Evaluation of a monolithic epoxy silica support for penicillin G acylase immobilization

In this work, a new type of penicillin G acylase (PGA)-based monolithic silica support was developed and evaluated for the chiral separation in HPLC. The preparation procedure consisted of two steps: preparation of an epoxy derivatized monolithic silica column and chemical modification of the epoxide groups with the enzyme chiral selector. The epoxy Silica-Rod column for the immobilization of PGA was prepared with the in situ modification process by using epoxy-silanes and the identification of the species bound to the surface was achieved by solid-state nuclear magnetic resonance. The enzyme was covalently immobilized to the surface of the derivatized monolithic column. The enantioselectivity and the performance of the developed column are discussed and compared to the corresponding experimental data obtained with a PGA-based microparticulate (5 microm) silica column.     

介孔材料的修饰及固定青霉素酰化酶的稳定性研究

利用扩孔剂的作用合成出较大孔径(12 nm)的介孔材料SBA-15, 并进行表面氨基修饰, 以此为载体, 以戊二醛为交联剂, 对青霉素酰化酶进行组装固定, 并对固定化青霉素酰化酶(PGA)的稳定性进行了深入的研究. 实验结果表明, PGA与载体交联后仍保持活性. 热稳定性研究结果表明, 制备的固定化青霉素酰化酶在低于60 ℃时保持稳定; pH在6~11范围内保持稳定; 固定化酶重复使用10次之后, 仍具有高达90%的残留活力.     

Recent progress in the development of immobilized penicillin G acylase for chemical and industrial applications: A mini‐review

Immobilized penicillin G acylase (PGA) as an important industrial catalyst can catalyze penicillin G potassium (PG) to 6‐aminopenicillanic acid (6‐APA). 6‐APA is an important intermediate for semisynthetic penicillin drugs, which occupies a huge market space in the anti‐inflammatory field; as a result, immobilized PGA occupies a huge market space in the pharmaceutical field. However, at present, there are different degrees of defects in the preparation and production process of immobilized PGAs on the market because of the huge demand; therefore, the performance of immobilized PGA and its productivity will bring huge economic benefits to enterprises. Therefore, research on immobilized PGA has always been a focus. This review first introduces the source, classification, structure, and catalytic mechanism of PGA and then studies the development of immobilization methods, immobilized carriers, reaction media, enzyme activity regeneration, and reactors of immobilized PGA in recent years.     

青霉素酰化酶在介孔分子筛MCM-41上的固定化研究

通过改变酶固定化时的pH值、温度、时间以及酶用量,研究了固定化条件对MCM-41载体上固定化青霉素酰化酶的酶活及其稳定性的影响,明确了酶活及其稳定性随固定化条件的变化规律,初步分析了青霉素酰化酶在介孔分子筛MCM-41上的固定化过程。     

Immobilization of Penicillin G Acylase on Calcined Layered Double Hydroxides

A hydrotalcite-like Mg2 /Al3 layered double hydroxide (LDH) material was prepared by means of amodified coprecipitation method involving a rapid mixing step followed by a separate aging process. LDH calcined at 500℃ , denoted as CLDH, was characterized by XRD, IR and BET surface area measurements.CLDH has a poor crystalline MgO-like structure with a high surface area and porosity. CLDH was used as asupport for the immobilization of penicillin G acylase(PGA). The effect of varying the immobilization conditions, such as pH, contact time and the ratio of enzyme to support, on the activity of the immobilized enzymein the hydrolysis of penicillin G has been studied. It was found that the activity of the immobilized enzyme decreased slightly with decreasing pH and reached a maximum after a contact time of 24 h. The activity of theimmobilized enzyme increased with increasing the ratio of enzyme to support. It was found that the adsorption of PGA inhibited the expected reaction of CLDH with an aqueous medium to regenerate a LDH phase. Itsoriginal activity(36%) after 15 cycles of reuse of the immobilized enzyme was retained, but no further loss in the activity was observed.     

Immobilization-stabilization of Penicillin G acylase fromEscherichia coli

We have developed a strategy for immobilization-stabilization of penicillin G acylase from E. coli, PGA, by multipoint covalent attachment to agarose (aldehyde) gels. We hve studied the role of three main variables that control the intensity of these enzyme-support multiinteraction processes: 1. surface density of aldehyde groups in the activated support; 2. temperature; and 3. contact-time between the immobilized enzyme and the activated support prior to borohydride reduction of the derivatives. Different combinations of these three variables have been tested to prepare a number of PGA-agarose derivatives. All these derivatives preserve 100% of catalytic activity corresponding to the soluble enzyme that has been immobilized but they show very different stability. The less stable derivative has exactly the same thermal stability of soluble penicillin G acylase and the most stable one is approximately 1,400 fold more stable. A similar increase in the stability of the enzyme against the deleterious effect of organic solvents was also observed. On the other hand, the agarose aldehyde gels present a very great capacity to immobilize enzymes through multipoint covalent attachment. In this way, we have been able to prepare very active and very stable PGA derivatives containing up to 200 International Units of catalytic activity per mL. of derivative with 100% yields in the overall immobilization procedure.     

Assessment of immobilized PGA orientation via the LC-MS analysis of tryptic digests of the wild type and its 3K-PGA mutant assists in the rational design of a high-performance biocatalyst

The mutant penicillin G acylase (PGA) 3K-PGA contains three additional Lys residues on the surface opposite the active site. This protein was designed to selectively drive its immobilization on aldehyde supports. We describe here a modified bottom-up proteomic method to assess the orientation of the immobilized wild-type and mutant proteins to verify our hypothesis of a driven immobilization induced by the mutations introduced. Tryptic digestion of the immobilized enzymes followed by liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis of released peptides was performed. This protocol generated peptides from the most accessible surface areas of the immobilized protein, thus not directly bound to the solid support, providing direct evidence of the areas involved in the linkage to the solid matrix. The results obtained suggest that 72 % of the wild-type PGA is immobilized on aldehyde agarose mainly through the Lys residues on the same side of the active site, whereas 3K-PGA reacted with the same support preferentially through the additional Lys residues introduced by mutation on the opposite side. This demonstrates that the active site of the 3K-PGA faces mostly (63 %) toward the reaction medium, resulting in significantly improved accessibility to the substrates. This finding is supported by the catalytic properties of the immobilized biocatalysts. The two immobilized preparations were tested in the synthesis of mandelyl-7-aminocephalosporanic acid (mandelyl-7-ACA) by N-acylation of the β-lactam nucleus (7-aminocephalosporanic acid) with mandelic acid methyl ester: upon immobilization, the synthetic properties of wild-type PGA strongly decreased, whereas those of 3K-PGA were unaffected. Furthermore, the activity of 3K-PGA was not influenced by the physicochemical nature of the support used for immobilization (glyoxyl agarose or aldehyde Sepabeads) unlike that of wild-type PGA, whose active site is close to the matrix. The results obtained from the analytical characterization correlate well with those obtained by investigation of the synthetic properties of the immobilized enzymes both in the synthesis of mandelyl-7-ACA and in the preparative synthesis of cefazolin. This work highlights the effect exerted by site-directed mutagenesis on the orientation of PGA upon immobilization on solid matrices and suggests how protein engineering tools can be exploited in a synergistic fashion to rationally develop efficient biocatalysts.

Optimization in the immobilization of penicillin G acylase by entrapment in xerogel particles with magnetic properties

Biocatalysis presents a sound alternative to chemical synthesis in the field of drug production, given the highly selective nature of biological catalysts. Penicillin G Acylase (PGA) from E. coli is currently used to hydrolyze penicillin G (PG) and catalyzes the synthesis of β-lactam antibiotics. In this work, particular emphasis is given to recent developments in penicillin G acylase immobilization, by entrapment simultaneously with nano-magnetic particles in a silica matrix. The sol–gel biocatalytic particles were prepared either by a conventional method (crushed powder) or by a more recent approach, based in an emulsion system using 150 mM AOT/isooctane, which allowed for the formation of spherical micro- and nanobeads. The effects on PGA activity of different sol–gel precursors, additives, enzyme concentration, aging, drying conditions and mechanical stability were evaluated. After these optimization studies, a mechanically stable carrier based on porous xerogels silica matrixes, starting from tetramethoxysilane (TMOS) with 65–67% PGA activity yield in these carriers allowed an immobilization yield of 74 mg protein g_{dry sol–gel}⁻¹ and 930 Ug_{dry sol–gel}⁻¹ for specific activity were obtained.     

Di-functional magnetic nanoflowers: A highly efficient support for immobilizing penicillin G acylase

The novel di-functional magnetic nanoflowers (DMNF) which had both epoxy groups and hydrophilic catechol as well as phthaloquinone groups capable of covalently coupling of penicillin G acylase (PGA) were characterized by scanning electron microscopy, transmission electron microscope (TEM), vibrating sample magnetometer, N₂ adsorption, and so on. The studies showed that DMNF possessed “hierarchical petal” structure of nanosheets had specific saturation magnetization of 39.7 emu/g and average pore diameter of 25.4 nm as well as specific surface area of 17.28 m²/g. For hydrolysis of penicillin G potassium catalyzed by the PGA immobilized on DMNF with enzyme loading of 106 mg/g-support, its apparent activity reached 2,667 U/g, which benefited from the “hierarchical petal” and large pore structure of the magnetic DMNF leading to high enzyme loading and fast diffusion of substrate molecules to the immobilized PGA to reaction. The apparent activity of the immobilized PGA could keep 2,408 U/g (above 90% of its initial activity) after repeating use for 10 cycles. The magnetic immobilized PGA exhibited excellent operational stability due to covalently coupling of the enzyme molecules between the support by covalent interaction of the amino groups of PGA and the reactive groups of epoxy, catechol, and phthaloquinone groups on DMNF. Furthermore, the PGA displayed good acid and alkaline resistance as well as thermal stability by immobilization using DMNF.     

A novel lipase-based stationary phase in liquid chromatography

In this paper, a new chiral stationary phase (CSP) based on Candida antarctica lipase B (CALB) bounding to the surface of macroporous silica gel was developed and its stereoselectivity in enantioseparation and asymmetrical hydrolysis was evaluated. Three CALB-based HPLC columns with different amounts of enzyme immobilized were prepared by employing the immobilization method, namely “in batch”. In this technique two chromatographic supports epoxy silica and aminopropyl silica were considered. This novel CSP was proven capable of hydrolyzing chiral esters asymmetrically as bioreactor and separating several aromatic alcohols and diniconazole enantiomers.     

Beaded reactive polymers 3 effect of triacrylates as crosslinkers on the physical properties of glycidyl methacrylate copolymers and immobilization of penicillin G acylase

Various glycidyl methacrylate (GMA) copolymers were synthesized by suspension polymerization, using pentaerythritol triacrylate (PETA), trimethylolpropane triacrylate (TMPTA), and trimethylolpropane trimethacrylate (TRIM) as crosslinking comonomers. These copolymers were evaluated for the immobilization of penicillin G acylase. Broad pore-size distribution that was observed was in the range 5-300 nm. Both surface area and pore volume increased with increase in the mole fraction of crosslinking comonomer (increasing crosslink density). The pore volume of the copolymers was more than doubled by including lauryl alcohol as porogen. Binding of penicillin G acylase (PGA) was quantitative on highly crosslinked copolymers. The expression of bound PGA was better on the relatively more hydrophilic GMA-TMPTA and GMA-PETA copolymer supports compared to the GMA-TRIM copolymers. Among the different copolymers studied, GMA-TMPTA copolymer 7411 exhibited highest activity of immobilized penicillin G acylase (167.4 IU/g) with 35.1% expression.     

介孔材料MCFs的合成及组装青霉素酰化酶的性质研究

介孔材料由于具有纳米级规则孔道和巨大的比表面积而在催化、吸附及分离等方面存在较大的应用价值．近年来,由介孔分子筛如MCM-41和SBA-15州等组装功能性材料已成为研究的热点．酶作为高效催化剂有许多优点,但在溶液中易失活,使用后无法回收,有的酶在溶液中还存在自水解问题：将酶组装在介孔材料中制成固定化酶则可解决上述问题．目前已成功地将辣根过氧化物酶     

乙烯基Ia3d介孔硅分子筛的环氧化及其固定化青霉素酰化酶(英文)

采用直接共聚法合成表面含有乙烯基的具有立方相Ia3d结构的介孔硅分子筛(V-ClMS),然后对乙烯基团进行环氧化制备得到表面环氧基功能化的介孔硅分子筛(E-CIMS),采用X射线衍射、N2吸附-脱附、透射电镜、热重分析和13C固体核磁共振对制备的介孔硅分子筛进行了表征.结果表明,表面含有乙烯基的V-ClMS介孔硅分子筛能被一步成功合成,并易于发生环氧化而获得表面环氧基功能化的E-CIMS介孔硅分子筛.将E-CIMS介孔硅分子筛作为载体用于固定化青霉素G酰化酶(PGA),研究了表面环氧基团对固定化PGA初活性和操作稳定性的影响.结果表明,随着表面环氧基团数量的增加,介孔硅分子筛孔径减小,表面疏水性增加,导致载酶量和初活性减小.但介孔硅分子筛表面适量的环氧基团能增强E-CIMS介孔硅分子筛与PGA之间的相互作用,从而提高固定化PGA的操作稳定性.     

Penicillin G acylase as chiral selector in CE using a pullulan-coated capillary

In the present study, penicillin G acylase (PGA), an enzyme belonging to the family of hydrolases, has been investigated as chiral selector in CE using the partial filling technique. Owing to the strong disposition of PGA to be adsorbed by the inner capillary wall, permanently coated capillaries were used to diminish both the protein-wall interactions and the EOF. In particular, the silica surface of the capillary was chemically coated by an antiadhesive and an hydrophilic layer of pullulan, a high-molecular-mass homopolysaccharide. The coating procedure consisted in the silanization with glycidoxypropyltrimethoxysilane and the subsequent coupling of the hydroxyl groups of pullulan onto the silanized capillary. Using this approach, a significant EOF suppression was obtained within a wide pH range (pH 3.0-9.0); this result was very important in order to find the suitable conditions for the application of partial filling technique. The optimization of partial filling was carried out by considering the effects of different experimental conditions (buffer pH, PGA concentration, and loading duration), on the migration time and enantioresolution of rac-ketoprofen. Under the selected conditions as: 100 mM sodium phosphate buffer (pH 5.5) containing 240 microM of PGA (partial filling of 120 s at a pressure of 50 mbar), a series of acidic compounds resulted to be enantioresolved in about 10 min. The long-term stability of the proposed coating was evaluated; more than 100 injections were performed without significant loss of reproducibility.     

Immobilization of modified penicillin G acylase on Sepabeads carriers

An approach to stable covalent immobilization of chemically modified penicillin G acylase from Escherichia coli on Sepabeads® carriers with high retention of hydrolytic activity and thermal stability is presented. The two amino-activated polymethacrylate particulate polymers with different spacer lengths used in the study were Sepabeads® EC EA and Sepabeads® EC HA. The enzyme was first modified by cross-linking with polyaldehyde derivatives of starch in order to provide it with new useful functions. Such modified enzyme was then covalently immobilized on amino supports. The method seems to provide a possibility to couple the enzyme without risking a reaction at the active site which might cause the loss of activity. Performances of these immobilized biocatalysts were compared with those obtained by the conventional method with respect to activity and thermal stability. The thermal stability study shows that starch-PGA immobilized on Sepabeads EC-EA was almost 4.5-fold more stable than the conventionally immobilized one and 7-fold more stable than free non-modified PGA. Similarly, starch-PGA immobilized on Sepabeads EC-HA was around 1.5- fold more stable than the conventionally immobilized one and almost 9.5-fold more stable than free non-modified enzyme.     

Optimized polymer-enzyme electrostatic interactions significantly improve penicillin G amidase efficiency in charged PEGA polymers

Hydrolytic yields as high as 80% were obtained by using penicillin G amidase (PGA) on substrates anchored on optimized positively charged PEGA polymers. By increasing the amount of permanent charges inside the polymer, electrostatic interactions between the positively charged PEGA⁺ and the negatively charged PGA (pI=5.2-5.4) were strengthened, thus favouring the accessibility of the bulky enzyme (MW=88 kDa) inside the pores. The effect of different amounts of charges on polymer swelling and protein retention inside the polymer was investigated and correlated to the enzyme efficiency demonstrating that electrostatic interactions predominate over swelling properties in determining enzyme accessibility.     

(3,5-二甲基苯基氨基甲酸酯)衍生的纤维素手性固定相

纤维素三（3,5-二甲基苯基氨基甲酸酯）是液相色谱中使用最广泛的手性柱。该文详细地研究了不同程度衍生的纤维素（3,5-二甲基苯基氨基甲酸酯）以及不同硅胶（粗制硅胶、氨丙基粗制硅胶、精制硅胶、氨丙基精制硅胶、大孔硅胶、氨丙基大孔硅胶）作为支撑体对该柱手性分离能力的影响。自制了13根手性色谱柱,分别考察了其对16种外消旋体的拆分,分离结果显示：三取代纤维素柱 > 二取代纤维素柱 > 纤维素柱;精制硅胶和大孔硅胶优于粗制硅胶,大孔硅胶的柱压更低;硅胶的氨丙基化对手性选择性有一定的影响;这些手性柱之间具有一定的互补性,尤其是纤维素柱。该文有助于人们更深刻地理解和更好地把握高效液相色谱手性柱的制备。     

Comparison of high-performance liquid chromatography of cyclotriphosphazene derivatives with one or two equivalent stereogenic centres

High-performance liquid chromatographic (HPLC) methods have been developed for investigating the stereogenic properties of two analogous series of dibenzylamino derivatives of cyclotriphosphazene containing either one or two equivalent stereogenic centres. Separation of the enantiomers of all the racemic compounds has been investigated by chiral HPLC using Whelk-01 and Chiralcel OD columns. In all cases, conditions for separation of enantiomers have been found using a Whelk-01 column with different ratios of tetrahydrofuran in n-hexane as the mobile phase. It is found that both the separation factor (alpha) and resolution factor (R(S)) of molecules with two equivalent stereogenic centres are greater than those for analogues with only one centre.     

Improved biotransformations on charged PEGA supports

PEGA supports functionalised with permanent charges show superior swelling properties in aqueous media when compared to neutral PEGA; a novel positively charged PEGA resin significantly improves penicillin G amidase (PGA) catalysed biotransformation on solid support, by favouring accessibility of the negatively charged enzyme.