固定化青霉素酰化酶的研究

将巨大芽孢杆菌胞外青霉素酰化酶通过共价键连接到醋酸纤维素载体上,制成的固定化青霉素酰化酶的表观活力达2000 u／g左右(PDAB法)。水解lO％(w／v)的青霉素G钾盐落液,使用30批,保留活力70％以上。6-氨基青毒烷酸(6-APA)总收率平均达88．37％。固定化青霉素酰化酶水解青霉素G的最适pH为9．95,最适温度为55℃,表观米氏常数为1.093×10-2mol／L,在pH 5．8-10．7,温度45℃以下酶的活力稳定。     

以环氧乙烷为活性基的多孔颗粒状固定化青霉素酰化酶的制备

报道了用以环氧乙烷为活性基的多孔颗粒状载体（Eupergit-C）制备固定由巨大芽孢杆菌（B．megaterium）产生的青霉素酰化酶的研究。用已二胺,赖氨酸对载体进行化学修饰后制备固定化酶,获得了较好的固定结果。用未修饰的载体制备固化酶,经24h固定反应,酶活力达176．5IU／g（wet）,酶活力总叫率达53．7％,酶蛋白的固定量为19＝7mg/g（dr）,酶蛋白的固定效率达87．5％。游离酶的酶浓度对制备固定化酶的活力无显影响。当加酶量从312IU/g（dry）上升到6250IU／g（dry）时,固定化酶活力从89IU／g(wet）上升到475IU／g（wet）,总收率和固定化效率分别从99％和99％下降到26．5％和32．5％,酶蛋白的固定量从6．9mg/g（dry）上升到112mg/g（dry）,酶蛋白的固定效率从99％下降至80．5％。以酶活力为155IU/g(wet）,酶蛋白固定量为22mg/g(dry）的固定化酶水解青霉素G钾盐,经过20批循环水解后,剩余酶活力为92．5％。     

以聚丙烯腈纤维为载体制备固定化青霉素G酰化酶的研究

以酸部分水解聚丙烯腈纤维为载体 ,以戊二醛为交联剂 ,共价键结合制备了固定化胞外青霉素G酰化酶。当水解后的载体中 NH2 基含量为 690 μmol g和含水量为 64%时 ,对酶蛋白的固定量达 1 0 0mg g以上 ,固定化酶的活力达 2 30 0IU g ,酶活力总产率为 30 % ,固定化效率为 56%。酶活力的总产率和固定化率随加酶量的增加而降低。该酶可以将浓度为 2 5%～1 2 5%的青霉素G钾盐水解 98%以上。批投青霉素G钾盐为 1 0g,酶负荷为 1 50IU g(PGK) ,经2 0批水解反应后 ,剩余酶活力为 80 %。用二硫基苏醣醇处理固定化酶 ,对水解青霉素G钾盐的操作稳定性有促进作用。固定化酶的室温保存半衰期为 1 30d。用戊二醛和硼氢化钠溶液处理固定化酶后 ,酶活力的室温保存稳定性有所降低。     

几种纤维素及无机吸附载体对青霉素酰化酶的吸附作用及其固定化

本文就几种纤维素和无机吸附载体对青霉素酰化酶的吸附作用及其固定化进行了研究,结果表明:DEAE-纤维素、EYI-纤维素、微晶纤维素、CM-纤维素、羟基磷灰石、中性氧化铝、硅藻土及粉末状膨润土对青霉素酰化酶都有很强的吸附能力,其固定化青霉素酰化酶的比活分别在1.47～19.43u/g之间,活力回收率为16.3％至84％,几种固定化酶的最适pH均较游离酶低,且其操作稳定性较好。     

聚丙烯腈纤维固定化青霉素酰化酶性质的研究

将巨大芽孢杆菌（Bacillusmegaterium）青霉素酞化酶连接到聚丙烯腈纤维载体上,制成固定化青霉素酰化酶。其表现活力约为2000u／g。水解青霉素G的最适温度为50℃;最适PH为9．0;在PHS．5～10．3、温度50℃以下酶的活力稳定;表观米氏常数Ka为1．33×10-8mol／L;最大反应速度Vm为2．564mmol·min-1;苯乙酸为竞争性抑制剂,抑制常数为0．16mol／L。水解10％的青霉素G钾盐溶液,使用20批,保留酶活力80％。     

颗粒状固定化青霉素酰化酶的研究

将巨大芽孢杆菌 (Bacillusmegaterium)胞外青霉素酰化酶通过共价键结合到聚合物载体EupergitC颗粒环氧基团上 ,制成的颗粒状固定化青霉素酰化酶表现活力达 1 40 0 μ/g左右。固定化酶水解青霉素的最适 pH8 0 ,最适温度为 55℃。在pH6 0～ 8 5、温度低于 40℃时固定化酶活力稳定。在 pH8 0、温度 37℃时 ,固定化酶对青霉素的表现米氏常数Ka为 2×1 0 - 2 mol/L ;苯乙酸为竞争性抑制剂 ,抑制常数Kip为 2 8× 1 0 - 2 mol/L ;6 APA为非竞争性抑制剂 ,抑制常数Kia为 0 1 2 5mol/L。固定化酶水解青霉素 ,投料浓度为 8% ,在使用 2 0 0批后 ,保留活力 80 %左右 ,6 APA收率平均达 89 48%。     

巨大芽孢杆菌青霉素G酰化酶在新型环氧载体ZH-HA上的固定化

巨大芽孢杆菌青霉素G酰化酶共价结合在新型环氧-氨基型载体ZH-HA 上,通过对酶浓度、固定化时间、pH以及缓冲液浓度等条件的考察,确定了最优固定化条件:50 mg比活力6000 U/g的巨大芽孢杆菌青霉素G酰化酶蛋白和1g ZH-HA悬浮于pH 9.01 mol/L磷酸缓冲液,室温搅拌6 h,制得固定化巨大芽孢杆菌青霉素G酰化酶,活力2126 U/g湿载体,活力回收率7.67%.比较研究了固定化酶与原酶性质,原酶最适温度45℃,最适pH为8.0.固定化酶则分别是50℃和9.0,分别比溶液酶偏移5℃、1.0个pH单位.经过40批连续水解青霉素G钾盐,固定化巨大芽孢杆菌青霉素酰化酶仍保持80%的活力,显示出良好的工作稳定性.     

聚丙烯腈纤维固定化青霉素酰化酶合成头孢氨苄的研究

将巨大芽孢杆菌胞外青霉素酰化酶通过共价键结合到聚丙烯腈纤维的衍生物上。制成的丝状固定化青霉素酰化酶表现活力达 1 5 3U g(湿重 )。固定化酶合成头孢氨苄的最适pH为 6 5 ,最适温度为 40℃。 7 ADCA的投料浓度以 4%为好 ,7 ADCA与PGME的投料量比率为1∶2 ,最佳用酶量为 1 70U g 7 ADCA。在pH6 5、温度 3 0℃时 ,固定化酶对 7 ADCA的表观米氏常数K7 ADCA为 0 1 6 2mol L ,对PGME的表观米氏常数KPGME为 0 3 6 4mol L ,最大反应速度Vmax为0 0 4 6 2mol·L- 1·min- 1,用固定化酶合成头孢氨苄 ,使用 5 0次保留酶活力 83 9%     

固定化青霉素V酰化酶的制备及性质

尖镰孢(Fusarium oxysporum)FP941青霉素V酰化酶经γ氧化铝吸附洗脱、硫酸铵沉淀和脱盐处理后,固定在环氧丙烯聚合物载体上,湿固定化酶表现活力为217 IU/g,固定化产率为53%。固定化酶作用最适温度为55℃,最适pH为80;在pH50～110及50℃以下稳定;37℃使用25次后,酶活力保留90%。     

活性炭吸附固定化粪产碱杆菌青霉素G酰化酶

目的：以活性炭为载体固定化粪产碱杆菌来源的青霉素G酰化酶,考察固定化酶的性质。方法：对影响酶固定化的因素优化筛选,确定有显著影响的因素：pH、离子强度、酶量、固定化时间进行L934的正交实验,获得最佳固定化条件,并对固定化酶的最适反应温度、pH及批次稳定性进行研究。结果：最佳固定化条件为：载体0.3g,酶量5mL,总反应体系为12mL,离子强度1mol/L,温度4℃,pH 7.0,固定化40h;最高固定化酶活性为135.9U/g湿载体。固定化酶性最适反应温度为55℃,最适pH为10,重复使用12次后没有活性损失。结论：活性炭吸附固定化青霉素G酰化酶的活性高,批次反应稳定,具有工业应用潜力。     

Covalent immobilization of penicillin acylase from Streptomyces lavendulae

Penicillin acylase from Streptomyces lavendulae has been covalently immobilized to epoxy-activated acrylic beads (Eupergit C). Consecutive modification of the matrix with bovine serum albumin leads to a new biocatalyst (ECPVA) with enhanced activity (1.5 fold) in the hydrolysis of penicillin V respect to its soluble counterpart. This biocatalyst had a K _m value of 7.6 mM, slightly higher than K _m for native acylase (3 mM). In addition, ECPVA can be recycled for at least 50 consecutive batch reactions without loss of catalytic activity.     

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.     

Immobilization of penicillin G acylase on aminoalkylated polyacrylic supports

A procedure is described for the immobilization of penicillin G acylase (PA) on Amberlite XAD7 modified by transamidation with 1,2-ethylenediamine and activated with glutaraldehyde. Reduction with sodium borohydride of the Schiff's bases formed between the amino groups of the protein and glutaraldehyde results in a dramatic improvement of the operational stability of the immobilized enzyme without affecting the catalytic activity. The enzyme kept in presence of the substrate, penicillin G, displays an increased stability with respect to that stored in pure phosphate buffer solution. The inactivation kinetics of the immobilized preparations of PA, determined in a continuous fixed bed reactor, as well as a discontinuous batch reactor, are reported.     

Hydrolysis of penicillin G by combination of immobilized penicillin acylase and electrodialysis

Phenylacetic acid, as inhibitory product, was formed from a hydrolysis of penicillin G by immobilized penicillin acylase. In this article, electrodialysis was applied to remove phenylacetic acid continuously from the reaction mixture and to enhance an efficiency of the reaction. When 268 and 537 mM of penicillin G solution were used as the substrate, the concentration of phenylacetic acid in the reaction mixture could be maintained at less than 81 and 126 mM, respectively, and eventually, 86% and 88% of phenylacetic acid produced were removed from the reaction mixture at the end of the hydrolysis, respectively. Times required to reach 96% and 94.8% conversion from 268 and 537 mM of initial penicillin G could be reduced to 65% and 64% respectively, by means of electrodialysis; while 3.0% and 4.3% of initial penicillin G of 268 and 537 mM were permeated out of the reaction chamber during the hydrolysis, respectively. However, a loss of penicillin G by permeation could be reduced from 4.3% to 3.4% by a repeated addition of penicillin G.     

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

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

Kinetic and microstructural characterization of immobilized penicillin acylase from Streptomyces lavendulae on Sepabeads EC-EP

Recombinant penicillin acylase from Streptomyces lavendulae was covalently bound to epoxy-activated Sepabeads EC-EP303®. Optimization of the immobilization process led to a homogeneous distribution of the enzyme on the support surface avoiding the attachment of enzyme aggregates, as shown by confocal electron microscopy. The optimal immobilized biocatalyst had a specific enzymatic activity of 26.2IUgwetcarrier^?1 in the hydrolysis of penicillin V at pH 8.0 and 40°C. This biocatalyst showed the highest activity at pH 8.5 and 65°C, 1.5 pH units lower and 5°C higher than its soluble counterpart. Substrate specificity of the derivative also showed its ability to efficiently hydrolyze other natural aliphatic penicillins such as penicillins K, F and dihydroF. The immobilized enzyme was highly stable at 40°C and pH 8.0 (t_1/2=625 h vs. t_1/2=397 h for the soluble enzyme), and it could be recycled for at least 30 consecutive batch reactions without loss of catalytic activity.     

Effect of complex nitrogen sources on the production of penicillin acylase byBacillus megaterium

The following complex nitrogen sources were evaluated for the production of penicillin acylase byBacillus megaterium: casein hydrolysate, corn steep liquor, stick water concentrate, blood meal and defatted sunflower meal. Experiments were run in shake flasks at 30‡C and pH 7.0. Best results were obtained with casein hydrolysate: 244 IU/I were produced with a productivity of 20.3 IU/l/h and yield of 717.6 IU/g of nitrogen. The lowest results correspond to sunflower meal with 39 IU/1.     

Encapsulation of crosslinked penicillin G acylase aggregates in lentikats: evaluation of a novel biocatalyst in organic media

The encapsulation of crosslinked enzyme aggregates (CLEA) of penicillin G acylase into a very rigid polymeric matrix based on polyvinyl alcohol (LentiKats) has been used successfully to improve the inadequate mechanical properties of CLEA. This encapsulation decreased CLEA activity by only around 40%. As compensation, a significant improvement in the stability of the CLEA in the presence of organic solvents was detected. This could be related to the highly hydrophilic environment inside the LentiKats biocatalysts: Partition experiments showed that the concentration of dioxane inside LentiKats was lower than in the reaction medium. In fact, thermal stability was about the same as in the corresponding CLEA. This permitted great improvement in the reaction rate for thermodynamically controlled synthesis of a model antibiotic (using phenylacetic acid and 7-amino-deacetoxycefalosporanic acid). Even more importantly, yields could be improved by using LentiKats-encapsulated CLEA, very likely by a favorable product/substrate partition. Thus, this very simple technique not only provides an efficient technique for solving the mechanical stability problem associated with CLEA, but also greatly improves the behavior of CLEA in organic media.