Directed evolution of Pseudomonas aeruginosa lipase for improved amide-hydrolyzing activity

A lipase from Pseudomonas aeruginosa was subjected to directed molecular evolution for increased amide-hydrolyzing (amidase) activity. A single round of random mutagenesis followed by screening for hydrolytic activity for oleoyl 2-naphthylamide as compared with that for oleoyl 2-naphthyl ester identified five mutants with 1.7-2.0-fold increased relative amidase activities. Three mutational sites (F207S, A213D and F265L) were found to affect the amidase/esterase activity ratios. The combination of these mutations further improved the amidase activity. Active-site titration using a fluorescent phosphonic acid ester allowed the molecular activities for the amide and the ester to be determined for each mutant without purification of the lipase. A double mutant F207S/A213D gave the highest molecular activity of 1.1 min(-1) for the amide, corresponding to a 2-fold increase compared with that of the wild-type lipase. A structural model of the lipase indicated that the mutations occurred at the sites near the surface and remote from the catalytic triad, but close to the calcium binding site. This study is a first step towards understanding why lipases do not hydrolyze amides despite the similarities to serine proteases in the active site structure and the reaction mechanism and towards the preparation of a general acyl transfer catalyst for the biotransformation of amides.     

Mathematical modeling of penicillin G Extraction by a bifunctional surfactant in a continuous extraction column

A bifunctional surfactant was used as a carrier of penicillin G for its continuous extraction by an emulsion liquid membrane without an extradant in a countercurrent extraction column of Oldshue-Rushton type. A permeation model was presented to describe transport mechanism of penicillin G in the continuous extraction system, including an axial dispersion model for the continuous phase and a mass transfer model for the dispersed emulsion phase. The mass transfer model describes the external mass transfer around the emulsion drop, the reaction at the external interface, the diffusion as well as the reaction equilibrium within the w/o emulsion drop, and the pH change of internal aqueous phase. Here, we considered three experimental variables: penicillin G concentration, pH of continuous phase and surfactant concentration. The calculated results from the permeation model fitted the experimental data well. This paper is dedicated to Dr. Youn Yong Lee on the occasion of his retirement from Korea Institute of Science and Technology.     

响应面法优化重组枯草芽孢杆菌发酵生产青霉素G酰化酶

利用响应面法优化重组枯草芽孢杆菌pAUB-BmPGA／BS168发酵生产青霉素G酰化酶的条件。通过Plackett-Burman设计法对碳源、氮源、无机盐、温度等19个因素对发酵产青霉素G酰化酶的影响进行评价。筛选出发酵温度和酵母膏为影响产酶的显著因素。采用了中心组合设计实验对两种显著影响因素进行了优化,应用响应面分析确定了显著因素的最佳水平。结果表明,当温度为34℃,酵母膏为8．8g／L时,最终的酶活达到28．2U／mL,比初始发酵条件提高了1．19倍。在最佳条件利用15L的发酵罐对重组枯草芽孢杆菌进行扩大培养,最终酶活可达51．0U／mL,相对于摇瓶发酵又有大幅度的提高。     

Engineering of Pseudomonas aeruginosa lipase by directed evolution for enhanced amidase activity: mechanistic implication for amide hydrolysis by serine hydrolases

A lipase from Pseudomonas aeruginosa was subjected to directed evolution for increased amidase activity to probe the catalytic mechanism of serine hydrolases for the hydrolysis of amides. Random mutagenesis combined with saturation mutagenesis for all the amino acid residues at the substrate-binding site successfully identified the mutation at the residue 252 next to the catalytic H251 as a hot spot for selectively increasing the amidase activity of the lipase. The saturation mutagenesis targeted for the oxyanion hole (M16 and H83) gave no positive results. The substitutions of Met or Phe for Leu252 significantly increased the amidase activity toward N-(2-naphthyl)oleamide (2), whereas the esterase activity toward structurally similar 2-naphthyl oleate (1) was not affected by the substitution. The triple mutant F207S/A213D/M252F (Sat252) exhibited amidase activity (k(cat)/K(m)) 28-fold higher than that of the wild-type lipase. Kinetic analysis of Sat252 and its parental clone 10F12 revealed that the amidase activity was increased by the increase in the catalytic efficiency (k(cat)). The increase in k(cat) suggested the importance of the leaving group protonation by the catalytic His during the break down of the tetrahedral intermediate in the hydrolysis of amides.     

随程离子交换法促进青霉素G水解生产6-氨基青霉烷酸

采用固定化青霉素酰化酶反应器进行青霉素G水解生产6-氨基青霉烷酸,同时与离子交换柱相组合以连续地去除苯乙酸而维持反应混合液中的低苯乙酸浓度.研究发现,采用随程离子交换法可提高青霉素G水解速率10.2%,同时可从反应混合液中去除53%的苯乙酸;通过确定苯乙酸、青霉素G在离子交换柱中的动力学行为和固定化酶的动力学参数,利用已建立的数学模型,可很好地对青霉素G水解生产6-氨基青霉烷酸过程进行计算机模拟.     

Ag/P(St-MMA)纳米复合高分子微球固定化青霉素酰化酶的研究

通过溶剂热法和无皂乳液聚合相结合,制备了P(St-MMA)高分子纳米微球.并以吸附沉积的方式在其表面沉积了Ag金属纳米粒子,最后将青霉素酰化酶共价连接在微球表面.初步研究了微球直径、银的质量分数等因素对固定化酶活力的影响.结果显示随着微球直径减小,固定化酶的偶联率和活力逐渐增加;银纳米粒子最多将固定化酶的偶联率和活力分别提高了42%和72%,固定化酶的最大表观活力(以干重记)达到了1 869 u/g,明显高于其它高分子载体固定化青霉素酰化酶的活力;实验证明银纳米粒子在青霉素水解过程中没有催化活力,但能大大提高青霉素酰化酶的催化活力.     

Membrane vis-LED photoreactor for simultaneous penicillin G degradation and TiO2 separation

The hybrid membrane photoreactor (MPR) combining a photoreactor irradiated with visible-light-emitting diode (vis-LED) and a cross-flow microfiltration (MF) membrane module was investigated in both closed-loop and continuous flow-through modes for the simultaneous degradation of penicillin G (PG) and separation of visible-light responsive TiO₂ particles, namely C-sensitized-N-doped TiO₂ (T300) and C-N-S tridoped TiO₂ (T0.05-450). The turbidity of permeate water was <0.2 NTU for both T300 and T0.05-450 suspensions in the MPR system operated at different transmembrane pressures (TMPs) and cross-flow velocities (CFVs), indicating effective separation of TiO₂ particles by the MF membrane. The operations at a higher TMP or lower CFV were more prone to induce TiO₂ deposition on the membrane surface without backwashing, which resulted in the membrane fouling, the loss of TiO₂ from the photoreactor and the decrease of PG photocatalytic degradation efficiency. 75% and 84% of PG were degraded in the closed-loop MPR without backwashing operated at 10 kPa and 0.15 m s⁻¹ after 4 h of vis-LED irradiation using 1.0 g L⁻¹ of T300 and T0.05-450, respectively. With backwashing of the membrane, the PG photocatalytic degradation efficiencies in the closed-loop MPR could be significantly enhanced to achieve 93% and 95% using 1.0 g L⁻¹ of T300 and T0.05-450, respectively, which were almost comparable to those achieved in the batch photoreactor. Due to its shorter hydraulic residence time in the photoreactor, the PG degradation efficiency in the continuous flow-through MPR with backwashing was lower than that achieved in the closed-loop MPR.