Immobilization of Penicillin G Acylase in Epoxy-Activated
Immobilization
APTEFF, 38, 1-190 (2007)UDC: 663.15:66.097:66.098DOI:10.2298/APT0738173ZBIBLID: 1450-7188 (2007) 38, 173-182Original scientific paper173IMMOBILIZATION OF PENICILLIN ACYLASE FROM Escherichia coliON COMMERCIAL SEPABEADS EC-EP CARRIERMilena G. Žuža, Slavica S. Šiler-Marinkoviü and Zorica D. KneževiüThis paper describes the covalent immobilization of penicillin G acylase from Escherichia coli on sepabeads EC-EP, an epoxy-activated polymethacrylic carrier and kinetic properties of the immobilized enzyme. The selected enzyme belongs to a class of biocatalysts whose industrial interest is due to their versatility to mediate hydrolysis of penicillins and semi-synthetic E -lactam antibiotics synthesis reactions.About 2.7 mg of the pure enzyme was immobilized onto each gram of sepabeads with an enzyme coupling yield of 96.9%. However, it seems that the activity coupling yield is not correlated with the amount of enzyme bound and the maximum yield of 89.4% can be achieved working at low enzyme loading (0.14 mg g -1). Immobilization of the penicillin acylase resulted in slightly different pH activity profile and temperature optima, indicating that the immobilization by this method imparted structural and conformational stability of this enzyme. It appears that both free and immobilized penicillin acylase followed simple Michaelis-Menten kinetics, implying the same reaction mechanism in both systems.KEYWORDS: Penicillin acylase; covalent immobilization; sepabeads carrier, Michaelis-Menten kineticsINTRODUCTIONPenicillin acylases (penicillin amidohydrolase; EC 3.5.1.11) catalyze the hydrolysis of penicillins into 6-aminopenicillanic acid (6-APA) and another organic acid whose structure depends on the type of penicillin hydrolyzed (1). Biotechnological applications of penicillin acylases (PA) have emerged as a serious alternative to traditional chemical procedures for the manufacture of E -lactam antibiotics, small peptides and pure isomers from racemic mixtures. However, penicillin acylases are involved mainly in industrial production of semi-synthetic penicillins (2).Milena G. Žuža, PhD student, Scholar of the Ministry of Science and Environmental Protection of Serbia, Dr. Slavica Šiler-Marinkoviü, Assoc. Prof., Dr. Zorica D. Kneževiü, Assist. Prof., University of Belgrade, Faculty of Technology and Metallurgy, Karnegijeva 4, 11000 Belgrade, Serbia, e-mail: milenazuza@They are classified in three sub-groups, according to their preferential substrate: pe-nicillin V acylase, penicillin G acylase and ampicillin amidase (3). Penicillin G acylase (PGA), catalyzing the hydrolysis of penicillin G, is the most important acylase because penicillin G is the cheapest raw material for the production of 6-APA, the nucleus from which a range of semi-synthetic penicillins are made. The standard industrial practice to produce semi-synthetic E-lactam antibiotics employs a chemical route, with protecti-on/de-protection of reactive groups, low temperatures (-30 o C or less), and organochlo-ride solvents. Hence, their enzymatic synthesis has received great attention as possible …green chemistry” alternative (4).To fully exploit the technical and economical advantages of highly cost penicillin acylase, it is recommended to use them in an immobilized form (5). This is because free enzyme, as a biocatalyst, is lacking long-term stability under the process conditions and is difficult to recover and recycle from the reaction mixture, making the reuse of the enzyme impossible. Hence, the idea of immobilizing the enzyme on a rigid solid support, enabling easy separation and the possibility of operation in a packed-bed or fluidized-bed reactor, has been of great industrial interest for many years (6). Among the immobi-lization methods, covalent attachment is more advantageous than the other methods since diffusional restrictions to substrate or products are decreased considerably. Moreover, the formation of the rigid enzyme-support linkage provides both kinetic and thermodynamic stabilization of the three-dimensional structure of the active catalytic site and often improves the enzyme thermal stability.The technology of PGA immobilization has been improved in the last decades, and PGA has been covalently immobilized onto various supports such as microparticulate and monolithic silica supports (2), poly(vinyl acetate–co-diviyl benzene) beads (7), activated agarose (8), grafted nylon membranes (9), etc. However, various problems associated with these supports were also reported. One common problem is the lack of active sites on the polymer. Consequently, most previous studies used glutaraldehyde as a non-spe-cific cross-linking agent to fix the enzyme on the polymeric matrix, but the results were often unsatisfactory, with low immobilization yield and low final enzyme activity. The-refore, the development of new techniques for PGA immobilization on inexpensive and industrially applicable carriers is of economical significance.Epoxy-activated supports are almost ideal to perform very easy immobilization of enzymes at both laboratory and industrial scale. Epoxide groups are convenient for the covalent binding of enzymes since they are able to directly react with amino, hydroxyl, or sulfhydryl groups of enzymes, depending on pH of the buffer used. Thus, it is not ne-cessary to activate the carrier or enzyme to achieve covalent immobilization. Moreover, the N-C, O-C or S-C bonds formed by the epoxide groups are extremely stable, so that the epoxide-containing commercial polymers such as Eupergit or Sepabeads can be suc-cessfully used for the immobilization of enzymes and proteins (10, 11).Although a number of studies have shown that epoxide-containing polymers are good carriers for enzyme immobilization, their potential for binding penicillin acylases has not been fully explored. In this paper, we investigated the covalent immobilization of peni-cillin G acylase from E. coli on Sepabeads EC-EP, a commercial polymethacrylic carrier that has introduced functional epoxy groups. The support is very stable and has good chemical, mechanical and other properties such as hydrophilicity, wide pore distribution, and almost ideal spherical beads that allow simple immobilization procedure, high pro-174tein binding capacity, low swelling tendency in high molar solution and in common solvents, high flow rate in column procedures, excellent performance in stirred batch reactors, etc. The first aim of the study was to investigate the effects of immobilization conditions such as initial enzyme concentration on enzyme and activity coupling yields. Having obtained the highly efficient immobilized PGA, it was then characterized its pH and thermal stability, as well as the reaction kinetics.EXPERIMENTALMaterialsPenicillin G acylase (E.C. 3.5.1.11) from E. coli (PGA) was a gift from DSM (The Netherlands). The enzyme was a crude preparation with specific activity of 167 U mg-1 protein and 90% (w/v) protein based on Lowry`s method for protein assay (12). Sepa-beads EC-EP (particle sizes 200-240 P m, average pore diameter 30-40 nm, water re-tention 55-65%, specific volume 2.8-3.3 cm3/dry g) was kindly donated by Resindion S.R.L. (Mitsubishi Chemical Corporation, Milan, Italy). 6-Aminopenicillanic acid (6-APA), penicillin G, p-dimethylaminobenzaldehyde (PDAB), Folin Ciocalteu’s Phenol Reagent were purchased from Sigma Chemical Co. (St. Louis, MO). All other chemicals were reagent-grade.Immobilization of penicillin G acylaseThe method for enzyme immobilization on Sepabeads EC-EP support involves the direct enzyme binding on polymers via epoxide groups (Fig. 1). Unmodified Sepabeads EC-EP (500 mg of wet carrier) was incubated with 10 cm3 of native enzyme attachment solutions in a shaking water bath (130 strokes per min) at 25 o C. Enzyme attachment so-lutions containing 7.5-120 U/g wet support were prepared in a 1.25 M potassium phos-phate buffer at pH 8.0. After incubation for 48 h, the beads were collected by vacuum filtration using a glass filter (Whatman), washed with 1 M NaCl (3x20 cm3), then with potassium phosphate buffer pH 8.0 (3x20 cm3), and stored in it at 4 o C until use. Samples of the filtrate and enzyme solution before immobilization, together with the washing solutions, were taken for protein content and enzyme activity assay.Fig. 1. Schematic illustration of the covalent method for the enzyme immobilization on Sepabeads EC-EP supportThe efficiency of immobilization was evaluated in terms of enzyme and activity coupling yields. The enzyme coupling yield, Y E (%) and activity coupling yield, Y A (%) were calculated as follows:175176 100%01E uP P Y [1] 100%12A u SA SAY [2]where P 1 is the amount of immobilized enzyme; P 0 the initial amount of enzyme; SA 2 the specific activity of immobilized penicillin acylase; and SA 1 is the specific activity of free penicillin acylase.Enzyme activity assayThe enzyme activity of penicillin acylase was determined by measuring the penicillin G enzymatic product 6-APA spectrophotometrically (13). One unit of penicillin acylase was defined as the amount of the enzyme required to produce 1 P mol of 6-APA per minute under the assay conditions (4% (w/v) penicillin G as substrate solvated in 0.1 M phosphate buffer, pH 7.92 at 37 o C). The amount of 6-APA was determined with the method of PDAB. The experimental procedures were as follows: firstly, the solutions of 20% (v/v) of acetic acid (A), 0.5% (w/v) of NaOH (B ) and 0.5% (w/v) of PDAB in methanol (C) were prepared; secondly, A,B and C were added to 0.5 ml of the sample solution in sequence to the volume ratio 2:1:0.5; finally, after reaction for 20 min at room temperature, the absorbance value of solution was determined at 415 nm.Determination of penicillin acylase concentrationPenicillin acylase concentration was determined spectrophotometrically at 210 nm by using bovine serum albumin as a standard. The penicillin acylase concentration was determined from a standard curve which was constructed for each measurement. The amount of bound enzyme was determined indirectly from the difference between the amount of enzyme introduced into the coupling reaction mixture and the amount of enzyme in the filtrate and in the washing solutions.Kinetics evaluationKinetic parameters of free and immobilized enzyme were determined by measure-ments of enzyme activity with various concentrations of substrates (0.25 to 4% of peni-cillin G in 0.1 M phosphate buffer, pH 7.9). The enzyme concentration was constant (0.1 g of immobilized enzyme or corresponding amount of free enzyme). All data are the averages of duplicate samples and were reproducible within r 5%. Mean and standard deviation of the results from at least two independent experiments were calculate using Microsoft Excel (Redmond, WA, USA) software.Assuming Michaelis-Menten type reaction kinetics, the apparent Michaelis-Menten constant, K m , and the maximum apparent initial rate, V max of the free and immobilized enzyme were calculated directly from the model by nonlinear regression analysis using the MATLAB software (version 6.5, Release 13, The MathWorks, Juc, Matick, MA, USA). The extent of mass-transfer control could be expressed by the effectiveness factor, EF (considered as the ratio between maximum rates of the reaction catalyzed by im-mobilized and free penicillin acylase under otherwise identical conditions).pH and temperature profileThe effect of pH on free and immobilized enzyme was studied by assaying the pre-parations at different pH values in the range from 4.5 to 9.3 at 37 o C. The effect of tem-perature on activities of free and immobilized penicillin acylase was also determined at temperatures from 8 to 75 o C under assay conditions (4% (w/v) penicillin G as substrate solvated in 0.1 M phosphate buffer, pH 7.9).RESULTS AND DISCUSSIONThe basic characteristics of an immobilized enzyme include the amount of immo-bilized protein, its activity and specific activity, thermal and pH profile as well as kinetic properties.The first aim of this study was to determine the relationship between penicillin acylase and support in respect to enzyme loading, specific activity of the immobilized enzyme and enzyme and activity coupling yields. Then, a comparative study involving free and immobilized penicillin acylase is performed in terms of reaction kinetics and activity profile as a function of pH and temperature.Optimization of immobilization conditionsAn attempt was made to achieve binding of high levels of enzyme with a high re-tention of its initial activity. Thus, the effect of varying penicillin acylase concentration in the attachment solution in the range of 2.25-36 10-3 mg/cm3 (7.5-120 U/g wet support) on the total protein loading on the sepabeads support and enzyme and activity coupling yields is investigated. The results are shown in Fig. 2. In each experiment, 0.5 g of wet polymer beads were immersed in 10 cm3 of enzyme attachment solution.It seems that the increase of the initial enzyme concentration in the attachment so-lution results in almost linear increase of the enzyme loading on support (Fig. 1a). It is interesting to notice that the enzyme coupling yield also increases as the initial enzyme concentration is increased to 9 10-3 mg/cm3 and levels off at about 97.5 r0.5%. Thus, the maximal obtained enzyme loading under this immobilization condition of approximately 2.7 mg of the protein/g of the dry support was equivalent to 96.9% enzyme coupling yield.The activity of the immobilized penicillin acylase increases rapidly with enzyme loading on supports up to about 1.7 mg g-1 and then becomes essentially constant (Fig.2.b). However, it seems that the activity coupling yield is not correlated with the amount of enzyme bound. For example, as the enzyme concentration in the attachment solution increased from 2.25 to 36 10-3 mg/cm3, the enzyme coupling yield increased slightly (by 22.4%), while the activity coupling yield decreased from 89.4 to 25.9%. The maximum activity coupling yield of 89.4% can be achieved working at low enzyme loading (0.14 mg g-1). The possible reason for the lower activity yields at high enzyme loadings may be close packing of the enzyme on the support surface, which could limit the access of substrates needed in the hydrolysis reaction. Nevertheless, the immobilized enzyme sho-wed good performance in considering that generally, covalent binding leads to significant inactivation of enzymes. Especially, the activity coupling yield achieved in this study177178(89.4%) was much higher than the values reported in the literature for covalently immo-bilized penicillin acylase (7-9).Initial enzyme concentration in attachment solution(mg cm -3)E n z y m e l o a d i n g o n S e p a b e a d s s u p p o r t (m g g -1 d r y s u p p o r t )Enzyme coupling yield (%)Enzyme loading on Sepabeads support (mg g -1)A c t i v i t y o f t h e i m m o b i l i z e d e n z y m e (U g -1)Activity coupling yield (%)(a) (b)Fig. 2. (a) Effects of initial enzyme concentration in attachment solution on the enzymeloading and enzyme coupling yield (b) Effects of enzyme loading on the activity of the biocatalyst and activity coupling yieldEffect of immobilization on optimum pH and temperatureThe activity of the immobilized penicillin acylase was investigated in the pH range 4.5-9.3 at 37 o C and compared with that of free enzyme. Results are presented in Fig. 3a. The pH activity profile of the immobilized enzyme was slightly narrower than that of the free enzyme having no essential effect on the pH optimum. The trends of the curves are similar in both cases. This similar behavior is related to the electrically neutral character of the support, which did not alter the pH of the microenvironment of the immobilized enzyme.R e l a t i v e a c t i v i t y (%)pHR e l a t i v e a c t i v i t y (%)Temperature (oC)(a) (b)Fig. 3. Effects of pH (a) and temperature (b) on activities of free and immobilized peni-cillin acylase. The enzyme activity was determined under assay conditions (4% (w/v) penicillin G as substrate solvated in 0.1 M buffer at indicated pH andtemperature).179However, the immobilized enzyme shows a higher activity at lower ( 5.5) and higher (!8.7) pH values than the soluble one, indicating that the immobilization of penicillin acylase imparted structural and conformational stability to this enzyme.The effect of temperature on activities of free and immobilized enzyme was also stu-died and the results are shown in Fig. 3b. It appears that the optimal reaction temperature shifted from 27.5 o C for the free penicillin acylase to 31.5 o C for the immobilized enzyme, suggesting a marginally better thermal stability of the immobilized enzyme.Kinetics propertiesThe reaction kinetics of immobilized penicillin acylase was compared with that of the free enzyme using penicillin G hydrolysis as a model reaction. The initial reaction rates for free and immobilized penicillin acylase were determined at different concentrations of substrate ranging from 0.25 to 4%, and the results are shown in Fig. 4. It seems that both free and immobilized penicillin acylase followed simple Michaelis-Menten kinetics. The values of kinetic parameters of free and immobilized enzyme are obtained directly by fitting the experimental data to the kinetic model.The nonlinear regression analysis indicated that the quality of the fit was quite good, with r 2 value of 0.960 and 0.949 for free and immobilized enzyme, respectively. The K m value for the free enzyme was determined to be 5.99r 1.46 mmol dm -3, whereas V max va-lue was 128.98r 5.37 P mol min -1 mg -1. The apparent value of K m for the immobilized en-zyme was 29.85r 8.7 mmol dm -3, approximately 5-fold higher than that of the free enzy-me, suggesting that enzyme immobilization by this method caused a decrease in the en-zyme-substrate affinity.I n i t i a l r a t e , v (P m o l mi n -1m g -1)[S] (mol dm -3)I n i t i a l r a t e , v (P m o l m i n -1m g -1)[S] (mol dm -3)(a)(b)Fig. 4. Effect of penicillin G concentration on initial rates of free (a) and immobilizedpenicillin acylase (b). The lines represent the best fits of the Michaelis-Menten kinetic model. Assay conditions: t=37 o C; 0.1 M phosphate buffer, pH 7.9; 0.1 g immobilized enzyme or the corresponding amount of free penicillin acylase.Similarly, the value of V m for the immobilized enzyme appeared to be approximately 9-fold lower than that of the free enzyme (V m =14.65r 1.55 P mol min -1 mg -1; EF =0.11), indicating that the catalytic property of the enzyme was significantly modified by theimmobilization process. Nevertheless, the immobilized penicillin acylase showed favo-rable kinetic properties in considering that the value of V m was generally considerably lower upon covalent immobilization (7, 9, 14).The increase in K m values for immobilized enzymes has also been reported by various authors (7, 14). Wang et al. (14) reported that K m value of immmobilized penicillin acylase was 7 times higher than that of the free enzyme owing to the influence of the support. These changes in kinetic parameters may be a consequence of either structural change in the enzyme occurring upon immobi-lization or lower accessibility of substrate to the active sites of the immobilized enzyme.CONCLUSIONThis work provides preliminary results on the use of Sepabeads EC-EP as a support matrix for covalent immobilization of penicilllin G acylase and application of the im-mobilized enzyme for penicillin G hydrolysis as a study model reaction. The immobi-lization procedure developed is quite simple, and easily reproduced, and involves the direct enzyme binding on polymers via epoxide groups.Penicillin G acylase from E. coli has been successfully immobilized on Sepabeads EC-EP using the covalent binding method. The highest activity coupling yield of 89.4% was achieved working at low enzyme loading (0.14 mg g-1). Both free and immobilized enzyme were characterized by determining the activity profile as a function of pH, temperature and reaction kinetics. The optimal pH for the immobilized enzyme activity was found to be 8.7. A slightly higher value for optimum temperature (31.5 o C) was found for the immobilized enzyme in comparison with that displayed by the free one (27.5 o C). The immobilized enzyme appears to have acceptable kinetic properties in the industrially feasible reaction system of penicillin G hydrolysis. The method is simple, inexpensive, and scaleable for industrial applications.ACKNOWLEDGEMENTThe authors are grateful to Resindion S.R.L. (Mitsubishi Chemical Corporation, Mi-lan, Italy) for donation of Sepabeads EC-EP carrier and DSM (The Netherlands) for providing Penicillin G acylase used in this study. Also, the authors thank the Ministry of Science and Environmental Protection of Serbia (Projects No. bth1008) for the financial support. M. Zuza also gratefully acknowledges the Ministry of Science and Environ-mental Protection of Serbia for Ph.D. fellowship.REFERENCES1.Torres-Guzman, R., I. Mata, J. Torres-B acete, M. Arroyo, M.P. Castillon and C.Acebal: Substrate specificity of penicillin acylase from Streptomyces lavendulae.Biochem. Biophys. Res. Commun. 291 (2002) 593-597.2.Calleri, E., C. Temporini, G. Massolini and G. Caccialanza: Penicillin G acylase-based stationary phases: Analytical applications. J. Pharmaceut. Biomed. Analysis 35 (2004) 243–258.1803.Giordano, R.C., M.P.A. Ribeiro and R.L.C. Giordano: Kinetics of E-lactam anti-biotics synthesis by penicillin G acylase (PGA) from the viewpoint of the industrial enzymatic reactor optimization. Biotechnol. Adv. 24 (2006) 27-41.4.Ribeiro, M.P.A., L.O. Andrea, R.L.C. Giordano and R. C. Giordano: Selectivity ofthe enzymatic synthesis of ampicillin by E.coli PGA in the presence of high concentrations of substrates. J. Molecular Cat. B: Enzym. 33 (2005) 81–86.5.Rajendhran J., and P. Gunasekaran: Recent biotechnological interventions for de-veloping improved penicillin G acylase. J. Biosci. Bioeng. 97, 1 (2004) 1-13.6.Chong, A.S.M., and X.S. Zhao: Design of large-pore mesoporous materials forimmobilization of penicillin G acylase biocatalyst. Catalysis Today 93–95 (2004) 293–299.7.Jianguo, L., C. Wei, W. Shuai and O. Fan: Studies of poly(vinyl acetate-co-diviylbenzene) beads as a carrier for the immobilization of penicillin acylase and the ki-netics of immobilized penicillin acylase. React. & Funct. Polym. 48 (2001) 75–84.8.Aguirre, C., P. Opazo, M. Venegas, R. Riveros and A. Illanes: Low temperatureeffect on production of ampicillin and cephalexin in ethylene glycol medium with immobilized penicillin acylase. Process Biochem. in press9.Eldin, M.S.M., M. Santucci, S. Rossi, U. B encivenga, P. Canciglia, F.S. Gaeta, J.Tramper, A.E.M. Janssen, C.G.P.H. Schroen and D.G. Mita: Non-isothermal cephalexin hydrolysis by penicillin G acylase immobilized on grafted nylon membranes. J. Molecular Cat. B: Enzym. 8 (2000) 221-232.10.Kneževiü, Z., N. Milosaviü, D. B ezbradica, Ž. Jakovljeviü and R. Prodanoviü:Immobilization of lipase from Candida rugosa on Eupergit C supports by covalent attachment. Biochem. Eng. J. 30 (2006) 269-278.11.Katchalski-Katzir, K., and D.M. Kraemer: Eupergit C, a Carrier for immobilizationof enzymes of industrial potential. J. Mol. Catal. B: Enzym. 10 (2000) 157-176.12.Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall: Protein measurement withthe folin phenol reagent. J. Biol. Chem. 193 (1951) 265-275.13.Shewale, J.G., K.K. Kumar and G.R. Ambekar: Evaluation of 6-aminopenicillanicacid by p-dimethylaminobenzaldehyde. Biotechnol. Tech. 1 (1987) 69-72.14.Wang, W., L. Deng, Z.H. Peng and X. Xiao: Study of the epoxydized magnetichydroxyl particles as a carrier for immobilizing penicillin G acylase. Enzyme Microb.Technol. 40 (2007) 255-261.ɂɆɈȻɂɅɂɁȺɐɂȳȺ ɉȿɇɂɐɂɅɂɇ-ȺɐɂɅȺɁȿ ɂɁ Escherichia coli ɇȺɄɈɆȿɊɐɂȳȺɅɇɈɆ SEPABEADS ɇɈɋȺɑɍɆɢɥɟɧɚ Ƚ. ɀɭɠɚ, ɋɥɚɜɢɰɚ ɋ. ɒɢɥɟɪ-Ɇɚɪɢɧɤɨɜɢʄ ɢ Ɂɨɪɢɰɚ Ⱦ. Ʉɧɟɠɟɜɢʄɍ ɪɚɞɭ ʁɟ ɢɫɩɢɬɚɧɚ ɤɨɜɚɥɟɧɬɧɚ ɢɦɨɛɢɥɢɡɚɰɢʁɚ ɩɟɧɢɰɢɥɢɧ-ɚɰɢɥɚɡɟ ɢɡ Escherichia coli ɧɚ ɤɨɦɟɪɰɢʁɚɥɧɨɦ ɩɨɥɢɦɟɬɚɤɪɢɥɚɬɧɨɦ ɧɨɫɚɱɭ ɫɚ ɟɩɨɤɫɢɞɧɢɦ ɮɭɧɤɰɢɨɧɚɥɧɢɦ ɝɪɭɩɚɦɚ (Sepabeads EC-EP) ɢ ɞɨɛɢʁɟɧɢ ɢɦɨɛɢɥɢɫɚɧɢ ɟɧɡɢɦ ʁɟ ɨɤɚɪɚɤɬɟɪɢɫɚɧ ɭ ɪɟɚɤɰɢʁɢ ɯɢɞɪɨɥɢɡɟ ɩɟɧɢɰɢɥɢɧɚ. ɂɡɚɛɪɚɧɢ ɟɧɡɢɦ ʁɟ ɨɞ ɜɟɥɢɤɨɝ ɢɧɞɭɫɬɪɢʁɫɤɨɝ ɡɧɚɱɚʁɚ ʁɟɪ ɤɚɬɚɥɢɡɭʁɟ ɪɟɚɤɰɢʁɟ ɯɢɞɪɨɥɢɡɟ ɩɪɢɪɨɞɧɢɯ ɩɟɧɢɰɢɥɢɧɚ ɢ ɫɢɧɬɟɡɟ ɩɨɥɭ-ɫɢɧɬɟɬɫɤɢɯ E-ɥɚɤɬɚɦɫɤɢɯ ɚɧɬɢɛɢɨɬɢɤɚ.181ɂɫɩɢɬɚɧ ʁɟ ɭɬɢɰɚʁ ɩɨɱɟɬɧɟ ɤɨɧɰɟɧɬɪɚɰɢʁɟ ɟɧɡɢɦɚ ɧɚ ɦɚɫɭ ɢ ɚɤɬɢɜɧɨɫɬ ɢɦɨɛɢ-ɥɢɫɚɧɨɝ ɟɧɡɢɦɚ, ɤɚɨ ɢ ɧɚ ɦɚɫɟɧɢ ɩɪɢɧɨɫ ɢɦɨɛɢɥɢɡɚɰɢʁɟ ɢ ɩɪɢɧɨɫ ɚɤɬɢɜɧɨɫɬɢ. ɇɚʁ-ɜɟʄɚ ɦɚɫɚ ɢɦɨɛɢɥɢɫɚɧɨɝ ɟɧɡɢɦɚ ʁɟ ɢɡɧɨɫɢɥɚ 2,5 mg ɩɨ ʁɟɞɢɧɢɰɢ ɦɚɫɟ ɧɨɫɚɱɚ, ɲɬɨ ɨɞɝɨɜɚɪɚ ɦɚɫɟɧɨɦ ɩɪɢɧɨɫɭ ɨɞ 96,9%. Ɇɟɻɭɬɢɦ, ɩɪɢɧɨɫ ɚɤɬɢɜɧɨɫɬɢ ʁɟ ɨɛɪɧɭɬɨ ɩɪɨ-ɩɨɪɰɢɨɧɚɥɚɧ ɦɚɫɢ ɢɦɨɛɢɥɢɫɚɧɨɝ ɟɧɡɢɦɚ ɬɚɤɨ ɞɚ ɫɟ ɦɚɤɫɢɦɚɥɧɢ ɩɪɢɧɨɫ ɨɞ 89,4% ɩɨɫɬɢɠɟ ɩɪɢ ɢɦɨɛɢɥɢɡɚɰɢʁɢ ɧɚʁɦɚʃɟ ɦɚɫɟ ɟɧɡɢɦɚ (0,14 mg/g ɧɨɫɚɱɚ). ɂɦɨɛɢɥɢ-ɡɚɰɢʁɚ ɟɧɡɢɦɚ ʁɟ ɩɪɨɭɡɪɨɤɨɜɚɥɚ ɦɚʃɟ ɩɪɨɦɟɧɟ ɭ pH ɩɪɨɮɢɥɭ ɚɤɬɢɜɧɨɫɬɢ ɢ ɨɩɬɢ-ɦɚɥɧɢɦ ɜɪɟɞɧɨɫɬɢɦɚ ɬɟɦɩɟɪɚɬɭɪɟ ɛɢɨɤɚɬɚɥɢɡɚɬɨɪɚ, ɲɬɨ ɭɤɚɡɭʁɟ ɧɚ ɧɟɡɧɚɬɧɭ ɫɬɚ-ɛɢɥɢɡɚɰɢʁɭ ɟɧɡɢɦɚ ɭɫɥɟɞ ɢɦɨɛɢɥɢɡɚɰɢʁɟ. Ʉɢɧɟɬɢɤɚ ɪɟɚɤɰɢʁɟ ɯɢɞɪɨɥɢɡɟ ɩɪɢɪɨɞɧɨɝ ɩɟɧɢɰɢɥɢɧɚ ɫɥɨɛɨɞɧɢɦ ɢ ɢɦɨɛɢɥɢɫɚɧɢɦ ɟɧɡɢɦɨɦ ɦɨɠɟ ɫɟ ɨɩɢɫɚɬɢ Ɇɢɯɚɟɥɢɫ-Ɇɟɧɬɟɧɨɜɨɦ ʁɟɞɧɚɱɢɧɨɦ ɢ ɨɞɪɟɻɟɧɟ ɫɭ ɜɪɟɞɧɨɫɬɢ ɤɢɧɟɬɢɱɤɢɯ ɤɨɧɫɬɚɧɬɢ ɡɚ ɨɛɚ ɫɢɫɬɟɦɚ.Received 16 April 2007Accepted 28 July 2007 182。
R-(-)-扁桃酸的生物合成研究进展
[3]Takahashi E,Nakamichi K,Furui M,et a1.R-(-)-Mandelicacid production from Racemic mandelic acids by pseudomonaspolycolor with asymmetric degrading activity[J].Journal ofFermentation and Bioengineering,1995,79(5):439-442.
R-(-)-扁桃酸的生物合成研究进展
扁桃酸又名α-羟基苯乙酸，是极其重要的手性药物中间体，具有消炎和杀菌的双重作用。目前国际市场上扁桃酸需求约以年均10％左右速度增长，尤其是R-(-)-扁桃酸早已成为国内外急需的产品[1,2]。R-(-)-扁桃酸及其衍生物是合成环扁桃酯、羟苄唑、匹莫林等血管扩张剂、杀菌剂、镇痉剂的重要药物中间体,且具有很好的生物分解性，是目前最受瞩目的酸性光学拆分剂，可使多数外消旋体胺类和氨基酸类经非对映体异构盐形成法进行光学拆分，如治咳药甲吗南的中间体八氢异喳琳衍生物可由R型扁桃酸拆分。
3水解酶生物催化合成R-扁桃酸
腈水解酶是一类可以将腈转化成相应酸及氨基的酶。当以扁桃腈为底物时，脂肪族水解酶立体选择性地生成R型扁桃酸，最重要的是其理论动力学反应产物收率为100％。具体作用机制如图4所示。
Endo等[22]将消旋体扁桃腈与Rhodococcus微生物反应，酶催化水解扁桃腈得到R-(-)-扁桃酸，R-(-)-扁桃酸的光学纯度达100％。Cesar等[23]采用来自木薯的有选择性的S-氧腈酶和来自荧光假单胞菌EBC191的无选择性的腈水解酶，分两步转化苯甲醛合成S-扁桃酸，产率很大，e.e值最高达98.0％。Yamamoto等[24]利用AlcaligenesfaecalisATCC8750菌株的静息细胞中腈水解酶催化外消旋扁桃腈得R-扁桃酸，产率为91％。Banerjee等[25]从假单胞菌中分离纯化得到的腈水解酶对扁桃腈同样具有立体选择性，可以将扁桃腈水解成R-扁桃酸，具有较高的对映体过量值。Banerjee等[25]报道海藻酸钙固定P．putida MTCC 5110细胞，利用腈水解酶水解扁桃腈，20个循环后固定化细胞仍具有88％的转化活性，e.e为98.8％。
培门冬酶静默失活在儿童急性淋巴细胞白血病治疗中的新进展
-专题论坛•培门冬酶静默失活在儿童急性淋巴细胞白血病治疗中的新进展梁石明文飞球中国医科大学深圳市儿童医院518000通信作者：文飞球，E m a i l:f w e n62@126.c o m【摘要】门冬酰胺酶包括大肠杆菌和欧文菌来源的门冬酰胺酶，以及培门冬酶。
与前二者相比，由于培门冬酶具有体内半衰期更长、免疫原性更低的优势，目前已成为儿童急性淋巴细胞白血病(A L L)的首选治疗药物。
但是，临床应用培门冬酶治疗A L L患儿过程中，可发生培门冬酶静默失活，从而导致A L L患儿治疗无效，并且严重影响其预后。
培门冬酶静默失活虽然属于亚临床超敏反应，但是与超敏反应相比，其具有一定的隐蔽性，临床难以识别。
因此，笔者通过介绍在儿童A L L治疗中培门冬酶静默失活的定义、发生机制、流行病学特征、危害及处理措施等方面的最新研究进展，以期探寻培门冬酶静默失活的有效识别方法及处理措施。
【关键词】前体细胞淋巴母细胞白血病淋巴瘤；药物疗法；药物过敏；药物监测；培门冬酶；静默失活基金项目：深圳市科技创新委基础研究学科布局项目（JCYJ20170817170110940)D O I：10. 3760/cma. j. cn511693-20200703-00140Advance on silent inactivation of pegasparaginase in treatment of pediatric acute lymphoblastic leukemiaLiang Shim i n g，Wen FeiqiuDepartment o f Hematologic O ncology»Shenzhen Children' s H o s p ita l»Shenzhen518000,Guangdong Province，ChinaCorresponding a u th o r：Wen Feiqiu •,Em ail：fw e n 62 @126. com【Abstract】Asparaginases include asparaginase derived from c’o/z. or ，andpegasparaginase. Compared with the former two, pegasparaginase has the advantages of longer half-life in vivo and lower immunogenicity, so it has become the first choice for the treatment of acutelymphoblastic leukemia ( ALL ) in children. However，during the clinical application ofpegasparaginase to treat children with ALL» silent inactivation of pegasparaginase may occur, whichleads to ineffective treatment of ALL children, and affects their prognosis seriously. Although thesilent inactivation of pegasparaginase is a subclinical hypersensitivity reaction，compared withhypersensitivity，it has a certain degree of concealment, which is hard to identify. This articleintroduces the latest research progress in the definition, mechanism，epidemiological characteristics,hazards and treatment measures of silent inactivation of pegasparaginase in the treatment of ALL inchildren, in order to find effective methods to identify and treat silent inactivation of pegasparaginase.【Key words】Precursor cell lymphoblastic leukemia-lymphoma ；Drug therapy；Drughypersensitivity；Drug monitoring；Pegasparaginase；Silent inactivationFund program：Basic Research Discipline Layout Project of Shenzhen Science and TechnologyInnovation Committee (JCYJ20170817170110940)DOI: 10. 3760/cma. j. cn511693-20200703-00140急性淋巴细胞白血病（a c u t e l y m p h o b l a s t i cl e u k e m i a，A L L)是儿童最常见的血液系统恶性肿瘤之一，其治疗药物包括糖皮质激素、长春新碱和门冬酰胺酶等[1]。
R-(-)-扁桃酸的生物合成研究进展
Tetrahedron:Asymmetry,2004,15(2):207-211.
[3]Takahashi E,Nakamichi K,Furui M,et a1.R-(-)-Mandelicacid production from Racemic mandelic acids by pseudomonaspolycolor with asymmetric degrading activity[J].Journal ofFermentation and Bioengineering,1995,79(5):439-442.
3水解酶生物催化合成R-扁桃酸
腈水解酶是一类可以将腈转化成相应酸及氨基的酶。当以扁桃腈为底物时，脂肪族水解酶立体选择性地生成R型扁桃酸，最重要的是其理论动力学反应产物收率为100％。具体作用机制如图4所示。
青霉素G酰化酶的研究进展
原理
特点 酶回收率比超声波处理方法高，而且酶的比活
较高。(Alves等,2003)
PGA的纯化
Zhou Z 等发酵所得的青霉素G酰化酶经硫酸铵分级沉淀和
welcome to use these PowerPoint templates, New DEAE-Sepharose CL-6B 两步纯化, 纯度提高86 倍, 活力回 Content design, 10 years experience 收率达到81 %,纯化后的PGA 活力为1. 469 u/ mg.(Zhou 等,2002)
上的固定化及后修饰，得到固定化酶表观酶活为177U/g, 固定化酶经巯基乙醇修饰后提高了操作稳定性及热稳定
性。(颜淑玮等，2006)
PGA的固定化
welcome to use these PowerPoint templates, 目前已有很多方法将含 PGA的大肠杆菌 E.coliNew 菌株及突 Content design, 10 years experience 变菌种的细胞进行固定化，如包裹在聚甲基丙烯酸酰胺 细胞内部，或者包裹在凝胶基质中。所得的固定化酶能 够有效地水解青霉素G,水解效果优于自由酶。
特点 酶比活力高,酶活力较超声波法低。(张贺迎等,2000)
PGA的分离提取
有机溶剂法
welcome use these PowerPoint templates, New 原理 to 本法是利用有机溶剂溶解细菌细胞内膜的脂肪 , Content design, 10 years experience 从而释放酶。不同浓度的有机溶剂对细胞内膜的作用 不同,释放出不同量的酶。
u/L。（杨志建等，2004）
PGA产生菌改造与发酵条件的优化
益赛普说明书
百济新特药房资深药师倾力制作千个药品专辑 安全、有效、合理用药之
【益赛普适应症】 用于治疗：1. 中度及重度活动性类风湿关节炎；2. 18岁及18岁以上成人中度至重度斑块状银屑病；3. 活动性强直性脊柱炎。 【益赛普用法用量】 成人推荐剂量为每次25mg，皮下注射，每周二次。 注射前用1毫升注射用水溶解，溶 解后可冷藏72小时。
【益赛普不良反应】 常见不良反应是注射部位局部反应，包括轻至中度红 斑、搔痒、疼痛和肿胀等，注射部位反应通常发生在 开始治疗的第一个月内，在随后的治疗中发生频率降 低。注射部位反应平均持续3－5天。在临床试验出现 的其它不良事件包括头痛、眩晕、皮疹、咳嗽、腹痛、 淋巴细胞增多白细胞减少、中性粒细胞减少、鼻炎、 发热、关节酸痛、肌肉酸痛、困倦、 面部肿胀、面部过敏、肝功能异常、 肾结石、左肺纤维化等。
【益赛普成份】 益赛普主要成份： 每瓶含重组人Ⅱ型肿瘤坏死因子受体－抗体融合 蛋白（rhTNFR:Fc）12.5毫克或25毫克，甘露醇 40毫克，蔗糖10毫克，三羟甲基氨基甲烷1.2毫克。 用1毫升灭菌注射用水溶解。
【益赛普性状】 益赛普为白色冻干粉针剂，加水溶 解后溶液为无色或微带黄色的澄清 透明液体。
【益赛普药理作用】
已知TNF是类风湿性关节炎（RA）病理过程中的 一个主要炎性介质，其参与调控的炎症反应可导致 关节的病理改变。益赛普的作用机制为竞争性地与 血中肿瘤坏死因子(TNF)－α结合，阻断它和细胞表 面TNF受体结合，降低其活性。
固定化青霉素华酶的新载体
第29卷第1期2002年北京化工大学学报JOURNAL OF BEI J IN G UN IV ERSIT Y OF CHEMICAL TECHNOLO GYVol.29,No.12002固定化青霉素酰化酶的新载体任玲玲 何 静 马润宇 段 雪3(北京化工大学可控化学反应科学与技术基础教育部重点实验室,北京 100029)摘 要:通过X 射线粉末衍射、红外光谱等手段考察了α2磷酸锆层柱材料作为载体固定青霉素酰化酶及其对载体结构影响。
结果表明无论是否加入戊二醛固定化酶后,层柱材料的结晶度都下降。
酶活性和回收率较高,分别为13915nmol/s 和2819%。
间歇操作稳定性较好,连续操作10次,酶活性下降24%。
关键词:α2磷酸锆;三乙烯四胺;青霉素酰化酶;插层组装中图分类号:Q939.92收稿日期:2001204212基金项目:国家自然科学基金资助项目(29973004);北京市自然科学基金资助项目(582014)第一作者:女,1970年生,讲师3通讯联系人 青霉素酰化酶(EC3.5.1.11,penicillin G acy 2lase ,P G A )是生产β2内酰胺类抗生素的重要工业用酶。
它不仅能催化青霉素G 水解生成62氨基青霉烷酸(62APA ),还能催化头孢霉素G 生成72氨基头孢烯酸(72ADCA )。
近5年来,固定化P G A 也在催化β2内酰胺类抗生素母核与新的酰基供体缩合,可产生一系列新青霉素和新头孢霉素。
在工业生产两种半合成头孢霉素上获得突破,促进了β2内酰胺类抗生素的新发展,也重新唤起了人们对P G A 的重视[1]。
由于固定化青霉素酰化酶具有新的工业应用前景,使各国研究者都在寻求更好的载体。
α2磷酸锆即α2Zr (HPO 4)2・H 2O (α2ZrP )是一种重要的阳离子型层状材料[2,3],具有较高的化学稳定性和热稳定性,层板耐酸、碱、电磁辐射。
通过插层组装,可以在层间引入可反应性基团,并且能够通过控制反应调节层间可反应性基团含量,使其对酶分子具有良好的亲和性,有利于酶活性的保持和寿命的延长。
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employed as an industrial catalyst for the manufacture 6-APA from penicillin G.8 A simple, safe, and biocompatible support for the enzyme immobilization is essential for successful biotechnology applications. Therefore, considerable efforts are still being made to immobilize PGA on solid supports, including gopolymer supports, and magnetic beads. Various immobilization methods have been used to improve the biocatalytic activity, stability, and recycling.8-12 It is noted that some biopolymers, such as aldehyde-agarose,13 inclusion in glatine,14 gelatin-chitosan,15 and concanavalin A,16 are satisfactory supports. Cellulose is an almost inexhaustible natural polymer with fascinating structure and properties such as biocompatibility, biodegradability, and safety, leading to increasing demands on its products.17,18 Usually, chemically modified cellulose is the primary candidate of cellulose-based materials.19 Therefore, cellulose derivatives have been used in enzyme immobilization, such as CMcellulose, TEAE-cellulose, DEAE-cellulose, and so on.9 However, direct utilization of pure regenerated cellulose as a support for enzyme immobilization has never been reported because many cellulose solvents such as N,N-dimethylacetamide (DMAc)/ LiCl, DMSO/triethylamine/SO2, ammonia/ammonium salt (NH3/ NH4SCN), and N-methylmorpholine-N-oxide (NMMO) are toxic to the organism system.
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Biomacromolecules 2010, 11, 2896–2903
Immobilization of Penicillin G Acylase in Epoxy-Activated Magnetic Cellulose Microspheres for Improvement of Biocatalytic Stability and Activities
Introduction
Enzymes are versatile biocatalysts having high specificity, and their applications have been found in areas of biomaterials and organic syntheses.1 However, the major drawbacks for their industrial applications are low thermal stability and solvent stability as well as the difficulty in recycling. Immobilized enzymes (IMEs) usually can enhance the stability and achieve ease recycling and have higher activity than native enzymes, whereas the support material is a very important factor.2 Immobilization of proteins onto solid supports is a promising biotechnological application because it is easy to separate proteins from the reaction media for repeated use. Therefore, IMEs have already been extensively investigated.3 For solid support, agarose gel beads have been chemically modified with aˆ-casein to display reactive glutamine (Gln) residues on the support surface.4 Novel calcium titanium phosphate-alginate (CTPalginate) and hydroxyapatite-alginate (HAp-alginate) microspheres have been used as enzyme delivery matrices and bone regeneration templates.5 Alginate-chitosan core-shell microcapsules as a biocompatible matrix for enzyme immobilization have been developed, and the enzyme loading efficiency is higher in the barium alginate core (100%) as compared with the calcium alginate core (60%).6 Films prepared from cellulose with polyamidoamine (PAMAM) dendrimers exhibit a better performance for immobilization of laccase than those prepared by simple mixing of the cellulose and the dendrimer.7
Penicillin G acylase (PGA) is a major industrial biocatalyst, and it has been used in the enzymatic production of 20 000 t a-1 of 6-aminopenicillanic acid (6-APA). PGA is initially
We prepared magnetic cellulose porous microspheres (MCM) with mean diameter of ∼200 µm by employing the sol-gel transition (SGT) method from a mixture of magnemite ferrofluid and cellulose dissolved in 7 wt % NaOH/12% urea aqueous solvent precooled to -12 °C. Subsequently, the cellulose microspheres were activated with epoxy chloropropane to enhance loading efficiency of biomacromolecules. Their morphology, structure, and properties were characterized by Fourier transform infrared spectroscopy, scanning electron microscopy, X-ray diffraction, and vibrating-sample magnetometer. The results indicated that the spherical magnetic γ-Fe2O3 nanoparticles with mean size of 10 nm were uniformly dispersed and embedded in the cellulose substrate of MCM, and the structure and nature of γ-Fe2O3 were conserved perfectly. Penicillin G acylase (PGA) as a biocatalyst was immobilized successfully in the porous microspheres, as a result of the existence of the cavity and affinity forces in the activated cellulose matrix. The immobilized PGA exhibited highly effective catalytic activity, thermal stability, and enhanced tolerance to pH variations. Furthermore, the cellulose microspheres loaded with the enzymes could be removed and recovered easily by introducing a magnetic field, leading to an acceptable reusability. Therefore, we have provided a simple and biocompatible support for the enzyme immobilization, which will be promising for the applications in the biomaterial fields.