含环氧基的交联聚合物载体的制备方法对固定化青霉素酰化酶活性的影响

 以甲基丙烯酸缩水甘油酯为单体，N，N′－亚甲基双丙烯酰胺为交联剂，采用两种方法合成了大孔、珠状的交联聚合物固定化酶载体．用红外光谱、扫描电子显微镜及N2吸附等方法测定了其结构、比表面积、孔径分布和表观活性．结果表明，以液体石蜡为主介质、甲醇水溶液为致孔剂合成的聚合物GM1（60）作载体时，固定化酶水解青霉素G的活性达537U／g；以正庚烷与四氯乙烯混合溶剂为介质、甲酰胺为致孔剂合成的聚合物GM2（60）作载体时，固定化酶的活性较低，为426U／g．在37℃下连续进行10次间歇操作（每次反应10min）后，前者活性降至487U／g，保持了初始活性的90．7％；后者活性降至378U／g，保持了初始活性的88．7％．二者催化活性的不同是由于两种方法制备的载体在结构与性能上存在着明显的差异．GM1（60）载体孔径大，水中溶胀性能好，对青霉素酰化酶的偶联作用强，固定化效果显著．     

功能基化聚丙烯酸甲酯固定化青霉素酰化酶

合成了一系列大孔丙烯酸甲酯－二乙烯苯交联共聚物,经酰肼化、叠氨后固定青霉素酰化酶,考察了反应条件对固定化酶的影响,当交联剂用量为３０％,至孔剂量为１３０％,混合致孔剂（正庚烷与乙酸乙酯）中正庚烷的质量分数为５５％时,所制的载体经活化后得到的固定化酶酶活较高,为９５ｕ／ｇ（湿）,用分批式反应器连续水解青霉素Ｇ钾盐,使用６３批次后仍保留酶活７９．４％。     

Novel approaches to the purification of penicillin acylase

Penicillin acylase ofE. coli NCIM 2400 has been purified to homogeneity using a combination of hydrophobic interaction chromatography and DEAE-cellulose treatment. A variety of substituted matrices were synthesized using D- or DL-phenylglycine, norleucine, ampicillin, or amoxycillin as ligands, all of which retained penicillin acylase at high concentrations of ammonium sulfate or sodium sulfate. The enzyme could be eluted nonbiospecifically by buffer of lower ionic strength with over 95% recovery of the activity. Ammonium chloride, ammonium nitrate, sodium chloride, sodium nitrate, and potassium chloride were ineffective in either adsorption or elution of the enzyme on these columns. Further purification of this partially pure enzyme with DEAE-cellulose at pH 7.0–7.2 yielded an enzyme preparation of very high purity according to electrophoretic and ultracentrifugal analyses, its specific activity being as high as 37 U/mg protein. The purifiedf enzyme has a molecular weight of 67,000 a sedimentation coefficient of 4.0S, and resolves into two forms upon isoelectric focusing. Overall recoveries ranged between 75 and 85%. Ease of operation, high recoveries, high purity of the enzyme and prolonged reuse of the conjugates make the process economically feasible and possibly of great commercial importance.     

微波辐射高效共价固定青霉素酰化酶

为提高青霉素酰化酶的共价固定化效率, 在微波辐射条件下将酶蛋白共价固定于介孔泡沫硅(MCFs)的孔道中. 通过正硅酸四乙酯水解缩合制备介孔泡沫硅, 再于微波辅助下将青霉素酰化酶共价固定在其孔道中. 以固定化酶相对活力和活力回收为指标, 考察了加酶量、固定化温度、微波辐射时间等条件对酶固定化效率的影响. 实验结果表明: 当加酶量为60 mg/g, 固定化温度为20 ℃, 微波辐射140 s, 固定化酶相对活力达到178.1%, 表观活力为1191.3 U/g(以湿重计). 与常规方法相比, 微波辅助固定化酶时, 固定化酶相对活力提高34.5%, 固定化时间亦大幅缩短至数分钟, 这为青霉素酰化酶的高效共价固定化提供了一条新的途径.     

聚丙烯酸载体用于青霉素酰化酶的固定

以反应性单体丙烯酸和交联剂二乙烯基苯,以石油醚为致孔剂,通过悬浮聚合制备固定化酶的载体,并用于对青霉素酰化酶的固定。研究了丙烯酸与二乙烯基苯以不同摩尔比对青霉素酰化酶固定活性的影响,以及悬浮聚合时水油相比例的不同所合成的载体对固定化酶性能的影响。当丙烯酸和二乙烯基苯摩尔比为84.2:4时合成的载体固定青霉素酰化酶的酶活为2784U/g,而水油相比为2.75:1（丙烯酸和二乙烯基苯摩洋比为84.2:5）时固定青霉素酰化酶活达到2183U/g。固定青霉素酰化酶可使青霉素转化,得到半合成青霉素的中间体6-氨基青霉烷酸,由此可制成高效、广谱、服用方便的新青霉素。     

酶作用下双金属复合氧化物记忆效应与其组成的关系

水滑石;青霉素酰化酶;固定化酶;酶作用下双金属复合氧化物记忆效应与其组成的关系     

高分子载体材料对青霉素酰化酶的固定化作用

介绍了天然高分子材料和合成高分子材料对青霉素酰化酶的固定化作用,着重讨论了高分子材料的制备、性质及其表面修饰对固定化酶活性和使用稳定性的影响。     

Immobilization of penicillin acylase in porous beads of polyacrylamide gel

A procedure is described for the immobilization of benzylpenicillin acylase from Escherichia coli within uniformly spherical, porous polyacrylamide gel beads. Aqueous solutions of the enzyme and sodium alginate and of acrylamide monomer, N,N'-methylene-bis-acrylamide, N,N,N,N'-tetramethylethylenediamine (TEMED) and sodium alginate are cooled separately, mixed, and dropped immediately into ice-cold, buffered calcium formate solution, pH 8.5, to give calcium alginate-coated beads. The beads are left for 30-60 min in the cold calcium formate solution for polyacrylamide gel formation. The beads are then treated with a solution of glutaraldehyde and the calcium alginate subsequently leached out with a solution of potassium phosphate. Modification of the native enzyme with glutaraldehyde results in a slight enhancement in the rate of hydrolysis of benzylpenicillin at pH 7.8 and 0.05M substrate concentration. The enzyme entrapped in porous polyacrylamide gel beads shows no measurable diffusional limitation in stirred reactors, catalyzing the hydrolysis of the substrate at a rate comparable to that of the glutaraldehyde-modified native enzyme. The immobilized enzyme preparation has been used in batch mode over 90 cycles without any apparent loss in hydrolytic activity.     

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.     

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.     

Supermacroporous hydrophobic affinity sorbents for penicillin acylase purification

Penicillin acylase (PA, EC 3.5.1.11) is used as a raw material in the production of semi-synthetic penicillins. Although there are many methods for PA purification, affinity chromatography is advantageous as it provides efficient one step purification. In this study, poly(2-hydroxyethyl methacrylate) based cryogel column containing hydrophobic N-methacryloyl-L-tryptophan (MATrp) functional monomer as a ligand was prepared. Interaction of MATrp with amino acids in PA structure is the basis of hydrophobic interaction chromatography in this study. PHEMA and PHEMATrp cryogel columns were characterized by surface area measurements, infrared spectroscopy, swelling tests, elemental analysis and scanning electron microscopy (SEM). Initial PA concentration, pH, effect of temperature, amount of ligand, flow rate, ionic strength and time on PA adsorption on PHEMATrp cryogel were investigated. Optimum pH was determined as 5.0 for PA adsorption and maximum adsorption capacity was obtained as 6.40 mg/g. It was observed that adsorption capacity increased with the increasing of temperature. Also, PA adsorption increased up to 0.25 M salt concentration and decreased in higher salt concentrations. Data obtained in this affinity system suggests that hydrophobic interactions are dominant. In the last stage of the study, PA was purified from Penicillium chrysogenum with 76.3% yield and 332.3 purification factor.     

A kinetic study of synthesis of amoxicillin using penicillin G acylase immobilized on agarose

We presenta kinetic model for the synthesis of amoxicillin from p-hydroxyphenylglycine methyl ester and 6-aminopenicillanic acid, catalyzed by penicillin G acylase immobilized on agarose, at 25°C. Michaelis-Menten kinetic parameters (with and without inhibition) were obtained from initial velocity data (pH 7.5 and 6.5). Amoxicillin synthesis reactions were used to validate the kinetic model after checking mass transport effects. A reasonable representation of this system was achieved under some operational conditions, but the model failed under others. Nevertheless, it will be useful whenever a simplified model is required, e.g., in model-based control algorithms for the enzymatic reactor.     

Immobilization of whole-cell penicillin g acylase by entrapping within polymethacrylamide beads

Escherichia coli ATCC 11105 containing the periplasmic penicillin G acylase was entrapped within a copolymer of methacrylamide andN,N’- methylenebisacrylamide. A solution of monomer that was made up from methacrylamide andN,N’-methylenebisacrylamide dissolved in buffer was mixed with lyophilized cells and ammonium persulfate. This suspension was then pumped drop by drop into in soybean oil supplemented with 0.06% (v/v) 3-(dimethylamino)-propionitril. During submerging in the oil phase, the droplets were hardened and induced to polymerize within the droplets. Particles with a volume ranging from 0.013–0.017 mL per bead containing a biomass concentration up to 38.0 g/L were prepared. The optimal condition for the deacylation of penicillin G to 6-aminopencillanic acid (6-APA) catalyzed by the immobilized whole-cell penicillin G acylase was found to be 45‡C and pH 8.0. Product inhibition of this enzyme by 6-APA could be eliminated by controlling pH value at 8 during the course of penicillin G hydrolysis using a pH-stat. Conversion determined by the pH-stat method were 0.3% higher than that by p-dimethylaminobenzaldehyde method. Cell concentration in the matrix was found to be an important factor influencing the maximum velocity and the specific activity retained in the matrix. A kinetic model, in which the mass transfer resistances as a result of external film mass transfer and pore diffusion were assumed to be negligible, could properly describe the hydrolysis of penicillin G by the cells entrapped within the polymethacylamide beads.     

An efficient synthesis of ampicillin on magnetically separable immobilized penicillin G acylase

<正>Penicillin G acylase(PGA) was immobilized on the magnetic hydrophilic polymer microspheres with average pore size of 17.1 nm,specific surface area of 128.2 m~2/g and saturate magnetization of 6.4 emu/g.The 96.7%ampicillin yield with 1.60 of the synthesis/hydrolysis(S/H) ratio from 6-aminopenicillanic acid(6-APA) and D-(-)-alpha-phenylglycine methyl ester(D-PGME) can be achieved using the resultant magnetic biocatalyst in ethylene glycol,where only 82.1%yield with 1.40 of the S/H ratio was obtained using the free PGA under the identical reaction conditions.The immobilized PGA can be separated magnetically and recycled for five times without obvious loss of its catalytic activity.     

Organically modified xerogels as supports for solid-phase chemistry

Organically modified xerogels (OMX_NH2) can be used as an easy to handle and chemically stable support in solid-phase chemistry and are compatible with enzymatic transformations.     

Organically modified xerogels as novel tailor-made supports for covalent immobilisation of enzymes (penicillin G acylase)

A novel application of organically modified silicates for covalent immobilisation of penicillin G acylase is reported. The immobilisation is efficient and the enzymatic preparation shows high specific activity and thermal stability. The technique opens new perspectives for the preparation of innovative tailor-made supports matching specific requirements of enzymatic processes.     

Immobilization of penicillin G acylase on poly[(glycidyl methacrylate)-co-(glycerol monomethacrylate)]-grafted magnetic microspheres

Poly[(glycidyl methacrylate)-co-(glycerol monomethacrylate)]-grafted magnetic microspheres were prepared by graft random copolymerization via ATRP from polymer microspheres with dispersed Fe(3)O(4) nanoparticles. Penicillin G acylase (PGA) was immobilized onto the polymer brush-grafted magnetic microspheres. The immobilized PGA prepared with initial glycidyl methacrylate/glycerol monomethacrylate ratios of 40/60 to 60/40 possessed higher catalytic activity than that prepared with higher proportions of glycidyl methacrylate in the initial monomer mixture. The immobilized PGA showed high thermal stability and enhanced tolerability to the pH variance.     

酶法拆分D,L-苯丙氨酸制备D-苯丙氨酸

在固定化青霉素酰化酶(IPA-750)存在下,通过N-苯乙酰-D,L-苯丙氨酸(2)的选择性水解完成了酶法拆分D,L-苯丙氨酸(1)制备D-苯丙氨酸(5)的过程。选择性水解的较适宜反应条件为:22.83g,m(2)∶m(IPA-750)=6∶1,pH7.0,于30℃反应5h,产物为N-苯乙酰-D-苯丙氨酸(4)和L-苯丙氨酸(3,收率63%,光学纯度99%)。4用6mol·L-1盐酸于120℃水解反应8h,经脱盐处理得5,收率67%,光学纯度91%。3在含醋酸酐的醋酸溶液中进行消旋化处理,得到100%消旋的1可继续进行下一轮酶法拆分。     

含环氧基团的聚合物载体合成方法的改进及其固定化青霉素酰化酶

 以 Span-60 和 Tween-20 为复合分散剂, 以 N,N′-亚甲基双丙烯酰胺为交联剂, 以甲基丙烯酸缩水甘油酯和烯丙基缩水甘油醚为功能性单体, 用反相悬浮聚合技术成功制备了含环氧基团的聚合物载体, 并用红外光谱和低温氮吸附对聚合物载体进行了表征. 以 Span-60 和 Tween-20 为复合分散剂, 替代原有的 Span-60 和硬脂酸钙复合分散剂, 大幅度减少了后处理过程中所需的时间和溶剂用量, 使固定化青霉素酰化酶的活性从 215 U/g 提高到 320 U/g. 与游离酶相比, 该固定化酶具有较好的操作稳定性, 在 pH = 5~11 和不高于 50 oC 的环境中具有较好的稳定性. 固定化酶的水解反应动力学过程与游离酶相同, 均遵循米氏反应动力学, 而且活性与底物浓度密切相关. 当底物浓度为 6.5% 时, 固定化酶的活性最高, 达到 353 U/g.     

胶原纤维固化黑荆树单宁吸附V(V)的研究

研究了胶原纤维固化黑荆树单宁对V(V)的吸附。采用不同温度、pH值等条件进行吸附研究,并进一步探讨了固化黑荆树单宁的吸附动力学和吸附柱动力学及其吸附机理。结果表明,该材料对V(V)的吸附平衡符合Freund lich方程,温度对吸附平衡的影响不明显;吸附动力学可用拟二级速度方程来描述,该材料同时具有良好的柱动力学特性;V(V)的吸附过程可能存在三个反应,即V(V)与吸附剂之间发生氧化还原反应生成V(IV),V(IV)和-COOH之间发生离子交换反应,以及V(IV)与单宁的邻位羟基发生螯合。