Reversible immobilization of cephalosporin C acylase on epoxy supports coated with polyethyleneimine

In this study, a recombinant cephalosporin C acylase (CCA) was covalently or physically immobilized on an epoxy-activated support LX1000-EPC4 (EP) or its derivatives, EP-polyethyleneimine (EP-PEI) and EP-ethylenediamine (EP-EDA) with cationic groups on the surface. Zeta potential was used as a tool for activated carrier analysis and immobilization analysis. The EP-PEI (the cationic polymer PEI grafted support) showed higher zeta potential than EP-EDA (the small diamine EDA modified support) and EP support. Among these three supports, immobilization of CCA on EP-PEI had the highest specific activity according to the range of enzyme loadings. Michaelis constant K_m values of EP-PEI-CCA and EP-EDA-CCA were 22?mM and 30?mM, respectively, which were lower than that of the free enzyme (43?mM), suggesting that the support’s zeta potential is related to the affinity of the enzyme for the substrate. The enzyme immobilized on EP-PEI showed a much higher thermal stability (stabilization factor of 32-fold compared with the free enzyme) than that on the EP-EDA (stabilization factor of 5.5-fold) and EP supports (stabilization factor of 1.7-fold). The adsorption of CCA on EP-PEI support was very strong and reversible. The CCA could be thoroughly desorbed using a high concentration of NaCl (e.g., 2 M) at low pH value (pH 3.0). The regenerated EP-PEI support could then be reused for enzyme immobilization.     

Catalytic properties of the immobilized Aspergillus tamarii xylanase

Xylanase from Aspergillus tamarii was covalently immobilized on Duolite A147 pretreated with the bifunctional agent glutaraldehyde. The bound enzyme retained 54.2% of the original specific activity exhibited by the free enzyme (120 U/mg protein). Compared to the free enzyme, the immobilized enzyme exhibited lower optimum pH, higher optimum reaction temperature, lower energy of activation, higher K_m (Michaelis constant), lower V_max (maximal reaction rate). The half-life for the free enzyme was 186.0, 93.0, and 50.0 min for 40, 50, and 60°C, respectively, whereas the immobilized form at the same temperatures had half-life of 320, 136, and 65 min. The deactivation rate constant at 60°C for the immobilized enzyme is about 6.0 × 10⁻³, which is lower than that of the free enzyme (7.77 × 10⁻³ min). The energy of thermal deactivation was 15.22 and 20.72 kcal/mol, respectively for the free and immobilized enzyme, confirming stabilization by immobilization. An external mass transfer resistance was identified with the immobilization carrier (Duolite A147). The effect of some metal ions on the activity of the free and immobilized xylanase has been investigated. The immobilized enzyme retained about 73.0% of the initial catalytic activity even after being used 8 cycles.     

Catalytic properties of the immobilized Aspergillus tamarii xylanase

Xylanase from Aspergillus tamarii was covalently immobilized on Duolite A147 pretreated with the bifunctional agent glutaraldehyde. The bound enzyme retained 54.2% of the original specific activity exhibited by the free enzyme (120 U/mg protein). Compared to the free enzyme, the immobilized enzyme exhibited lower optimum pH, higher optimum reaction temperature, lower energy of activation, higher K_m (Michaelis constant), lower V_max (maximal reaction rate). The half-life for the free enzyme was 186.0, 93.0, and 50.0 min for 40, 50, and 60°C, respectively, whereas the immobilized form at the same temperatures had half-life of 320, 136, and 65 min. The deactivation rate constant at 60°C for the immobilized enzyme is about 6.0 × 10⁻³, which is lower than that of the free enzyme (7.77 × 10⁻³ min). The energy of thermal deactivation was 15.22 and 20.72 kcal/mol, respectively for the free and immobilized enzyme, confirming stabilization by immobilization. An external mass transfer resistance was identified with the immobilization carrier (Duolite A147). The effect of some metal ions on the activity of the free and immobilized xylanase has been investigated. The immobilized enzyme retained about 73.0% of the initial catalytic activity even after being used 8 cycles.     

Stabilization of enzymes by multipoint immobilization of thiolated proteins on new epoxy-thiol supports

The controlled and partial modification of epoxy groups of Eupergit C and EP-Sepabeads with sodium sulfide has permitted the preparation of thiol-epoxy supports. Their use allowed not only the specific immobilization of enzymes through their thiol groups via thiol-disulfide interchange, but also enzyme stabilization via multipoint covalent attachment. Penicillin G acylase (PGA) from Escherichia coli and lipase from Rhizomucor miehei were used as model enzymes. Both enzymes lacked exposed cysteine residues, but were introduced via chemical modification under very mild conditions. In the first moments of the immobilization, a certain percentage of immobilized protein could be released from the support by incubation with DTT; this confirms that the first step was via a thiol-disulfide interchange. Moreover, the promotion of some further epoxy-enzyme bonds was confirmed because no enzyme release was detected after some immobilization time by incubation with DTT. In the case of the heterodimeric PGA, it was possible to demonstrate the formation of at least one epoxy bond per enzyme subunit by analyzing with SDS-PAGE the supernatants obtained after boiling the enzyme derivatives in the presence of mercaptoethanol and SDS. Thermal inactivation studies showed that these multipoint enzyme-support attachments promoted an increase in the stability of the immobilized enzymes. In both cases, the stabilization factor was around 12-15-fold comparing optimal derivatives with their just-thiol immobilized counterparts.     

金属螯合载体定向固定化木瓜蛋白酶的研究

以磁性金属螯合琼脂糖微球为载体,利用金属螯合配体（IDACu²⁺）与蛋白质表面供电子氨基酸相互作用的原理,定向固定了木瓜蛋白酶。固定化最适条件为Cu²⁺1.5×10^-2mol/g载体、固定化时间4h、固定化pH7.0、给酶量30mg/g载体。固定化酶的最适反应温度70℃、最适反应pH8.0,固定化酶的热稳定性明显高于溶液酶,固定化酶活力回收为68.4％,且有较好的操作稳定性,载体重复使用5次后固定化酶酶活为首次固定化酶79.71%。     

Application of ammonium bicarbonate buffer as a smart microenvironmental pH regulator of immobilized cephalosporin C acylase catalysis in different reactors

In a stirred tank reactor, during catalysis with immobilized cephalosporin C acylase (CCA), the microenvironmental pH dropped to 7.2 in a nonbuffered system (with the pH maintained at 8.5 by adding alkali) due to the existence of diffusional resistance. Moreover, the immobilized CCA only catalyzed five batch reactions, suggesting that the sharp pH gradient impaired the enzyme stability. To buffer the protons produced in the hydrolysis of cephalosporin C by CCA, phosphate and bicarbonate buffers were introduced. When CCA was catalyzed with 0.1 M ammonium bicarbonate buffer, no obvious gradient between the bulk solution and intraparticle pH was detected, and the catalysis of 15 batch reactions was achieved. Accordingly, with 0.2 M ammonium bicarbonate buffer in a packed bed reactor, the immobilized CCA exhibited continuous catalysis with high conversion rates (≥95%) for 21 days. Reactions with ammonium bicarbonate buffer showed significant increases in the stability and catalytic efficiency of the immobilized CCA in different reactors compared to those in nonbuffered systems.     

Stabilization of enzymes by multipoint attachment via reversible immobilization on phenylboronic activated supports

In this work, we have used supports activated with m-amino-phenylboronic groups to “reversibly” immobilize proteins under very mild conditions. Most of the proteins contained in a crude extract from E. coli could be immobilized on Eupergit C-250 L activated with phenylboronic and then fully desorbed from the support by using mannitol or SDS. This suggested that the immobilization of the proteins on these supports was not only via sugars interaction, but also by other interaction/s, quite unspecific, that might be playing a key role in the immobilization of the proteins. Penicillin acylase from E. coli (PGA) was also immobilized in Eupergit C activated with m-amino-phenylboronic groups. The enzyme could be fully desorbed with mannitol immediately after being immobilized on the support. However, longer incubation times of the immobilized preparation caused a reduction of protein elution from the boronate support in presence of mannitol. Moreover, these immobilized preparations showed a higher stability in the presence of organic solvents than the soluble enzyme; the stability also improved when the incubation time was increased (to a factor of 100). By desorbing the weakest bound enzyme molecules, it was possible to correlate adsorption strength with stabilization; therefore, it seems that this effect was due to the rigidification of the enzyme via multipoint attachment on the support.     

Improvement of the functional properties of a thermostable lipase from alcaligenes sp. via strong adsorption on hydrophobic supports

Lipase QL from Alcaligenes sp. is a quite thermostable enzyme. For example, it retains 75% of catalytic activity after incubation for 100 h at 55 °C and pH 7.0. Nevertheless, an improvement of the enzyme properties was intended via immobilization by covalent attachment to different activated supports and by adsorption on hydrophobic supports (octadecyl-sepabeads). This latter immobilization technique promotes the most interesting improvement of enzyme properties: (a) the enzyme is hyperactivated after immobilization: the immobilized preparation exhibits a 135% of catalytic activity for the hydrolysis of p-nitrophenyl propionate as compared to the soluble enzyme; (b) the thermal stability of the immobilized enzyme is highly improved: the immobilized preparation exhibits a half-life time of 12 h when incubated at 80 °C, pH 8.5 (a 25-fold stabilizing factor regarding to the soluble enzyme); (c) the optimal temperature was increased from 50 °C (soluble enzyme) up to 70 °C (hydrophobic support enzyme immobilized preparations); (d) the enantioselectivity of the enzyme for the hydrolysis of glycidyl butyrate and its dependence on the experimental conditions was significantly altered. Moreover, because the enzyme becomes reversibly but very strongly adsorbed on these highly hydrophobic supports, the lipase may be desorbed after its inactivation and the support may be reused. Very likely, adsorption occurs via interfacial activation of the lipase on the hydrophobic supports at very low ionic strength. On the other hand, all the covalent immobilization protocols used to immobilize the enzyme hardly improved the properties of the lipase.     

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).     

Immobilization of penicillin acylase on acrylic carriers

Penicillin acylase obtained from E. Coli (E. C. 3.5.1.11) was covalently bound via glutaric aldehyde to acrylic carriers crosslinked with divinylbenzene or ethylene glycol dimethacrylate. The best enzymatic preparation was obtained by using ethyl acrylate/ ethylene glycol dimethacrylate copolymer. 1 cm³ of the carrier bound 6.4 mg of protein, having 72% activity in relation to the native enzyme. The preparation lost only 10% of its initial activity after 100 d of storage at 4°C. A negligible effect of immobilization on the enzyme activity at different temperatures or pH as well as significant increase of the stability of the immobilized enzyme at elevated temperatures were observed.Abbreviations BA butyl acrylate - AE ethyl acrylate - PA penicillin acylase - 6-APA 6-aminopenicillanic acid - EGDMA ethylene glycol dimethacrylate - DVB divinylbenzene     

Immobilization of invertase via carbohydrate moiety on chitosan to enhance its thermal stability

A new technique using chitosan as support for covalent coupling of invertase via carbohydrate moiety improved the activity and thermal stability of immobilized invertase. The best preparation of immobilized invertase retained 91% of original specific activity (412 U mg^–1). The half-life at 60°C was increased from 2.3 h (free invertase) to 7.2 h (immobilized invertase). In contrast, the immobilization of invertase via protein moiety on chitosan or using Sepharose as support resulted in less thermostable preparations. Additionally, immobilization of invertase on both supports caused the optimal reaction pH to shift from 4.5 to 2.5 and the substrate (sucrose) concentration for maximum activity to increase from 0.5 M to 1.0 M.     

An approach for the improved immobilization of penicillin G acylase onto macroporous poly(glycidyl methacrylate‐co‐ethylene glycol dimethacrylate) as a potential industrial biocatalyst

The use of penicillin G acylase (PGA) covalently linked to insoluble carrier is expected to produce major advances in pharmaceutical processing industry and the enzyme stability enhancement is still a significant challenge. The objective of this study was to improve catalytic performance of the covalently immobilized PGA on a potential industrial carrier, macroporous poly(glycidyl methacrylate‐co‐ethylene glycol dimethacrylate) [poly(GMA‐co‐EGDMA)], by optimizing the copolymerization process and the enzyme attachment procedure. This synthetic copolymer could be a very promising alternative for the development of low‐cost, easy‐to‐prepare, and stable biocatalyst compared to expensive commercially available epoxy carriers such as Eupergit or Sepabeads. The PGA immobilized on poly(GMA‐co‐EGDMA) in the shape of microbeads obtained by suspension copolymerization appeared to have higher activity yield compared to copolymerization in a cast. Optimal conditions for the immobilization of PGA on poly(GMA‐co‐EGDMA) microbeads were 1 mg/mL of PGA in 0.75 mol/L phosphate buffer pH 6.0 at 25°C for 24 h, leading to the active biocatalyst with the specific activity of 252.7 U/g dry beads. Chemical amination of the immobilized PGA could contribute to the enhanced stability of the biocatalyst by inducing secondary interactions between the enzyme and the carrier, ensuring multipoint attachment. The best balance between the activity yield (51.5%), enzyme loading (25.6 mg/g), and stability (stabilization factor 22.2) was achieved for the partially modified PGA. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:43–53, 2016     

Active site titration as a tool for the evaluation of immobilization procedures of penicillin acylase

Native and immobilized preparations of penicillin acylase from Escherichia coli and Alcaligenes faecalis were studied using an active site titration technique. Knowledge of the number of active sites allowed the calculation of the average turnover rate of the enzyme in the various preparations and allowed us to quantify the contribution of irreversible inactivation of the enzyme to the loss of catalytic activity during the immobilization procedure. In most cases a loss of active sites as well as a decrease of catalytic activity per active site (turnover rate) was observed upon immobilization. Immobilization techniques affected the enzymes differently. The effect of increased loading of penicillin acylase on the average turnover rate was determined by active site titration to assess diffusion limitations in the carrier.     

Immobilization of penicillin G acylase on macro-mesoporous silica spheres

In this study, macro-mesoporous silica spheres were prepared with a micro-device and used as the support for the immobilization of penicillin G acylase (PGA). To measure the enzymatic activity, the silica spheres with immobilized PGA were placed into a packed-bed reactor, in which the hydrolysis of penicillin G was carried out. The influences of the residence time, the initial concentration of the substrate, the accumulation of the target product 6-aminopenicillanic acid, and the enzyme loading amount on the performance of the immobilized PGA were investigated. The introduction of macropores increased the enzyme loading amount and decreased the internal mass transfer resistance, and the results showed that the enzyme loading amount reached 895 mg/g (dry support), and the apparent enzymatic activity achieved up to 1033 U/g (dry support). In addition, the immobilized PGA was found to have great stability.     

Silanized palygorskite for lipase immobilization

Lipase from Candida lipolytica has been immobilized on 3-aminopropyltriethoxysilane-modified palygorskite support. Scanning electron micrographs proved the covalently immobilization of C. lipolytica lipase on the palygorskite support through glutaraldehyde. Using an optimized immobilization protocol, a high activity of 3300 U/g immobilized lipase was obtained. Immobilized lipase retained activity over wider ranges of temperature and pH than those of the free enzyme. The optimum pH of the immobilized lipase was at pH 7.0–8.0, while the optimum pH of free lipase was at 7.0. The retained activity of the immobilized enzyme was improved both at lower and higher pH in comparison to the free enzyme. The immobilized enzyme retained more than 70% activity at 40 °C, while the free enzyme retained only 30% activity. The immobilization stabilized the enzyme with 81% retention of activity after 10 weeks at 30 °C whereas most of the free enzyme was inactive after a week. The immobilized enzyme retains high activity after eight cycles. The kinetic constants of the immobilized and free lipase were also determined. The K_m and V_max values of immobilized lipase were 0.0117 mg/ml and 4.51 μmol/(mg min), respectively.     

Stabilization of penicillin G acylase from Escherichia coli: site-directed mutagenesis of the protein surface to increase multipoint covalent attachment

Three mutations on the penicillin acylase surface (increasing the number of Lys in a defined area) were performed. They did not alter the enzyme's stability and kinetic properties; however, after immobilization on glyoxyl-agarose, the mutant enzyme showed improved stability under all tested conditions (e.g., pH 2.5 at 4 degrees C, pH 5 at 60 degrees C, pH 7 at 55 degrees C, or 60% dimethylformamide), with stabilization factors ranging from 4 to 11 compared with the native enzyme immobilized on glyoxyl-agarose.     

磁性纳米颗粒固定化乙醇脱氢酶的研究

本研究采用3-丙氨基三乙氧基硅烷(APTES)和戊二醛修饰包裹有SiO2磁性Fe3O4纳米颗粒表面,将其作为固定化载体固定化乙醇脱氢酶,研究固定化条件对固定化效率的影响,并对固定化酶性质进行分析。研究发现,当Fe3O4@SiO2纳米颗粒修饰上氨基和醛基后依然具有良好的水分散性和胶体稳定性,适合作为固定化载体。通过单因素优化,发现当最适给酶量为11. 3U/100 mg,搅拌转速为150 r/min,固定化p H和固定化温度分别控制在6. 5和5℃～15℃,固定化时长为45 min时,具有较好的固定化效果,固定化率可达到60. 2%。在此条件下制备得到的固定化酶与游离酶相比,固定化酶具有良好的耐高温和耐碱性。所得固定化乙醇脱氢酶在连续使用8次后,固定化率仍保留在57%左右,表明该固定化酶具有较好的操作稳定性,可为连续生产NADH提供技术依据。     

Immobilization of <Emphasis Type="Italic">Alcaligenes faecalis</Emphasis> penicillin G acylase on epoxy-type supports

Alcaligenes faecalis penicillin G acylase has several desired features over other penicillin G acylases and its use in industry requires immobilization. In this work, two novel supports ZH-EP (epoxy type) and ZH-HA (epoxy-amino type) were used to immobilize Alcaligenes faecalis penicillin G acylase (AfPGA) with Eupergit C as reference. The saturation of immobilized protein on ZH-EP (269 mg/g, 116 h) and ZH-HA (296 mg/g, 15 h) was obtained more rapidly than Eupergit C (197 mg/g, 260 h). And the activity of immobilized AfPGA on ZH-EP (520 U/g) and ZH-HA (2200 U/g) was higher than that on Eupergit C (310 U/g). The properties of three immobilized enzymes were compared and no obvious difference was observed, which indicated that ZH-EP and ZH-HA were promised in industry.     

二氧化硅纳米材料固定中性脂肪酶的条件优化及其特性

以二氧化硅纳米材料为载体,采用吸附法对脂肪酶进行固定化,研究了不同条件对固定化脂肪酶的催化活性的影响,得到最佳的固定化条件:给酶量为28300U/g,固定化温度为45oC,pH值为7.5,时间为10h,此时固定化酶的活力约为3867U/g载体。固定化酶的最适反应温度为45oC,比游离酶的反应温度高5oC,最适pH下降到5.5,低于游离酶的反应pH(pH7)。固定化酶的热稳定性和pH稳定性较游离酶有了很大的提高,其在70oC以下能保持70%以上的酶活力,而游离酶在50oC下残余酶活力仅为30%。在pH5～8的范围内,固定化酶的酶活力能保持50%以上,而游离酶只能保持20%左右。用固定化的中性脂肪酶催化不同的油品,即大豆油、菜籽油及泔水油生产生物柴油,菜籽油的酯化率最高。     

Evaluation of the performance of immobilized penicillin G acylase using active-site titration

Penicillin G acylase from Escherichia coli was immobilized on Eupergit C with different enzyme loading. The activity of the immobilized preparations was assayed in the hydrolysis of penicillin G and was found to be much lower than would be expected on the basis of the residual enzyme activity in the immobilization supernatant. Active-site titration demonstrated that the immobilized enzyme molecules on average had turnover rates much lower than that of the dissolved enzyme. This was attributed to diffusion limitations of substrate and product inhibition. Indeed, when the immobilized preparations were crushed, the activity increased from 587 U g-1 to up to 974 U g-1. The immobilized preparations exhibited up to 15% lower turnover rates than the dissolved enzyme in cephalexin synthesis from 7-ADCA and D-(-)-phenylglycine amide. The synthesis over hydrolysis ratios of the immobilized preparations were also much lower than that of the dissolved enzyme. This was partly due to diffusion limitations but also to an intrinsic property of the immobilized enzyme because the synthesis over hydrolysis ratio of the crushed preparations was much lower than that of the dissolved enzyme.