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Brazilian Journal of Chemical Engineering

Print version ISSN 0104-6632On-line version ISSN 1678-4383

Braz. J. Chem. Eng. vol. 14 no. 4 São Paulo Dec. 1997 




1Departamento de Engenharia Química– Universidade Federal de São Carlos
C.P. 676 - CEP 13565-905 São Carlos – SP – Brazil– Fax number: (016) 274-8266;
2Unidad de Biocatalisis – Instituto de Catalysis y Petroleoquimica/CSIC-UAM
Cantoblanco, 28049 Madrid – Spain


(Received:June 11, 1997; Accepted: October 30, 1997)


Abstract - Multi-point immobilization, by an intense enzyme-support attachment, may increase the operational stability of a biocatalyst. Penicillin G acylase has many applications, from the hydrolysis of penicillin G (production of 6-aminopenicillanic acid) to the synthesis of semi-synthetic antibiotics. The application of this technique in macroporous silica involves support activation with 3-glycidyloxypropyltrimetoxysilane, followed by acidic hydrolysis and oxidation with sodium periodate. The aldehyde-glyoxyl groups so formed react subsequently with the enzyme. The degree of activation affects the yield and stability of the enzyme immobilization. For 20 UI of enzyme, the results show an immobilization yield equal to 100%, whenever there are more than 140 m Eq of aldehyde groups/g of dry silica. The immobilized enzyme half-life is 23 minutes at 60ºC; under the same conditions, the soluble enzyme has no residual activity after a few minutes. The increase in the degree of activation does not lead to higher stability, which indicates the negative influence of sub-products, formed during the activation of the support.
Multi-point immobilization, biocatalyst, silica.




The intrinsic properties of enzymes - high catalytic efficiency and specificity - make enzymatic process a good alternative to traditional chemical methods. Penicillin G acylase has many applications, from the hydrolysis of penicillin G or V - in order to produce 6-aminopenicillanic acid (6-APA), an intermediate in the synthesis of semisynthetic penicillins - to the resolution of racemic mixtures, the production of aromatic esthers and the synthesis of semi-synthetic antibiotics. The hydrolysis of penicillin G using immobilized Penicillin G acylase is one of the few industrial processes where the enzymatic route is used. Generally, water solubility and low operational stability limit the use of enzymes at the industrial scale.

The immobilization of enzymes on insoluble supports allows their reutilization, avoiding losses and reducing the cost of recuperation. Enzyme immobilization using enzyme-support multi-point attachment may cause a significant increase in its stability (Guisán, 1988). Within this procedure, aldehyde groups previously added to the support are linked to amino groups of the enzyme, forming Schiff bases. Reduction with borohydride converts these links into very strong bonds and the tertiary structure of the enzyme becomes more stable. The amino groups (present in the lysine residues available on the enzyme surface, plus the amino terminal group) are very reactive when unprotonated. This reactivity causes a very intense multi-interaction with the activated support and allows a multi-point attachment.

There is a wide range of supports that can be used for enzyme immobilization. Macroporous silica was chosen in this work because it is cheap and can be regenerated. The formation of glyoxyl (aldehyde) groups on the silica is achieved by silanization with 3-glycidyloxypropyltrimetoxysilane (GPTMS), followed by acidic hydrolysis to open the oxirane ring and to obtain vicinal hydroxy groups. Finally, the aldehyde groups are formed by oxidation with sodium periodate. The degree of activation of the support (i.e., the number of aldehyde groups on the silica surface) is related to the intensity of the multi-interaction between enzyme and support. Consequently, different values for the thermal stability and recovered activity may be obtained when the silanization conditions are changed. Using GPTMS (an exception among other silanes in view of its highly reactive oxirane ring), this work analyzes the influence of silanization conditions on the immobilization of penicillin G acylase on macroporous silica.




Agarose 10-CL was donated by Hispanagar S.A. (Burgos, Spain). Macroporous silica with a mean pore of diameter 273 Å , a BET specific area of 56,6m2/g, an adsorption surface of 53,51m2/g, a pore volume of 0,3262cm3/g, and a particle diameter between 150-250 m m, was prepared in the Department of Technological Chemistry and Applications of the University of the State of São Paulo, Araraquara (Trevisan et al., 1993). Penicillin G acylase from E.coli with nominal activity of 520 UI/ml (1 UI is the amount of enzyme which hydrolyzes 1 µ mol of 5% penicillin G at 37ºC, pH=8.0, 0.1M phosphate buffer) was generously donated by Antibióticos S.A. (Madri, Spain). All reagents used are of an analytical grade of different commercial brands.

Activation of Macroporous Silica

Glyoxyl-silica supports are obtained by silanization with GPTMS under different conditions of silane concentration, solvent (water or toluene), pH, temperature, time of silanization and ratio in solution/g silica, always under gentle stirring. After silanization, the supports are washed with toluene (or water, depending on the solvent used in the reaction) and acetone and dried at 40ºC for 1 hour. The hydrolysis of epoxy groups is done with 0.1M sulphuric acid during 2 hours at 85ºC, with the same ml solution/g silica ratio as that used for silanization. After filtration, washing with water/acetone and drying, oxidation proceeds for different periods of time at room temperature, with an initial ratio of 5ml of solution (3ml of water and 2ml of 0.1M sodium periodate solution)/g silica, by adding small volumes of 0.1M sodium periodate solution. Uni-point derivatives are prepared with 10% of the periodate used in the preparation of the multi-point support. The amount of periodate that is not consumed in the reaction is measured by titration of the filtrate with KI (Guisán, 1988).

Immobilization of Penicillin G Acylase

A solution of 100mM phenylacetic acid (PAA) is prepared in a 0.1M bicarbonate buffer at pH = 10,0. After the adjustment of the pH, the enzyme (20UI/g dry silica) is added at 4ºC and the resulting solution is added to the wet activated silica at the ratio of 20ml solution/g dry silica. It reacts under a gentle stir at 20ºC for 3 hours and then is reduced with borohydride (1mg/ml) during 30 minutes at the same temperature as that of immobilization. Afterwards, the derivatives are washed with a 5mM phosphate buffer at pH = 7.0 and 0.01% sodium azide. Samples of the supernatant and of the suspension are taken periodically.

Activity of Penicillin G Acylase

This activity of the soluble enzyme is checked by measuring the increase of absorbance at 348 nm, which accompanies the hydrolysis of p-nitrophenyl phenylacetate - PAONP- (Wang et al, 1986) at 25ºC. For the immobilized enzyme, aliquots are taken from a stainless steel basket immerged in the reactor. This procedure prevents the withdrawal of silica during the sampling. The activity of the immobilized enzyme is studied by measuring the amount of 6-APA released during the hydrolysis of 5% penicillin G at 37ºC and pH = 8.0 at different points in time, by reaction with p-aminobenzaldehyde (5), with absorbance measured at 415nm.

Thermal Stability

Soluble or immobilized enzyme is incubated at 60ºC in a 0.05M phosphate buffer and pH = 8.0. At different points in time the residual activity is measured with PAONP.



Influence of Silanization Conditions on Degree of Activation

The support silanization is conducted under different experimental conditions - table 1. At 25ºC and a ratio of 1g silica/5ml solution of 15% GPTMS, 24 hours in toluene results in activation identical to that obtained with water at pH = 8.5 for 1 hour, which indicates that a polar solvent shows the best performance. In the case of toluene, an increase in silanization temperature would be difficult to handle experimentally due to the great degree of inflammability of this solvent, and consequently, it is not tested. This result agrees with the one obtained by Porsch, 1993. This author says that in toluene an "ether" rather than a "glycol" bonded phase is obtained with high epoxide loss. The best results at pH = 8.0 are also in accordance with this work, which says that the pH of the solution must be greater than 4.0 to avoid the oxirane ring opening to glyceryl ether. It also says that the mechanism of oxirane ring opening is different in acidic and basic media. Alkyloxiranes form corresponding ether and primary hydroxy groups capable of further addition of another oxirane ring during acid catalysis; basic catalysis produces mainly secondary hydroxy groups with low reactivity towards other epoxide groups. Hence, the basic catalysis might decrease the formation of higher adducts.

The best result obtained for a low concentration of GPTMS is a consequence of the high reactivity of this reagent, which can lead to its polymerization instead of a reaction with silica when high concentrations are used. The increase in degree of activation with the increase of temperature and reaction time is kinetically expected. Finally, notice that the oxidation occurs slowly in samples with low degrees of activation (up to 140 µ moles CHO/g silica). This is the reason why, in the majority of the experiments, when time and temperature of silanization are compared, the oxidation is not followed until completion.

Immobilization Yield of Penicillin G Acylase and Recovered Activity for Different Degrees of Activation of Silica-glyoxyl

Table 2 shows the results of immobilization yield and recovered activity of penicillin G acylase in activated macroporous silica for different conditions. Immobilization yield is the ratio between the amount of enzyme which disappears from the supernatant and the enzyme initially added to the support. Recovered activity is the ratio between the amount of enzyme in the silica after immobilization and the enzyme which disappears from the supernatant (and was adsorbed). Uni-point derivatives have a low degree of activation and, consequently, a minimum possible number of bonds with the enzyme. In the point of view of stability, the uni-point derivatives must show behavior similar to that of the soluble enzyme. Since they are prepared with an immobilized enzymatic load similar to that of the soluble enzyme, they are used here as a base of comparison with respect to the stabilization effects of the multi-point attachment.


Table 1: Degree of activation of silica (µ moles-CHO/g dry silica) under different experimental conditions

Time (h)
Solvent g silica/ml
Time (h)
µ moles
60 1 3.3 15 Water 1/5 1 34.3
25 1 3.3 15 Water 1/5 1 8
60 1 8.5 15 Water 1/5 1 30
25 24 3.3 15 Water 1/5 1 25
25 24 - 15 Toluene 1/5 1 28
60 1 3.3 15 Water 1/5 0.5 25
60 3 3.3 15 Water 1/5 0.5 31
60 5 3.3 15 Water 1/5 0.5 32
60 3 3.3 15 Water 1/5 5 70
25 1 8.5 5 Water 1/30 5 85
60 1 8.5 5 Water 1/30 5 140
60 3 8.5 5 Water 1/30 5 180
60 5 8.5 5 Water 1/30 5 200
90 5 8.5 5 Water 1/30 5 2900


Table 2: Immobilization yield (%) and recovered activity of penicillin G acylase (PGA) immobilized in silica-glyoxyl with different degrees of activation. Uni: PGA immobilized in low-activated support

  Degree of activation of support (µ moles-CHO/g dry silica)
  4 (Uni) 40 70 140 600 2900
Immobilization Yield (%) 0.05 5.0 70.0 100.0 100.0 100.0
Recovered Activity (%) 100.0 100.0 100.0 100.0 100.0 100.0


Figure 1: Thermal stability for different degrees of activation of the support; a) 70 µ moles CHO/g dry silica; b) 140 µ moles CHO/g dry silica; c) soluble enzyme; d) 2900 µ moles CHO/g dry silica.


According to table 2, for degrees of activation above 140 µ moles CHO/g dry silica, the immobilization yield is always equal to 100%. For a low degree of activation, however, the available number of aldehyde groups is insufficient to allow the correct alignment of the enzyme amino groups that would make the multi-point attachment possible. The recovered activity of the immobilized enzyme is always 100%, which is expected due to the presence of an inhibitor that protects the enzyme, avoiding bonding with the support which damages the active site. The uni-point derivatives have low immobilization yield due to the low number of activated groups in the support.

Thermal Stability of Silica Derivatives with Different Degrees of Activation

The results of thermal stability for soluble enzyme and derivatives incubated in a phosphate buffer at pH = 8.0 for different periods of time at 60ºC appear in the figure 1.

The results obtained for thermal stability of PGA derivatives with different degrees of activation show that there is an optimum activation degree of the support. Above that optimum, the simultaneous formation of sub-products prevents the enzyme from making extra bonds with the support to increase its rigidity and, consequently, its stability. The final distance between the enzyme and the silica-glyoxyl support probably does not permit a correct alignment of amino groups with aldehyde groups of the support, or else this distance is too great to confer stability. In this case, although a great number of multi-point attachments have been made, the enzyme has sufficient mobility to vibrate with the increase in temperature, thus losing activity. Although a significant increase in stability, in comparison with that of the soluble enzyme and of the uni-point derivative, is obtained, the increase is lower than that reported by Guisán, 1988, for activated agarose with glycidol, whose derivatives maintain 100% activity during 3 hours at 60ºC. It is evident that the lower stability presented by the derivatives with higher degrees of activation, when compared to the intermediate ones, is caused by an increase in the formation of sub- products. The best derivative tested - 140 µ moles CHO/g silica - is obtained with 5 hours of silanization at 60ºC, pH = 8.5 and 1 hour of oxidation. It is possible that the same degree of activation, if attained at lower temperatures and/or lower times of silanization, will lead to better results for thermal stability, which will be examined in the future.



A minimum degree of activation of the support is necessary to achieve 100% immobilization yield. Nevertheless, higher degrees of activation may lead to lower thermal stability, despite of the improvement in immobilization and the higher recovery of activity that occurs in this situation. This is probably due to the development of sub-products during activation. The best immobilization parameters are obtained performing the silanization at 60ºC, pH = 8.5, during 5 hours. An increase in the stability of derivative compared to that of the soluble enzyme is observed.



This work has been supported by PADCT/CNPq and CNPq/RHAE.



Guisán, J.M., Aldeyde-Agarose Gels as Activated Supports for Immobilization-Stabilization of Enzymes. Enzyme Microb. Technol. 10, 375-382 (1988).         [ Links ]

Porsch, B., Epoxy- and Diol-Modified Silica: Optimization of Surface Bonding Reaction. Journal of Chromatography A., 653, 1-7, (1993).         [ Links ]

Trevisan, H.C.; Mei, L.H.I. and Zanin, G.M., Hydrometal Treatment of Silica Gels Aiming the Preparation of Supports for Enzyme Immobilization. Annals of the Sixth European Congress on Biotechnology, TU 259, Florence (1993).         [ Links ]

Wang, Q.C.; Fei, J.; Cui, D.F.; Zhu, S.G. and Xu, L.G., Application of an Immobilized Penicillin Acylase to the Deprotection of N-phenylacetyl Insulin. Biopolymers 25, 5109-5114 (1986).         [ Links ]

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