads as described earlier.

Ihe imnobilized snores were allowed to qerminate bv allcuing them

to grow in a mediun containing 2% casein acid hvdrolvsate (CAH) and

18 glucose and the growth of the mycelitni inside the beads were

mitored by observing the cmsa-sections of the beads daily. After 7

days (when the cross-section of the beads shaved qood wcelial

growth), the beads were filtered, washed extensively with distilled

water and wcked in a pecked bed reactor with recycle mode for the

~roduct ion of CYC A.

PsdPJng af the reactor and media feeding:

The design of the pecked bed reactor with recycle mode used for

the ~rcductiorl of cyc A is shown in Pig.10. For the wckinq of the

reactors, ha& mixed with water was cared into the coltmm and the

water & l l d to drain out. In the case of wcked bed reactor under

Page.. 68

Page.. 69

butch model ~ i m 1 was added to the column to the level of the

beads and allowed to remain for biotransfonnation. In the case of

packed bed reactor under continuous mode with substrate recycle,

medium 1 was Demoed into the colunm in the reverse flow mode using a

~eristalic oumo at a flow rate of O.%l/min. The outlet of the column

was recvcled after mantitation of the level of cvc A as shown in

Estimation of cyclosporine A:

Thc yield of cvc A ~roduced by both the systems were monitored

using a Shimadzu LC 6A HPLC (Japan) by the method described earlier.

Samples frun the reactors were mixed with equal volume of C

acetonitrile, filtered and injected into a C18 calm for the

quantification of cyc A level.

Effect of Precursor amino acids:

The effect of the Drecursor amino acids, valine and leucine at

different concentration on the yield of cvc A when added individually

and in combination in a wcked bed reactor was studied by feedinq

different biotransformat ion media containinq various concentrat ions

of the precursor amino acids and estimating the yield of cyc A.

Page.. 70

Determination of the half-life of the reactor:

The half-life of the reactor was calculated bv estimating the

yield of cvc A/ml of the media for manv batches routinelv. The

half-life was expressed as the time for an activitv loss of 50% of

its original value (Klein and Waqner, 1983). In the Dresent studv

the half-life of a oacked bed reactor under batch mode was

determined.

Determination of the viability of the itmobilized funqus:

The viability of the imnobilized funqus was determined

periodically by observinq the growth of the imnobilized funqus under

reincubation with nutrients in a liquid medium (Sanerville gal.,

1977). Beads from the column were crushed and mixed with qrwth

medium and the qrowth of the funqus was monitored.

Page.. 71

Results:

The experimental model shown in Fiq.9 which vas used for the

~oduction of immbilized biocatalvst was cawble of oroducing 2-3 kq

of the biocatalyst w r hour of uniform size of 3-5 mn (Plate 21 under

sterile cmditims.

Innrobilization of the swres in alginate followed bv incubation

in liquid medium resulted in good growth of the mycelim inside the

alginate matrix as seen by the cross-section of the beads taken on

different davs (Plate 3 ) .

The yield of cyc A in a packed bed reactor after 24 and 46 h of

media addition is shown in Table 6. The table shows that the vield

of cyc A in six other d i a (2-7) is higher than that obiained in

medium 1 indicating the positive effect of orecursor amino acids on

the synthesis.

Table 7 shows the vield of cyc A in a packed bed reactor under

substrate recvcle mode packed with ~mrcelirnr-imnobilized

biocatalvsts. From the table, it can be noted that the vield of

Cyc A increases when the substrate is recvcled for three times.

Table 8 shaws the vield of cyc A in a wcked bed reactor under

recycle w&a packed with spore imnobilized biocatalvst. It is

Plate 1: Mode? for the rxoductim of imwbilized biocatalycr+_a

Plate 3: Cross section of stmre inmobilized himatalvsts on incubation with median

A: 0 Qy 6: 4th dav C: 7th dav D: Outer surface on 7th day

Page. . 72

Table: 6.

Cvc A concentration at 24 and 48 h after

sdaition of media t o wcked bed reactors.

(Batch mode)

M i a cycAleve1

No. ( i n pg/ml)

24 h 48h

* Mean of three batches.

Page.. 73

Cyc-rine A lwels in mycelium-inmobilized wcked bed reactors

under continwus subatrate feeding.

* Cycle Time of contact Cyc A level

no. ( i n min. ) ( i n pg/ml)

* Mean of three batches.

Page.. 74

Table: 8.

Cycloswrine A levels in score-imnobilized ~acked bed reactors under

continuaus substrate feeding.

t

Cycle Time of contact Cyc A level

no. (in min.) ( in pg/ml)

* Hean of three batches.

Page.. 75

found that tho yield of cyc A increases as the substrate is recycled

indicating the better utilizatidn of the substrate when it was

Viability studies done on the biocatalvst wcked in wcked bed

reactor showed that the imnobilized myceliun vas viable for m r e than

one year.

Studies on the half-life of the wcked bed reactor packed with

mycelium-imnobilized biocatalvst indicate that the half-life of the

biocatalvst was between 5 and 7 months after which the vield of cyc A

obtained fran it was reduced by 50% of the original vield.

Discussion

Imnobilization of the swres followed bv their incubation in

medium resulted in a good q r m h of wcelium inside the beads in 7

days and these biocatalvsts were able to svnthesize cvc A when they

were incubnted in a medium containing the Drecursor amino acids.

Packed-bed reactors have the advantaqe of simplicitv of

o ~ e r ~ t i ~ n , high mass-transfer rates, and hiqh reaction rates. In the

case of imnobilized living cells, oxygenation and ca&-di-oxide

removal are neceasarv. Under these conditions, a wcked bed reactor

is liable to w s e some ~roblem such as removal of gas. In the

Page.. 76

laboratory scaler this moblem can be circumvented to a laiqc extent by prior ox~enation of the substrate. Recycle-cell reactors find

amlication when reaction rates are too slow or when high bulk

mass-transfer coefficient values are necessarv. In this t m of

reactor, a oortion of the outflow is recvcled and mixed with the

inlet stream of the reactor. This Dennits owration of the reactor

at high fluid velocities, which minimises bulk mass transfer

resistance to the transwrt of substrate to the catalvst surface.

Even thoqh high flaw-rates reduce the contact time of the substrate

in the reactor ( w r cycle), the recycling Dmcess effectively

provides sufficient contact time to achieve desired convertions.

Table 6 shows the yield of cyc A obtained fran a packed bed

reactor under batch mode after 24 and 48 h and the effect of

precursor amino acid concentrations on tne vield. Increasing the

concentration of the precursor amino acids narnelv, DL-leucine and

DL-valine fran 750 mg/l to 2 g/l increases the vield of cyc A

signif icantlv: however increasing the concentration of both the amino

acids together does not lead to a svnerqistic effect on the vield.

von Wartburq and Traber (1986) have rewrted that in the sutmeqed

cultivation of T.inflatum, increasing the concentration of L-valine

to 8 g/l in the medium resulted in very significant increases in the

yield of total cvcloswrines as well as in the vield of cvclosmrine

C and D. Likewise Chun and Agathos (1989) have also reoarted increase

in the vield of cvc A by the exogenous feeding of L-valine in both

Page. . 77

aubmerqod and imnobilizd cultivation of the fungus r-inflatum.

Huu$ver, it 18 to be notd that not all the added amino acids are

utilized for the oroductim of cyc A and hence it can be assumed that

higher levels of valine and leucine may act as inducers or

activators of the cvcloscorine synthetase canolex and may induce the

specific incorooration of the added amino acids resulting in the

production of the desired wcloswrine and suwression of other

cyclos~orine tycles.

~adle 7 sh- the yield of cyc A obtained £ran a wacked bed

reactor under rubstrate rocfcle mode which was wcked with mycelium C

imnobilized biocatalyst . Recycling of the substrate for three cycles was found to increase the yield of cyc A from 18.99 pq/ml to 45.95

@/ml. However, further recycling did not lead to increase in the

productivitv of cvc A which may be due to feed back inhibition of the

product.

The half-life of the wcked bed reactor under batch mode was

found to be 180 days bv which time there was a 50% reduction in the

yield of cvc A when it was estimated after 24 h after the addition of

media to the reactor.

Table 8 shcm the yield of cyc A from a oacked bed reactor under

continuous substrate feeding which was packed with smre idilized

biocatalvst, at the end of each of the three cvcles of media feeding.

Page.. 78

Tk contact time of the media with the biocatalyst during each cycle

meintained as 180 min by carefully regulatinq the feeding of the

media. From the table it can be seen that the vield of cyc A

increasd at the end of each cvcle reachinq a level of 37 ug /d which

is nearly equal to the yield obtained fran a veqetative mycelium

imnobilized system. The vield can be further increased bv increasinq

the contact time of the media with the biocatalvst bv requlatinq the

flow rate of the media and also by increasing the dimensions of the

reactor.

These studies show that inmobilized reactors can be used for the

production of cyc A and the yield can be enchanced bv manioulating

the dimensions of the reactorr media feeding straterqy, directed

biosynthesis of cvc A by increasing the concentration of the

precursor amino acids, valine and leucine in the media. Further

study in this area many lead to the wrfection of an imnobilized

reactor for the ~r~duction of CYC A.

Page.. 79

3.2.3 Studies on the chemical identity and biological activity of

cycloswrine A obtained fran Tolypocladium so.

In the present study, the chemical identitv and biological

poten~? of the cyc A obtained from the indiqenous strain of

Tolypocladium so. was evaluated. The identitv of the cocmmd was

checked by HPLC analysis, IR s-ral analysis and canoaris>on of the

amino acid profile of the hydrolyzed canpound after running a thin

layer chranatogram. The biological potencv of the camound was

evaluated in canoarision with the standard by ( a ) evaluating its

antifunqal activity against Aspergillus (b) oerforminq skin

grafting experiments in rats and ( c ) by studvinq the augmentation of

delayed tvpe hypersensitivity (DTH) response to toleroqenic dose of

human red blood cells (HRBC) in mice.

Material & Methods

HPLC analysis of cvc A:

HPLC analysis of the standarad and test oreoarations of cvc A

were performed according to the method of Kreuziq (1984) as described

in section 3.1.1.

Thin layer chmtograDhy of q c A hydrolysate:

I he standard and test preparation of cyc A (2 mg) were

Page. . 80

hydrolyzed in boiling 6N HCl for 48 h under vacuum in sealed vials.

The s m l e a were then neutralized with NaOH and made upto 10 ml with

distilled water and swtted on a silica coated plate and the

chranatogram develowd in butanol : acetic acid : water (5:1:4)

solvent system. The s w t s were visualized by soraving with a

modified ninhydrin reagent which is used for the visualization of

methyl amino acids.

Determination of antifungal activity:

m e antifungal activity of the test premration of cvc A was

evaluated against As~eryillus +. Filter w w r discs soaked in

the test preparation of cyc A dissolved in methanol and air dried 0

were placed on A.niqer spore plates and left at 37 C overnight.

Growth inhibition zone around the filter Dawr disc was taken as an

indication of antifungal activity.

Animals used:

Laboratory bred Lewis male rats (3-4 months old) were used for

the skin grafting study. Male mice (4-6 weeks, 20-25 qm) were used

for the study of augumentation of delayed t w h-rsensitivity bv 0

wc A. The animals were maintained at 28 C and received standard

rat feed with water ad libitum.

Page.. 81

;Mugs used:

(1 Reference weparation - Standard cvclosporine i .v.

(bndoz Ltd.t Swizterland), 5 ml vial containing 2X) mg cyc A was

purchased f m m the druq ,stores.

( 2 ) Test preuarat ion of. cyc A Droduced from

Tolypocladium so. of Vector Control Research Centre (VCRC).

For skin graftinq exoeriments, the test meoaration of cyc A

was evaluated in can~rision to the standard ~rewration. The two

preparations were given in two regimens of 15 mq/kg and 30 -/kg bodv

weight after dissolving in olive oil. The control qrou~ of animals

received onlv olive oil.

For study of augmentation of delayed type hywrsensitivity, the

test Drewration of cyc A was reconstituted in ethanol and was

administered intraperitoneally (i.p) at a concentration of 2OO-nrg/ kg

bcdy weight in approximately 0.2 ml ethanol. Control group of mice

received onlv 0.2 ml of the vehicle. Cyc A was adninistered 48 h

prior to the imnization of the animals with HRBC.

Experimental model for skin grafting:

Grafting was performed according to the method of Steinmuller

Page. . 82

(1984). Donor rats were sacrificed by cervical dislocation, and the

hair on the ventral side cleanly shaved and an area of the skin

removed. The fatty layer beneath the dermis was acraped off and the

skin cut into buttons of 1 an diameter. T h w were then w u h d

thoroughly in phosphate buffered saline (pH 7.4) and suspended in the

same buffer with the hair side above. The recipient were

anaesthetlzed with chloral hydrate (30 mg/kg bodv weight) , the hair

on the lateral wall of the thorax was removed and the skin cut down

to the vascular fascia lying just above the subcutaneous muscle.

Skin buttons cut from the donors were placed on the graft bed of the

recipient with the hairy side above and sutured. Vaseline

impregnated gauze was placed above the graft and bandaged with

adhesive tape.

Signs of rejection criteria:

Five criteria were selected to determine the onset of rejection

raspoMe, ie. the onset of erythema in the visible layers of the

allograftd skin, size of the graft, crustirq of the graft ,cplrolent

d$I#cbrge and status of the hair on the graft.

Clinical evaluation:

l b a animals were monitored regularly for weiqht gain, fever,

diarrhoea and gingivitis. m e s e m levels of the enzymes, s e w

Page.. 83

glutamate oxaloacetate transaminase (SCOT) and sennn glutamate

pyruvsts transaminase (SGPT) were monitored weekly to assess

hepatotoxicity (Reitman & Prankel, 1957). Blood urea (Natelm g

a1 . , 1951 ) and creatinine (Bonsnes 6 Tausskv, 1945) levels were - checked weekly for nephrotoxicity. Circulatinq cyc A levels were

monitored by reversed phase HPLC using a C 18 column according to the

method of Kreuzig ( 1984).

Stat istical analysis:

The data obtained on the acceptance of the skin graft were

subjected to Fisher's exact test , while that concerninq biochemical

parameters such as creatinine, urea, SGOT, and SGFT and cyclosporine

A level in the blood serum were subjected to analvsis of variance

test.

Imnunization of the mice:

H m blood (15 ml) was collected in Alsever's solutiqn fran L

which the RBC4s were obtained by centrifugation and waahed three

times in phoschate buffered saline (PBS) cH 7.2. Experimental and 9

the control group of animals received 10 HRBC in 0.2 ml PBS

intravenously (i.v) through the tail vein 48 h after cvclos~rine A

administration.

Page. . 84

DTH assav:

8 Mice were challenqed 4 days after imnunization with 10 HRBC

in 0.05 ml PBS under the right hind foot tad. The left foot pad

received PBS alone. DTH reactions were assessed after 24 h by

measurinq the increase in the dorso-ventral thickness of the test

foot oad over the control foot pad using a dial calipers. All

measurements were conducted bv the same individual and the results

were expressed as specific increases in the foot oad thickness -1

(10 mn mean fi s.d. 1. An increase in foot oad thickness of 0.2 mn

or more was considered significant at 1% level (Collins and

Mackaness , 1970).

Assay of circulating antibody:

Serum total haemagglutinin titres to HRBC were estimated on

heat-inactivated samples as described by Hudson and Hay (19803.

Page.. 85

Results

The HPU: profile of the test preparation of cyc A of Vector

Control Research Centre, Pondicheny and the standard meparation were

identical in relation to the retention time (Fiq.11) and their IR

spectra were su~erimposable (Fig. 12)

TLC ~rofiles of the hydrolysates of the standard and test

preparations of the cyc A were found to be matching.

Plate 4 shows the antifungal activity of cyc A against

Aspergillus a. The antifungal activity is visible as zone of

growth inhibition around the filter paper disc.

The data regarding acceptance of skin grafts are given in Table

9. All the animals of the control group showed qraft rejection as

evidenced by shrinking and crusting of the qraft and absence of

vascularization and hair growth. (2 the other hand, 83% of the animals

administered with the reference preparation at both 15 and 30 mq/kg/dav

showed acceptance of graft as evidenced f m vascularization and hair

growth on the qraft. In the test preparation treated animal grout3 the

acceptance rate was 67% at 15 mg/kg/day while it was 83% at 30

mg/kg/day (Plate 5). Statistical analysis of this data shoved that the

acceptance rate of skin qraft in animals of all the ex~erimental qroups

receiving cyc A differed significantly from control (D ( 0.05). Also,

the differences in the acceptance rate of skin qraft between animals

Fig.11. HPLC profiles of Cyclosporine A.

A. Standard Cyc A B. VCRC preparation of Cyc A

Analytical conditions:

Column : RP LC-18 (Dupont) 4 . 6 m x 25 cm. Temperature : 60°C

Mobile Phase: ACN:HZO (80:20) Flov rate : ? ml/min. Detector : UV 216 nm (0.01 AUFS)

I. 0 a 5 Z,

Q 4 > w

E 9 C -d 0 Y

-4 0 u n. In m I. 0 Iu # n. U

' < p al c < %

.d I . W E g, -5 .3 UI I. .4J 0 0 - 4 P Y u m r d

n. U Q v I. lu Xn. 0 U

g x H b 4 Q u v L. u c w 9) saw 3s 2

Plate 4: Antifunqal activity of VCRC ~rewration of Cvc A aqainst Amergillus niger

Page.. 88

Table 9: The acceptence / rejection of skin grafts

No.of No.of Graft Percenta-

rats rats Acct Reje- ge a&

treated alive ptcd cted tance

Control 6 6 nil 6 nil

Standard precmration 6 6 5 1 83

15 mg/kq

Standard weparation 6 6 5 1 83

30 mg/kq

Test preparation 6 6 4 2

15mg/kg

Test premrat ion 6 6 5 1 83

30 mg/kq

Plate 5: Skin qrafting i n rats wing VCRC mparation of Cvc A (30 nqhq bodv weight)

A: dn the dav of qraftinq B: ac-ance of mft ( 14th Qv)

Page.. 89

t r w d with the reference and teat preparations at both the dases were

not significant (p 0.5). The data indicate that once the threeihold

level of cvc A is reached in the serum the acceptance of graft is not

dose related.

The results of the biochemical assays are oresented in Tables 10

and 11. CANOVA test' of the data showed that there was no significant

shift in the level of creatinine, urea, SGUT and SGW in the animals of

control group fran dav 0 to day 14. All the e x w r i m t a l animal groum

showed significant increase in the level of creatinine (p 0.01)

from day 0 to dav 7 , except that treated with the reference prewration

at 15 mq/kq/dav: in this case significant increase was noticed between

day 7 and day 14. Regarding the level of blood urea, all the

experimental animal grou~s shared significant increase fran day 0 to.

day 7 and f m day 7 to day 14 ( D 0.01), excedt that treated with

15 mg/kg/day of the test preparation; in this case significant increase

was observed between day 7 and day 14.

C

The data obtained on the activity of liver enzvmes sh& that the

influence of cyc A is more on SGOT than on SGFT. ministration of the

reference and test preparations of cyc A at the lower dose did not

alter the level of SGPT significantly on different days of ssmplinq.

However, at the higher dose there was significant increase in its level

from day 7 to day 14 (D 0.01). In the case of SGOT, all the animal

groupa showed siqnif icant increase in its level (o 0.01) from day 0

Page.. 90

Table: LO. @~~fwfpcyCf ;tg;pFinAand blood urea i n r a U

Treatments Creatinine (mg/dl)* Urea (mg/dl)* b day 7th day 14th dav 0 day 7th day 14th day

Control 0.655 0.750 0.732 25.25 26.00 25.25

Standard reparation 0.592 0.783 0.980 24.85 29.72 36.66 15 q / k q

Standard oremration 0.615 1.045 1.210 24.89 34.07 38.09 24 wkq

Test preparation 0.655 1.045 1.105 25.25 26.51 35.02 15 mg/b

Test preparation 0.710 1.100 1.199 26.25 28.80 35.05 30 mg/k¶

- --

* A l l values mean of six observations.

%blet l le Sanm lwd Of and SGW i n ra t s treated vith cyclosporine-A '

R.stamts SGOT units* XPT units * ( ns6 0 day 7th day 14th dav 0 day lth dav 14th day

Control 42.10 43.68 42.51 24.05 24.80 25.53

Standard memra t im 43.10 47.52 50.51 23.10 23.81 24.80 15 mg/kg

Standard rn%o6ration 40.25 55.68 58.55 24.80 24.70 29.52 30 mg/W

Test preparation 42.89 44.64 49.25 21.44 24.80. 26.25 15 -/kg

Test prewration 42.90 48.00 56.25 21.61 24.80 28.35 30 -/kg

* A l l values mean of slx obsetvatrons.

Page.. 92

to day 7 and from day 7 to day 14, except the one treated with 15

mq/lrg/&lv of the test preparation; in this case significant incream

was discernible from day 7 to day 14.

The. data on the serum level of cyc A are qiven in Table 12.

Analysis of the data s h a ~ e that there are significant differences (W

0.01) in the serum level of cyc A between preoarations, doses and days.

In both the reference and test preparation treated animal groupsr there

was an increase in the serum level of cyc A from day 0 to day 7 and

fran day 7 to day 14. And the serum level in the animal group

administered with the test preparation was alwavs higher than in the

animal g r o w treated with the reference oreoaration for any given

dosage.

m e experimental animals showed no sign of qinqivitis, diarrhoea,

fever and behavioural abnormalities. All the animals resmnded to

stimuli normallv. Table 13 shows the data on the body weight of

animals, in the control and experimental grou~s during the study ceriod.

m e r e were significant differences in the weight gain of animals

administered with both the preparations and doses compared to the

control on different days of observation (Fig.13).

DTH reactions:

9 Drug vehicle treated animals innunired with 10 HRBC and

RestmntS Cycloswrine A (nq/rnl)

(n-6) 0 day 7th day 14th day

Control ni 1 nil nil

Standard preparation nil 55.0 80.0

15 -/kg - +1.85 22.40

Standard orewration nil 110.0 127.7

30 &kg - +3.82 2 3 . 0 1

Test reo oar at ion nil 80.0 95.0

15w/kq - +2.55 22.83

Test prewretion nil 118.7 141.7

30 -/kg - + 2.92 2 2.67

Page.. 94

Tuble:13

Effect of cyclosparine A on the body weight

Group

( n a )

Body weiqht lq) Weight gain *

Day8 (¶)

0 3 6 9 1 2 1 5

Control 83 93 103 115 122 130 47

Test preparation 103 116 117 122 128 136 33

(15 m3/kg)

Standard preparation 88 100 104 110 115 121 33

(30 mg/kg)

Test preparation 105 114 118 125 130 137 32

(30 mg/kg)

* All values rnean of six obsenrations.

Page.. 95

Post Grafting Period in Days

F i g . 13 . Effec t of Cyc. A on the body weight Rain.

V - Control group - Reference preparation ~f Cyc. A ( 1 5 rnglkg.1 - Standard preparation of Cyc. A ( 1 5 mgIkg.1

0 - Reference preparation of Cyc. A (30 mg/kg.) 0 - Standard reparation of Cyc. A (30 mg/kg.)

Page. . %

Control Test

Fig.16. Ef fec t of Cyc. A a t 200 mg/kg. on DTLl reac t ions . Results are mean t SD nbtained from groups of 6 mice.

Page.. 97.

challsnp.8 96 h later failed to exhibit lYRl r e m s e (mean footDad -1

welling ( 4 2f1-m 1. Administration of 200 mq/kg of cyc A 48 h before

imnunization resulted in good footpad swelling reactions (fig.14).

Serum antibody titers:

I n the drug vehicle treated grouD, the serum antiboby level was

elevated as seen by the agglutination at 1/1200 dilutions. Hovever, in

the cast of w c A treated group, the circulating level of the

antibodies were reduced as seen by reduction in the antiboby titre

(1/W dilution).

Discussion

Results of the HPLC analysis as well as the IR soectral data of

the standard and test oreparations of cvc A, clearly show the

similarity of the two ~reparations. Further, analysis of the amino

acid profile of the two campounds after acid hydrolysis and TLC,

confirms the similarity between these comunds in their amino acid

canposition also. These studies clearly indicate that the structure

and composition of cyc A obtained f r m the indigenous TolyDocladium so.

is identical to that of standard cyc A.

Page.. 90

Cyc, A ~ 8 e s s a n a m spectnm of antifungal activity. T h y

t w i t the growth of M X ~ ~ Q swcies of yearts, mcorales, ascanycetes,

and fmi i a i e c t i (Curwlaria lunatal Neurospora C T M M I etc) - ( ~ s y f ~ 8 1 1976). The test preparation of cvc A was found to possass

antifungal activity as evidenced by a zone of growth inhibiticm around

the filter mpar disc impreqnated with cyc A and placed on Aspergillus

niger plates.

Results on the skin qraftinq experiments with the test and

standard ~rewrations of cyc A indicate that the test premration,

obtained from Tolypocladium SD., is as efficient as the standard

preparation in Dreventinq rejection of skin qrafts in rats. All the

animals of the control group showed qraft rejection. On the other

hand, 83% of the animals administered with the standard oremration at

both 15 and 30 mq/kq/day showed acceptance of qraft. Ihe test group

showed an acceptance rate of 67% at 15 mq/kq/day and 83% at 30

nq/kq/dav (Table), The acceptance rate in all the exwrimental grouos

receivitq cyc A differed significantly from the control (P< 0.051.

Alsol the differences in accewtance rate between the animals treated

with Standard and test preparations of cyc A at both the do- &re not

significant ( P > 0 . 5 ) .

At high doses, cyc A is known to cause neohrotoxicity (Devinefli

et e., 1984: Whiting et al. , 1982) and he~atotoxicitv (Klintmalm - -- al. I 1981 : b u m c i s et al., 1981 6 Atkinson 5 G., 1984). The data of - --

Page. . 99

the WfMmt study, thouqh show a qeneral increase in the level of serum

creatinine and blood urea in the animal groups treated with the test

and reference ~reoarations, a dose dewndent elevation was noticed only

in the case of blood urea. Serum level of SGPT was not significantly

altered at the lover dose. Significant increase could be observed after

14 days of treatment with the higher dose in both the groups. (XI the

other hand, a dose related response for SGOT was observed in both the

groups. According to willebrand et al. (19841, circulating cyc A level -- of 1X)e-49 ng/ml did not cause nephrotoxicity in human subjects, but the

levels of 195235 nq/ml have caused nephrotoxicitv. In the present

study, even a level of IlOfl8 nq/ml was resoonsible for nephrotoxicity

as evidenced bv marked increase in serum creatinine and blood urea

levels. This mav be due to differences in the threshold level of

circulatinq cvc A reauired for causing ne~hrotoxicitv in different

experimental animals. Thus, the results are indicative of n e ~ h r b and

hepatotoxicitv, as observed in other animal svstems.

Farthinq 5 g. ( 1981 1 have remrted that hiqh doses of cvc A

dministration (50 mq/kg body weight/day) over lonq wriods of time

resulted in loss of body weight which may be due to qeneral toxicity.

In the present study, there were significant reductions in the body

weight gains in the animals treated with both the oremrations of

cyc A. These results along with toxicitv s ~ t m s observed in the

liver enzymes indicate that the reduct ion in the body weight gain may

be due to impairment of the liver function.

Page.. 100

WC A Ore-treatment is known to prevent hiqh dose SRBC-induced

s u w s l s i m of DTH (Webster 6. Thanson, 1987) and reduce the antiboby

praduciW earncity. In the Dresent study, it was found that,

administration of the test reparation of cyc A at 200 mg/kg body

weight resulted in good DTH response accompanied by reduction in the

circulating antibody level. This study clearly shows that the test

preparaticn of cyclosoorine obtained from the indigenous strain of

Tolypocladiirm so. is as potent as the standard cvc A in terms of its

biological potency.

Page.. 101

3.9 gkudir on the biosvntherris of cyclosmrine A.

3.3 .l W#81:aCteri2ation of S-Menosvlmethionine svnthetase

" (1Y11P:bmthionina S-admosyltransferaw, E.C.2.5.1.6) and

cvclorrobrine synthetaw from Tolmladium SD.

lntroduct ion

W e A is a C V C ~ ~ C oartially N-methvlated undecawDtide

containing the unusual amino acids .(-amino butvric acid, Palanine,

and (2St 3R, 4Rt 6E)-3-hvdroxy-4-metfivl-2-methvl-amino-6-octenoic

acid (C9-amino acid). These facts, alona with the broad suectm of

congeners indicate a non-ribosomal biosvnthetic oathwav involving

enzynrtl svstems as in the case of qramicidin S (Kleinkauf and

Koiachuitz, 19781, and enniatin (Zocher e., 19821.

Studio8 canducted by Kobe1 &. ( 1983) using labeled acetate

and methionine indicate that the C9 amino acid is derived by the head

to tail ccdon~tion of 4 acetate units and the methvl qrouw in

cyclo@xxina A originate from S-adenosvl methionine (SAM). SAM acts

as mbthvl donor for the methylation of amino acids during the

biaiynthaais of cvclosrmrine as in the case of other oe~tide

antibiotics (Zocher g g., 1982). This indicates that for the

W t h ~ i 8 of cyclosmrine, an en- system should owrate to provide

the -1 methimine rmired for the rnethvlation of amino acids

Wun5 in wclotmorine.

+ W the pressnt study, two enzymes, S-ladenosylmethionim,

mthrta~ (SW aYnthetasa/methionine activating en-) and

cyc5owwrine svnthetase fran our isolate of Tolwocledim so. were

chrfacterizsd and the probable mechanism of cvclos~orine synthesis

elucidatad. Cvclosmrine svnthetase was detected by measuring the 14

thiol binding of C-leucine to the svnthetase based on the method

of Zinmccr and Laland (1975).

Materials and methods

Chranatagraohv materials:

Saphacrvl - S300 Suoer fine Pharmacia, Sweden.

Sephadex G I 0 Pharmacia, Sweden.

Zorbax bioseries GF-250 qel filtration

HPLC mlunm DuPont, U.S.A

Standard orotein kit for molecular LKB orodukter ABt

Weight detennination bv gel filtration Sweden.

and HPt.C'.

Enz~me inhibitor.

Sodim fluoride

*amtic acid

9-MOnc~yl methionine

Alkaline phosDhatase

Pantathonate

Adanosim trichmhate

Standad amino acid kit

Jercoaine

Methyl leucine

Methyl valine

Phenyl isothiocvanate

rotein in seauencinq qrade)

Radioactive amino acid used:

SQeCific activity 58.7

Pluka AC, Buchs.

Siqma Chemical Co,, USA.

Siqma Chemical Co., USA.

Siqma Chemical Co., USA.

Sicpa Chemical Co., USA.

Sigma Chemical Co., USA.

Sigma Chemical Co., USA.

S i w Chemical Co. USA.

Siqma Chemical Co., USA.

Siqm Chemical Co. , USA.

Bhabha Atomic Research

Centre, India

All other chemicals used were of analvtical grade.

Estimation of SAEI mthetasc enzvme activitv:

SAM synthetaJe activity was estimated bv the method of Cantoni

(1955). The assay is based on the amount of DvrophosDhate liberated

fmIl &TP in the DMtUmCe and absence of methionine.

Page.. 104

No K -A!@ 0.06 N (obtained by the neutralisation 2 2

of the d i d i u m salt 1.

Tris (hv8mxvmethyl) 0.5 M ( recrvstallized frcm ethanol 1.

m i n o methane buffer DH 7.4

W l 1.0 M 2

Reduced qlutathionine 25 rq/ml neutrallized imnediately before

(GSH) use

The assay mixture contained 0.15 ml of ATPI 0.1 ml of water for

the blank or 0.1 ml of L-methimine for the testl 0.2 ml of buffer,

0.25 ml of MgCl , 0.1 ml of GSH and 0.2 ml of the enzyme. The 2

reaction was started by the addition of the en- to the assav 0

mixture at 37 C. After 30 min of incubation, the reaction was

arrested by the addition of 1 ml of 10 8 TCA. The ortho-phoJphate

was estimated in tho test and blank rotei in-free filtrates. The

WZylIm activity was determined by estimtinq the increase ii? &unt

of ortheosphate liberated f m ATP in the presence and abisence of

One unit of enzyme activity is defined as that amount which in the

D t s s e n c e of methionine caused an increased formation of 3y9 of

Paqe. . 105 from ATP in 30 min. Swcific activity was exoreseed as

units Dor nrg of orotein.

~.~.nnination of cvloswrine svnthetase activitv:

CYclwoorine smthetase activity was determined based on the 14

thiol bindinq of C-leucine to the e n z w c m l e x (Zimner and

The reaction mixture contained 100 micromole of

triethanolamincHC1 adjusted to DH 7.6 with NaOH, 4 umle of DTP, 2.5

mole of ATP, 50 mole of maqnesim acetate, 0.2 omole of EDTA and 1 14

mole of the labelled amino acid ( C L-leucine). The reaction was

started bv the addition of the enzyme and all- to continue for 30 0

min at 37 C. The reaction was arrested bv the addition of 1 ml of

10 \ cold TCA containinq 0.5 % sodium tunqstate and 1 millimole of

the unlabelled amino acid (leucine) was added. After centrifugation,

the ~reci~itate was washed twice with 3 ml of 5 % TCA containing 0.25

\ sodium tungstate, once with 3 ml of 2 % sdium sulohate and finallv

with 3 ml of methanol and dried in vacuum. The ~recipitate was

di8~olved in 99 % formic acid (250 ul/rq of orotein) and counted in a

acintillstion counter (RackBeta 1123, LKB) after dissolving in a

toiwna based cocktail.

Page.. 106

Cyc A was estimated by a sliqhtlv modified method of Kreuziq

(1984) usinq a Shimadzu LCdA HPK system. The column used for the

analv8fs was a rWersad ohase C18 bonded silica column maintained at 0

a constant temoerature of 60 + 2 C bv means of an column oven (CPO

6A). Acrtonitrile : water containing 0.1% trifluoro acetic acid

(80:20) was used as the mobile ohase for the elution at a flow rate

of 2 ml/min.

Determination of molecular weight by gel filtration:

For the determination of the molecular weiqht of the enzymes,

Sephacwl-S300 gel filtration column (2.5 x 36 an, P h a ~ c i a K26) was

used accordinq to the method of Andrews (1965). The colurm was

equilibrated with 0.05 M Tris-HC1 buffer containinq 0.1 M NaCl at oH

7.8. The colurm was connected to a Shimadzu LC-6A pum via the

Rheodyne injector ( 2 ml samle low). The outlet of the column was

connected to Shimadzu SPPdAV UV-VIS detector which was set at 280 run

and the chrmtoqram was recorded usinq a Shimadzu CR-6A recorder.

The column was run at a constant f l w rate of 1 ml/min. The elution

volumr, (Ve) for the s m l e and the marker rotei ins and for blue

dextran (which is used for the determination of the void volume ie

Vo) is ' determind from the retention time (1 min = 1 ml as the flow

Page.. 107

i J ~ 1 mL/min). The molecular weiqht standards useB for the

~ S i b m t i a m of the c o l m were: RNase-A (13,700 W),

c2wioLminogan-A (25,000 MW) , Ovalbumin (43,000 HW), Albumin

(67~000 B W ) r Aldolase (158,000 MW), Catalase (232,000 MW), Ferritin

(442rQOQ HW) and ~vroglobulin (668,000 MW) .

The K value was calculated using the follcuinq relationship av

K = Ve-Vo/Vt-Vo av

where Ve is the elution volume of the sample,

Vo is the elution volume of blue dextran (void volume),

Vt is the total bed volume.

The K values of the standard molecular weight markers were av

olottad aqainst their log molecular weiqhts and the graph thus

oMained was used for the determination of the molecular weight of

the unknown orotein.

Molecular weight determination by HPLC on GF-250 colunm:

Protein purification and molecular weiqht determinations were

pl.tf0d by HPLC on a GF-250 column. When comwred to SDS-PAGE,

this method is easy to perform, very fast and highly reproducible for

the Beteminstion o&the molecular weights. Zorbax Bioseriea GF-250

hydroohific qel filtration c o l m (9.4ux 25 cm) was used d t h a

t3hiladzU CC-6A NPIX: svstm. The separation of the different moteins

ir b a d the winciple of qel filtration and the elution time

IretarLSm t i m ) for a qiven molecule is inverselv ~romrtional to

0 it# mlrculsr weight. The elutim was oerformd at 30 C using 0.2

M Na HPO buffer, QH 7.0 at a flow rate of 2 ml/minc with the 2 4

d.tOCtOr wt at 254 ~KII (0.04 AUFS) .

Isolation and wrification of the enzymes from the fungal mycelium:

The funqua, Tolvwxladium so. was cultivated in KX) ml Erlenmeyer

flasks containinq 100 ml of medium with dextrose 4%, wDtone l%,

qlycerol 40, malt extract 20, casein acid hvdrolvsate 3%,

DL-thrmine 0.01% and DL-valine 0.01% (DH 5.2). Cultures were 0

maintainad in a rotarv shaker at 150 nm at 25 C. Samples were

taken at different time intervals and analvsed for biomass (g/l), MAE

activitv (units/nq protein) and cvc A levels (mg/l).

Bianasa yield was worked out by centrifuginq the culture at 5000 nm

for 15 min and expressed as wet weight (g/l).

Prewration of the crude enzvme:

Rte late growth ~hase mycelium (10 daVs old ) was harvested by

mtrifwgatim at ran for 20 min. The mycelium was washed 4

QSrY. vfth normal baline and rmicatd in a buffer containing 0.2

%?Lr#C1, pH 8.0, 0.3 M KClr 0.5 M EDTAI 1 mM M~nyIntharacMul-1-

f 1 . u o t i k r 10 nM MgCl I 10 nN lYIT and 40% (w/v) glycerol. h i s wss 2

cmKrf Luq.d at lot000 nm for 30 min to remove the cell debris and

i Jm d~tmmstant was taken as the crude enzw.

0 To the c N d e e n m 1 solid amnonium sulohate was added at 4 C

slowly with constant stirrinq to a final concentration of 60 $

(w/v). This was stirred in the cold overnight after which it was

centrifuqed at 15000 m for 45 min. The precioitate was redissolved

in minimum volume of the buffer and dialysed against distilled water

overnight. The dialysed en- was lyoohilized and used for further

curification on s Sephactvl S 300 gel filtration column.

Chranatograthic procedures:

Por the wrification of the enzymes bv gel filtration on C

8.clracN1 S300, the cho~matograDhic media were wcked on a PhaMcia

K-26 d m with end fittings. The colunm (2.6 x 36 an) was

oquilikated with 50 mM Tris-HC1 buffer containing 0.1 H NaCl (gH

7.5). The dialysed ammican sulohate precioitate reconstituted in a

knovn volume of buffer was loaded onto the column ( 2 ml/injection)

a d the elution carried out with the same buffer at a f l ~ rate of 1

Page., 110

&/@in. %llPle addition, elution of the ~ o t e i n 8 ~ maintainance of

mutant flaw rate, mitorinq at 280 nm of the elution of the

pootei~ and r-rding of the elution profile were all wrfonned

wing a S h i d z u 6A HPLC svatem. Fractions of 5 ml (5 min/fraction)

were collected usinq a Haake Buchler fraction collector (mcdel-1001

USA:.

SAM svnthetase activity and cvcloseorine svnthetase activity were

determined in the different fractions by the methods of Cantmi

(1955) and Zimner and Laland (1975) rewctively as described

earlier. Active fractions of both the enzymes were pooled sewratelv

and lvochilized for further purification on a GF-250 HPLC qel

filtration column.

Purification by gel filtration using HPLC on a GF-250 calm?

Both the enzvmes (SAM synt hetase and cvclos~orine synthetase)

were further purified bv gel filtration on a GF-250 column using a

m. Zorbax Bioseries GF-2% gel filtration column (9.4 mn x 25 an)

Mas used in a Shimadzu LC-6A HPK system. S m l e s were loaded onto

the calm by means of a Rheodyne injector (200 ul) and the elution

Was carried out using 0.2 M Na HPO buffer, OH 7.0 at a constant 2 4

flaw rate of 2 ml/min with the detecior set at 254 nm (0.04 AUFS).

Page.. 111

Mia fractions ~ t e led^ lyorrhilized, and used for further

bioetmical characterization.

~iochrmical characterization of SAM synthetaae enzyme:

All biochemical characterizations were carried out usinq the HPLC

purified enzvme orewration.

me enzvme activity was determined based on the method of Cantoni

(1955).

For the determination of the enzyme activity at different DH, the

lyoohilized en- was reconstituted in individual buffers having

variws OH values. For the determination of the enzvme activity at

different temoerature, the assay mixture was incubated for a constant

time (30 min) in water baths maintained at different temwratures.

Metal ions and inhibitors were premred at 20X concentration and

their influence on the enzyme activity was studied bv adding the

inhibitors and metal ions (5 N 4 ) alonq with the assay mixture and

estimating the residual enzyme activity after a constant time (M

min). C

Biochemical characterization of cyclosporine svnthetase en-:

Th. biochemical characterization was carried out using the HPLX:

=if ied enwm prenarat ion.

Page.. 112

IPh. enzwe activity was mitored according to the -had of 14 Z ~ Q ~ R M 8nd Laland (1975) b a d on the bindinq of C-leucine to the

ensym.

Effect of DH an the thiol binding of amino acids to the synthetase:

In order to study the influence of pH on the extent of thiol

bindinq of amino acids to the cyclosporine svnthetase, the enzyme 14

assay using C-leucine was done at different pH and the amount of

radioactivitv ' bound to the enzyme was estimated bv counting the

radioactivity using a liquid scintillation counter.

14 Influence of methvl leucine on the binding of C-leucine to the

cyclosporine synthetase:

The cametition betwen methyl leucine and leucine for the thiol

bindinq sites on the cyclosmrine synthetase camlex was studied by 14

mrforminq the thiol binding of C-leucine in the Dresence and

absence of methyl leucine (unlabelled) at two different

concentrations of 50 uM and 100 uM. If methyl leucine ccmpetes with

leucine for the bindinq sites on the enzvme, there would be a

decrease in the radioactive count in the enzvme after orecipitation.

Page.. 133

l W *nrynw activity was mitorod according to the method of 14

Zirar)r M kland (1975) baaed on the binding of C-leucine to the

mzyar*

effect of PH on the thiol binding of amino acids to the svnthetase:

In otdcr to study the influence of DH on the extent of thiol

binding of amino acids to the wclos~arine svnthetaee, the en- 14

atmay ursinq C-leucine was d m e at different DH and the amount of

radioactivity bound to the enzyme was estimated by counting the

radioactivity u~inq a liquid scintillation counter.

14 Influancc of methyl leucine on the binding of C-leucine to the

cyclcmgxrine synthetaae:

Ihe canoetition between methyl leucine and leucine for the thiol

binding sites on the cyclosporine svnthetase conwlex was studied bv 14

perfonninq the thiol binding of C-leucine in the Qresence and

~bsence of methyl leucine (unlabelled) at two different

concentrations of 50 uM and 100 uM. If methvl leucine c-tes with

leucine for the binding sites on the enzyme, there would be a

&crease in the radioactive count in the en- after oreci~itation.

14 ~ # Q h t i C m of bound C-leucine to cycloawrine emthetam! enzyme:

m i 6 wss done based on the method of ~wkoski G. (1970) with 1 ~ # , laodifications. Reaction mixture containinq 200 ul of the en-,

14 200 u l of 0.1 M Trisr Dn 7.5 and 100 moles of C-leucine were

0 incubated at 37 C for 30 min. The mixture was chilled in i~er

throuqh a Seohadex G 1 0 column (35 X 0.8 cm) and eluted with

h i s buffer. The eluate containing the e n z w rotei in was collected,

0.1 m l of it was used for determination of radioactivity and the

remaining treated with trichloroacetic acid (TCA final concentration

109). Carrier rote in (albumin, 0.21111 of 0.5%) was added to aid in

the carplete ~recioitation of the e n z w rote in. After 20 min, the

precipitate was centrifuged at 8000 rcm for 30 min, washed once with 14

=A, lyonhilized and the enzyme bound C-leucine estimated using a

scintillation counter.

Determination of 4'~hosphowntetheine in the cvcloswrine svnthetase

enzyme:

lbe premce of 4'ohas~hooantetheine in the en- c w l e x was

..tiamtad by the method of Zocher g. (1982) wigh 'sane

modifications. Enzyme fractions were heated with KOH (final

atmmntration of IN) in a steam bath for 1 h to aerate the 1

4 SJwJsphmetheine fran the enzyme canolex. The . VH of the'

Page.. 115

G i & b Van adc~ted to 8.a with conc. HC1 and treated with alkaline

DhOBOhsta~ for 3 h at 37 C to liberate the free oantothenate. lhis

was estimated bv a HPLC on a C 18 column reversed ohase banded silica

cohnm I 4 . h X 25cm) using 0.1 M KH PO , OH 5.4 as the mobile 2 4

chaw at a flw rate of 2 ml/min with the detector set at 214 nm.

Role of thiol directed aqent, Iodoacetic acid on the enzyme activity:

Thc effect of Iodoacetic acid on the thiol binding of 14 C-leucine to the enzyme was carried out bv incubating 5 nt4

cancentration of idacetic acid (IAA) alonq with the assay mixture

and estimatinq the bound radioactive leucine to the enzyme. For the

estimation of the extent of inhibition and time taken for maximum

inhibition, IAA (5 mM) was oreincubated with the enzvme for various 14

time intervals and the amount of C-leucine bound to the e n z m

after the e n z w assav was estimated (exmessed as cun of 14 C-leucine).

Detection of amino acid methyltransferase activitv in the e n z w

c m l e x :

Reagents:

S-Me-1 mthionine (SAM) : 5 mg in 2 ml HC1 DH 3.0 (for stability)

*is (hytlroxv wthvl) amino

methane : 0.5 H OH 8.0

Page.. 116

A&NW1 tr.iUhWfhte (ATPI : 0.06 f l OH 7.4 (adjwtad with IUMJ - % *,

A ' 'ho wthvltransferase activity of the e n z w was observed by

detwtfng th. conversion of amino acids into their corresponding.

methyl mino acid8 in the presence of S-adenosvl methionine (SAM).

TVm amino acids and their corresconding methyl amino acids after

hrivsti~tion to PTH-amino acids (Lamaire g., 1988) were

detected by HPLC using a PTH-colum.

m e assay mixture contained the 200 ul of the e n z w , 50 ul of

SAM, l m ul of adenosine triGhosphate (ATPI, along with 100 ul of the

amino acid (individual amino acids present in cyclosoorine molecule) 0

with the OH adjusted to 7.5 and was incubated at 37 C for 30 min.

'Ihe reaction was started bv the addition of SAM hich was added only

at the last. After incubation, the enzyme was precipitated with TCA

( f i ~ 1 concentration 10% centrifuged and the methyl amino acids

estimated in both the suwrnatent and the orecioitate. The enzyme

bound amino acids were extracted for derrvat~sation and detection,

b a d on the method of Froyshov & &. (1970).

Cleavage of the bound amino acid from the enzyme c m l e x bv wrformic

acid:

14 Th. C-leucine bound to the enzyme was released from the

enzyme canolex by verformic acid cleavaqe based on the method of

FW~shov c. (1970).

Page.. 117

"4% llllSMU Drecipitate was dis6olved in 99 % fonnic acid and left v &i%ti4! 10 min. 100 ul of wrformic acid w r ml of formic acid

0 t& 'Wed t0 it and the reaction allwed to ~roceed at 0 C for a

further oeriod of 2.5 h. Performic acid was reo oared freahlv before

It 0r-d by mixing 1 ml of hvdroqen wroxide and 9 ml of

99 % .formic acid and allwing the mixture to stand in a dark closed

bottla at roan temDeratur* for 2 h before use. The mixture was

di&d 10 times with ice cold water and freeze dried. Thf residue

wsr extracted 3 times with a mixture of ethanol/0.2 N HC1 (9 : 1,

v/v). The rote in residue was dried in vacuum and subnitted twice to

the above extraction ~rocedure. The ccmbined extract was evmrated

to drvnem, dissolved in a small volume of ethanol/HCl and used for

the detection of the amino acids by a HPLC procedure.

Datection of methvl amino acids bv HPLC after derivatization with

phenyl ieathiocvanate.

Derivatieat ion reagent: This was prewred bv mixing Phenvl

isathiowanate (protein sequencinq grade), ethanol, triethanolamine

and water in the ratio of 1: 7: 1: 1.

110 ul of this derivatiaation reagent was added to the saple and 0

t$l. reaction was all& to take place at 22 C for 20 min under

nStr;oem. Thm, 1 ml of methanol was added and the mixture dried

Paqe.. 118

d' hi41 vacuum. This steo was receated once mare to remove the 3lmootllcti.s. The vacuum dried sample was redissolved in mininun

Mu#, at distilled water and taken for HPLC analvsis.

Ekt.ct ion of PTH-amino acids by HPLC:

For the detection of PTH-amino acids, Shimadzu LC 6A (Jamn) High

oressure liquid chraatqraob was used. Derivatized s m l e s were

loaded on a PTH column (Zorbax PTH column, 4.6 m x 25 an, mPont)

and eluted with a qradient solvent svstem consistinq of acetonitrile

: water containinq 0.1 $ WA (80 : 20) and 0.1 M amnium acetate OH

3.5: water (15 : 35) at a flow rate of 2 mllmin at ambient

temrerature (qradient oroqram shown in the fiq. 1. The methyl amino

acids were detected based on their retention time as comared with

that of the standard amino acids.

Detection of methyl amino acids in the amino acid ~ool of the

mvccliun:

At any stsge in the growth of the fungus Tolv~ocladium so., the

rmmnce of the corre-nding methyl amino acids of cyclosporine

molecule, was checked after extraction of the m l i a l ami'no acids

according to the method of Sutherland and Wilkinsm (1971) and

&t&& after derivatiaatian with Phenylisothiocvanate.

Page.. 119

tha extraction of the amino acids fran the mycelium, the

~ 1 i U f R micated in 10 % TCA after which the cellular debris

uu ram0v.d bv centrifugation at 15,000nxn for 1 h. The clear

swrrnetmt was extracted with ether several times until the OH of

the sQueou8 ~ h a w reached 5.5. The aaueous phase was then

lyoOhilized, rediseolved in minimum volume of distilled water and

taken for the estimation of the amino acid w a l by HPLC after

~ ~ c o l u m derivatization with ohenyl isothiowanate.

Results

Synthesis of SAM svnthetase during the growth of Tolwocladium so.

m e results on the cell qrawth, biomass ~roduction, yield of cvc

A and mycelial SAM synthetase activitv are resented in Fig.15. As

the m$weliurn enters into active qrowth phase, the swcific activity

of SAM mthetase also increased with corresmdinq increase in the

total cvc A levels.

Purification of the enzvmes.

The s t e w involved in the ourif ication of SAM mthetase yielded

an m z h e which was 50 fold w r e than the crude enzvme and the yield

was 399, The crude e n z w shaved many waks on GF-250 HPLC calm.

Page.. 120

0 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3

Tirce of Fermentation in days

Fig. 15. Dynamics of Cyclosporine A synthesis during the fermentation of Tolypocladium sp.

Page.. 121

orecioitatim and further ourif ication on Seohactyl

SKX) gel filtration column vielded three fractions (Fig. 16) of vhich

th8 fraction 2 of molecular weight 29,000 shoved SAM synthetase

rnzynw activitv and the first fraction of mlecular wight of above

MO,OOO shaved cycloswrine synthetase activity (Table 14) (based an 14

the binding of C-leucine). The fraction showing SAM mthetaae

activity, on further ~urification (on a GF-250 HPLC gel filtratian

colum) vielded an enzyme 50 fold w r e than the crude en- with an

yield of 39%. Cyclosmrine synthetase enzyme also showed a highe;

arritv level after ourification on GF-250 HPLC column (mrity b a d

on GF-250 ~rofile) t h q h the ~urification fold was not calculated as

done for SAM evnthetase. Figure 17 shows the GF-250 profiles of

cyclosoorine svnthetase before and after the ourification stem.

Table 15 gives the ourification details of SAM svnthetase enzyme and

Fig.18 show the GF-250 gel filtration ~rofiles of the enzyme before

and after the ourification st-.

Molecular weight of SAM svnthetase enzyme and cvcloswrine synthetase

enzyme:

The mlecular weight of SAM synthetase enzyme as determined by

qelfiltration on Sechracryl S 300 column was found to be 29,000

(Fig.19). By gel filtration on a Zorbax Bioseries c o l m using a

HPX, it was estimated to be 28,850 (Fiq.20).

Page. . 122

Fig. 16. %paration prof i le of Ammoni~lmsulphate precipitated crude enzymc nn rephacryl S3OO colurn.

~ n i ~ ~ ~ ~ s u l p h a t e precipitate was dissolved in 50 mF( Tris - HC1, pH 7.5 containing 0.1 M NaC1. 2 ml of the sample was loaded on to the coluan. Elution was performed in the same buffer.

Pdak 1 exhibited Cyclosporine synthetase act iv i ty - P m k 2 exhibited SAM synthetase activity -

Page.. 123

14 Fatfwtion of C-leucine bound to different e n z w fractions:

14 C-Leucine bound

en- Control Test Test-Control

fraction (CPM) (CPM) (CPM)

Fraction 2 6000

(IW 29,000)

v d u w O X D T ~ a5 counts per milligram of the orotein

CFW - Counts Per Minute.

Anal

ytic

al co

ndit

ions

:

Coluom

: Zor

bax-

Bioseries

CF-250

Mobile Phase : Na2

HP04

- 0.m.

pn:7.0

Flow Rac

e : 2 mllain.

Detector

: 2

54 ~

m, (O

.W m

~)

Page.. 128

Retention Time (Xin . )

F ig .20 . Molecular weight determination by HPLC on GF - 250 column. P r o f i l e of prote in t e s t m i x t ~ l r e .

Analyt ical Conditions: Peak Ident i ty

Column : Zorbax - ~ i o s e r i e s , GF-2.50 1 . Thyroglobulin Plobi lePhase : N a H P O - 0 . 2 M . p H : 7 . 0 2 . Ferr-itin Flow Rate 2 4

: 2 ml/rnln. 3 . Albumin Detector : UV 254 nm (0 .04 AUFS) 4 . Ovalbumin

5 . RNase

Page.. 129

'Rm nrolecular weiqht of c v c l o s w r i n e svn the ta se was es t imated to

be 700,000-720,000 bv g e l f i l t r a t i o n on Seohacrvl S300 chrcmatograDW

( 2 and more than 700,000 bv GF-250 HPLC q e l f i l t r a t i o n

&ranatograahv (Piq .22) .

m r a t u r e and OH ootinnnn of SAM svnthetase enzvme:

The enzvme was found t o have ootinnnn a c t i v i t v over t h e OH range

7-9 wi th maximum a c t i v i t y a t 8.0 (Fiq.23). The inf luence o f 0 0

temperature on t h e e n z w over t h e range 4 C t o 70 C indicated 0

t h a t t h e e n z w was not s t a b l e a t higher t emwra tu res (above 40 C) 0

and had a o o t i m a c t i v i t v a t 37 C (Fiq.24).

E f f e c t of i n h i b i t o r s on t h e a c t i v i t v of SAM svnthetase e n z w :

Addition of NaF and EDTA a t 5 I'M concentration inh ib i t ed t h e

enzvme a c t i v i t v cormlete lv and IAA a t 5 mM concentration inh ib i t ed

33% o f t h e a c t i v i t y (Table 16 ) .

+t .++ Requirement of Mg and Mn f o r SAM svnthetase enzvme a c t i v i t v :

++ A 3 i n t h e c a s e of o t h e r ATP r e q u i r i q enzvmes, Mq is r w i r e d

u

f o r ootinnnn enzyme a c t i v i t v . Subs t i tu t ion of Mn res tored some

a c t i v i t y but n o t camletelv (Fig.25). This s tudv a l o q with * i n h i b i t o r s t u d i e s shows t h a t t h i s e n z w is a W reqoir inq

, Page.. 130

Retent ion time b i n . )

6'

5 . 5 -

5-

4 . 5 -

4 -

3.5

FIg.ZOL22. Determination of molecular ve ight by HPLC on GF - 250 colwrm.

Log molecular weight vs. Retention time

Thy 0

SAM synthetase enzyme

I I I I I c

Thy - Thyroglobul i n Fer - F e r r i t i n BSA - Bovine Serum Albumir Oval - Oval bumin RNase-Ribonuclease A

4 4 . 5 5 5 . 5 6

Page.. 131

Fig.23. pH a c t i v i t y p r o f i l e o f SAM synthetase enzyme.

Lyophi l ized enzyme was disaolved i n buffer s o l u t i o n s o f var ious pH va lues . The a c t i v i t i e s were determined by us ing the same buf f er . The buffer used for the study were 0 . 2 M a c e t a t e buffer (pH 3-61, 0 . 2 M Tris - HC1 buf f er (pH 7-91. and 0 . 2 M Carbonate buf f er (pH 9-11).

Page.. 132

h m Y

'S too 1 -

Temperature ( O C .

Fig.24.Temperature activity profile of S k . synthetase enzyme. The enzyme activity was determined at different temperatures as describpd under Materials and Methods.

Page.. 133

mler 16

eifect of inhibitors on the activity of S M synthetase.

Inhibitor Concentration % Inhibition

-

Sodium flouride 5 mM 100

Page.. 134

NO m e t a l ion ng++

Pig . 25. Ef fec t of lwta l ions on SAM synthetase a c t i v i t y .

A l l metal ions were added a t 50 mH concentration.

crrlw. Inhibition of the enzyme by IAA which is a thiol directed

.gmt, indicate that -SH q r w w are nresent in the enzyme which are

required for the activity.

14 ImlatiaI of C-leucine fran Cycloswrine svnthetase:

14 When the e n z m was incubated with C-leucine followed bv gel

filtration on a Sethadex G10 colum and ~recioitation with TCA,

radiosctivitv was detected in both the TCA suoernatant and

oreci~itate.

pH of cvclosvorine synthetase:

14 #he DH o~timum for the thiol binding of C-leucine to the

cyclosobrine synthetase was found to be between 6.0-8.5 with a

maximnr at 7.5. Below and above these OH, the bindinq of the amino-

acid was found to be reduced (Fiq.26).

The e n w w s h d the oresence of 4'DhosDhowntotheine (Fig.27).

Inhibition of cvclos~orlne synthetase bv iodo acetic acid:

Cvcloswrine synthetase was inhibited bv the thiol directed

agent, IAA. Addition of IAA along with the incubation mixture

inhibited 90 6 of the activitv. When it was re incubated with the

Page. . 136

F i g . 2 6 . pH a c t i v i t y p r o f i l e of Cyclospor ine s y n t h e t a s e enzyme

L y o p h i l i z e d enzyme was d i s s o l v e d i n b u f f e r s o l u t i o n a t v a r i o u s pH v a l u e s . The a c t i v i t i e s were determined by u s i n g t h e same b u f f e r . The b u f f f r used were 0.2H a c e t a t e b u f f e r (pH 3-6). 0.2M T r i s - HC1 b u f f e r (pH 7-91 and 0 . 2 1 Carbcna te b u f f e r (pH9-11) . Enzyme a c t i v i t y was de te rmined a s d e s c r i b e d under M a t e r i a l s and Methods.

Page.. 137

Analytical Conditions:

Collrmn: RP C18 bonded silica ( 2 5 cm x o.6 m) Flowrate: 2 ml/min. Mobile phase: 0.1 M KH2POG, pH 5 . 4 Dcteccor: U V 2lL nm.

@WW#O tor different time weriods, the activitv decreased as the time ~CKW- (Pfq.28). This shown that the e n z m contains active -SH

group. which are e s ~ t i a l for the enzyme activitv.

Amino acid methvl transferase activitv in cvclosmrine svnthetase

enzyme:

Amino acid methvltransferase activitv was detected in the

cycloawrine svnthetase complex based on the conversion of the amino

acid. glvcine, leucine and valine to their corresoondinq methyl amino

derivatives. The correswndinq methyl amino acids were found to be

bound to the enzyme when it was orecioitated with TCA (Fiq.29) and no

methyl amino acid was found in the TCA supernatant.

Presence of methyl amino acids in the mvcelial amino acid wol:

Wnen the oresence of methyl amino acids in the mvcelial wol was

checked after their extraction from late loq ohase mvcelium, none of

the methvl amino acids Dresent in cycloswrine molecule (M-lycine,

M-valine, M-leucine) were oresent in it (Fig.30).

Ccnpctiticm between leucine and methvl leucine for bindinq sites in

cyclosrmrine svnthetase enzvme:

0 10 20 30

Time of Incubation i n Min.

F i g . 2 8 . E f f e c t of Iodoacetic a c i d ( IAA) on Cyclosporine synthetase a c t i v i t y .

Enzyme a c t i v i t y based on 14 binding was estimated C-leucine

a f t e r preincubating the enzyme with ULA (5 mM) f o r d i f f e r e n t per iods of t ime.

Paqe.. 140

Fig.29(a) . P le thy l t ransferase a c t i v i t y of cyclosporine synthetase enzyme. Conversion of g lyc ine t o sa rcos ine : A. Standard sa rcos ine B. Enzyme c o n t r o l C. Enzyme t e s t ~howing t h e presence of sa rcos ine

Analy t ica l condi t ions :

Co 1 umn : Zorbax PTH column x 4 . 6 1 4

Mobile Phase: A. A c e t o n i t r i l e : water (0.1% TFA) (80 : 20)

B. APrmonium a c e t a t e , 0.1M, pH:3.5 : water ( 3 : 7 )

Gradient : Time (min) B concent ra t ion 0.01 50

10.00 50 Flow Rate : 2 ml/min. Dctec tor : 254 nm (0.01 AUFS)

Page.. 141

Page.. 142

Paqe.. 143

F i g . 3 0 ( a ) . Detection of methyl amino ac ids in the mycel ia l amino ac id pool of Tolypocladium sp.

A . Stardard methyl g lyc ine (RT 3 .89 ) B. Hycel ia l amino ac id pool

Analyt ical condit ions:

Sam a s given in F ig .29 (a ) .

Fig,3O(b). Detection of methyl amino acids ln the mycelial amino acid pool of Tolypocladium sp. A. Standard methyl amino acids

Methyl valine (RT 6.602) Nethyl keucine (RT 8.317)

B. m c a l i a l amino acid pool An.lytic.1 conditionr: Same a s in Fig.3Ob)

Page. . 145

14 When the * I I I O ~ blridinq of C-leuclne to the cvcloswrine

synthstssa was 1.3rried out in the presence of methvl leucine, there

was no s iqnf f i~~br l t reduction in the radioactive leucine bound t o the

rnzym i n the :,resence o f methyl leucine a s evidenced bv no

difference i n the rcdioactive count between the smles (Table 17).

Page.. 146

Table: 17

14 Competition between C-leucine and methyl leucine for binding

sites in cvcloaaorine synthetase:

14 C-Leucine bound

Amino acid (s) Control Test Test-Control

'he results on the growth, SAM mthetase enzyme activity and

cYc A yield that as the SAM mthetase activitv increases there

i 8 a ~ d l l m m d i n q increase in the yield of cvc A. This study

establishes a role for SAM synthetase in the svnthesis of cyc A. SAM

synthotase $6 involved in the synthesis of S-adenosyl raethionine

(SIW) which is the methyl donor for the methvlation of amino acids of

cyc A. Hance it may be concluded that SAM is suoolied by the action

of this enzyme for the methvlation of amino acids in cvc A. Also it

is found that SAM svnthetase activity is not associated with the

cyclomrine s~nthetase activity which indicates that this enzyme is

cumartmentalized at some other site in the mvcelim.

The ~urification of the SAM svnthetase from'the funqal mycelium

of Tolyaocladium so. yielded an enzyme which was 50 fold wrified

than the crude e n z w vith an overall yield of 39%. The molecular

weight of this e n z w as determined by Sechacntl S-300 gel filtration

was 29,000 and by qel filtration by HPLC on GF-250 column was

28,850. The e n z m was inhibited by sodium fluoride, EDTA and

iodoacetic acid which indicates that it reauires metal ions and

active -SH qror~ce for its activity. Further studies s h m that the ++

en- requires Pig for its activity but its action was restored +t

to sane extent by the addition of Mn . These reeults are in close C

Page.. 148

s g m t with the vrooarties of SAM svnthetase fran rat liver

(Cantoni, 1955).

Ih. emme showed a OH optimum between 7 and 9. Ma x i m activity 0

we8 notad at DH 8.0 and at 37 C.

Zochet e. ( 1986) have reported an enzyme fraction frqm the

crude extracts of :.inflaturn that was able to catalvze the activation

of the constituent amino acids of cyc A. They have demonstrated the

N-methylation of the amino acids present in the peotide chain as in

the case of emiatin (Zocher & g., 1982) but t h w were not able to

demonstrate the c m l a t e synthesis of cvc A. Billich ad8 Zocher

(1987) have subsequently remrted an enzyme fraction fran T.inflatun

with a molecular weight of 700,000 which was able to synthesize

complete cvc A molecule in vitro in the presence of the constituent

amino acids, S-adenosyl methionine and adenosine triohosdate. The

enzyme was detected based on the D-alanine dewndent ATP/PPi exchange

( W a r s g., 1968). Lawen 2. (1989) have recently reported

the cell-free svnthesis of new cyclosoorine analoqs with the helo of

the multifunctional enzvme canolex.

In the oresent study Cycloswrine synthetase was wrified fm

the Tolwocladium sp. and the enzyme activitv for the total synthesis

of cyclcmporine resided in one polywptide chain with a molecular

weight of about 700 K% as elready reported (Billieh and Zocher,

1987) in contrast to other antibiotic synthetases, eq., qrmicidin S

Page.. 149

Tho e n z w activity warr detected based on the thiol binding of 14 C-leucine to the enzyme carplex. The thiol binding of the mino

acida wa8 further confinned by the Dresence of rsdioactivity in both

the TCA ruoernatant and ~recioitate. These results are similar to

thoae fwnd in tho qramicidin S system (Kloinkauf and Gevms, 1969)

and are consistent with the notion that the amino acids are bound to

the e n z w s both noncovalentlv as minoawl adenvlates and covalentlv

by thictester linkaqes. As in tyrocidine svnthesis, each enzyme

fraction reacts with one molecule of the substrate amino acid and one

mlecule of ATP to form an intermediate aminoacvl-enzvme carplex and

inorganic Dvroohos~hate. The adenylate bound amino acid is then

transferred to an enzvme-bound sulfhydryl qrouD to form a thio-ester

with the 1 iberat ion of adenosine mono ~hoschate (AMP). Precipitation

of the e n z m with TCA would be exwcted to discharqe the adenylate

but not the amino acid bound as thio-ester, which results in the

presence of radioactivity in both the TCA ~reci~itate and summatent

in our studv. Hence, for further studies on the activity of the 14

cyclosoorine synthetase, the thio-ester binding of C-leucine to

the e n z w was measured.

The gH o o t i m of the enzyme was found to be 6.0-8.5 with a DH

maxinm at 7.5. This correlates with the observatim that the

w ~ & ~ i c m of cvc A increases raoidly during the late qrowth Dhasa

and stationam ~hase of the fungus when the oH of the culture

incrc~os ebwe 7.0.

Measurament of thio-ester bindinq of radioactive leucine in the

p r w of methly leucine shws that methvl leucine did not c w t e

with lwcine indicating the specificity of the binding sites on the

enzynw. Thus, the amino acids of cyclosmrine bind to the enzvme

through thio-ester linkages where t h w are methylated either

imnediately or after the cwletion of w ~ t i d e svnthesis. However,

the r m by Zocher al, (1986) that a diketopiperazine

cyclo(D-ala-N-methyl-L-leu) was obtained £ran D-alanine and L-leucine

in the presence of ATP and S4M suggests that methvlation occurs

imnediatelv after bindinq of the amino acid to the enzyme by

thio-eater linkage.

Methvltransferase activity vas detected in the cyclosoorine

synthetase and it was found that the methyl amino acids were bound to

the enzvme when precioated with TCA. The e n z w bound methyl amino

acids were detected only after liberation of them fran the enzyme by

~rfonnic acid cleavaqe. This clearly indicates that methylation

takes dace at the stage of thio-esterif ication, vielding N-nethyl

amino acids as in the case of enniatin svnthetase re~orted from

F u m r i m oxyswnsn (Zocher r)_ g., 1962). -

indicates that the amino acids are bound to the synthetase

by thiol bods here thcy are methylated to their comesoonding

-thy1 .imlitI0 acids by the svecific methyltransferases oresent in the

WIthet68a and the methylatd amino acids remain bound to it for

further W i d e elangation.

Further stdies on the presence of free N-methyl amino acids in

the mycelial amino acid pool shows the absence of any of the methyl

mino acids present in the cvclosmrine molecule which further

confinas that free amino acids are not c resent in the myceliun and

that methylation of the amino acids takes place onlv after bindinq to

the e n z w canolex.

The oresence of the 4'&08&1aJantetheine residue (s) covalently

attached to the enzyme is not clear but may be wrt of the elonqation

system involved in the oeotide svnthesis.