Journal of Chemical Technology and Biotechnology J Chem Technol Biotechnol 81:136–145 (2006)DOI: 10.1002/jctb.1370

Synthesis of silk sericin peptides–L-asparaginase bioconjugates and theircharacterizationYu-Qing Zhang, Mei-Lin Tao, Wei-De Shen,∗ Jian-Ping Mao and Yu-hua ChenSilk Biotechnology Laboratory, School of Life Science, Soochow University, Suzhou 215006, People’s Republic of China

Abstract: The natural silk sericin, recovered from Bombyx mori silk waste by degumming and degrading,is a water-soluble peptide with different molecular masses, ranging from 20 to 60 kDa. It is composed of 15sorts of amino acids, among which the polar amino acids with hydroxyl, carboxyl and amino groups suchas aspartic acid, serine and lysine account for 72%. The covalent attachment of the silk sericin peptides toL-asparaginase (ASNase) produces silk sericin peptides–L-asparaginase (SS–ASNase) bioconjugates thatare active, stable, have a lower immune response, and have extended half-lives in vitro in human serum.The modified enzyme coupled with sericin protein retains 55.8% of the original activity of the nativeenzyme. The optimal pH of SS–ASNase derivatives shifts considerably, to 5.0 in comparison with pH6.0–8.0 of the native form. The thermostability and resistance to trypsin digestion of the modified enzymeare greatly enhanced as compared with ASNase alone. The Michaelis constant (Km) of SS–ASNase is 65times lower than that of the enzyme alone. This suggests that the affinity of the enzyme to its substrateL-asparagine greatly increases when bioconjugated with silk sericin. The in vivo experiments also showthat the silk sericin peptides have no immunogenicity, and the antigenicity of the enzyme is obviouslydecreased when coupled covalently with the silk sericin peptides. 2005 Society of Chemical Industry

Keywords: L-asparaginase; silk sericin; bioconjugates; modification

INTRODUCTIONL-Asparaginase (EC 3.5.1.1; ASNase), a tetramericprotein with a molecular weight of 138–141 kDa,is often selected as a multimeric model enzymeto test the strategy of immobilization, modificationor bioconjugation. The enzyme, derived largelyfrom two bacterial sources (Escherichia coli andErwinia chrysanthemi), a well-known and effectivechemotherapeutic agent in the management of acutelymphoblastic leukemia (ALL), is able to hydrolyze L-asparagine into L-aspartic acid and ammonia, resultingin low plasma and cerebrospinal fluid levels ofthis amino acid. In spite of its high therapeuticefficacy, prolonged use leads to hypersensitivity,ranging from mild allergic reactions to life-threateninganaphylaxis,1 due to the high molecular mass of theenzyme and its bacterial origin.2 Moreover, antibodiesagainst ASNase greatly accelerate its clearance fromthe circulation and thus reduce its therapeuticeffectiveness.3,4 Therefore, the enzyme circulates inthe blood system for only a short time before beingtaken in and broken down by native proteases.

In order to reduce the immunological response,prolong the action time and enhance the drug’seffects in blood, the native ASNase has oftenbeen immobilized with various kinds of insolublebiocompatible polymers and modified chemically orbioconjugated with soluble polymers to producevarious immobilized and modified ASNases. Themodification or immobilization of the enzyme notonly reduced its immunity and toxicity to humansbut also greatly improved its resistance to proteolysisin comparison with native ASNase. Some attemptshave been made to prepare insoluble, matrix-supportsfor ASNase immobilization, such as collagen,5

CM-cellulose,6 polyacrylamide,7 poly (2-hydroxyethylmethacrylate) gels,8,9 and PEG–BSA hydrogels.10

This type of derivative may be appropriate forextracorporeal devices in the clinical treatmentof ALL. On the other hand, there have beenmany reports on soluble biocompatible polymersfor ASNase modification or bioconjugation. Thesepolymers include mainly both natural and artificialpolymers such as albumin,11 dextran,12 polyethylene

∗ Correspondence to: Wei-De Shen, Silk Biotechnology Laboratory, School of Life Science, Soochow University, Suzhou 215006, People’sRepublic of ChinaE-mail: shenwd@suda.edu.cnContract/grant sponsor: State 863 Project of China; contract/grant number: 2004AA2Z1020Contract/grant sponsor: University Natural Science Funds of Jiangsu Province, People’s Republic of China; contract/grant number:04KJB180122(Received 16 April 2005; revised version received 4 June 2005; accepted 8 June 2005)Published online 23 September 2005

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Synthesis of silk sericin peptides–L-asparaginase bioconjugates

glycol13 and polyvinyl alcohol,14 in which a solublebiocompatible polymer polyethylene glycol (PEG)used for enzyme modification, called bioconjugation,has received most extensive attention in recent years.Polyethylene glycol- L-asparaginase (PEG–ASNase)or pegaspargase (Oncaspar, ENZON, Piscataway,NJ, USA and Rhone-Poulenc Rorer Pharmaceuticals,Collegeville, PA, USA) is a form of ASNase that hasbeen commercially available since 1994.15 When PEGwith a molecular weight of 5 kDa was conjugated tothe enzyme, PEG–ASNase prevented the uptake ofthe enzyme by the reticuloendothelial system. Thisin turn decreased the risk of developing antibodiesto asparaginase and prolonged the circulating half-life of the drug from 1 day to 5–7 days afterintramuscular administration.16,17 • Due to thedecreased immunogenicity, PEG–ASNase can alsobe substituted for native unmodified L-ASNase whenhypersensitivity has occurred. However, pegaspargasehas adverse effects similar to the native enzyme.A recent report shows a possible higher incidenceof pancreatitis associated with pegaspargase.18 Silkfilament derived from the silkworm Bombyx moricomprises two proteins, sericin (the outer coating)and fibroin (the inner brins), which are both naturalmacromolecular biopolymers. The former is removedby a process called degumming in the silk industryand is mostly discarded as a wastewater fromsilk processing. Sericin is a globular and water-soluble glue protein, unlike silk fibroin which isan oriented fiber protein. When the silk filamentwas subjected to degumming treatments in boilingwater, alkaline solution or to proteolysis, sericinwas easily isolated from the inner brins fibroin anddegraded into sericin peptides and its hydrolyzates,ranging widely from about 10 to over 300 kDain molecular weight. The sericin peptides havinga lower molecular weight of less than 60 kDa,commonly less than 5 kDa, are characterized byexcellent moisture absorption and release, and a lot ofbiological activities such as antioxidation, tyrosinaseactivity inhibition,19 and pharmacological functionssuch as anticoagulation,20 anticancer activities,21,22

cryoprotection23 and promotion of digestion.24,25 Therest, having a higher range of molecular weight from60 to over 300 kDa, is poorly soluble in cold waterbut soluble in boiling water, thus it is called hotwater-soluble sericin protein. The protein could beapplied in many fields such as degradable biomaterials,biomedical materials, synthetic polymer materials orforming articles, functional biomembrane materials,functional fibers, fabrics and articles.26 Not long ago, itwas reported that poorly soluble microparticles of silksericin, having an average size of about 10 µm, wereused as a vector for ASNase immobilization. Whenit was immobilized on the sericin microparticles, theimmobilized enzyme improved considerably its affinitywith its substrate L-asparagine, its thermostability andthe resistance to trypsin digestion.27 • However, thistype of immobilized ASNase may be only appropriate

for extracorporeal devices in clinical treatment. Todate, there have been no reports on the water-soluble silk sericin protein used for the modificationor bioconjugation of ASNase or even other enzymes.

Here, we have prepared easily soluble sericinpeptides with molecular masses of less than 60 kDa.When used for conjugation with the enzyme, thesoluble sericin is better than the poorly solublemicroparticles of sericin because it could conjugatecovalently with more enzyme molecules and couldbe used as an injection in vivo in clinical use. Thebiosynthesis, characterization and activities in vitro ofthe silk sericin peptides–ASNase (SS–ASNase forshort) bioconjugates are described in detail.

MATERIALS AND METHODSMaterialsThe cocoons of silkworm Bombyx mori were providedby the Sericulture Department, Soochow University.ASNase from E. coli with a specific activity of271.8 IU mg−1 was manufactured by ChangzhouQianhong Bio-Pharma Co., Ltd, Jiangsu Province,People’s Republic of China. Human serum wasa gift from No. 1 Peoples’ Hospital of SoochowUniversity. Trypsin, asparagine and glutaraldehydewere purchased from Sigma (St Louis, MO, USA).KI, HgI2, trichloroactic acid and other reagents wereall analytical reagent grade, purchased from ShanghaiChemicals Factory (People’s Republic of China).

Preparation of water-soluble silk sericin powderThe hot water-soluble sericin protein powder with ahigher molecular mass was prepared using the previ-ously reported procedure.28 When washed repeatedlywith plenty of hot water, 1 kg cocoon shells wereimmersed in 15–30 L distilled water overnight. Whenthey were put into a high-pressure boiler at 120 ◦C and2 atm for 1 h, the degumming solution was filteredwith an activated carbon filter to remove impuritiesor precipitates. The resulting sericin solution waseasy to agglomerate into a gel at room temperaturedue to its higher molecular mass. So, the hot solu-tion of sericin protein was adjusted to pH 10.0 by1.0 mol L−1 NaOH solution at once, put it into a high-pressure boiler and maintained for 2 h at 120 ◦C and2 atm.29 The resulting degraded solution of sericinpeptide was slightly alkaline and neutralized by adding1.0 mol L−1 H2SO4 solution. When the peptide solu-tion was filtered through a 10 kDa ultrafilter, thesericin peptides of less than 10 kDa, free amino acidsand some salts were removed. The resulting sericinpeptides of more than 10 kDa in molecular mass weremade into a water-soluble powder with a spray-dryer(Shanghai Trustech Co. Ltd, People’s Republic ofChina) at temperatures ranging from 70 to 120 ◦C.The sericin peptide powder is easily soluble in coldwater and was used for enzyme bioconjugation in thefollowing experiments.

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Amino acid compositionA known amount of sericin powder was hydrolyzedin 6.0 mol L−1 HCl at 110 ◦C for 24 h. When HClin the hydrolyzed sericin solution was removed, thehydrolyzed solution was used for determination ofamino acids composition with a Hitachi (Tokyo,Japan) 825–50 model autoanalyzer.

Sodium dodecyl sulfate–polyacrylamide gelelectrophoresisWhen a given amount of sericin protein wasdissolved in hot water, the range of molecu-lar weight of sericin was determined by sodiumdodecyl sulfate–polyacrylamide gel electrophoresis(SDS–PAGE) according to the method described pre-viously by Laemmli.30 with 12% acrylamide gel and5% condensing gel, which was stained with 0.25%Coomassie Brilliant Blue R-250 (Aldrich, Milwau-kee, USA).

Preparation of SS–ASNase derivativesAfter 1.0g of sericin powder was added into a plasticflask, and mixed with 10 mL of ASNase solution, aknown volume of 25% glutaraldehyde solution wasadded. After mild homogenization of the mixture,10 mL of 5 mg mL−1 L-asparagine solution was addedfor the protection of the active center of the enzyme,and the flask was stoppered tightly and placed inan orbital shaker at 4 ◦C for 1–10 h. After sometime, a known amount of glycine was added toend the reaction. Then the reaction mixture wasdialyzed against flowing distilled water in a 16/32dialysis membrane (Union Carbide Corporation,Tokyo, Japan) for 24 h to remove impurities andunreacted cross-linking reagents. Subsequently theSS–ASNase bioconjugate solution was volumedwith distilled water. The resulting SS–ASNasebioconjugate solutions were maintained at 4 ◦C forthe following purification experiments.

Determination of the enzymatic activityThe activity analysis for native or bioconjugatedASNase was performed using the method of Mash-burn and Wriston, with slight modifications.31 Theimproved procedure comprises the following steps.After 0.10–0.20 mL of native or modified ASNasehad been added to 0.2 mL 0.05 mol L−1 Tris–HClbuffer (pH 8.6) in a test tube containing a magneticrod and incubated at 37 ◦C for 10 min, 1.7mL of0.05 mol L−1 L-asparagine was added to 0.05 mol L−1

Tris–HCl buffer in the tube and stirred for the enzy-matic reaction to occur. After 10 min, the enzymaticreaction was interrupted by adding 1.5 mol L−1 oftrichloroacetic acid solution. After centrifugation at15 000 rpm for 5 min, 1.0 mL supernatant of the sam-ple was mixed with 1.0 mL Nessler’s reagent and 7mLwater, standing for 10 min. The reaction activities ofthe native and modified enzymes were measured froma change in optical density (absorbance) for 10 min at450 nm using a spectrophotometer (Hitachi U-3000,

Japan). According to the relationship between the con-centration and absorbance, a standard curve of intactASNase vs absorbance was established for calculatingactivities of modified enzymes. The activities or rela-tive activities of various samples present in the paperwere average values of three-repeated measurements.

In vitro experimentsUnmodified ASNase or SS–ASNase bioconjugates(100 µL) were added to 900 µL normal human serum.After mild homogenization, the serum mixture wasincubated at 37 ± 1 ◦C in an incubator. Small amountsof serum mixture, taken at a given time-interval, weremeasured repeatedly three times and the survivalactivities of ASNase in the serum mixture werecalculated.

Immunization testIn order to prepare antisera against silk sericin, freeASNase and SS–ASNase in rabbits, three samplesolutions (5.0 mg mL−1 sericin, 0.5 mg mL−1 intactASNase and 0.5 mg mL−1 SS–ASNase derivatives inPBS) were filtered with a filter (0.22 µm in diameter)to obtain sterilized samples. The experimental animalswere divided into three groups, silk sericin, freeASNase and SS–ASNase (each group comprised threerabbits). Male adult rabbits (about 1 kg; purchasedfrom the Experimental Animal Center at SoochowUniversity, Suzhou, People’s Republic of China) wereinjected intravenously (ear vein) on day 0 with 2 mLof three sample solutions, respectively. Subsequently,these rabbits were injected intravenously for a secondand third time on days 21 and 31 with 3 mL of thesesame solutions, respectively. These rabbits were bledfrom the heart on day 10 after the third injection.Blood samples (50 mL) were then placed into aseptictubes for 2 h, and then the serum was placed intoother aseptic tubes containing 0.1% sodium azide(NaN3) which were kept frozen at −20 ◦C for theimmunogenic determination of silk sericin, free andmodified ASNases.

According to the method of Edwards,32 counterimmunoelectrophoresis (CIE) was carried out using1% agarose in 0.02 mol L−1 barbital buffer, pH8.6. The electrophoresis was conducted at roomtemperature at 10 mA per slide for 90 min; knownpositive and negative control sera were also runwith each test. The slides were then washed, dried,stained in Coomassie Blue and finally de-stainedready for analysis and scanned. Positive reactionswere defined by precipitation lines between antigenand hyperimmune serum.

RESULTS AND DISCUSSIONSynthesis of SS–ASNase bioconjugatesAmino acid compositionsIn order to understand some of the properties of thesericin protein required for enzyme bioconjugation,the amino acid composition of the water-soluble

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protein was measured using a Hitachi 825–50 modelautomatic analyzer. Table 1 shows that the aminoacid composition of the protein was constituted by15 kinds of amino acids, among which the polaramino acids with hydroxyl and amino groups suchas aspartic acid, serine and lysine account for 72%.These strongly polar side groups of the aminoacids are suitable to couple covalently with ASNaseby using glutaraldehyde. Therefore, the modifiedmaterials do not need the activating treatment prior tobioconjugation.

Molecular weight rangesWhen a given amount of sericin powder was dissolvedin cold water, the molecular weight of the protein wasmeasured by SDS–PAGE with 12% gel. As shown inFig. 1, the Marker lane and Sericin lane indicate thestandard protein ladder from 10 to 200kDa (GibcoCo., BRL, Rockville, MD) and the molecular weightof silk sericin, respectively. The water-soluble sericinwas a degradable product, characterized by the widerange of molecular weight from about 20 kDa to over60 kDa.

Purification of SS–ASNase bioconjugatesThe crude derivatives obtained in the section‘Preparation of SS–ASNase derivatives’ probably

Table 1. Amino acid composition of the water-soluble sericin (mol%)

Amino acid Content Amino acid Content

Asp 20.55 Met 0.00Thr 7.96 Ile 0.88Ser 25.28 Leu 1.36Glu 7.90 Tyr 4.47Pro 0.00 Phe 0.91Gly 10.51 Lys 4.68Ala 3.97 His 1.75Cys 0.72 Arg 5.26Val 3.79 Try —

MW Marker Sericin

10K

20K

30K

40K

50K

70K

200K

Figure 1. SDS–PAGE (12% gel) of water-soluble silk sericin.

contained some by-products such as polymerized silksericin peptides. The crude derivatives must be furtherpurified to remove these conjugates with no or loweractivity. The bioconjugates solubilized in 0.05 mol L−1

phosphate buffer (pH 8.00) were subjected toSephadex G 200 gel filtration chromatography inthe same buffer. When absorption of the protein wasdetected from flowing elution buffer, 5 mL of theelution buffer was fractionally collected in each tube.ASNase activity of the elution buffer was measuredby using the method described above. The highestactivity in these elution samples was set as 100% ofrelative activity. All of the elutes with less than 10%relative activity were removed. Those elution sampleswith more than 10% relative activity were harvestedtogether as a sample of SS–ASNase bioconjugates forthe following experiments.

Glutaraldehyde concentrationIn order to understand the optimal conditionsfor ASNase bioconjugation with silk protein, wefirst investigated the effect of the glutaraldyhydeconcentration on the modification of ASNase. Asshown in Fig. 2, the glutaraldyhyde concentration inthe reaction mixtures had a great influence on therelative activity of the modified ASNase. A peak ofrelative activities of the modified enzyme appearsat a glutaraldehyde concentration of nearly 0.7%.Therefore, the modified enzymes were prepared using0.7% glutaraldehyde concentration in the followingexperiments.

BuffersThree buffers of 0.05 mol L−1 citrate, phosphate andTris–HCl were prepared and adjusted to pH 8.6 with0.1 mol L−1 HCl or NaOH solution. The activities ofSS–ASNase bioconjugates were measured in the threebuffers by the same method, respectively. As shown

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.40

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Rel

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tiviti

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%)

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Figure 2. Effect of glutaraldehyde concentration on the activity ofSS–ASNase conjugates. The cross-linking reaction between ASNaseand sericin protein was done in an orbital shaker at 4 ◦C in50 mmol L−1 Tris–HCl buffer (pH 8.6).

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in Table 2, the enzymatic reactions of SS–ASNaseconjugates did not suffer obviously in three buffers.Therefore, 0.05 mol L−1 Tris–HCl buffer (pH8.6) wasused in the following experiments.

Activity recoveriesA series of native ASNases with different concen-trations was determined to obtain their correspondingabsorbance values. Then, a standard curve of the intactASNase was established between the concentrationsof the free ASNase vs absorbance values. The linearequation is:

y = 0.53361x − 0.05351

and the correlation coefficient (γ ) is 0.99475 (Fig. 3).The activity recovery of the modified enzyme can becalculated according to the linear equation. The resultis illustrated in Table 3. The activity recoveries ofSS–ASNase bioconjugates declined gradually with theincrease of ASNase content in sericin derivatives, andwhen the content of the ASNase in sericin powder wasat 0.55 U mL−1, the activity recovery of the ASNasederivative was at its maximum value, 55.8%.

Degree of enzyme modificationIn general, the degree of modification of an enzymemodified or bioconjugated with another molecule isexpressed by the changes of the free amino groupsof the enzyme. Here, degrees of modification of ε-amino groups in ASNase and silk sericin peptideswere determined by using trinitrobenzene sulfonate(TNBS).33 The results show that the amount of ε-amino groups in the SS–ASNase bioconjugates aftermodification decreased to 50.9% of the original levelin both ASNase and silk sericin before modification(Table 4). In other words, the degree of modificationof ASNase with silk sericin protein reached 49.1%.

Table 2. Effect of various buffers (50 m mol L−1, pH 8.6) on

sericin–ASNase activity

Buffer ABS ± SD Relative ABS (%)

Citrate 0.756 ± 0.070 100Phosphate 0.746 ± 0.047 98.63Tris–HCl 0.655 ± 0.025 86.61

Table 3. Activity recoveries of the sericin–ASNase conjugates

Sample ASNase(U mL−1)

ASNasecontent (U) Activity (U) Recovery/%

0.55 0.165 0.092 ± 0.003 55.81.10 0.330 0.174 ± 0.009 52.82.20 0.660 0.342 ± 0.012 51.8

11.00 3.300 1.571 ± 0.034 47.622.00 6.600 2.878 ± 0.021 43.6

The activity values in the table were average values of three-repeatedmeasurements.

0.0 0.5 1.0 1.5 2.0

0.0

0.2

0.4

0.6

0.8

1.0

1.2

Abs

orba

nce

(450

nm)

Native ASNase (IU)

Linear equation: y = 0.53361x - 0.05351R2 = 0.99475

Figure 3. Standard curve of native ASNase (U) vs absorbance(λ450 nm).

Table 4. ε-Amino groups of sericin and ASNase before and after

bioconjugation

ε-Amino groupBefore bio-conjugation

After bio-conjugation

Modificationrate (%)

Absorbance (ABS) 0.322 0.164 50.93

Note: In the bioconjugation of sericin and ASNase, concentration ofthe enzyme is 0.025 mg mL−1.

Kinetic properties of SS–ASNase bioconjugatesThe Michaelis constant (Km)Figure 4 shows the Lineweaver–Burk plots of intactand modified ASNases based on the results obtainedat pH 8.6 at 37 ◦C. The concentrations of the substrateL-asparagine in 0.05 mol L−1 Tris–HCl buffers variedbetween 0.4 and 2.0 mmol L−1. Two linear equationsof SS–ASNase derivatives and ASNase alone wereobtained from the plots y = 1.1176x + 7.0458 andy = 13.026x + 1.2477, respectively. The Michaelisconstant (Km) of SS–ASNase conjugates (1.59 ×10−4 mol L−1) decreased 65-fold compared of thenative enzyme (1.04 × 10−2 mol L−1). This showsthat the affinity between ASNase and its substrateasparagine was greatly increased when ASNase wasconjugated with silk sericin.

pH effectThe effect of pH on the activities of native andmodified ASNases was studied by changing the pHof the Tris–HCl buffer from 3.0 to 10.0. As shown inFig. 5, the activity pattern of modified enzymes withthe variations of pH is clearly different from nativeenzyme. The optimal pH value of unmodified enzymealone ranged widely from 6.0 to 8.0, while that of theenzyme modified with silk sericin was at 5.0, whichsuggested that after the enzyme was coupled covalentlywith sericin protein, the optimal pH range narrowedand the optimal pH evidently shifted towards the acidicdirection, in comparison with native enzyme.

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y = 13.026x + 1.2477 y = 1.1176x + 7.0458

0

5

10

15

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25

30

35

-7.5 -5.0 -2.5 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0

[S]−1 (mM−1)

[V]-1

((A

BS

/min

)-1)

SS-ASNase

Native ASNase

Figure 4. Lineweaver– Burk plots for native ASNase and SS–ASNase conjugates. The data in the figure were average values of three-repeatedmeasurements. The enzymatic reactions of native and modified ASNases with various concentration of substrate L-asparagine were carried out at37 ◦C with Tris–HCl buffer (pH8.6) for 10 min.

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SS-ASNase conjugatesNative ASNase

2 3 4 5 6 7 8 9 10

Figure 5. Effect of pH on activities of native and bioconjugatedASNases. Relative activities were calculated by using the highestabsorbance value of native and bioconjugated ASNases as 100%,respectively.

Reaction temperatureAs shown in Fig. 6, the effect of the enzymatic reactiontemperature on the activity of modified ASNase wasinvestigated from 30 ◦C to 80 ◦C. In a profile of relativeactivity vs reaction temperature, the optimal reactiontemperature at a maximal activity showed no apparentshift between both native and modified enzymes,but the optimal range of reaction temperature ofthe modified enzyme became broader. When thereaction temperature rose to 70 ◦C, the enzymaticreaction of native enzyme almost stopped and theenzyme lost its activity, while the modified enzymestill remained 80% of its original activity. Even thoughthe reaction temperature rose to 80 ◦C, the survivalactivity of modified enzyme was still about 60%.The bioconjugation of ASNase with sericin proteinwidened enormously the optimum temperature rangeof the enzymatic reaction.

30 40 50 60 70 80

0

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%)

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ASNase conjugatesNative ASNase

Figure 6. Effect of temperature on the activity of ASNase alone andbioconjugates. Relative activities were calculated by using the highestabsorbance value of ASNase alone and bioconjugates as 100%respectively.

The stability of SS–ASNase bioconjugatesThermostabilityWhen the native and modified enzymes wereincubated in 0.05 mol L−1 Tris–HCl buffer (pH 8.6)at 40–80 ◦C for 30 min, respectively, their survivalactivities were measured as described in ‘Materialsand Methods’. The effects of the heat treatment on thetwo enzymes are shown in Fig. 7. The thermostabilityof the modified ASNase was obviously enhanced incomparison with native enzyme. When incubatedindividually from 40 to 80 ◦C for 30 min, the survivalactivity of the free enzymes decreased rapidly and theiractivity was almost lost at 80 ◦C. The thermostabilityof the modified ASNase is very good. When incubatedat 80 ◦C for 30 min, the SS–ASNase bioconjugatesretained more than 80% of their original activity.

Resistance to trypsin digestionA given amount of native or modified ASNase in0.2 mL 0.05 mol L−1 Tris–HCl buffer (pH 8.6) was

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mixed with 30 µL trypsin solution (1.0 mg mL−1)for proteolysis. After the sample solution had beenincubated for known time intervals, its activity wasdetermined. Figure 8 shows the resistances of freeASNase and bioconjugates to trypsin digestion. It isobvious that the resistance of the modified ASNase totrypsin digestion was greatly improved in comparisonwith that of the native form. After being hydrolyzed for10 min by trypsin, the activity of the modified enzymeretained more than 70% of the original activity whilethe unmodified enzyme retained no activity. Eventhough the incubation time in trypsin solution waslengthened to 60 min, SS–ASNase bioconjugates hadretained more than one-half of their original activity.

In vitro half-lifeA1.0 mL aliquot of native ASNase or SS–ASNasederivatives in 0.05 mol L−1 Tris–HCl buffer (pH

40 50 60 70 80

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Rel

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%)

Temperature (°C)

ASNase conjugates ANSase alone

Figure 7. Thermal stability of ASNase alone and bioconjugates.Relative activities were calculated by using the highest absorbancevalue of ASNase alone and bioconjugates as 100%, respectively.

0

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Time (min)

ASNase bioconjugatesASNase alone

0 20 40 60 80 100 120

Figure 8. The resistances of ASNase alone and bioconjugates totrypsin digestion. Relative activities were calculated by using thehighest absorbance value of free and immobilized ASNases as 100%,respectively.

8.6) was mixed with 4 mL of human blood serum.After mild homogenization, the mixture solution wasincubated at 37 ◦C in a water bath for a long time.As shown in Fig. 9, the survival activity of 0.1 mLmixture serum solution was measured at certain timeintervals. The survival activity of native or modifiedASNase was calculated by means of the ASNasestandard curve from a change in optical densityfor 10 min at 450 nm by using a spectrophotometer(Hitachi U-3000). Then, the time (in vitro half-life)for the activity of the native or modified ASNase todecrease to half of the original activity was measured.The result shows that in vitro the half-life of themodified ASNase lengthened to nearly 84 h, abouttwo times that of the native enzyme. This suggeststhat the covalent conjugation of enzyme with silksericin peptides considerably enhanced the resistanceto trypsin digestion as well as other proteases in humanserum.

0

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Hours

ASNase bioconjugatesASNase alone

10 20 30 40 50 60 70 80 90 1000

Figure 9. The in vitro half-lives of ASNase alone and bioconjugates.Relative activities were calculated by using the highest absorbancevalue of ASNase alone and bioconjugates as 100%, respectively.

0 2 4 6 8 10 120

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60

80

100

Stored time (days) for ASNase alone

Stored months (months) SS-ASNase bioconjugates

Rel

ativ

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ctiv

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%)

SS-ASNase bioconjugates

ASNase alone

0 2 4 6 8

Figure 10. The storage stabilities of ASNase alone andbioconjugates. Relative activities were calculated by using the highestabsorbance value of ASNase alone and bioconjugates as 100%,respectively.

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Synthesis of silk sericin peptides–L-asparaginase bioconjugates

Storage stabilityIn general, there were no significant differencesbetween the retention activities of both the native andmodified ASNases after a long storage period at a lowertemperature in lyophilized form. The native enzymein liquid form easily lost its activity after a longer timeof storage. Figure 10 is a comparison of the survivalactivities of both native and modified ASNases whenstored in 0.05 mol L−1 Tris–HCl buffer (pH 8.6) at4 ◦C. Having been stored for 8 days, the native enzymewent gradually down to less than 40% of its originalactivity. However, after being stored for 12 monthsat the same conditions, SS–ASNase bioconjugatesretained over 60% of the original activity, and morethan 50% of the activity was still maintained even after2 years’ storage (data not shown). It is evident thatthe enzyme enhanced considerably its stability whenbioconjugated with silk sericin peptides.

Immunogenicity of silk sericin and antigenicity ofSS–ASNase conjugatesIt is well known that the silk protein from Bombyx moriis a biocompatible material owing to its long-term useas a surgical suture.34,35 • The immunogenic studieson silk proteins focus mainly on silk fibroins36,37 ratherthan the silk sericin protein. The results indicatethat silk fibers are largely immunologically inert inshort- and long-term culture with RAW 264.7 murinemacrophage cells while insoluble fibroin particlesinduced significant release of tumor necrosis factor(TNF).38 Soluble sericin proteins extracted fromnative silk fibers did not induce significant macrophageactivation. Apart from this, there have been no morestudies on the immunogenicity of the sericin protein.

The antisera of silk sericin peptides, free andmodified ASNases were prepared in male rabbitsfor immunoreactions for three times according tothe procedure given in the section ‘ImmunizationTest’. The antigen antibody binding ability of thesilk protein was measured by CIE. The result showsthat there were no precipitation lines between theantigen (sericin) and hyperimmune serum (Fig. 11).This indicated that the aqueous silk sericin proteindoes not induce the occurrence of the antibody ofsericin in rabbits, like the other silk protein fibroindoes. Therefore, the aqueous silk sericin with arange of molecular weight from 20 to 60 kDa is notimmunogenic and is also a biocompatible protein.

It is well known that seven or eight antigenicrecognition sites exist on the surface of ASNase thatresult in severe immune sensitive reactions. In general,the antigenicity of an enzyme is reduced by chemicalmodification or bioconjugation. As shown in Fig. 11,there are apparent precipitation lines between nativeASNase (antigen, Ag) and hyperimmune serum (Ab)in the rabbit injected repeatedly with plenty of nativeASNase. The combined activity of Ag (native ASNase)and Ab (hyperimmune serum) is very strong. Whenthe antibody titer was 24, the precipitation line stillappeared between native ASNase and hyperimmuneserum. This indicated that the unmodified enzymeinduced severe immune sensitive reaction in rabbit.When the amino groups of the enzyme were bondedcovalently with the amino groups of silk sericinpeptides, the free amino groups present on thesurface of SS–ASNase conjugates were obviouslydecreased by about a half of the total amount beforebioconjugation. Correspondingly, the precipitationreaction between SS–ASNase bioconjugates (antigen,Ag) and hyperimmune serum was evidently weakenedby CIE. The precipitation line disappeared whenthe antibody (against SS–ASNase) titer was 22.The bioconjugation of silk sericin peptides greatlyreduced the immunogenicity of the enzyme alone. Thebioconjugation of ASNases with silk sericin peptidescould mask these recognition sites of the enzyme andso greatly reduce their antigenicity. In fact, after beingcoupled covalently, silk sericin peptides acted as asteric barrier to retard the interaction of the enzymewith antibodies as well as protease hydrolysis.

CONCLUSIONS1. The natural silk sericin protein, obtained by

processing twice in water and slightly alkalinesolution at high-temperature and high-pressure, isa water-soluble peptide with different molecularmasses ranging from 20 to 60 kDa. The degradingprotein comprises largely the polar amino acidswith hydroxyl, carboxyl and amino groups such asaspartic acid, serine and lysine which occupy 72%of the total amount of the protein. The resultsderived from in vivo immunoassay in rabbit havedemonstrated that the silk sericin protein has noimmunogenicity.

Silk Sericin Titer Native ASNase Titer SS-ASNase Titer

1:25

1:22

1:20

1:25

1:24

1:20

1:25

1:22

1:20

− Ag Ab + − Ag Ab + − Ag Ab +

Figure 11. Antibodies detected by counter immunoelectrophoresis.

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Y-Q Zhang et al

2. The silk sericin peptides with hydroxyl, carboxyland amino groups could couple covalently withASNase. About 49% of the ε-amino groups of theprotein were modified with the enzyme. The result-ing SS–ASNase bioconjugates are characterizedby their higher thermostability, widener optimalranges of enzymatic reaction and narrower optimalpH. The modified enzyme coupled with sericinprotein retains 55.8% of the original activity. TheMichaelis constant (Km) of SS–ASNase is 65 timeslower than that of the enzyme alone. This suggeststhat the affinity of the enzyme to its substrateL-asparagine extremely increased when bioconju-gated with silk sericin.

3. When the enzyme is attached covalently withsilk sericin, which is a globular gum protein, itsresistance to trypsin digestion is greatly enhanced,and its storage stability in liquid form is greatlylengthened. The SS–ASNase bioconjugates haveconsiderably extended in vitro half-lives in humanserum. The in vivo experiments also show that theantigenicity of the modified enzyme is obviouslydecreased as compared with ASNase alone.

In general, the processing of ASNase bioconjugationwith silk sericin protein greatly improved both thephysicochemical and biological stability and decreasedits antigenicity. Silk sericin bioconjugation technology,characterized by no activation prior to coupling, offersa simple and promising strategy for the enhancementof the therapeutic value of the enzyme.

ACKNOWLEDGMENTSThis work was partly supported by ‘State 863 Project’(2004AA2Z1020) and University Natural ScienceFunds of Jiangsu Province (04KJB180122), People’sRepublic of China.

REFERENCES1 Reynolds JEF, Asparaginase, in Martindale: The Extra Phar-

macopoeia, 30th edn. The Pharmaceutical Press, London,pp 456–457 (1993).

2 Ruyssen R and Lawers A, Asparaginase, in PharmaceuticalEnzymes: Properties and Assay Methods, Story-Scientia, Gent,pp 191–199 (1978).

3 Peterson RG, Handschumacher RE and Mitchell MS,Immunological responses to L-asparaginase. J Clin Invest50:1080–1090 (1971).

4 Wahn V, Fabry U, Korholz D, Reinhardt D, Jurgens H andGobel U, Modified pharmacokinetics of E. coli by formation ofspecific antibodies to L-asparaginase of different immunoglob-ulin classes in children with acute lymphocytic leukemia.Pediatr Pharmacol 3:303–311 (1983).

5 Jefferies SR, Richards R and Bernath FR, Preliminary studieswith L-asparaginase bound to implantable bovine collagenheterografts: a potential long-term, sustained dosage, anti-tumor enzyme therapy system. Biomat Med Dev Art Org5(4):337–354 (1977).

6 Hasselberger FX, Brown HD, Chattopadhyay SK, Mather AN,Stasiw RO, Patel AB and Pennington SN, The preparationof insoluble, matrix-supported derivatives of L-asparaginasefor use in cancer therapy. Cancer Res 30(11):2736–2738(1970).

7 Edman P and Sjoholm I, Acrylic microspheres in vivo. II.The effect in rat of L-asparaginase given in microparticlesof polyacrylamide. J Pharmacol Exp Ther 211:663–667(1979).

8 Mori T, Tosa T and Chibata I, Preparation and properties ofL-asparaginase entrapped in the lattice of polyacrylamide gel.Cancer Res 34(11):3066–3068 (1974).

9 O’Driscoll KF, Korus RA, Ohnuma T and Walczack IM,Gel entrapped L-asparaginase: kinetic behavior andantitumor activity. J Pharmacol Exp Ther 195:382–388(1975).

10 Jean-Francois J, D’Urso EM and Fortier G, Immobilization ofL-asparaginase into a biocompatible poly (ethylene glycol)-albumin hydrogel: evaluation of performance in vivo. BiotecholAppl Biochem 26:203–212 (1997).

11 Poznansky MJ, Shandling M, Salkie MA, Elliott J and Lau E,Advantages in the use of L-asparaginase-albumin poly-mer as an antitumor agent. Cancer Res 42(3):1020–1025(1982).

12 Wileman TE, Properties of L-asparaginase-dextran conjugates.Advanced Drug Delivery Reviews 6(2):167–180 (1991).

13 Ashihara Y, Kono T, Yamazaki S and Inada Y, Modification ofE. coli L-asparaginase with polyethylene glycol: disappearanceof binding ability to anti-L-asparaginase serum. BiochemBiophys Res Commun 83(2):385–391 (1978).

14 Nambu M, Process for producing immobilized L-asparaginasepreparations for the therapy of leukemia. US Patent4617271 (1986).

15 Manuel SM, 1994 Biotechnology drug approvals. Am PharmNS 35:12–13 (1995).

16 Keating MJ, Holmes R, Lerner S and Ho DH, L-asparaginaseand PEG asparaginase: past, present, and future. Leuk-Lymphoma 10(Suppl):153–157 (1993).

17 Berg SL, Balis FM, McCully CL, Goodwin KS andPoplock DG, Pharmacokinetics of PEGL-asparaginase andplasma and cerebrospinal fluid L-asparagine concentrationin rhesus monkey. Cancer Chemother Pharmacol 32:310–314(1993).

18 Alvarez OA and Zimmerman G, Pegaspargase-induced pan-creatitis. Medical and Pediatric Oncology 34:200–205(2000).

19 Kato N, Sato S, Yamanaka A, Yamada H, Fuwa N andNomura M, Silk protein, sericin, inhibits lipid peroxidationand tyrosinase activity. Biosci Biotech Biochem 62:145–147(1998).

20 Tamada Y, Anticoagulant and its production. Japan Patent 09-227402A (1997).

21 Sasaki M, Kato N, Watanabe H and Yamada H, Silk protein,sericin, suppresses colon carcinogenesis induced by 1,2-dimethylhydrazine in mice. Oncol Rep 7(5):1049–1052(2000).

22 Zhaorigetu S, Sasaki M, Watanabe H and Kato N, Supple-mental silk protein, sericin, suppresses colon tumorigenesisin 1, 2-dimethylhydrazine-treated mice by reducing oxida-tive stress and cell proliferation. Biosci Biotechnol Biochem65(10):2181–2186 (2001).

23 Kazuhisa T, Takagi H, Takahashi M, Yamada H and NakamoriS, Cryoprotective effect of the serine-rich repetitive sequencein silk protein sericin. J Biochem 129(6):979–986 (2001).

24 Sasaki M, Kato N and Yamada H, Consumption of silk proteinsericin elevates intestinal absorption of zinc, iron, magnesiumand calcium in rats. Nutrition Research 20(10):1505–1511(2000).

25 Sasaki M, Yamada H and Kato N, A resistant protein, sericin,improves atropine-induced constipation in rats. Food SciTechnol Res 6(4):280–290 (2000).

26 Zhang Y-Q, Application of natural silk sericin protein inbiomaterials. Biotechnology Advances 20:91–100 (2002).

27 Zhang Y-Q, Tao M-L, Shen W-D, Zhou Y-Z, Ding Y, Ma Yand Zhou W-L, Immobilization of L-asparaginase on themicroparticles of the natural silk sericin protein and itscharacters. Biomaterials 25:3751–3759 (2004).

144 J Chem Technol Biotechnol 81:136–145 (2006)

Synthesis of silk sericin peptides–L-asparaginase bioconjugates

28 Zhang Y-Q, Comparison analysis of degumming procedures forsilkworm silk. Acta Sericologica Sinica 28(1):75–79 (2002).

29 Zhang Y-Q, Shen W-D, Tao M-L, Zhou W-L, Ma Y andDing Y, Process for preparing water-soluble powder of silksericin protein. China Patent 1477141A (2004).

30 Laemmli UK, Cleavage of structural proteins during the assem-bly of the head of bacteriophage T4. Nature 227:680–685(1970).

31 Mashburn LT and Wriston JC, Tumor inhibitory effectof L-asparaginase. Biochem Biophys Res Comm 12:50–56(1963).

32 Edwards EA, Counter immunoelectrophoresis and doubleimmunodiffusion, in Rapid Diagnosis in Infectious Disease,ed by Ryfel W. CRC Press, Boca Raton, FL, pp 19–37(1979).

33 Habeeb AFSA, Determination of free amino group in proteinsby trinitrobenzenesulfonic acid. Anal Biochem 11:328–336(1996).

34 Sakabe H, Ito H, Miyamoto T, Noishiki Y and Ha WS, In vivoblood compatibility of regenerated silk fibroin. SEN-IGAKKAISHI 45:487–490 (1989).

35 Kurosaki Sadayuki, Otsuka Hirokuni, Kunitomo Mayumi,Koyama Mamoru, Pawankar Ruby and Matumoto Koshi,Fibroin allergy: IgE mediated hypersensitivity to silk suturematerials. J Nippon Med Sch 66(1):41–44 (1999).

36 Oyama F, Shimizu S and Shimura K, Study on immunologicalproperties of fibroin heavy and light chains. J Biochem96:1689–1694 (1984).

37 Santin M, Motta A, Freddi G and Cannas M, In vitro evaluationof the inflammatory potential of the silk fibroin. J BiomedMater Res 46:382–389 (1999).

38 Panilaitis B, Altman GH, Chen J, Jin H, Karageorgiou JV andKaplan DL, Macrophage responses to silk. Biomaterials24:3079–3085 (2003).

J Chem Technol Biotechnol 81:136–145 (2006) 145