The presence of thiolated compounds allows the immobilization of enzymes on glyoxyl agarose at mild pH values: New strategies of stabilization by multipoint covalent attachment

Highly activated glyoxyl-supports rapidly immobilize proteins at pH 10 (where the -amino groups of the Lys groups of the protein surface are very reactive), and stabilize them by multipoint covalent attachment. However, they do not immobilize proteins at pH 8. This paper shows that the enzyme immobilization at this mild pH value is possible by incubation of the enzymes in the presence of different thiolated compounds (dithiothreitol, DTT; was selected as optimal reagent). The thiolated compounds (even the not reducing ones) stabilized the imino bonds formed at pH 8 between the aldehydes in the support and the amino groups of the protein. However, thiolated compounds are unable to reduce the imino bonds or the aldehyde groups and a final reduction step (e.g., using sodium borohydride) was always necessary. After enzyme immobilization through the most reactive amino group of the protein, the further incubation of this immobilized enzyme at pH 10 would improve the reactivity of the -amino groups of the Lys residues of the protein surface. Then, an intense multipoint covalent reaction of the enzyme with the dense layer of glyoxyl groups in the support could be obtained, increasing the stability of the immobilized enzyme. Using three different industrially relevant enzymes (penicillin G acylase from Escherichia coli (PGA), lipase from Bacillus thermocatenulatus (BTL2) and glutaryl acylase from Pseudomonas sp. (GA)), new immobilized-stabilized biocatalysts of the enzymes were produced. After reduction, the preparations incubated at pH 10 were more stable than those that were only immobilized and reduced at pH 8. In the case of the PGA, this preparation was even 4–5-fold more stable than those obtained by direct immobilization at pH 10 (around 40,000–50,000-fold more stable than the soluble enzyme).     

Positive effects of the multipoint covalent immobilization in the reactivation of partially inactivated derivatives of lipase from Thermomyces lanuginosus

Different immobilized preparations of lipase from Thermomyces lanuginosus (TLL) have been inactivated by exposure to high temperatures, guanidine or 95% of dioxane. The studied preparations were: non-stabilized cyanogen bromide (CNBr-TLL), aminated CNBr-TLL (CNBr-TLL-A), and two stabilized preparations of aminated TLL by immobilization on glyoxyl support, Gx(9/10)-TLL-A (TLL-A immobilized at pH 9 and later incubated at pH 10) or Gx(10)-TLL-A (directly immobilized at pH 10). The reactivation of the partially inactivated immobilized enzymes under mild conditions by incubation in aqueous buffer, allowed recovery of some of the original activity, which was improved when it was pre-incubated in guanidine. Amination produced a fairly negative effect on the reactivation of the enzyme, but the multipoint covalent attachment of this aminated enzyme reversed the effect (e.g., recovered activity increased from 20% for CNBr-TLL to 80% for Gx(9/10)-TLL-A). The negative effect of the amination was clearer when the inactivation was caused by exposure to high temperatures, although the multipoint attachment of aminated enzyme was able to improve the recovered activity. The determination of enzyme activity in the presence of hexadecyltrimethylammonium bromide slowed the inactivation rates of all preparations and improved the recovery of activity after incubation under mild conditions, suggesting that the opening mechanism of the lipase could be a critical step in the TLL inactivation/reactivation. The use of multipoint attached TLL preparations did not only improve enzyme stability, but it also increased activity recovery when the preparation was incubated under mild conditions.     

Purification, immobilization and stabilization of a highly enantioselective alcohol dehydrogenase from Thermus thermophilus HB27 cloned in E. coli

This paper shows the purification and immobilization of a very interesting thermophilic alcohol dehydrogenase from Thermus thermophilus HB27 cloned in Escherichia coli. The purification was based on a first thermal treatment of the crude extract, that leaves the target enzyme in the supernatant, followed by the adsorption of most contaminant proteins in a IMAC column (the target protein did not adsorb on these columns due to the poorness of His residues). Final purification factor was around a 9-fold factor (no other protein bands were detected in SDS-PAGE gels) with an overall yield around 80%. Covalent immobilization of the enzyme on very different supports only permitted to improve the enzyme stability by a 5–10-fold factor, very similarly to the results obtained by the adsorption of the enzyme on polyethyleneimine coated supports. This enzyme adsorbed by ionic exchange maintained the activity unaltered during immobilization which was a very rapid process, and was more stable than the covalent preparations in the presence of organic solvents, and the enzyme was quite strongly adsorbed on the support. Therefore, it was proposed as a good option to prepare industrial biocatalysts of the enzyme. This preparation was utilized in the asymmetric reduction of acetophenone to produce (S)-(−)-1-phenylethanol, with an enantiomeric excess of more than 99%.     

Immobilization–stabilization of the lipase from Thermomyces lanuginosus: Critical role of chemical amination

This paper describes the immobilization and stabilization of the lipase from Thermomyces lanuginosus (TLL) on glyoxyl agarose. Enzymes attach to this support only by the reaction between several aldehyde groups of the support and several Lys residues on the external surface of the enzyme molecules at pH 10. However, this standard immobilization procedure is unsuitable for TLL lipase due to the low stability of TLL at pH 10 and its low content on Lys groups that makes that the immobilization process was quite slow. The chemical amination of TLL, after reversible immobilization on hydrophobic supports, has been shown to be a simple and efficient way to improve the multipoint covalent attachment of this enzyme. The modification enriches the enzyme surface in primary amino groups with low pKb, thus allowing the immobilization of the enzyme at lower pH values. The aminated enzyme was rapidly immobilized at pH 9 and 10, with activities recovery of approximately 70%. The immobilization of the chemically modified enzyme improved its stability by 5-fold when compared to the non-modified enzyme during thermal inactivation and by hundreds of times when the enzyme was inactivated in the presence of organic solvents, being both glyoxyl preparations more stable than the enzyme immobilized on bromocyanogen.     

Enhancing the functional properties of thermophilic enzymes by chemical modification and immobilization

The immobilization of proteins (mostly typically enzymes) onto solid supports is mature technology and has been used successfully to enhance biocatalytic processes in a wide range of industrial applications. However, continued developments in immobilization technology have led to more sophisticated and specialized applications of the process. A combination of targeted chemistries, for both the support and the protein, sometimes in combination with additional chemical and/or genetic engineering, has led to the development of methods for the modification of protein functional properties, for enhancing protein stability and for the recovery of specific proteins from complex mixtures. In particular, the development of effective methods for immobilizing large multi-subunit proteins with multiple covalent linkages (multi-point immobilization) has been effective in stabilizing proteins where subunit dissociation is the initial step in enzyme inactivation. In some instances, multiple benefits are achievable in a single process.Here we comprehensively review the literature pertaining to immobilization and chemical modification of different enzyme classes from thermophiles, with emphasis on the chemistries involved and their implications for modification of the enzyme functional properties. We also highlight the potential for synergies in the combined use of immobilization and other chemical modifications.     

Design of β‐galactosidase/silica biocatalysts: Impact of the enzyme properties and immobilization pathways on their catalytic performance

The use of heterogeneous biocatalysis in industrial applications is advantageous and the enzyme stability improvement is a continuous challenge. Therefore, we designed β‐galactosidase heterogeneous biocatalysts by immobilization, involving the support synthesis and enzyme selection (from Bacillus circulans, Kluyveromyces lactis, and Aspergillus oryzae). The underivatized, tailored, macro‐mesoporous silica exhibited high surface area, offered high enzyme immobilization yields and activity. Its chemical activation with glyoxyl groups bound the enzyme covalently, which suppressed lixiviation and conferred higher pH and thermal stability (120‐fold than for the soluble enzyme), without observable reduction of activity/stability due to the presence of silica. The best balance between the immobilization yield (68%), activity (48%), and stability was achieved for Bacillus circulans β‐galactosidase immobilized on glyoxyl‐activated silica, without using stabilizing agents or modifying the enzyme. The enzyme stabilization after immobilization in glyoxyl‐activated silica was similar to that observed in macroporous agarose‐glyoxyl support, with the reported microbiological and mechanical advantages of inorganic supports. The whey lactolysis at pH 6.0 and 25°C by using this catalyst (1 mg ml^?1, 290 UI g^?1) was still 90%, even after 10 cycles of 10 min, in batch process but it could be also implemented on continuous processes at industrial level with similar results.     

One-step purification, covalent immobilization, and additional stabilization of poly-His-tagged proteins using novel heterofunctional chelate-epoxy supports.

Epoxy supports covalently immobilize proteins following a two-step mechanism; that is, the protein is physically adsorbed and then the covalent reaction takes place. This mechanism has been exploited to combine the selectivity of metal chelate affinity chromatography with the covalent immobilization capacity of epoxy supports. In this way, it has been possible to accomplish, in a simple manner, the purification, immobilization, and stabilization of a poly-His-tagged protein. To fulfill this objective we developed a new kind of multifunctional epoxy support (chelate epoxy support [CES]), which was tested using a poly-His-tagged glutaryl acylase as a model protein (an alphabeta-heterodimeric enzyme of significant industrial interest). The selectivity of the immobilization in CES toward poly-His-tagged proteins was dependent to a large extent on the density and nature of the chelated metal. The highest selectivity was achieved by using low-density chelate groups (e.g., 5 micromol/g) and metals with a low affinity (e.g., Co). However, the rate of covalent immobilization of the protein by its reaction with the epoxy groups on the support significantly increased at alkaline pH values. The multipoint attachment to the CES also depended on the reaction time. The immobilization of both glutaryl acylase subunits was achieved by incubation of the enzyme derivative at pH 10 for 24 h, with the best enzyme derivative 100-fold more stable than the soluble enzyme. By taking advantage of the selectivity properties of the novel support, we were able to immobilize up to 30 mg of protein per gram of modified Eupergit 250 using either pure enzyme or a very crude enzyme extract.     

Concomitant selective adsorption and covalent immobilization of a His-tagged protein switch with silica-based metal chelate-epoxy bifunctional adsorbents

A series of silica-based bifunctional adsorbents containing both metal-chelating groups and epoxy groups for the concomitant purification and immobilization of His-tagged protein switch RG13, a potential bioreceptor for developing maltose biosensors, were prepared by controlling the ratio of iminodiacetic acid-conjugated silane (GLYMO-IDA) and silane (GLYMO) used for surface modification. The bifunctional adsorbent prepared with a [GLYMO-IDA]/[GLYMO] ratio of 0.2, containing a [metal chelating group]/[epoxy group] ratio of 1.42, was shown to exhibit a metal chelating capacity of 88.42 ± 15.91 μmole Cu²⁺/g, a protein adsorption capacity of 1.81 ± 0.19 mg/g and a superior selectivity over the other bifunctional adsorbents. Results of kinetic studies showed that selective adsorption and covalent bond formation at 4 °C were achieved in 1 h and 15 h, respectively, which allowed the sequential adsorption and covalent immobilization of protein switch RG13. A protein immobilization yield of 94.6 % and a global activity yield of 63.4 % were obtained, giving an immobilized protein switch RG13 with an enzymatic activity of 4.57 ± 0.19 U/g, under optimal conditions at pH 8.0 and 40 °C. In the repeated-batch operation, the bifunctional adsorbent-immobilized RG13 retained 91 % of the original activity after 20 cycles, 39 % higher than the counterpart prepared with monofunctional metal chelate adsorbent mediated solely by coordinate linkages.     

Covalent immobilization of pure isoenzymes from lipase of Candida rugosa

Covalent immobilization of pure lipases A and B from Candida rugosa on agarose and silica is described. The immobilization increases the half-life of the biocatalysts ( ) with respect to the native pure lipases ( ). The percentage immobilization of lipases A and B is similar in both supports (33–40%). The remaining activity of the biocatalysts immobilized on agarose (70–75%) is greater than that of the enzymatic derivatives immobilized on SiO₂ (40–50%). The surface area and the hydrophobic/hydrophilic properties of the support control the lipase activity of these derivatives. The thermal stability of the immobilized lipase A derivatives is greater than that of lipase B derivatives. The nature of the support influences the thermal deactivation profile of the immobilized derivatives. The immobilization in agarose (hydrophilic support) gives biocatalysts that show a greater initial specific reaction rate than the biocatalysts immobilized in SiO₂ (hydrophobic support) using the hydrolysis of the esters of (R) or (S) 2-chloropropanoic and of (R,S) 2-phenylpropanoic acids as the reaction test. The enzymatic derivatives are active for at least 196 h under hydrolysis conditions. The stereospecificity of the native and the immobilized enzymes is the same.     

极端嗜热菌Thermus thermophilus HB8中天冬氨酸转氨酶在大肠杆菌中的表达、纯化及酶学性质研究

为获得具有热稳定性的天冬氨酸转氨酶,从极端嗜热细菌Thermus thermophilus HB8中克隆得到天冬氨酸转氨酶基因aspC,并在大肠杆菌BL21(DE3)和Rosetta(DE3)中进行表达,发现在Rosetta(DE3)中具有较高的表达量。重组酶的最适反应pH是7.0,37 ℃下在pH8～10的缓冲液中保温1 h酶活几乎不改变。重组酶反应的最适温度为75 ℃,酶活稳定的温度范围为25～55℃。重组酶在65℃时半衰期为3.5h,75℃时为2.5h。重组酶的K_m^KG为7.559mmol/L,V_max^KG为0.086mmol/(L·min),K_m^Asp为2.031mmol/L,V_max^Asp为0.024mmol/(L·min)。Ca²⁺、Fe³⁺、Mn²⁺等金属离子对酶活性有微弱抑制作用。     

极端嗜热菌Thermus thermophilus HB8中天冬氨酸转氨酶在大肠杆菌中的表达、纯化及酶学性质研究

为获得具有热稳定性的天冬氨酸转氨酶,从极端嗜热细菌Thermus thermophilus HB8中克隆得到天冬氨酸转氨酶基因aspC,并在大肠杆菌BL21(DE3)和Rosetta(DE3)中进行表达,发现在Rosetta(DE3)中具有较高的表达量。重组酶的最适反应pH是7.0,37 ℃下在pH8～10的缓冲液中保温1 h酶活几乎不改变。重组酶反应的最适温度为75 ℃,酶活稳定的温度范围为25～55℃。重组酶在65℃时半衰期为3.5h,75℃时为2.5h。重组酶的K_m^KG为7.559mmol/L,V_max^KG为0.086mmol/(L·min),K_m^Asp为2.031mmol/L,V_max^Asp为0.024mmol/(L·min)。Ca²⁺、Fe³⁺、Mn²⁺等金属离子对酶活性有微弱抑制作用。     

Stabilization of the hexameric glutamate dehydrogenase from Escherichia coli by cations and polyethyleneimine

The enzyme glutamate dehydrogenase (GDH) from Escherichia coli is a hexameric protein. The stability of this enzyme was increased in the presence of Li⁺ in concentrations ranging from 1 to 10 mM, 1 M of sodium phosphate, or 1 M ammonium sulfate. A very significant dependence of the enzyme stability on protein concentration was found, suggesting that subunit dissociation could be the first step of GDH inactivation. This effect of enzyme concentration on its stability was not significantly decreased by the presence of 10 mM Li⁺. Subunit crosslinking could not be performed using neither dextran nor glutaraldehyde because both reagents readily inactivated GDH. Thus, they were discarded as crosslinking reagents and GDH was incubated in the presence of polyethyleneimine (PEI) with the aim of physically crosslinking the enzyme subunits. This incubation does not have a significant effect on enzyme activity. However, after optimization, the PEI-GDH was found to almost maintain the full initial activity after 2 h under conditions where the untreated enzyme retained only 20% of the initial activity, and the effect of the enzyme concentration on enzyme stability almost disappeared. This stabilization was maintained in the pH range 5–9, but it was lost at high ionic strength. This PEI-GDH composite was also much more stable than the unmodified enzyme in stirred systems. The results suggested that a real adsorption of the PEI on the GDH surface was required to obtain this stabilizing effect. A positive effect of Li⁺ on enzyme stability was maintained after enzyme surface coating with PEI, suggesting that the effects of both stabilizing agents could not be exactly based on the same mechanism. Thus, the coating of GDH surface with PEI seems to be a good alternative to have a stabilized and soluble composite of the enzyme.     

Influence of different silica derivatives in the immobilization and stabilization of a Bacillus licheniformis protease (Subtilisin Carlsberg)

Alcalase 2T, a commercial preparation of Subtilisin Carlsberg, was covalent immobilized onto physiochemically characterized silica supports. The effect of mean pore diameter and surface chemistry on enzyme activity in the hydrolysis of casein has been examined. Two sets of chemically distinct silica supports were used presenting terminal amino (S_APTES) or hydroxyl groups (S_TESPM-pHEMA). The percentage of immobilized protein was smaller in S_APTES (31–39%) than in S_TESPM-pHEMA (62–71%), but presented higher total and specific activity. Silicas with large pores (S₁₀₀₀, 130/1200 Å) presented higher specific activities relative to those with smaller pore sizes (S₃₀₀, 130/550 Å). The influence of glutaraldehyde concentration and the time of enzyme coupling to the S₁₀₀₀S_APTES supports was examined. The apparent K_m value for the S₁₀₀₀S_APTES immobilized enzyme is lower than the soluble one which may be explained by the partitioning effects of the substrate. No intraparticle diffusion limitations were observed for the immobilized enzyme and therefore the substrate diffusion does not influence the observable kinetics. Finally, the optimum pH, optimum temperature, thermal stability, operational stability, and storage stability of the immobilized and freely soluble enzymes were compared.     

Modulation of a lipase from Staphylococcus warneri EX17 using immobilization techniques

This research describes the immobilization on glyoxyl, cyanogen bromide or octyl agarose beads of a purified lipase from Staphylococcus warneri strain EX17 (SWL), and the effect on its properties. The immobilization on glyoxyl-agarose at pH 10 and 25 °C, conditions in which the enzyme is readily inactivated, required the stabilization of the soluble enzyme. This was attained by the addition of 25% glycerol. Using this additive, immobilization on glyoxyl-agarose beads proceeded very quickly with good activity retention around 80%. This was the most stable preparation under thermal inactivation at pH 5, 7 and 9, in the presence of either cosolvents or detergents. This preparation was hyperactivated by concentrations of Triton X-100, which would produce negative effects over enzyme activity when using the other SWL preparations. Immobilized SWL preparations hydrolyzed different chiral esters, such as (±)-methyl mandelate, (±)-2-O-butyryl-2-phenylacetic acid, and (±)-2-hydroxy-4-phenyl-butyric acid ethyl ester, being its specificity depended on the immobilization protocol. The enantiospecificity was also strongly modulated by the immobilization. Thus, using HPBEt as substrate, octyl-SWL exhibited an opposite enantiospecificity to the other two biocatalysts. This preparation was the most enantioselective in the hydrolysis of (±)-2-O-butyryl-2-phenylacetic acid (E = 56.3).     

Site-directing an intense multipoint covalent attachment (MCA) of mutants of the Geobacillus thermocatenulatus lipase 2 (BTL2): Genetic and chemical amination plus immobilization on a tailor-made support

Immobilized enzymes are preferred over their soluble counterparts due to their robustness in harsh industrial processes; the most stable enzyme derivatives are often produced through multipoint covalent attachment (MCA). However, most enzymes are unable to establish optimal MCA to electrophile-type supports given the heterogeneous distribution and/or low content of primary amino groups on their surfaces; this restricts both the diversity of areas prone to react and the number of attachments to the support. To overcome this we propose combining site-directed immobilization and protein engineering to increase the number of bonds between a specific enzyme surface and a tailor-made support. We applied this novel strategy to engineered mutants of the lipase 2 from Geobacillus thermocatenulatus with one Cys exposed residue, that after genetic amination and/or chemical amination, were immobilized on glyoxyl-disulfide support using a site-directed MCA protocol. Two highly stabilized derivatives of chemically aminated lipase variants, in which site-directed MCA implied the surrounding surface of residues Cys344 or Cys40, were produced: the first one was 2.4-fold more productive than the reference derivative (648 g of hydrolyzed ester); the second derivative was 40% more selective (EPA/DHA molar ratio) and as active (1 μmol g catalyst⁻¹ min⁻¹) as the reference in the production of PUFAs.     

Purification, immobilization, and characterization of a specific lipase from Staphylococcus warneri EX17 by enzyme fractionating via adsorption on different hydrophobic supports

Staphylococcus warneri strain EX17 produces three lipases with different molecular weights of 28, 30, and 45 kDa. The 45 kDa fraction (SWL-45) has been purified from crude protein extracts by one chromatographic step based on the selective adsorption of this lipase by interfacial activation on different hydrophobic supports at low ionic strength. The adsorption of SWL-45 on octyl-Sepharose increased the enzyme activity by 60%, but the other lipases were also adsorbed on this support. Using butyl-Toyopearl, which is a lesser hydrophobic support, the purification factor was close to 20, and the only protein band detected on the sodium dodecyl sulfate-polyacrylamide electrophoresis analysis gel was that corresponding to the SWL-45, which could be easily desorbed from the support by incubation with triton X-100, producing a purified enzyme. SWL-45 was immobilized under very mild conditions on cyanogen bromide Sepharose, showing similar activities and stability as for its soluble form but without intermolecular interaction. The effects of different detergents over the activity of the immobilized SWL-45 were analyzed, which was hyperactivated by factors of 1.3 and 2.5 with 0.01% Tween 80 and 0.1% Triton X-100, respectively, while ionic detergents produced detrimental effects on the enzyme activity even at very low concentrations. Optimal reaction conditions and the effect of other additives on the enzyme activity were also investigated.     

巴西橡胶树胶乳磷脂酶A2的分离纯化及部分酶学性质鉴定

通过丙酮沉淀、DEAE-纤维素离子交换柱层析和Sephadex G-100凝胶过滤柱层析等分离纯化技术,对巴西橡胶树胶乳C-乳清磷脂酶A2进行分离纯化。用SDS-PAGE测定其亚基的相对分子量。测定该酶最适温度和pH,动力学常数Km和Vmax。并测定Ca2+和La3+对酶活性的影响。结果显示:该酶被纯化了49.47倍,产率为5.12%。SDS-PAGE检测为单一条带,其亚基相对分子量约43kDa。最适反应温度为37℃,最适反应pH为8.0,Km为0.44mmol·L-1,Vmax为7.22μmol.(mL.min)-1。最适Ca2+浓度为50μmol·L-1,稀土元素La3+离子对磷脂酶A2活性有抑制作用,但加入Ca2+后可缓解La3+对磷脂酶A2活性的抑制作用。胶乳C-乳清磷脂酶A2与其他植物磷脂酶A2在Ca2+的依赖性上存在差异。研究结果为今后探索橡胶树胶乳磷脂酶A2的催化机理、调节机理及生理功能等奠定了基础。     

Purification and properties of thermostable lipase from a thermophilic Bacillus stearothermophilus MC 7

Extracellular thermostable lipase produced by the thermophilic Bacillus stearothermophilus MC 7 was purified to 19.25-fold with 10.2% recovery. The molecular weight of the purified enzyme determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was shown to be 62 500 Da. The purified enzyme expressed maximum activity at 75–80 °C and its half life was 30 min at 70 °C. The K_m and V_max were calculated to be, respectively, 0.33 mM and 188 μM min⁻¹ mg⁻¹ with p-nitrophenyl palmitate (pNPP) as a substrate. Enzyme activity was inhibited by divalent ions of heavy metals, thiol and serine inhibitors, whereas calcium ion stimulated its activity. The most advantageous method for immobilization was found to be ionic binding to DEAE Cellulose. The enzyme was able to hydrolyze both soluble and insoluble emulsified substrates and was classified as a lipase, expressing some esterase activity as well.     

High-level production and covalent immobilization of Providencia rettgeri penicillin G acylase (PAC) from recombinant Pichia pastoris for the development of a novel and stable biocatalyst of industrial applicability

A complete, integrated process for the production of an innovative formulation of penicillin G acylase from Providencia rettgeri(rPAC(P.rett))of industrial applicability is reported. In order to improve the yield of rPAC, the clone LN5.5, carrying four copies of pac gene integrated into the genome of Pichia pastoris, was constructed. The proteinase activity of the recombinant strain was reduced by knockout of the PEP4 gene encoding for proteinase A, resulting in an increased rPAC(P.rett) activity of approximately 40% (3.8 U/mL vs. 2.7 U/mL produced by LN5.5 in flask). A high cell density fermentation process was established with a 5-day methanol induction phase and a final PAC activity of up to 27 U/mL. A single step rPAC(P.rett) purification was also developed with an enzyme activity yield of approximately 95%. The novel features of the rPAC(P.rett) expressed in P.pastoris were fully exploited and emphasized through the covalent immobilization of rPAC(P.rett). The enzyme was immobilized on a series of structurally correlated methacrylic polymers, specifically designed and produced for optimizing rPAC(P.rett) performances in both hydrolytic and synthetic processes. Polymers presenting aminic functionalities were the most efficient, leading to formulations with higher activity and stability (half time stability >3 years and specific activity ranging from 237 to 477 U/g (dry) based on benzylpenicillin hydrolysis). The efficiency of the immobilized rPAC(P.rett) was finally evaluated by studying the kinetically controlled synthesis of beta-lactam antibiotics (cephalexin) and estimating the synthesis/hydrolysis ratio (S/H), which is a crucial parameter for the feasibility of the process.