Simple strategy of reactivation of a partially inactivated penicillin g acylase biocatalyst in organic solvent and its impact on the synthesis of β‐lactam antibiotics

Some reactions of organic synthesis require to be performed in rather aggressive media, like organic solvents, that frequently impair enzyme operational stability to a considerable extent. We have studied the option of developing a reactivation strategy to increase biocatalyst lifespan under such conditions, under the hypothesis that organic solvent enzyme inactivation is a reversible process. Glyoxyl agarose immobilized penicillin G acylase and cross‐linked enzyme aggregates of the enzyme were considered as biocatalysts performing in dioxane medium. Reactivation strategy consisted in re‐incubation in aqueous medium of the partly inactivated biocatalysts in organic medium, best conditions of reactivation being studied with respect to dioxane concentration and level of enzyme inactivation attained prior to reactivation. Best results were obtained with glyoxyl agarose immobilized penicillin G acylase at all levels of residual activity studied, with reactivations up to 50%; for the case of a biocatalyst inactivated down to 75% of its initial activity, full recovery of enzyme activity was obtained after reactivation. The potential of this strategy was evaluated in the thermodynamically controlled synthesis of deacetoxycephalosporin G in a sequential batch reactor operation, where a 20% increase in the cumulative productivity was obtained by including an intermediate stage of reactivation after 50% inactivation. Biotechnol. Bioeng. 2009;103: 472–479. © 2009 Wiley Periodicals, Inc.     

Catalytic activity of poly(acryloyl morpholine)-immobilized horseradish peroxidase in organic/aqueous solvent mixtures

The immobilization of horseradish peroxidase by covalent coupling within an expanded poly(acryloyl morpholine) gel network is described. The activity of the immobilized horseradish peroxidase was compared with that of the native enzyme in aqueous buffer and in buffered mixtures of dimethyl-formamide/water, ethanediol/water, methanol/water and tetrahydrofuran/water of varying solvent ratios at pH 6.1. On increasing the organic solvent concentration in the substrate solution, active immobilized enzyme retained its activity much better than an equivalent amount of the native enzyme. The oxidation of ferrocene (water-insoluble) and ferrocene derivatives to the corresponding ferricinium ions, was accomplished efficiently by the immobilized enzyme in buffered 50% methanol/water solution. The immobilized enzyme exhibited superior resistance to thermal denaturation.     

N‐terminal fusion of a hyperthermophilic chitin‐binding domain to xylose isomerase from Thermotoga neapolitana enhances kinetics and thermostability of both free and immobilized enzymes

Immobilization of a thermostable D ‐xylose isomerase (EC 5.3.1.5) from Thermotoga neapolitana 5068 (TNXI) on chitin beads was accomplished via a N‐terminal fusion with a chitin‐binding domain (CBD) from a hyperthermophilic chitinase produced by Pyrococcus furiosus (PF1233) to create a fusion protein (CBD‐TNXI). The turnover numbers for glucose to fructose conversion for both unbound and immobilized CBD‐TNXI were greater than the wild‐type enzyme: k_cat (min^?1) was ～1,000, 3,800, and 5,800 at 80°C compared to 1,140, 10,350, and 7,000 at 90°C, for the wild‐type, unbound, and immobilized enzymes, respectively. These k_cat values for the glucose to fructose isomerization measured are the highest reported to date for any XI at any temperature. Enzyme kinetic inactivation at 100°C, as determined from a bi‐phasic inactivation model, showed that the CBD‐TNXI bound to chitin had a half‐life approximately three times longer than the soluble wild‐type TNXI (19.9 hours vs. 6.8 hours, respectively). Surprisingly, the unbound soluble CBD‐TNXI had a significantly longer half‐life (56.5 hours) than the immobilized enzyme. Molecular modeling results suggest that the N‐terminal fusion impacted subunit interactions, thereby contributing to the enhanced thermostability of both the unbound and immobilized CBD‐TNXI. These interactions likely also played a role in modifying active site structure, thereby diminishing substrate‐binding affinities and generating higher turnover rates in the unbound fusion protein. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Cross-linked aggregates of penicillin acylase: robust catalysts for the synthesis of β-lactam antibiotics

A novel method for the immobilization of penicillin G acylase (penicillin amidohydrolase, E.C. 3.5.1.11) is reported. It involves the physical aggregation of the enzyme, followed by chemical cross-linking to form insoluble cross-linked enzyme aggregates (CLEAs). Compared with conventionally immobilized penicillin G acylases, these CLEAs possess a high specific activity as well as a high productivity and synthesis/hydrolysis (S/H) ratio in the synthesis of semi-synthetic antibiotics in aqueous media. Moreover, they are active in a broad range of polar and apolar organic solvents.     

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.     

An efficient immobilizing technique of penicillin acylase with combining mesocellular silica foams support and <Emphasis Type="Italic">p</Emphasis>-benzoquinone cross linker

To improve the performance of covalently immobilized penicillin acylase (PA), the immobilization was carried out in mesocellular silica foams (MCFs) using p-benzoquinone as cross linker. The characterizations of the immobilized enzyme were studied carefully. The results showed that the relative activity of the immobilized PA was increased to 145% of that of free enzyme. The activity was 3.7 folds of that of PA on the silica nanoparticles. The enzyme in MCFs presented a turnover equal to that of free enzyme. It was also found that the optimum pH of the immobilized PA shifted to pH 7.5 and the optimum reaction temperature rose from 45 to 50 degrees C. Furthermore, the stability of PA was ameliorated greatly after immobilization. Fourier transform infrared spectroscopy showed no major secondary structural change for PA confined in MCFs. The proposed covalent immobilizing technique would rank among the potential strategies for efficient immobilization of PA.     

Hsp104 facilitates the endoplasmic‐reticulum–associated degradation of disease‐associated and aggregation‐prone substrates

Misfolded proteins in the endoplasmic reticulum (ER) are selected for ER‐associated degradation (ERAD). More than 60 disease‐associated proteins are substrates for the ERAD pathway due to the presence of missense or nonsense mutations. In yeast, the Hsp104 molecular chaperone disaggregates detergent‐insoluble ERAD substrates, but the spectrum of disease‐associated ERAD substrates that may be aggregation prone is unknown. To determine if Hsp104 recognizes aggregation‐prone ERAD substrates associated with human diseases, we developed yeast expression systems for a hydrophobic lipid‐binding protein, apolipoprotein B (ApoB), along with a chimeric protein harboring a nucleotide‐binding domain from the cystic fibrosis transmembrane conductance regulator (CFTR) into which disease‐causing mutations were introduced. We discovered that Hsp104 facilitates the degradation of ER‐associated ApoB as well as a truncated CFTR chimera in which a premature stop codon corresponds to a disease‐causing mutation. Chimeras containing a wild‐type version of the CFTR domain or a different mutation were stable and thus Hsp104 independent. We also discovered that the detergent solubility of the unstable chimera was lower than the stable chimeras, and Hsp104 helped retrotranslocate the unstable chimera from the ER, consistent with disaggregase activity. To determine why the truncated chimera was unstable, we next performed molecular dynamics simulations and noted significant unraveling of the CFTR nucleotide‐binding domain. Because human cells lack Hsp104, these data indicate that an alternate disaggregase or mechanism facilitates the removal of aggregation‐prone, disease‐causing ERAD substrates in their native environments.     

Redirecting the inactivation pathway of penicillin amidase and increasing amoxicillin production via a thermophilic molecular chaperone

We have previously shown that a single-subunit thermosome from Methanocaldococcus jannaschii (rTHS) can stabilize enzymes in semi-aqueous media (Bergeron et al., 2008b). In the present study, rTHS was used to stabilize penicillin amidase (PGA) in methanol-water mixtures. Including methanol in the reaction medium for amoxicillin synthesis can suppress unwanted hydrolysis reactions but inactivate PGA. Inactivation and reactivation pathways proposed for PGA illustrate the predictability of enzyme stabilization by rTHS in co-solvents. Calcium was necessary for reversible dissociation of the two PGA subunits in methanol-water and the presence of calcium resulted in an enhancement of chaperone-assisted stabilization. rTHS also acted as a stabilizer in the enzymatic synthesis of the beta-lactam antibiotic amoxicillin. rTHS stabilized PGA, increasing its half-life in 35% methanol by fivefold at 37 degrees C. Stabilization by rTHS was enhanced but did not require the presence of ATP. Including rTHS in fed-batch reactions performed in methanol-water resulted in nearly 4 times more amoxicillin than when the reaction was run without rTHS, and over threefold higher selectivity towards amoxicillin synthesis compared to aqueous conditions without rTHS. The thermosome and other thermophilic chaperones may thus be generally useful for stabilizing enzymes in their soluble form and expanding the range of conditions suitable for biocatalysis.     

Enzyme catalyzed biotransformations in aqueous two-phase systems with precipitated substrate and/or product

Biotransformations catalyzed by free and immobilized enzymes have been carried out in aqueous suspensions with up to 25% (w/w) precipitated substrate or product. For the kinetically controlled synthesis of N-Acetyl-Tyr-Arg-NH(2) with up to 0.8 M insoluble activated substrate N-Acetyl-TyrOEt catalyzed by alpha-chymotrypsin (EC3.4.21.1) the dipeptide yield was found to be >90%. This and the space-time yields were higher than those observed for one-phase aqueous systems and much higher than in systems where the insoluble substrate had been solubilized by addition of organic solvents. In the equilibrium controlled hydrolysis of 0.4 M D-phenylglycine-amide catalyzed by immobilized penicillin amidase (EC 3.5.1.11) the product precipitates. The enzyme immobilized in the support with the smallest pores could be reused without reduction in the rate due to precipitation in the pores. This decreases the number of immobilized enzyme molecules that can be used as biocatalysts. The latter was observed for supports with larger pores as the solubility decreases with increasing particle size. These results demonstrate that biotransformations with insoluble substrates or products using free or immobilized enzymes can be easily carried out in aqueous two-phase systems, without organic solvents, provided that the pore sizes of the supports are sufficiently small and that the rate of mass transfer from the precipitated substrate is large. The latter increases with decreasing particle size. (c) 1995 John Wiley & Sons, Inc.     

Enantioselective synthesis of (S)‐naproxen using immobilized lipase on chitosan beads

S‐naproxen by enantioselective hydrolysis of racemic naproxen methyl ester was produced using immobilized lipase. The lipase enzyme was immobilized on chitosan beads, activated chitosan beads by glutaraldehyde, and Amberlite XAD7. In order to find an appropriate support for the hydrolysis reaction of racemic naproxen methyl ester, the conversion and enantioselectivity for all carriers were compared. In addition, effects of the volumetric ratio of two phases in different organic solvents, addition of cosolvent and surfactant, optimum pH and temperature, reusability, and inhibitory effect of methanol were investigated. The optimum volumetric ratio of two phases was defined as 3:2 of aqueous phase to organic phase. Various water miscible and water immiscible solvents were examined. Finally, isooctane was chosen as an organic solvent, while 2‐ethoxyethanol was added as a cosolvent in the organic phase of the reaction mixture. The optimum reaction conditions were determined to be 35 °C, pH 7, and 24 h. Addition of Tween‐80 in the organic phase increased the accessibility of immobilized enzyme to the reactant. The optimum organic phase compositions using a volumetric ratio of 2‐ethoxyethanol, isooctane and Tween‐80 were 3:7 and 0.1% (v /v/v), respectively. The best conversion and enantioselectivity of immobilized enzyme using chitosan beads activated by glutaraldehyde were 0.45 and 185, respectively.     

Directed self‐immobilization of alkaline phosphatase on micro‐patterned substrates via genetically fused metal‐binding peptide

Current biotechnological applications such as biosensors, protein arrays, and microchips require oriented immobilization of enzymes. The characteristics of recognition, self‐assembly and ease of genetic manipulation make inorganic binding peptides an ideal molecular tool for site‐specific enzyme immobilization. Herein, we demonstrate the utilization of gold binding peptide (GBP1) as a molecular linker genetically fused to alkaline phosphatase (AP) and immobilized on gold substrate. Multiple tandem repeats (n = 5, 6, 7, 9) of gold binding peptide were fused to N‐terminus of AP (nGBP1‐AP) and the enzymes were expressed in E. coli cells. The binding and enzymatic activities of the bi‐functional fusion constructs were analyzed using quartz crystal microbalance spectroscopy and biochemical assays. Among the multiple‐repeat constructs, 5GBP1‐AP displayed the best bi‐functional activity and, therefore, was chosen for self‐immobilization studies. Adsorption and assembly properties of the fusion enzyme, 5GBP1‐AP, were studied via surface plasmon resonance spectroscopy and atomic force microscopy. We demonstrated self‐immobilization of the bi‐functional enzyme on micro‐patterned substrates where genetically linked 5GBP1‐AP displayed higher enzymatic activity per area compared to that of AP. Our results demonstrate the promising use of inorganic binding peptides as site‐specific molecular linkers for oriented enzyme immobilization with retained activity. Directed assembly of proteins on solids using genetically fused specific inorganic‐binding peptides has a potential utility in a wide range of biosensing and bioconversion processes. Biotechnol. Bioeng. 2009;103: 696–705. © 2009 Wiley Periodicals, Inc.     

Immobilization of Papain on the Mesoporous Molecular Sieve MCM‐48

The immobilization of papain on the mesoporous molecular sieve MCM‐48 (with a pore size of 6.2 nm in diameter) with the aid of glutaraldehyde, and the characteristics of this immobilized papain are described. The optimum conditions for immobilization were as follows: 20 mg native free enzyme/g of the MCM‐48 and 0.75 % glutaraldehyde, 2 h at 10–20 °C and pH 7.0. Under these optimum conditions for immobilization, the activity yield [%] of the immobilized enzyme was around 70 %. The influence of the pH on the activity of the immobilized enzyme was much lower compared to the free enzyme. The thermostability of the immobilized enzyme, whose half‐life was more than 2500 min, was greatly improved and was found to be significantly higher than that of the free enzyme (about 80 min). The immobilized enzyme also showed good operational stability, and the activity of the immobilized enzyme continued to maintain 76.5 % of the initial activity even after a 12‐day continuous operation. Moreover, the immobilized enzyme still exhibited good storage stability. From these results, papain immobilized on the MCM‐48 with the aid of glutaraldehyde, can be used as a high‐performance biocatalyst in biotechnological processing, in particular in industrial and medical applications.     

Conversion of benzylpenicillin to 6-aminopenicillanic acid in a batch reactor and continuous feed stirred tank reactor using immobilized penicillin amidase

A rate equation has been derived to describe the hydrolysis of benzylpenicillin to 6-aminopenicillanic acid by penicillin amidase. The integrated from of the rate equation has been shown to predict satisfactorily the progress of the reaction in a batch reactor using either soluble or immobilized penicillin amidase. The rate equation was also used to predict the performance of a continuous feed stirred tank reactor containing immobilized enzyme. There was good agreement with experimental measurements.     

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％。     

Studies on improved techniques for immobilizing and stabilizing penicillin amidase associated with E. coli cells.

A method for catalyst development has been suggested for immobilizing whole E. coli cells containing penicillin amidase. Conventional methods have limitations, such as permeation of substrate and product through cellular membranes, leaching of protein and other cellular components into the reaction phase, lower specific activity compared to immobilized enzyme system, etc. The whole cell immobilization technique has been optimized for different process parameters. The most suitable conditions for this process were pH, 4.25; cell concentration, 3.75%; concentration of glutaraldehyde, 1.5%; level of bovine serum albumin as additional support, 2 mg ml-1. The reaction was continued for 2 h. The granular catalyst has good mechanical strength, low protein leachability, and high retention of penicillin amidase activity.     

Immobilization of a thermostable α‐amylase by covalent binding to an alginate matrix increases high temperature usability

Thermostable α‐amylase was covalently bound to calcium alginate matrix to be used for starch hydrolysis at liquefaction temperature of 95°C. 1‐ethyl‐3‐(3‐dimethylamino‐propyl) carbodiimide hydrochloride (EDAC) was used as crosslinker. EDAC reacts with the carboxylate groups on the calcium alginate matrix and the amine groups of the enzyme. Ethylenediamine tetraacetic acid (EDTA) treatment was applied to increase the number of available carboxylate groups on the calcium alginate matrix for EDAC binding. After the immobilization was completed, the beads were treated with 0.1 M calcium chloride solution to reinstate the bead mechanical strength. Enzyme loading efficiency, activity, and reusability of the immobilized α‐amylase were investigated. Covalently bound thermostable α‐amylase to calcium alginate produced a total of 53 g of starch degradation/mg of bound protein after seven consecutive starch hydrolysis cycles of 10 min each at 95°C in a stirred batch reactor. The free and covalently bound α‐amylase had maximum activity at pH 5.5 and 6.0, respectively. The Michaelis‐Menten constant (K_m) of the immobilized enzyme (0.98 mg/mL) was 2.5 times greater than that of the free enzyme (0.40 mg/mL). The maximum reaction rate (V_max) of immobilized and free enzyme were determined to be 10.4‐mg starch degraded/mL min mg bound protein and 25.7‐mg starch degraded/mL min mg protein, respectively. The high cumulative activity and seven successive reuses obtained at liquefaction temperature make the covalently bound thermostable α‐amylase to calcium alginate matrix, a promising candidate for use in industrial starch hydrolysis process. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Novel grafted agar disks for the covalent immobilization of β‐D‐galactosidase

Novel grafted agar disks were prepared for the covalent immobilization of β‐D‐galactosidase (β‐gal). The agar disks were activated through reacting with ethylenediamine or different molecular weights of Polyethyleneimine (PEI), followed by glutaraldehyde (GA). The modification of the agar gel and the binding of the enzyme were verified by Fourier Transform Infrared (FTIR) and elemental analysis. Moreover, the agar's activation process was optimized, and the amount of immobilized enzyme increased 3.44 folds, from 38.1 to 131.2 U/g gel, during the course of the optimization process. The immobilization of β‐gal onto the activated agar disks caused its optimum temperature to increase from 45°C to 45–55°C. The optimum pH of the enzyme was also shifted towards the acidic side (3.6–4.6) after its immobilization. Additionally, the Michaelis‐Menten constant (K_m) increased for the immobilized β‐gal as compared to its free counterpart whereas the maximum reaction rate (V_max) decreased. The immobilized enzyme was also shown to retain 92.99% of its initial activity after being used for 15 consecutive times. © 2015 Wiley Periodicals, Inc. Biopolymers 103: 675–684, 2015.     

Release of periplasmic penicillin amidase from Escherichia coli by chloroform shock

Penicillin amidase is a periplasmic enzyme in Escherichia coli. Conventionally, the periplasmic enzymes are released into the medium by osmotic shock which is tedious involving a number of centrifugation steps. The present communication deals with a simple technique for the release of penicillin amidase by chloroform shock. Experimental findings show that the periplasmic penicillin amidase does not show any variation by the chloroform treatment. This analysis was also extended to the E. coli cells grown at various concentrations of phenylacetic acid, optimal concentration of phenylacetic acid plus glucose and lactic acid.     

Production of immobilized penicillin acylase using aqueous polymer systems for enzyme purification and in situ immobilization

A novel approach for the isolation and purification of penicillin acylase (PA), which couples aqueous two-phase partitioning and enzyme immobilization has been investigated.A PA yield of 90% was achieved by treating E. coli cells with 4% butyl acetate, freeze-thawing step, and pressure homogenization. PA purification (93% recovery) was achieved by (1) removing cell debris via precipitation with polyethylene glycol (PEG 2000); (2) aqueous two-phase partitioning using a PEG 2000 + phosphate system (87% recovery).An in situ enzyme immobilization approach, using oxirane acrylic or aldehyde-agarose beads dispersed in the PEG-rich phase, was explored for the conversion of penicillin G to 6-aminopenicillanic acid. An appropriate immobilization reaction time was found. The catalytic performance of the enzyme, when immobilized, was found not to be affected by recycling of the phase-forming components.     

Biocatalytic properties of thermolysin immobilized on polyvinyl alcohol cryogel

Preparations with different contents of thermolysin were obtained by the immobilization of the enzyme on granulated polyvinyl alcohol cryogel. Their activity and stability in an aqueous medium and in mixtures of polar organic solvents of different composition were investigated. The catalytic properties of the preparations in reactions of peptide bond formation were studied, and the optimal amount of the biocatalyst, the concentrations of initial reagents, and the ratios of organic solvents and water necessary for effective enzymatic peptide synthesis catalyzed by immobilized thermolysin were determined. A series of peptides of the general formula Z-Ala-Ala-Xaa-pNA, where Xaa = Leu, Ile, Phe, Val, or Ala, were synthesized, and the immobilized enzyme was shown to retain substrate specificity in an organic medium.