adsorption of trypsin on commercial silica gel

Research Article

Adsorption of trypsin on commercial silica gel

The immobilization of trypsin onto various commercial silica gels was studied.Silica gels were used directly and characterized by mercuric porosimetry. Agitationrates (100–740 rpm) and particles size (35–75 to 250–500mm) of silica gels did notaffect the trypsin immobilization capacity. The pore size (3 to 15 nm) is a limitingfactor of the trypsin adsorption onto the mesopores structure of silica gels. Theadsorption of trypsin was determined as a function of their initial concentrationand multilayer formed at high trypsin concentration.

Keywords: Enzyme / Immobilization kinetic / Silica-gel / Trypsin

Received: March 03, 2009; revised: April 28, 2009; accepted: May 06, 2009

DOI: 10.1002/elsc.200900018

1 Introduction

Proteases are widely used in industrial and biomedical appli-cation, among which trypsin is the most extensively concerned.However, application of native enzyme is limited in industrialprocesses in regard to the problem of its instability and rapidlosing of biocatalytic activity during the operation and storageperiods resulting from its autolysis effect, protein unfoldingand aggregation. Besides that, it is difficult to remove theenzyme from the substrate solution and might lead to acontamination of the product [1–3]. The immobilization ofenzymes on supports has been the subject of considerableresearch for over 40 years and consequently different meth-odologies have been suggested, including crosslinking, covalentattachment, physical entrapment and physical adsorption. Theimmobilization technique should allow the enzyme to main-tain its catalytic activity while diminishing other processes thatare detrimental to the enzyme, such as autolysis. Manyresearchers have manifested that immobilized enzyme havegreat advantages over the native enzyme such as the stabilityand its recovery [4–6]. Of these methods, physical adsorptionis the most widely used.

It is evident, from the studies carried out that both the typeof surface and the method of immobilization could haveprofound effects on the resulting biological activity of thebound enzymes. Hydrophobicity, charge, and chemical make-up of the material could affect the stability of the protein [7]. Itis well known that the characteristics of the support (i.e. shape,particle size, porosity, chemistry, and mechanical strength)may strongly affect basic characteristics of the immobilizedenzyme [8, 9]. Therefore, the selection of the matrix is a key

factor influencing the activity and the applicability of theresulting bioreactor. Meanwhile, the immobilization methodcould also affect the enzyme activity through the chemicalmodification of the amino acids involved in coupling stepsespecially when the coupling place is much closed to the activeplace of an enzyme [10]. The solid supports used are almostalways polymeric resins, natural polymeric derivatives, organicgels, fibers, zeolite and mesoporous molecular sieves. However,inclusion of enzymes in the pores of microporous structures(i.e., zeolites) is an impossible task since the pore size of thesematerials is too small (o2 nm) [11]. The pore dimensions ofmesoporous materials offer the possibility of accommodatingenzymes within the channels.

Enzymatic reactions have been widely employed in variousindustrial processes [12, 13]. For example, trypsin iscommonly used in the hydrolysis of casinomacropeptide [12].The hydrolytic product (peptides) is of immense importancein the food industry and it is recognised as a functionalfood. The design and development of processes for thecontinuous production of the peptide on a large-scale at acompetitive cost is of interest. Another important applica-tion of immobilized hydrolytic enzyme like trypsin is inthe manufacture of hypoallergenic infant food [13]. Thisproduct contains peptides obtained from natural proteinsthat are treated with proteolytic enzymes to limit proteinhydrolysis thereby destroying the allergenic epitopes of naturalprotein. To manufacture these peptides, bioprocess withenzymes such as trypsin can be successfully used. Also, semi-synthesis of human insulin [14, 15], resolution of enantiomericO- or N, O-derivatized amino acids [16] and transesterifica-tion [17] have been investigated. Trypsin is a globular enzymewith molecular diameter 3.8 nm [11] and molecular mass23,800 Da [18] that could be incorporated into the porematerials.

The present work describes the immobilization of trypsinon commercial silica gels. Different parameters such as the

Jose M. Gomez

Ma. Dolores Romero

Gassan Hodaifa

Elena de la Parra

Departamento de Ingenierıa

Quımica, Universidad

Complutense de Madrid,

Spain

Correspondence: Jose M. Gomez ([email protected]), Depar-

tamento de Ingenierıa Quımica, Universidad Complutense de Madrid,

Spain.

& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim http://www.els-journal.com

336 Eng. Life Sci. 2009, 9, No. 4, 336–341

stirring rate, the particle and pore size of silica gels werestudied. Finally, the adsorption isotherm was obtained andfitted to the model.

2 Materials and methods

2.1 Reagent and materials

Commercial silica gels (Sigma-Aldrich) of different pore sizesin the range of 3–15 nm and silica gel 6 nm of different size ofparticles 35–75, 75–150, 150–250, and 250–500 mm were used.Lyophilized powder of porcine pancreatic trypsin 1,000-2,000BAEE units/mg solid, from Sigma Aldrich was used withoutfurther purification.

2.2 Mercury porosimetry

Mercury porosimetry (Thermo Electron Corporation, Pascal440 and 140 Series) was employed to characterize the porosityof silica gels by applying various levels of pressure to a sample(0.3 g approximately) immersed in mercury and at environ-mental temperature. The pressure required to intrude mercuryinto the pore sample is inversely proportional to the size of thepores. The analysis was carried out loading the sample into apenetrometer, which consists of a sample cup connected to ametal-clad, precision-bore, glass capillary stem. The penet-rometer is sealed and placed in a low pressure port, where thesample is evacuated to remove air and moisture. The cup ofpenetrometer and capillary stem are then automatically back-filled with mercury. As the pressure on the filled penetrometerincreases, mercury penetrates into the pores, beginning withthose pores of largest diameter. The instrument automaticallycollects low pressure measurement over the range of pressuresspecified (300 kPa). Then, the penetrometer is moved to thehigh pressure chamber, where high pressure measurements aretaken (400 MPa).

2.3 Enzyme adsorption

Enzyme adsorption was carried out by mixing enzyme(2.5–42 mg mL�1) with a silica gel suspension (final concen-tration of 10 mg mL�1) in 125 mL reactor with stirring at 251Cand pH 5 6.5 (using 25 mM phosphate buffer solution). Theamount of trypsin adsorbed has been determined by spectro-photometry at 280 nm by difference in the contents of trypsinbefore and after the adsorption, where the concentration oftrypsin after the enzyme immobilization has been determinedin the supernatant obtained by centrifugation.

The influence of the liquid-phase mass transfer on theperformance of a stirred immobilization of trypsin in silica-gelswas studied. Experiments with different speed agitation wereperformed at 100, 340, 500 and 740 rpm. The experiments with500 rpm were carried out in tube with 10 mL to study thechange of adsorption systems between batch reactor and tube.

Moreover, immobilized experiments with different pore andparticle sizes of silica gels were studied. To do that the pore size

between 3–15 nm, and 35–75 to 250–500 mm for the particlesize it have been varied. The immobilization of native trypsinhas been made with an initial concentration of trypsin of2.5 mg mL�1 and the concentration of commercial silica gelsolution as support 10 mg mL�1. Also, different enzymeconcentrations have been studied maintaining the sameconcentration in the support (10 mg mL�1). The immobiliza-tion experiments and analytical methods were made at least inthree times each. The calculation and statistical methods usedwere available in the OriginPro 7.5 program.

2.4 Experimental device

All the experiments were made in a batch reactor that wasagitated with Heidolph type RZR-1 motor. The 2-bladedimpellers, 0.0255 m in diameter, the vertical distance betweenthe impellers was 0.0355 m and the lower impeller was located0.0092 m from the bottom of the reactor.

The reactor vessel was 0.0519 m in diameter and its overallheight was 0.11 m. The working volume and the overallvolume of the reactor were 0.125 L and 0.210 L, respectively.The static liquid height was 0.0697 m in all experiments. Theimpellers were agitated with a 77 W motor (220 V).

3 Results and discussion

3.1 The influence of the external mass transfer

The liquid-phase mass transfer phenomenon on the reactorperformance was evaluated by varying the agitation rates from100 to 740 rpm using the following immobilization conditions:trypsin: 2.5 mg mL�1; silica-gel: 10 mg mL�1 (diameterpore 5 15 nm, particle size 5 75–150 mm) 25 mM bufferphosphate solution, pH 6.5 and temperature equal to 251C.The experiments were focused on evaluating the influence of

Figure 1. Influence of the liquid-phase mass transfer on theperformance of a stirred immobilization of trypsin in silica-gels:Experiments in batch reactor with 125 mL: J 100 rpm, ~ 340rpm and ^ 740 rpm. Experiments in tube with 10 mL: � 500 rpm.

Eng. Life Sci. 2009, 9, No. 4, 336–341 Adsorption of trypsin on commercial silica gel 337


the agitation rates on trypsin adsorption and on the cycle timerequired to reach the maximum trypsin adsorption (Fig. 1).

A mean value of 161 mg of trypsin adsorption equilibrium(g of silica gel)-1 was obtained when the agitation speed variedfrom 100 to 740 rpm (p 5 0.999, with a standard deviation ofSD 5 3), indicating no influence of external mass transfer.Also, in all the cases studied the maximum trypsin adsorptionwas obtained after 60 min (Fig. 1).

Comparing the adsorption system with other experimentscarried out in tube with 10 mL, no change was detected in theequilibrium concentration of the immobilized enzyme (Fig. 1).In the experiments carried out in the tube of 10 mL withagitation a break was detected in the support (it is difficult toseparate the support by centrifugation). For this reason, it isnot advisable to operate more than 60 min in this system.

3.2 Trypsin adsorption

The characteristics of the commercial silica-gels used in thisstudy are summarized in Table 1. SG indicates commercialsilica-gel and the number indicates the pore size of thecommercial material.

In Table 1, it can be see that the porosity of support hasbeen separated in three different ranges, pores with diameterinferior to 4 nm (o4% of total specific volume of pores), poreswith diameter between 4 and 200 nm (varied between 10% and61% of total specific volume pores), and pores with diametermore than 220 nm (varied between 36 and 88% of total specificvolume pores), Fig. 2.

Adsorption of enzyme on silica-gel is depending on thestructural and morphological features of the support. Thecommercial silica-gel is a material with micropores (porewidth up to 2 nm), mesopores (pore width ca.2 to ca.50 nm)and macropores (pore width450 nm) (Table 1). This struc-ture allows its use in enzyme adsorption (and other separationtechniques). The pore and particle sizes could be important tocontrol the immobilization process [11, 19, 20]. Firstly, theinfluence of the particle size on the immobilization of trypsinon silica-gel was studied. Four commercial supports with poresize of 6 nm and different particle sizes were used in this study(SG-6A, SG-6B, SG-6C and SG-6D). The characteristics ofthese materials are summarized in Table 1. Figure 3 shows theimmobilization of trypsin on silica-gel with different particlesizes and the same pore size.

The variation in the equilibrium trypsin adsorption values,qe, with the particle sizes (Fig. 3) was not significant (p 5 1, themean value of equilibrium trypsin adsorption 5 139 mg g�1,with a standard deviation of SD 5 9). Therefore, the particlesize had no influence on the immobilization of trypsin on thesilica-gel and the adsorption would be mainly carried out onthe internal surface of the supports. These results are similarwith those obtained by Lyubinskii et al. [21] working with S-80aminoorganosilokhrom activated with glutaraldehyde. In thiscase the amount of trypsin immobilized per gram was aroundof 20 mg g�1 which is lower than the amount obtained withsilica-gel.

The influence of the pore size on the trypsin adsorption wasstudied carrying out the immobilization on silica gels withpore size in the range from 3.0 to 15.0 nm. Fig. 4 shows the

Table 1. Characteristics of the commercial silica-gels.

Support Particle

size (mm) Diameter (nm)

Total cumulative

volume (cm3 g�1)

Pore diameter

ranges (nm)

Specific volume

(cm3 g�1)

%Specific

volume

SG-3 75–150 3 0.61 o4.0 0.009 2

4.0–220 0.06 10

217–8� 106 0.54 88

SG-4 63–200 4 1.1 o4.0 0.02 2

4.0–220 0.3 29

255–8� 106 0.7 69

SG-6A 35–75 6 1.3 o4.0 0.0 0

4.5–220 0.5 38

211–3� 106 0.8 62

SG-6B 75–150 6 1.2 o4.0 0 0

4.0–220 0.3 24

233–8� 106 0.9 76

SG-6C 150–250 6 1.1 o4.0 0.02 2

4.0–220 0.4 38

200–5� 106 0.7 60

SG-6D 250–500 6 0.86 o4 0.03 4

4.0–220 0.5 61

223–15� 106 0.3 36

SG-10 63–200 10 1.3 o4 0 0

4–220 0.3 21

215–8� 106 1.0 80

SG-15 75–150 15 1.5 o4 0 0

4–220 0.3 19

207–8� 106 1.2 81

338 J. M. Gomez et al. Eng. Life Sci. 2009, 9, No. 4, 336–341


immobilization of trypsin on silica-gel with different pore

sizes. Moreover, the experimental data were fitted to thepseudo-first-order Lagergren equation (Eq. 1), where k1 is therate constant of pseudo-first-order adsorption (min-1), qe andqt are the adsorbed amount on the support (mg g�1) in theequilibrium and at time t, respectively.

qt ¼ qeð1� e�k1tÞ ð1Þ

The study of the adsorption kinetics is desirable since itprovides information about the mechanism of adsorption anddiffusion of trypsin in the pores, which is important to opti-mising the efficiency of the process and their application inindustrial catalysis. The kinetic parameters are shown in Table 2.

Figure. 4 shows the experimental results of the adsorptionon silica-gel and its fitting to a pseudo-first order kineticmodel (Eq. 1).

Adsorption of trypsin was not carried out on the SG-3 sincethe pore size 3.0 nm was smaller the molecular size of theenzyme 3.8 nm; there are only 10% of the specific volume of

pores with diameter between 4 to 220 nm (Table 1). So thetrypsin could not enter in the pore (pore diameter is a limitingfactor) and the adsorption was only on the external surface(13 mg g�1). This structure indicated that the adsorption wascarried out at the second range of diameter (trypsin moleculediameter 3.8 nm), while the third range are the pores withdiameter larger than 200 nm and they can be considered as apart of the external surface of the silica-gel and it is notpossible to be used for adsorption. It has been previouslyreported that the protein size is a limiting factor for theadsorption occurs [12]. This fact confirmed the hypothesis thatno adsorption enzyme takes place in the macropores (Fig. 2).In the other supports the pore size is bigger than the molecularsize of the trypsin and it could come into the pores, being theadsorption on the internal surface. In general, the adsorptionof trypsin in the equilibrium was augmented as the pore size ofthe silica gel was increased (Fig. 4). When the pore size is4.0 nm the amount of trypsin adsorbed was clearly increased(without limiting factor) in respect to the silica-gel with poresize of 3.0 nm. However, when the pore size was increased from6.0 to 15.0 nm the amount adsorbed scarcely varied between

Figure 4. Influence of the pore size of the silica-gel in the trypsinadsorption. Supports: & SG-3, J SG-4, ~ SG-6D, & SG-10and * SG-15. Immobilization conditions: trypsin: 2.5 mg mL�1;silica-gel: 10 mg mL�1, 25 mM; buffer solution: 25 mM phos-phate and pH 6.5; temperature: 25 1C.

Table 2. Parameters of the pseudo-first order kinetic model: poresize influence.

Support Pore

size

(nm)

qe;experimental

(mg g�1)

Pseudo-first order

kinetic model

qe

(mg g�1)

k1

(min�1)

R2

SG-3 3 13 – – –

SG-4 4 107 110 0.09 0.97

SG-6D 6 133 135 0.23 0.99

SG-10 10 142 142 0.90 0.99

SG-15 15 158 159 1.55 0.99Figure 3. Equilibrium trypsin adsorption with different particlesizes of commercial silica-gels. Immobilization conditions:trypsin: 2.5 mg mL�1; silica-gel (6 nm): 10 mg mL�1, 25 mMbuffer phosphate solution and pH 6.5; temperature: 25 1C.

Figure 2. Schematic porous diameter ranges on commercialsilica-gel particle.



139 mg g�1 and 158 mg g�1. On the other hand, it is interestingto indicate that the increase in the value of k1 (Table 2) withthe augment of pore size indicated clearly the influence of theinternal diffusion process (exit and entry of molecules tothe internal surface). This limiting factor has no influence onthe diffusion rate when the pore diameter is larger than 10 nm(Fig. 3).

Similar results were obtained by another authors in theimmobilization of trypsin on other supports such as MCM-48(2.4 nm), MCM-41 (3.5 nm), SBA-15 (5.6 nm), SBA-15M2(10.0 nm), CNS (18.0 nm), COS (6.0 nm), MCM-41/33(3.3 nm), MPS-F127 (5.1 nm) and MCM-41/28 (2.8 nm)[22, 23]. In terms of the amount of trypsin per gram of silica-gels, similar values have been obtained to those cited in theliterature but using a commercial support at shorter immo-bilization time [24]. On the other hand, the amount of trypsinimmobilized per gram is lower than the amount obtained withsilica-gel.

3.3 Adsorption isotherm

In order to determined the adsorption isotherm the initialtrypsin concentration was varied in the interval from 2.5 to42 mg mL�1. SG-15 was selected as support to determinate theisotherm equilibrium. Firstly, equilibrium time for theadsorption was determinate. Figure 5 shows the variation inenzyme immobilized over time for three experiments withdifferent initial trypsin concentrations.

The adsorption proceeds at a high rate (E2 min) and theadsorption equilibrium is attained in 60 min (Fig. 5) beingachieved in the first two minutes around 70% of the totaladsorbed by the support.

Figure 6 shows the adsorption isotherms of trypsin oncommercial silica gel (SG-15). The experimental data were

adjusted to the Langmuir model described in Eq. 2

qe ¼qmaxKCTrypsin

1þ KCTrypsinð2Þ

where qe is the load of enzyme (mg) adsorbed per gram ofsupport on equilibrium and Ctrypsin is the enzyme concentration(mg mL�1) on equilibrium. The maximum adsorption expressedin terms of enzyme was qmax 5 3102 mg trypsin (g SG-15)�1 andthe model constant is K 5 0.0583 (mg mL�1)�1 and R2 5 0.999.

Figure 6 shows the proper adjustment of Langmuir to theexperimental data up to 16.7 mg mL�1 (1500 mg/g silica gel). Fromthis value the second layer adsorption appears. This second layerbegins before reaching the maximum capacity of the model. Themaximum adsorption expressed in terms of enzyme which deter-mines the mathematical model (Eq. 2) is an apparent value (dashline Fig. 6). Really, the second layer starts forming before it reachesthe maximum adsorption corresponding to the first layer (Fig. 6).The real maximum value of trypsin adsorbed (1527 mg)/g of silica(SG-15) in the monolayer corresponds a trypsin equilibriumconcentration in the liquid phase CTrypsin 5 16.7 mg trypsin mL�1.The immobilization of trypsin on commercial silica-gel occursboth in a monolayer (solid line in Fig. 6) and in a multilayer form.The maximum real value of trypsin adsorbed is higher than othervalues registered with other supports as CNS (18 nm), COS(6 nm), MCM-41/33 (3.3 nm), MPS-F127 (5.1 nm), MCM-41/28(2.8 nm) and MCC [23, 25].

The pore size of the silica-gel with an average pore diameterof 15 nm, allows the enzyme molecules to access easily to thepores leading to a high immobilization. Multilayer adsorptionis largely confined to the macropores (pore diameter4220nm) representing 81% of specific volume (Table 1).

4 Conclusions

The physical immobilization of trypsin in the mesoporestructure of silica gels has been demonstrated. The agitation

Figure 5. Effect of the initial trypsin concentrations in theenzyme adsorbed on commercial silica-gel support, * 16, n 24,^ 32 mg trypsin mL�1 (Conditions: 10 mg SG-15 mL�1, porediameter of particle dP = 15 nm, 25 mM buffer phosphatesolution, pH = 6.5, temperature 251C).

Figure 6. Adsorption isotherm of trypsin to commercial silica-gel(SG-15), * Experimental data, -Langmuir. Conditions: 10 mgSG-15 mL�1, 25 mM buffer phosphate solution, pH = 6.5,temperature 251C

340 J. M. Gomez et al. Eng. Life Sci. 2009, 9, No. 4, 336–341


speed (4100 rpm) does not affect the immobilization capacityof silica gels. In all the experiments made in this study with adifferent pore and particle size of silica gels the trypsin hasbeen adsorbed, only in the case when the pore diameter was of3 nm the trypsin has not been adsorbed in a satisfactory form.These results confirm the very strong interaction betweentrypsin and commercial silica-gel. The maxima adsorption(159 mg g�1) has been reached with a pore size of 15 nm afterrelatively short time equal to 4 min. In comparison, in all theexperiments the equilibrium phase has been reached in acontact time inferior to 25 min. The particles size does nothave any effect on the trypsin immobilization. Adsorptionisotherm is described by the Langmuir equation.

Acknowledgements

Financial support was provided by the Santander group andthe Complutense University of Madrid with the project No.PR34/07-15825.

Conflict of interest statement

The authors have declared no conflict of interest.

References

[1] R. Villalonga, M. L. Villalong, V. Gomez, Preparation and

functional properties of trypsin modified by carbox-

ymethylcellulose. J. Mol. Catal. B-Enzym. 2000, 10, 483–490.

[2] Z. Zhang, Z. He, M. He, Stabilization mechanism of MPEG

modified trypsin based on thermal inactivation kinetic

analysis and molecular modeling computation. J. Mol. Catal.

B-Enzym. 2001, 14, 85–94.

[3] E. Ruckenstein, W. Guo, Cross-linking mercerized cellulose

membranes and their application to membrane chromato-

graphy. J. Membrane Sci. 2001, 187, 277–286.

[4] M. Nouaimi, K. Moschel, H. Bisswanger, Immobilization of

trypsin on polyester fleece via different spacer. Enzym.

Microbial. Technol. 2001, 29, 567–574.

[5] A. Kumar, M. N. Gupta, Immobilization of trypsin on an

enteric polymer Eudragit S-100 for the biocatalysis of

macromolecular substrate. J. Mol. Catal. B-Enzym. 1998, 5,

289–294.

[6] T. N. Krogh, T. Berg, P. Hojrup, Protein analysis using

enzymes immobilized to paramagnetic beads. Anal. Biochem.

1999, 274, 153–162.

[7] M. Martin, L. Anders, Immobilization of trypsin on porous

glycidyl methacrylate beads: effects of polymer hydro-

philization. Coll. Surf. B-Biointerf. 2000, 18, 277–284.

[8] Z. Bılkova, A. Castagna, G. Zanusso, A. Farinazzo et al.,

Immunoaffinity reactors for prion protein qualitative

analysis. Proteomics 2005, 5(3), 639–647.

[9] A. Podgornik, T. B. Tennikova, Chromatographic reactors

based on biological activity. Adv. Biochem. Eng. Biotechnol.

2002, 76, 165–210.

[10] M. M. E1-Masry, A. D. Maio, P. L. Martelli, Influence of the

immobilization process on the activity of b-galactosidase

bound to nylon membranes grafted with glycidyl metha-

crylate. Part 1. Isothermal behaviour. J. Mol. Catal. B-Enzym.

2001, 16, 175–189.

[11] J. F. Jr. Diaz, K. J. Balkus, Enzymes immobilized in

MCM-41 Molecular Sieves. J. Mol. Catal. B: Enzym. 1996, 2,

115–126.

[12] A. Margot, E. Flaschel, A. Renken, Development of a

continuous operated reactor for the limited hydrolysis of

whey protein by trypsin. Process Biochem. 1998, 33 (2),

125–131.

[13] V. M. Prata, S. Bouhallab, G. Henry, P. Aimar, An experi-

mental study of caseinomacropeptide hydrolysis by trypsin

in a continuous membrane reactor. Biochem. Eng. 2001, 8

(3), 195–202.

[14] K. Ueno, K. Morikawa, Use of immobilized trypsin for

semisynthesis of human insulin. Biotechnol. Bioeng. 1989,

33(1), 126–128.

[15] K. Morihara, K. Ueno, A new procedure for enzymatic

semisynthesis of human insulin by hydrolysis of single-chain

des-(b-30)- insulin precursor with lysyl endopeptidase.

Biotechnol. Bioeng. 1991, 37(7), 693–695.

[16] K. Kasai, Trypsin and affinity chromatography. J. Chroma-

togr. 1992, 597(1– 2), 3–18.

[17] J. M. Gomez, J. Deere, D. Goradia, J. Cooney, E. Magner, B.

K. Hodnett, Transesterification catalyzed by trypsin

supported on MCM-41. Catal. Lett. 2003, 88(3– 4), 183–186.

[18] R. M. Stroud, L. M. Kay, R. E. Dickerson, The structure of

bovine trypsin: electron density maps of the inhibited

enzyme at 5 Angstrom and at 2-7 Angstron resolution.

J. Mol. 1974, 83, 185–208.

[19] Y. J. Han, J. T. Watson, G. D. Stucky, A. Butler, Catalytic

activity of mesoporous silicate-immobilized chloroperox-

idases. J. Mol. Catal. B 2002, 17, 1–8.

[20] K. L. Washmon, V. L. Jr. Jimenez, K. J. Balkus, Cytochrome c

immobilized into mesoporous molecular sieves. J. Mol.

Catal. B: Enzym. 2000, 10, 453–469.

[21] G. V. Lyubinskii, E. A. Kalinichenko, V. A. Tertykh, Porous

structure and trypsin capacity of an activated silica matrix.

Theor. Experim. Chem. 2004, 28(3), 216–219.

[22] H. P. Y. Humphrey, P. A. Wright, P. N. Botting, Enzyme

immobilisation using siliceous mesoporous molecular sieves.

Micropor. Mesopor. Mat. 2001, 44– 45, 763–768.

[23] D. Goradia, J. Cooney, B. K. Hodnett, E. Magner, The

adsorption characteristics, activity and stability of trypsin

onto mesoporous silicates. J. Mol. Catal. B: Enzym. 2005, 32,

231–239.

[24] H. Takahashi, B. Li, T. Sasaki, C. Miyazaki et al., Immobilized

enzymes in ordered mesoporous silica materials and

improvement of their stability and catalytic activity in an

organic solvent. Micropor. Mesopor. Mat. 2001, 44– 45,

755–762.

[25] N. E. Kotel’nikova, A. S. Mikhailova, E. N. Vlasova, Immo-

bilization of proteolytic enzymes trypsin and a-Chymo-

trypsin to cellulose matrix. Russian. J. Appl. Chem. 2007,

80(2), 322–329.



adsorption of trypsin on commercial silica gel

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