ENZYMA'l'IC REMOVAL PAINTINGS Frantisek Makes

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LINING PASTE FROM

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gin was adsorbed on polyethylene glycol 1000. This thick mass with the amylase was used for removing the paste from the surface of the picture. The final inhibition was performed by means of menadion in a solution of propanol by spraying into the back of the picture.

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Introduction

'l'he conservation of pa nt.Ln s has been hitherq to concerned with the removal of earlier conservation materiaIs. For example pastes used \,~ for the lining of paintings. This lining tech~ nique uses severa 1 paste mixtures which peneSUMMARY ~ ( trate into each paint layer of the painting The paste used for the lining of paintings iS.~ t~r~ugh heat.an~ pre~su~e. Therefore~ in t~e absorbed by the canvas, by the ground and ll.nl.ng pal~tl.ngs l.t l.Snot o~ly ~ questl.oTl of often also by the surface of the paint layr. y of st~engthenl.ng the canvas by lron1n~ on,a . ~ new llnen cloth, but also of a consoll.datl.on I It cannot ~e ~emoved.completely by scrapl.ng:" of the paint and ground layers which have only, but at, lS po ssLble to remove most o~ p. I flaked off. There s no panacea in the form ' by m7ans of an enzymatl.c reactl.~n. The paste ~ ~of a paste that can be used indiscriminate1y cons7s~ed.of meal, glue and.resln. M~uld for every painting. The choice of paste is I (Pen1c7ll1um sp. and Aspergl.llus fuml.gatus), determined by the technical character of each i bac t er.aand yeasts taken from the paste were .. 1 b I t t f h d ! inocu1ated on a Sabouraud agar with 1ining ~al~tlng ane y ~,e ex en o t e amage on I paste and peptone water. A filtrate was inl.t. Not cvcry ~nste materi~l (starch, glue, " .' dextrl.ne, casel.n, wax, reSl.ns and so on) can oculated on two speclal suspenslons: a Saboub d' 1 d tI b t t b b' d ' raud agar with glucose and a Sabouraud agar cluse llnCtCdPen.ethn Yth' u mbus e comhl.nhe I,' t with Metocel and starch. Two filtrates from ane emu ga.e Wl o. er su s ances w l.C t these suspensions were separated chromatoc~ange thel.r propertles. How7ver~ these f~rI graphical1y on Sephadex G-l00. The bio1ogica1 e1gn substance~ cause.a t7nsl.on l.n the pa7nt- f ng, makes l.t.l.nelastl.c~ activity of the enzymes found in the filtrates 7 1nduces changes.1n t d t . d f 11 l.tshygroscoplC propert1es and further m1cro- r was e e rtru ne as o ows: b 1 t Lv .t, i proteases 11.3 U = 11.3 ~g/min. l.a ac l.Vl. . y r a-amylase 5.1 U = 5.1 micro-equivalent/min. The removal of pastes of different composicellulase 1.lxl0-2 U/ml. tions is not easy and due attention has not been paid to it. Pastes are removed from the By moistening the paste the cellulase is acback of the painting by scraping. This gives ff. tivated biologically so that it damages the a smooth surface on the back of the painting original canvas of the painting. Therefore, so there i5 no relief that penetrates into the glucose was used as a specific inhibitor of pa n t; J oyc r du rnq Lhe .rollowing ironing. llow-t,' cellulase. ever, a consideralJlc quantity of the old paste ~ is left behind in the painting and a new paste ~ For removal of the paste two systems were of a different character is ironed on the used: pain ting. System I, consisting of wax, benzin, toluene and turpentine were used to prevent This conservation method gives the painting I'. infiltration of system II into the in the course of time a worse condition. This painting, and appears often in a very short time (about 10 System II,consisting of 170 U/mg of lyophilisyears), if a painting is put in unfavourable ed a-amylase of bacterial origin + conditions. This environment creates, however, 20 mAnson units/mg of lyophilised favourable conditions for the development of i.,. proteinase K of funga 1 origin + glumicro-organisms. A painting placed in a more l cose 300mg/l00ml. favourable environment will also be damaged in time, but the process is much longer. '.:'.' The enzymes and the added glucose were adsorbed on carboxymethylcellulose in a phosln our research we have paid special attention phate buffer at pH 7.0. This resulted in a to the remova 1 of pastes by means of enzymatic thick mass used to remove the paste from the reactions. Such reactions can penetrate deeply back of the painting. After the removal of the into the painting. A typical characteristic.of paste the remainder of the glucose was removed these reactions is that they cause relatively by means of a thick suspension consisting of easily a lower activity of the enzyme present / ...... glucose o~idase and catalase, both adsorbed in the pastes. This depends on the complicated on cellulose powder of the type CF 11 in a structure and the specificity of their effect. phosphate buffer at pH 7.5 - 8.0. The inactivation of the pastes can be induced for instance by denaturation, viz. by altering their chemical structures by acting upon t The pain~ layer was consolidated by means of such external factors as the temperature or System I. The back of the picture was moistened with turpentine and benzine. e~r~~~~:ing the environment in which they are A filtration paper impregnated with the same mixture of solvents A successful removal of pastes depends on the was attached to the back of the pieture. The solvents and a presknowledge of paiDting technic and the detailed investigation of every component of the picsure of their vapours prevented thus penetration from the paint ture. layer of products resulting from an enzymatic breakdown of the paste. System lI, consisting of 170 U/mg of lyophilised a-amylase of bacterial ori-

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Examination oE the painting

A new canvas was put on the back oE the painting: The photo shows severa1 spots where the canvas has E1aked oEE, owing to the action oE the enzymes produced by the mou1d which have cleft the cellu1ose. Solubi1ity oE pastes in distilled water

The picture was painted in the 17th century by an unknown Master with oi1 on canvas. According to prior inEormation the picture has been 1ined. ln 1969 tne picture was 1ined again. The photo shows the white spots where the paste is present on the surface of the painting. The paste consisted oE glue, Venet an ba1sam a,,,! mea L.

250 mg oE a paste were put mechanical1y on diffcrent parts oE a ?ainting and the solubility of the paste in distilled water was examined in several samples. The results showed that the solubility varied according to the extent oE mou1d damage on the paste. The highest degree of mou1d damage was found in the lower part of the painting, where 14% of the paste was dissolved aEter 1 hour at 200C. ln the upper part af the painting 6.0% of the paste was dissolved at constant conditions. ln the middle part of the painting 5.2% of the paste was dissolved at constant conditions. The solubility of the paste was determined by means oE a protein from the paste in the Brdicka solution. The guantity af protein was assessed by means of the calibration curve. (1) Test conditions The polarographic experiments were performed with the model E 261 R Po1arecord made by Metrohm Ltd. Switzer1and; the ga1vanometer sensitivity was 4,1.10-9A/mm. Starting voltage: -O.BV. Voltage range: -2.0V. ln experiments with Eree1y dropping capillaries the droptime was adjusted" by choosing a suitable mercury pressure to 2 sec at the voltage of -1.525V, at which the second protein wave a~pears. The Co + ammoniacal buffer solution had the following composition: O.lM NH4Cl, 1M NH3, O.OOlM CoCl~.

Due to the presenc0 oE humidity mould deve 1oped. The mould Erom the paste grew into the cracks oE the paint 1ayer an the back of the picture.

Mou1d cultivaticn The mould present in the paste was found to consist of Penicillium sp, Aspergillus fumigacus, some bacteria and yeasts. The extracel1ular enzymes praduced by them were studied as wcll as t he r power of hydrolyzing pastes, lhe

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canvas and the paint layer. The mould was inoculated on a Sabouraud agar with glucose and peptone water at a buffer pH 7.0 where they grew. The filtrate was.inoculated on two special suspensions: a) A Sabouraud agar with lining paste and b) A Sabouraud agar with Metocel and starch After 5 days, when the growth of the microorganisms was maximum, the microbial mass was removed from the filtration substrate and the filtrate was used for isolation of the enzymes. Method of determining the activity of the proteases present in the filtrate 1. As an operation of essential importance the proteases were separated chromatographically on Sephadex G-l00. The extract was prepared by extracting the pulverized dry substance of the filtrate during 30 mino in a 0.05M buffer with 5 ml sodium acetate and 5 rnl calcium acetate for each gram of dry substance. 3 ml of this solution were put on the colurnn and eluted at a constant buffer. The enzymes were purified by means of ion exchange chromotography on DEAE cellulose of the type DE-52. 2. Since proteases catalyze the degradation of nitrated casein in the sarne way as the degradation of natural casein, 1 ml of the proteases isolated according to point 1 above was mixed with 20 mg of nitrated casein in a solution of 1 ml of 0.07M phosphate buffer at pH 7.4. The reaction occurred at 370e during 60 mino 3. 2 ml of O.lM trichloroacetic acid (0.2M acetic acid + 0.2M sodium acetate) was mixed with the product of the reaction according to point 2 above. This mixture was centrifugalized. To the supernatant a 0.3M solution of sodium carbonate was added and the intensity of the yellow colour was measured spectrophotometrically. 4. The 6A436 in the supernatant was measured spectrophotometrically. Result: The proteolytic activity of the proteases was expressed in protease units (U), 1U being defined as the amount of nitrated casein in ~g which reacts with the proteases under the above described test conditions during 1 mino In the present case it was found that the activity was 11.3 U = 11.3 ~g of nitrated casein/min. (2,3,4) Method of determining the activity of the aamylase present in the filtrate 1. As an operation of essential importance the a-amylase were separated chromatographically on Sephadex G-l00. The extract was prepared by extracting the pulverized dry substance of the filtrate during 30 mino in a 0.05M buffer with 5 ml sodium acetate and 5 ml calcium acetate for each gram of dry substance. 3 ml of this solution were put on the column and eluted at a constant buffer. 2. 1.0ml Zulkowsky starch in a solution of 0.016M sodium acetate buffer (10mg/ml)at pH 6.0 was mixed with 1 ml of the amylases isolated according to point 1 above. The reaction occurred at 250e during 15 mino 3. 2.0ml 0.04M 3.5-dinitrosalicylic acid, dissolved in 0.4N NaOH(1.06M potassium so-

dium tartrate), keep at 1000e for 5 minutes then make up to 20.Oml. 4. The A490 in the supernatant was measured spectrophotometrically by measuring the intensity of coloration of the alkalized supernatant. Result: The activity of the amylase was expressed in amylase units(U), 1 U being defined as the amount of amylases which under the above described test conditions releases 1 micro-eguivalcnt of rcducing groups during por minutc(calculated as maltose). In the present case it was found that the activity was 5.1 U = 5.1 micro-eguivalent/ mino (5) Method of ~ctermining the activity of the cellulase present in the filtrate 1. As an operation of essential importance the cellulase were separated chromatographically on Sephadex G-l00. The extract was prepared by extracting the pulverized dry substance of the filtrate during 30 mino in a 0.05M buffer with 5 ml sodium acetate and 5 ml calcium acetate for each gram of dry substance. 3 ml of this solution were put on the column and eluted at a constant buffer. 2. 3.10-5M of resorufin acetate were mixed with O.OlM tris buffer at pH 7.0 and 1 ml of the cellulase isolated according to point 1 above. The reaction occurred at 250e during 2 mino 3. The 6A540 in the supernatant wa~ then measured spectrophotometrically by measuring the intensity of coloration of the supernatant. Result: Thc actlvity af thc ccllulases was expressed in cellulase units(U), lU being the amount of cellulase which causes resorufin acetate to change into resorufin during a minute. In the present case it was found that the activity was 1,1.10-2U/ml.(6) The examination of the action of enzymes isolated fram the paint layer and the canvas of the picture has given the following results: The paint layer was not hydrolyzed by the above mentioned enzymes. Between the canvas and the ground layer there was no proteinic isolation that could be damaged at the given conditions. The original canvas of the painting was damaged by the enzymes produced by the cellulase. The composition of the original paint layer did not act upon the above mentioned enzymes of the inhibition. New cements effected a partia 1 inhibition, owing to the chalk and the red cadmium used for retouch. Further inhibition was induced by the anil!n colour stuffs of the text added on the back of the painting which had penetrated into the paste. eellulase caused damage on the canvas of the picture. The cellulase was examined separately from the other enzymes. Dur!ng the enzymatic hydrolysis the presence of humidi ty was neces~~~~~yb~~ ~~: ~~~i~i!~c~n~~~~~f~~~~Oi!c:!9acnecessary to inhlbit the cellulase, in order to prevent a more serious damage on the canvas of the picture.

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If the system is permeated with a liquid or solid substance, an increasing liquid state arises at certain conditions on the liquid or solid substance resulting in an increasing volume of the whole system. Us1ng this premise the second system, aimed at removing the paste, was prevented from penetrating into the p~int layer. The pressure of system I can be controlled(7). If remova 1 of the paste requires a longer time and the pressure of system I diminishes considerably, it is necessary to use system I once again. The paste was removed by means of 170 U/mg lyophilised a-amylase of bacterial origin(Enzyme unit 1 U is defined as that quantity of enzyme which under test conditions liberates 1 micro-equivalent of reducing groups per minute calculated as maltose), 20 mAnson units/mg lyophilised proteinase K of funga I origin(Enzyme unit 1 Anson unit is defined as that quantity of enzyme which under test conditions liberates 1 ~mole of FOlin-positive amino acids per minute calculated as tyrosine) and 300 mg/l00ml glucose bound to carboxymethylcellulose dissolved in a buffer of sodium acetate at pH 7.0. Glucose was used as specific inhibitor of the cellulase(8). In the above mentioned concentration the inhibitoring effect of glucose was imm~diate. After the paste had been removed from the linen painting a part of the paste' remained in the deep layers of the canvas together with glucose. The removal of the remainder was easy to perform by means of glucose oxidase and catalase. Since the formation of gluconic acid lowers the pH values with a unit the pH value used at the beginning was adjusted to 7.5 in the course of the reaction. The enzymatic activity was determined polarographically. Glucose oxidase catalyzes the oxidation of S-D-glucose on D-gluconic acid. In the presence of cata lase hydrogen peroxide is cleft and at the same time molecular oxygen is released. The whole course of both reactions can be characterized by the consumption of 1 atom of oxygen for the oxidation of 1 molecule of glucose. The activity of glucose oxidase and catalase was determined by evaluation of the two polarographic curves. The first curve corresponds to the reduction of oxygen on hydrogen peroxide, and the second one to the reduction of hydrogen peroxide on water. The limit current of the first reduction wave was characterized by a potential between -0.4V and -0.5V, and the limit current of the second reduction wave by a potential between -1.35 and -1.45V on calumel electrode. For removal of the glucose the following enzymes were used: 12 U/mg of lyophilised glucose oxidase, where 1U is defined as that quantity of enzyme which under test conditions catalyses the transformation of 1 ~mole of substrate per minute. (1 ~mole of oxygen = 32 ~g corresponding to 1 ~mole of glucose = 180 ~g.) 550 U/mg of lyophilised catalase, where lU is defined as that quantity of Cnzyme which under test conditions catalyses the transformation of 1 ~mole of substrate per minc.tc. The removal of the glucose from the painting by means of the above mentioned enzymes was controlled on the back of the painting by measuring the changes of the pH values. The glucose oxidase and catalase were adsorbed on cellulose powde r of the type CF 11 in a phosphate buffer at pH 7.5 - 8.0. The time required for the remova 1 of glucose was 1 hour. The paint layer was consolidated the following systems:
84.2.29

Photo of the cellulase ture filtrate, 450 x ,

isolated from Lhe cul-

In order to determine the cellulase activity the behaviour of the cellulase was examined at the same conditions as during the removal of the paste. Removal of the paste The results obtained conccrning the cellulasc makes it possible to draw some conclusions fur the removal of the paste: Change of pH values cannot be used for removaI of the paste, because cellulase at pU 8. O has a 10'/,ctivity. At this pI!value or a greater the paste cannot be removed, as this would cause damage on the canvas layer in sue an alkaline range. An analogous situah tion arises in the acid range. At pll 4.O cellulase has a 20% activity, and such a highly acid environment, pH 4.0 or lower, would induce damage on the linen painting. Change of temperature can bc used for removal af lhe paste. From the results obtained it appears that cellulase does not have any activity below 150C or over 400C. In this case t would be po ssi.b e to use for the hydroL lysis of the paste bacterial amylase which shows a 80% activity between 400C and 500C. Yet, the problem remains how lo prcvent, at sueh temperatures, the painting surface from being damaged by the intruding products of the enzymatic hydrolysis. The besl method is to use glucose as specific inhibitor of the cellulase and to work at the environment temperature 200C.

For removal of the pastc the following systems were used: System I: wax, turpentinc, benzine and toluene. System II:a-amylase, proteinase K and glucose. The enzymes were adsorbed on carboxymethylcellulose in a phosphate buffer at pH 7.0. Japanese ricepaper was attached to the paint layer using wax, turpentine, benzine and toluene(system I). The painting strengthened in this way was then coated with melinex. Thus, the painting was prepared for the removal of the paste from the back of the painting. The proportions in each system are very important.

by means of

System I.

The back of the picture was moistened with turpentine and benzine. A filtration paper impregnated with the same mixture of solvents was attached to the back of the picture. The solvents and apressure of their vapours prevented thus penetration from the paint layer of products resulting from an enzymatic breakdown ,of the,glue.

Figure 2 Cellulase activity from the canvas

Cellulase

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System 11. Some quantity' of a-amylase was adsorbed on polyethylene glycol 1000. This thick mass with the amylase was used for removing the paste from the surface of the picture. Polyethylen glycol 1000 was dissolved in a sodium acetate buffer at pH 7.0. lmg of a-amylase was added to 1 9 polyethylene glycol. The time required for remova 1 of the paste was 15 mino Polyethylene glycol 1000, a-amylase and the products of the hydrolysis of the paste were then removed from the surface of the picture by means of turpentine. Figure 1 Cellulase optimal activity

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The intensity of cellulase hydrolysis was measured at various temperatures. The activity maximum was reached at a temperature of 280c. At 400C no enzymatic activity could be measured. At 150C the activity was minimal ~ From these results it appears that when the paste was moistened at temperatures below 150C [.~. or over 400C no hydrolysis of the original can- .' vas by cellulase occured. The whole inhibition was performed by menadion (vitamin K3) and was considerably effective. Some pastes to which menadion had been added, were examined. It was proved that such pastes did not get mouldy. It is known that naphthoquinones are easily subjected to photochemical cleavage. Therefore, during the examinations light was screened off. The inhibition on the back of the picture was performed by spraying menadion in a solution of isopropanol in a ratio of 2 mg of menadion to 100 ml isopropano1.(9) ,
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Refcrences Makes,F. ,Enzymatic Consolidation of Paintings, Goteborg 1979 Pfleiderer,G.and Krauss,A.,Biochem.Z.342,85 (1965) Kunitz,M. ,J.Gen.Physiol.30,291 (1946) Davidek,J.,Analyza potravin, Praha 1977 Sandstedt,R.M.,Kneen,E.and Blish,M.J., Cereal Chemistry 16, 712(1939) Makes,F.,Enzymatic Consolidation of Paintings,ICOM Committee for Conservation 6th Trienneal Meeting Ottava 1981 Makes,F.,Enzymatic Consolidation of Paintings, Goteborg 1979 Makes,F.,Science and Technology in the Service of Conservation Washington Congress, 3-9 Septembcr 1982 Makes,F.,Polarographic Research upon Proteases produceo by Aspergillus glaucus,Penicillium verrucosum,Rhizopus stolonifer,bacteria and yeasts in leather book covers(Nordisk tidskrift for bok&biblioteksvasen,Nr 1 1984, Skoklosterstudie Nr 17) l' \

The cellulase produced by the above mentioned micro-organisms was found to have its optimal activity in the range of pH5.0 - 6.5. By extending the op t Lmu.n range to pH 4.5 - 8.5 a 65% activity was still observed. By a further lowering of the optimum range to pH 4.0 - 8.5 the activity fell to 10%.

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84.2.30

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