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.     

Enhancement of penicillin G hydrolysis using penicillin acylase to 6-aminopenicillanic acid by simultaneous chromatographic separation

Penicillin G (Pen-G) was hydrolyzed to 6-aminopenicillanic acid (6-APA) and phenylacetic acid (PAA) in a chromatographic reactor-separator using the mixture of immobilized Escherichia coli cells and a macroporous adsorbent as stationary phase and a phosphate buffer of pH 7.8 as eluant. Pen-G conversion of 98% was observed without adjustment of the eluant pH due to the effective separation of 6-APA from Pen-G and PAA. At a sample load of 600 mg Pen-G, the volume overload gave higher Pen-G conversion (86%) than the mass overload (68%), while their difference in product resolution (0.9 and 1.0, respectively) was insignificant.     

Biosynthesis of penicillin V acylase by Fusarium sp.: effect of culture conditions

Penicillin V acylase was produced, both intracellularly and extracellularly, by Fusarium sp. SKF 235 grown in submerged fermentation. When neopeptone was added to the medium, >95% of the penicillin V acylase was extracellular. In the absence of a complex organic nitrogen source, the fungus produced low levels of totally intracellular penicillin V acylase. MgSO₄ was essential for synthesis of the enzyme, which was induced by phenoxyacetic acid and penicillin V. The maximum yield of penicillin V acylase was 430 IU/g dry cell wt. The optimum pH value and temperature for the penicillin V acylase were 6.5 and 55°C, respectively.     

Rapid continuous colorimetric enzyme assay for penicillin G acylase

A rapid, continuous, colorimetric enzyme assay for penicillin G acylase has been developed. The assay measures the formation of the acidic products of penicillin G hydrolysis by following the decrease in pH using Phenol Red as an indicator. The activity measured is directly proportional to the amount of enzyme added to the assay, having a linear relationship with an R ² value of 0.9994.     

Substrate specificity of penicillin acylase from Streptomyces lavendulae

The kinetic parameters of several substrates of penicillin acylase from Streptomyces lavendulae have been determined. The enzyme hydrolyses phenoxymethyl penicillin (penicillin V) and other penicillins with aliphatic acyl-chains such as penicillin F, dihydroF, and K. The best substrate was penicillin K (octanoyl penicillin) with a k(cat)/K(m) of 165.3 mM(-1) s(-1). The enzyme hydrolyses also chromogenic substrates as NIPOAB (2-nitro-5-phenoxyacetamido benzoic acid), NIHAB (2-nitro-5-hexanoylamido benzoic acid) or NIOAB (2-nitro-5-octanoylamido benzoic acid), however failed to hydrolyse phenylacetil penicillin (penicillin G) or NIPAB (2-nitro-5-phenylacetamido benzoic acid) and penicillins with polar substituents in the acyl moiety. These results suggest that the structure of the acyl moiety of the substrate is more determinant than the amino moiety for enzyme specificity. The enzyme was inhibited by several organic acids and the extent of inhibition changed with the hydrophobicity of the acid. The best inhibitor was octanoic acid with a K(i) of 0.8 mM. All the results, taking together, point to an active site highly hydrophobic for this penicillin acylase from Streptomyces lavendulae.     

Modifying the substrate specificity of penicillin G acylase to cephalosporin acylase by mutating active-site residues

The penicillin G acylase (PGA) and cephalosporin acylase (CA) families, which are members of the N-terminal (Ntn) hydrolases, are valuable for the production of backbone chemicals like 6-aminopenicillanic acid and 7-aminocephalosporanic acid (7-ACA), which can be used to synthesize semi-synthetic penicillins and cephalosporins, respectively. Regardless of the low sequence similarity between PGA and CA, the structural homologies at their active-sites are very high. However, despite this structural conservation, they catalyze very different substrates. PGA reacts with the hydrophobic aromatic side-chain (the phenylacetyl moiety) of penicillin G (PG), whereas CA targets the hydrophilic linear side-chain (the glutaryl moiety) of glutaryl-7-ACA (GL-7-ACA). These different substrate specificities are likely to be due to differences in the side-chains of the active-site residues. In this study, mutagenesis of active-site residues binding the side-chain moiety of PG changed the substrate specificity of PGA to that of CA. This mutant PGA may constitute an alternative source of engineered enzymes for the industrial production of 7-ACA.     

巨大芽孢杆菌青霉素G酰化酶的定点突变及其动力学性质研究

为了提高青霉素G酰化酶（PGA）在酸性及有机溶剂中的稳定性,以大肠杆菌的晶体结构为模板,用软件PMODELING同源模建巨大芽孢杆菌青霉素G酰化酶的三维结构结构并且选择PGA分子表面的合适碱性氨基酸突变为丙氨酸,通过三种不同的快速PCR介导定位突变的方法,将位于PGA的α亚基21位、128位和β亚基492位、512位的赖氨酸残基分别突变为丙氨酸,获得四个突变酶Kα021A、Kα128A、Kβ492A和Kβ512A。其中Kα128A和Kβ512A保持与野生型相近的酶活力,其动力学性质如最适温度、最适pH,Km及Kcat没有明显变化;突变酶Kα021A和Kβ492A则丧失 了酶活力。上述结果表明,PGA分子表面非活性中心的赖氨酸→丙氨酸点突变使突变子的性状发生了分化,突变效应呈现出丰富的多样性。该有理设计不但可以提高酶的稳定性,而且为揭示PGA结构和功能的关系提供了一个新的研究模型。     

微波对青霉素酰化酶催化反应性能的影响

运用动力学方法研究了微波对青霉素酰化酶(pK1和pK2分别为5.69-6.06和11.56)催化反应性能的影响。结果显示:使用微波解冻档对青霉素酰化酶进行一定时间的预处理后,能够加速酶的水解反应。酶液的最适处理时间为15 s,微波处理后,酶的最适温度为从原来的37℃上升到40℃,操作稳定性基本不变。对最适微波条件处理后的青霉素酰化酶pH值依赖性催化反应进行研究,从logVm和log(Vm/Km)与pH值关系曲线计算得到该酶的pK1和pK2分别为5.66-6.55和11.05。     

Continuous penicillin G hydrolysis in an electro-membrane reactor with immobilized penicillin G acylase

Penicillin G (2%, w/v in phosphate buffer, pH 8) was hydrolysed in a flow-through, miniature electro-membrane reactor with the penicillin G acylase immobilized in 5% (w/v) polyacrylamide (diam. 10 mm, thickness 2.6 mm, enzyme activity 24 U ml^–1). The conversion of penicillin G increased from 0.15 to almost 0.5 when the electric current applied to the reactor was changed from –600 to +600 A/m² with a substrate residency of 1 h. Symbols and abbreviations c _j ^p & concentration of component j in product stream (M) c _j ^s & concentration of component j in substrate stream (M) c _s ^o & substrate concentration at reactor inlet (M) C _j ^p=c _j ^p/c _S ⁰ & scaled concentration of component j in product stream C _j ^s=c _j ^s/c _S ⁰ & scaled concentration of component j in substrate stream i & electric current density (A/m²) j & reaction component, j P, Q or S P & main reaction product (6-aminopenicillanic acid) PGA & penicillin G acylase Q & side reaction product (phenylacetic acid) S & substrate (penicillin G) Y _s=C _P ^s+C _P ^p & substrate conversion & mean residence time of substrate and product streams in reactor (h) =C _Q ^s+C _Q ^p+C _S ^s+C _S ^s & check-sum of scaled concentrations =C _P ^p/(C _P ^s+C _P ^p) & separation factor of 6-aminopenicillanic acid (0 1)     

Chromogenic analogues of penicillin dihydroF and penicillin K for the continuous spectrophotometric determination of aliphatic penicillin acylase activity

The synthesis of 2-nitro-5-[(hexanoyl)-amino]-benzoic acid and 2-nitro-5-[(octanoyl)-amino]-benzoic acid as chromogenic substrates for the determination of aliphatic penicillin acylase activity is described. During enzymatic hydrolysis, the released chromophore, 2-nitro-5-amino-benzoic acid, was detected at 405 nm. Penicillin acylase from Streptomyces lavendulae had an appreciable activity towards these substrates, which can then be used to detect penicillin acylases able to cleave hexanoyl and octanoyl residues off synthetic amides as well as penicillin dihydroF and penicillin K, their natural analogues.     

雷氏普罗威登斯菌青霉素G酰化酶基因在大肠杆菌中的克隆与表达

利用PCR和分子克隆技术从雷氏普罗威登斯菌（Prouidencia rettgeri)(ATCC29944)的基因组DNA中获得一个青霉素G酰化酶（penicillinGacylase,PGA)基因并将其装入表达质粒pET24a。携带有重组质粒pETPGA的Escherichia coli基因工程菌BL21（DE3）/pETPGA实现了PGA的高效表达,对发酵条件的研究表明基因工程菌在24℃,添加5g/L甘油条件下以1.0mmol/LIPTG诱导1.5h酶活力即达到993.4U/L,比野生菌酶活力（15U/L）提高了66倍。     

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.     

pH stability of penicillin acylase from <Emphasis Type="Italic">Escherichia coli</Emphasis>

The inactivation kinetics of penicillin acylase from Escherichia coli have been investigated over a wide pH range at 25 and 50 degrees C. The enzyme was very stable in neutral solutions and quickly lost its catalytic activity in acidic and alkaline solutions. In all cases, the inactivation proceeded according to first order reaction kinetics. Analysis of the pH dependence of enzyme stability provides evidence that stable penicillin acylase conformation is maintained by salt bridges. Destruction of the salt bridges due to protonation/deprotonation of the amino acid residues forming these ion pairs causes inactivation by formation of the unstable "acidic" EH(4)(3+), EH(3)(2+), EH(2)(+) and "alkaline" E(-) enzyme forms. At temperatures above 35 degrees C penicillin acylase apparently undergoes a conformational change that is accompanied by destruction of one of these salt bridges and change in the catalytic properties.     

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.     

A new method for spectrophotometric assay of activity of cross-linked penicillin acylase aggregates

A new method for monitoring reactions catalyzed by an immobilized enzyme, cross-linked penicillin acylase aggregates (PA CLEA), is suggested. Appropriate chromogenic substrates for spectrophotometric assay of catalytic activity of immobilized enzyme were chosen and their kinetic parameters determined. Active sites in PA CLEA preparations were titrated by the suggested method; it is shown that almost all active sites are retained during immobilization. This method is characterized as highly expressive, simple, and precise and may be used for control of PA immobilization efficiency as well as for study of operational, thermal, and pH stability of immobilized enzyme preparations.     

Enhanced production of penicillin G acylase from a recombinant Escherichia coli

Penicillin G acylase (pac) gene was cloned into a stable asd ⁺ vector (pYA292) and expressed in Escherichia coli. This recombinant strain produced 1000 units penicillin G acylase g^–1 cell dry wt, which is 23-fold more than that produced by parental Escherichia coli ATCC11105. This enzyme was purified to 16 units mg^–1 protein by a novel two-step process.     

青霉素酰化酶在新型复合载体上的固定化研究

通过γ-氯丙基三甲氧基硅烷的媒介,将聚乙烯亚胺(PEI)化学偶联在硅胶微粒表面,制备了新型复合载体PEI/silica gel,然后通过双官能团试剂戊二醛的作用,将青霉素酰化酶固定在复合载体上;考察了戊二醛用量、pH值、固定化温度、固定化时间及给酶量等条件对固定化青霉素酰化酶表观活力、活性回收率等性能的影响;并通过测定复合载体在固定化前的ζ电位,探索了复合载体PEI/silica gel固定化酶的作用机理。研究结果表明,由于PEI分子链中含有大量胺基,共价键联与物理吸附相结合,使青霉素酰化酶被快速稳定地固定化,并具有高的催化活性与活力回收率。复合载体PEI/silica gel(0.5 g)固定青霉素酰化酶的适宜固定化条件为:固定化温度为30℃;固定化时间为14~15 h;戊二醛用量为1.2 mmol/g;pH=7.92;给酶量为0.1 mL/g。     

重组青霉素G酰化酶拆分制备（S）-邻氯苯甘氨酸

对产青霉素G酰化酶的重组枯草芽胞杆菌发酵产酶条件进行优化,确定优化后的发酵条件：可溶性淀粉10g/L、蛋白胨12g/L、酵母粉3g/L、NaCl10g,／L;pH7．5、培养温度37℃、装液量80mL（500mL三角瓶）、培养28h,青霉素G酰化酶的表达水平由最初的7．34U／mL提高至18．23U／mL。以表达青霉素G酰化酶的枯草芽胞杆菌发酵液为酶源,在水相中对映选择性催化N-苯乙酰-（R,S）-邻氯苯甘氨酸制备（S）-邻氯苯甘氨酸,当底物浓度为100mol／L时转化4h,转化率达44．2％。对底物浓度为80mmoL／L反应液中的（S）-邻氯苯甘氨酸进行分离,达到理论收率的94．29％（以N-苯乙酰-（R,S）-邻氯苯甘氨酸的0．5倍摩尔量为理论产率）,e．e．值大于99．9％。170℃条件下,N-苯乙酰-（R）-邻氯苯甘氨酸与苯乙酸共熔消旋为N-苯乙酰-（R,S）-邻氯苯甘氨酸可用于循环拆分。     

Kinetically controlled acylation of 6-APA catalyzed by penicillin acylase from Streptomyces lavendulae: effect of reaction conditions in the enzymatic synthesis of penicillin V

Abstract

Enzymatic synthesis of penicillin V (penV) by acylation of 6-aminopenicillanic acid (6-APA) was carried out using methyl phenoxyacetate (MPOA) as activated acyl donor and soluble penicillin acylase from Streptomyces lavendulae (SlPVA) as biocatalyst. The effect of different reaction conditions on penV synthesis was investigated, such as enzyme concentration, pH, molar ratio of 6-APA to MPOA, as well as presence of DMSO as water-miscible co-solvent at different concentrations. Time-course profiles of all reactions followed the typical pattern of kinetically controlled synthesis (KCS) of β-lactam antibiotics: penV concentration reached a maximum (highest yield or Y_max) and then decreased gradually. Such maximum was higher at pH 7.0, observing that final penV concentration was abruptly reduced when basic pH values were employed in the reaction. Under the selected conditions (100?mM Tris/HCl buffer pH 7.0, 30?°C, 2.7% (v/v) DMSO, 20?mM MPOA, 0.3 UI/ml of SlPVA), Y_max was enhanced by increasing the substrate molar ratio (6-APA to MPOA) up to 5, reaching a maximum of 94.5% and a S/H value of 16.4 (ratio of synthetic activity to hydrolytic activity). As a consequence, the use of an excess of 6-APA as nucleophile has allowed us to obtain some of the highest Y_max and S/H values among those reported in literature for KCS of β-lactam antibiotics. Although many penicillin G acylases (PGAs) have been described in kinetically controlled acylations, SlPVA should be considered as a different enzyme in the biocatalytic tool-box for novel potential synthetic processes, mainly due to its different substrate specificity compared to PGAs.