Invertase reversibly immobilized onto polyethylenimine-grafted poly(GMA–MMA) beads for sucrose hydrolysis

The epoxy group containing poly(glycidyl methacrylate-co-methylmethacrylate) poly(GMA–MMA) beads were prepared by suspension polymerisation and the beads surface were grafted with polyethylenimine (PEI). The PEI-grafted beads were then used for invertase immobilization via adsorption. The immobilization of enzyme onto the poly(GMA–MMA)–PEI beads from aqueous solutions containing different amounts of invertase at different pH was investigated in a batch system. The maximum invertase immobilization capacity of the poly(GMA–MMA)–PEI beads was about 52 mg/g. It was shown that the relative activity of immobilized invertase was higher then that of the free enzyme over broader pH and temperature ranges. The Michaelis constant (K_m) and the maximum rate of reaction (V_max) were calculated from the Lineweaver–Burk plot. The K_m and V_max values of the immobilized invertase were larger than those of the free enzyme. The immobilized enzyme had a long-storage stability (only 6% activity decrease in 2 months) when the immobilized enzyme preparation was dried and stored at 4 °C while under wet condition 43% activity decrease was observed in the same period. After inactivation of enzyme, the poly(GMA–MMA)–PEI beads can be easily regenerated and reloaded with the enzyme for repeated use.     

Stability and kinetic behavior of immobilized laccase from Myceliophthora thermophila in the presence of the ionic liquid 1‐ethyl‐3‐methylimidazolium ethylsulfate

The use of ionic liquids (ILs) as reaction media for enzymatic reactions has increased their potential because they can improve enzyme activity and stability. Kinetic and stability properties of immobilized commercial laccase from Myceliophthora thermophila in the water‐soluble IL 1‐ethyl‐3‐methylimidazolium ethylsulfate ([emim][EtSO₄]) have been studied and compared with free laccase. Laccase immobilization was carried out by covalent binding on glyoxyl–agarose beads. The immobilization yield was 100%, and the activity was totally recovered. The Michaelis‐Menten model fitted well to the kinetic data of enzymatic oxidation of a model substrate in the presence of the IL [emim][EtSO₄]. When concentration of the IL was augmented, the values of V_max for free and immobilized laccases showed an increase and slight decrease, respectively. The laccase–glyoxyl–agarose derivative improved the laccase stability in comparison with the free laccase regarding the enzymatic inactivation in [emim][EtSO₄]. The stability of both free and immobilized laccase was slightly affected by small amounts of IL (<50%). A high concentration of the IL (75%) produced a large inactivation of free laccase. However, immobilization prevented deactivation beyond 50%. Free and immobilized laccase showed a first‐order thermal inactivation profile between 55 and 70°C in the presence of the IL [emim][EtSO₄]. Finally, thermal stability was scarcely affected by the presence of the IL. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:790–796, 2014     

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.     

Efficient Synthesis and Antioxidant Evaluation of 2‐Aryl‐3‐(Pyrimidin‐2‐yl)‐Thiazolidinones

In the present study, we reported the efficient synthesis of 11 3‐(pyrimidin‐2‐yl)‐thiazolidinones in good yields using molecular sieve as the desiccant agent. In addition, we have evaluated the antioxidant capacity of the synthesized compounds by the 2,2‐diphenyl‐2‐picrylhydrazyl hydrate (DPPH?) and the 2,2‐azinobis(3‐ethylbenzothiazoline‐6‐sulfonic acid) diammonium salt (ABTS^+?) radicals scavenging assay. Six compounds showed antioxidant activity towards DPPH? (EC₅₀ between 16.13 and 49.94 µg/mL) and also demonstrated excellent activity regarding ABTS^+? (TEAC: 10.32–53.52). These results showed that compounds 3‐(pyrimidin‐2‐yl)‐thiazolidinones may be easily synthesized by a less expensive procedure and could be a good starting point to the development of new antioxidant compounds. © 2013 Wiley Periodicals, Inc. J BiochemMol Toxicol 27:445‐450, 2013; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.21506     

Purification and characterization of a metalloprotease from Chryseobacterium indologenes Ix9a and determination of the amino acid specificity with electrospray mass spectrometry

The heat-stable protease from Chryseobacterium indologenes Ix9a was purified to homogeneity using immobilized metal affinity chromatography. The enzyme was characterized as a metalloprotease with an approximate relative molecular mass of 24,000, a pH optimum of 6.5, and a high temperature optimum (50 degrees C). The metal chelator EDTA and the Zn2+-specific chelator 1,10-phenanthroline were identified as inhibitors and atomic absorption analysis showed that the enzyme contained Ca2+ and Zn2+. The activity of the apoenzyme could be restored with Ca2+, Zn2+, Mg2+, and Co2+. Phosphoramidon and Gly-d-Phe did not inhibit Chryseobacterium indologenes Ix9a protease. Heat inactivation did not follow first order kinetics, but showed biphasic inactivation curves. The protease has a Km of 0.813 microg. ml-1 for casein as substrate. Amino acid analysis showed that the protease contains a high amount of small amino acids like glycine, alanine, and serine, but a low concentration of methionine and no cysteine at all. Electrospray mass spectrometry of proteolysis fragments formed when insulin B chain was hydrolyzed showed cleavage at the amino terminal of leucine, tyrosine, and phenylalanine. A hydrophobic amino acid at the carboxyl donating side seems to increase the rate of reaction.     

三乙醇胺基强碱性聚苯乙烯树脂共价固定胰酶的研究

用多孔强碱型三乙醇胺基聚苯乙烯阴离子交换树脂做为载体,用CNBr与载体上的多羟基作用共价偶联了胰酶。红外光谱表明:其共价偶联反应机理与用CNBr活化多糖类载体并接酶的机理相类似。最适偶联条件研究表明:CNBr用量增多,酶蛋白载量增加。但比活下降。偶联pH为10时,固定化酶有适宜的载量和较高的比活。由于胰酶水解蛋白反应释放出H~+质子,这些质子在载体内积累,使微环境内H~+质子浓度增加,进而使得固定化胰酶的pH—活性曲线在pH9～11范围内未出现下降。在变温和60℃恒温下对固定化酶的热稳定性测试表明:固相酶的热稳定性比天然酶的热稳定性有所提高。     

Kinetic modeling of native Cassava starch thermo‐oxidative degradation using Weibull and Weibull‐derived models

A new approach in kinetic modeling of thermo‐oxidative degradation process of starch granules extracted from the Cassava roots was developed. Based on the thermoanalytical measurements, three reaction stages were detected. Using Weibull and Weibull‐derived (inverse) models, it was found that the first two reaction stages could be described with the change of apparent activation energy (E_a) on conversion fraction (α(T)) (using “Model‐free” analysis). It was found that first reaction stage, which involves dehydration and evaporation of lower molecular mass fractions, can be described with an inverse Weibull model. This model with its distribution of E_a values and derived distribution parameters includes the occurrence of three‐dimensional diffusion mechanism. The second reaction stage is very complex, and it was found to contain the system of simultaneous reactions (where depolymerization occurs), and can be described with standard Weibull model. Identified statistical model with its distribution of E_a values and derived distribution parameters includes the kinetic model that gives the variable reaction order values. Based on the established models, shelf‐life studies for first two stages were carried out. Shelf‐life testing has shown that optimal dehydration time is achieved by a programmed heating at medium heating rate, whereas optimal time of degradation is achieved at highest heating rate. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 41–57, 2014.     

Improved activity and stability of an immobilized recombinant laccase in organic solvents

Laccase (E.C. 1.10.3.2) from Trametes versicolor was immobilized (adsorbed) by drying on various supports (glass, glass powder, silica gel, and Nylon 66 membrane). The enzyme activity and stability were determined in diethyl ether, ethyl acetate, and methylene chloride. The initial rate for the oxidation of syringaldazine varied up to 245-fold depending on the solvent and support, the best results being obtained with Nylon 66 membrane. No inactivation of immobilized laccase over 72 h was observed in diethyl ether and ethyl acetate, while exposure to methylene chloride resulted in significant activity decreases regardless of the support material.     

Tyrosinase inactivation in organic solvents.

The inactivation of the catecholase activity of mushroom tyrosinase was investigated under nonaqueous conditions. The enzyme was immobilized on glass beads, and assays were conducted in chloroform, toluene, amyl acetate, isopropyl ether, and butanol. The reaction components were pre-equilibrated for 2 weeks with a saturated salt solution at a water activity of 0.90. The initial reaction velocity varied between 1.3 x 10(3) mol product/((mol enzyme)(min)) in toluene and 8.7 x 10(3) mol product/((mol enzyme)(min)) in amyl acetate. The turnover number varied between 8.1 x 10(3) mol product/mol enzyme in toluene and 7.2 x 10(4) mol product/mol enzyme in amyl acetate. In each solvent, the tyrosinase reaction inactivation parameters were represented by a probabilistic model. Changes in the probability of inactivation were followed throughout the course of the reaction using a second model which relates the reaction velocity to the amount of product formed. These models reveal that the inactivation rate of tyrosinase decreases as the reaction progresses, and that the inactivation kinetics are independent of the quinone concentration in toluene, chloroform, butanol, and amyl acetate. Significant effects of quinone concentration were, however, observed in isopropyl ether. The likelihood of inactivation of the enzyme was found to be greatest toward the beginning of the reaction. In the latter phase of the reaction, inactivation probability was less and tended to remain constant until the completion of the reaction.     

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.     

Thermostability of soluble and immobilized alpha-amylase from Bacillus licheniformis

In view of a possible application of the alpha-amylase from Bacillus licheniformis as a time-temperature integrator for evaluation of heat processes,(11) thermal inactivation kinetics of the dissolved and covalently immobilized enzyme were studied in the temperature range 90-108 degrees C. The D-values (95 degrees C) for inactivation of alpha-amylase, dissolved in tris-HCl buffer, ranged from 6 to 157 min, depending on pH, ionic strength, and Ca(2+) and enzyme concentration. The z-value fluctuated between 6.2 and 7.6 degrees C. On immobilization of the alpha-amylase by covalent coupling with glutaraldehyde to porous glass beads, the thermoinactivation kinetics became biphasic under certain circumstances. For immobilized enzyme, the D-values (95 degrees C) ranged between 17 and 620 min, depending largely on certain environmental conditions. The z-value fluctuated between 8.1 and 12.9 degrees C. In each case of biphasic inactivation, the z-value of the stable fraction (with the higher D-values) was lower than the z-value of the labile fraction. (c) 1992 John Wiley & Sons, Inc.     

Cyclo(phenylalanine‐proline) induces DNA damage in mammalian cells via reactive oxygen species

Cyclo(phenylalanine‐proline) is produced by various organisms such as animals, plants, bacteria and fungi. It has diverse biological functions including anti‐fungal activity, anti‐bacterial activity and molecular signalling. However, a few studies have demonstrated the effect of cyclo(phenylalanine‐proline) on the mammalian cellular processes, such as cell growth and apoptosis. In this study, we investigated whether cyclo(phenylalanine‐proline) affects cellular responses associated with DNA damage in mammalian cells. We found that treatment of 1 mM cyclo(phenylalanine‐proline) induces phosphorylation of H2AX (S139) through ATM‐CHK2 activation as well as DNA double strand breaks. Gene expression analysis revealed that a subset of genes related to regulation of reactive oxygen species (ROS) scavenging and production is suppressed by the cyclo(phenylalanine‐proline) treatment. We also found that cyclo(phenylalanine‐proline) treatment induces perturbation of the mitochondrial membrane, resulting in increased ROS, especially superoxide, production. Collectively, our study suggests that cyclo(phenylalanine‐proline) treatment induces DNA damage via elevation of ROS in mammalian cells. Our findings may help explain the mechanism underlying the bacterial infection‐induced activation of DNA damage response in host mammalian cells.     

Kinetic characterisation of enzymatic esterification in a solvent system: adsorptive control of water with molecular sieves

The kinetics of enzymatic esterification of glycerol with oleic acid, in equimolar ratio, catalyzed by immobilized Mucor miehei lipase in a solvent system in the presence of the molecular sieves was carried out at 37°C at different Lipozym and solvent (n-hexane) concentrations and the molecular sieve contents were studied in a batch stirred-tank reactor (BSTR). The reactions were followed by the determination of reaction conversions during 45 h. The experimental data of enzymatic esterification of glycerol with oleic acid in a solvent system in the presence of molecular sieves showed minimal deviation from the calculated value in the irreversible second order kinetic model. On the basis of the experimental data, we found an empirical correlation between concentrations of Lipozym, concentrations of solvent (n-hexane), contents of the molecular sieve and the reaction rate constant, k₁.     

Wax ester synthesis by lipase-catalyzed esterification with fungal cells immobilized on cellulose biomass support particles

Esterification reactions between long-chain alcohol and oleic acid were performed for producing wax esters. The reaction can be catalyzed efficiently by cell-bound lipase of Rhizopus niveous fungal cells immobilized within cellulose biomass support particles. Carrying out the reaction in a solvent-free system is feasible by adding a molecular sieve for dehydration purposes. To optimize the yield, addition of a molecular sieve should be performed gradually during the whole course starting from the beginning of the reaction. The influence of reaction conditions such as temperature and substrate concentrations on reaction rates and yields were investigated; however, this reaction system is under the influence of both internal and external mass transfer resistance. Conducting the reaction in an organic solvent system with hexane or heptane as the solvent can eliminate diffusional effects. Reaction kinetics were subjected to detailed study in this system. The kinetics of the reaction can be represented satisfactorily by a Ping-Pong Bi Bi mechanism with deadend inhibition by alcohol.     

An amperometric enzyme biosensor for real‐time measurements of cellobiohydrolase activity on insoluble cellulose

An amperometric enzyme biosensor for continuous detection of cellobiose has been implemented as an enzyme assay for cellulases. We show that the initial kinetics for cellobiohydrolase I, Cel7A from Trichoderma reesei, acting on different types of cellulose substrates, semi‐crystalline and amorphous, can be monitored directly and in real‐time by an enzyme‐modified electrode based on cellobiose dehydrogenase (CDH) from Phanerochaete chrysosporium (Pc). PcCDH was cross‐linked and immobilized on the surface of a carbon paste electrode which contained a mediator, benzoquinone. An oxidation current of the reduced mediator, hydroquinone, produced by the CDH‐catalyzed reaction with cellobiose, was recorded under constant‐potential amperometry at +0.5 V (vs. Ag/AgCl). The CDH‐biosensors showed high sensitivity (87.7 µA mM^?1 cm^?2), low detection limit (25 nM), and fast response time (t_95% ～ 3 s) and this provided experimental access to the transient kinetics of cellobiohydrolases acting on insoluble cellulose. The response from the CDH‐biosensor during enzymatic hydrolysis was corrected for the specificity of PcCDH for the β‐anomer of cello‐oligosaccharides and the approach were validated against HPLC. It is suggested that quantitative, real‐time data on pure insoluble cellulose substrates will be useful in attempts to probe the molecular mechanism underlying enzymatic hydrolysis of cellulose. Biotechnol. Bioeng. 2012; 109: 3199–3204. © 2012 Wiley Periodicals, Inc.     

Automated docking studies provide insights into molecular determinants of ligand recognition by N‐acetyl‐1‐d‐myo‐inosityl‐2‐amino‐2‐deoxy‐α‐d‐glucopyranoside deacetylase (MshB)

The metal‐dependent deacetylase N‐acetyl‐1‐d ‐myo‐inosityl‐2‐amino‐2‐deoxy‐α‐d ‐glucopyranoside deacetylase (MshB) catalyzes the deacetylation of N‐acetyl‐1‐d ‐myo‐inosityl‐2‐amino‐2‐deoxy‐α‐d ‐glucopyranoside (GlcNAc‐Ins), the committed step in mycothiol (MSH) biosynthesis. MSH is the thiol redox buffer used by mycobacteria to protect against oxidative damage and is involved in the detoxification of xenobiotics. As such, MshB is a target for the discovery of new drugs to treat tuberculosis (TB). While MshB substrate specificity and inhibitor activity have been probed extensively using enzyme kinetics, information regarding the molecular basis for the observed differences in substrate specificity and inhibitor activity is lacking. Herein we begin to examine the molecular determinants of MshB substrate specificity using automated docking studies with a set of known MshB substrates. Results from these studies offer insights into molecular recognition by MshB via identification of side chains and dynamic loops that may play roles in ligand binding. Additionally, results from these studies suggest that a hydrophobic cavity adjacent to the active site may be one important determinant of MshB substrate specificity. Importantly, this hydrophobic cavity may be advantageous for the design of MshB inhibitors with high affinity and specificity as potential TB drugs. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 406–417, 2014.     

Immobilized horse liver alcohol dehydrogenase as an on‐line high‐performance liquid chromatographic enzyme reactor for stereoselective synthesis

Horse liver alcohol dehydrogenase (HLADH) has been non‐covalently immobilized on an immobilized artificial membrane (IAM) high‐performance liquid chromatography (HPLC) stationary phase. The resulting IAM‐HLADH retained the reductive activity of native HLADH as well as the enzyme's enantioselectivity and enantiospecificity. HLADH was also immobilized in an IAM HPLC stationary phase prepacked in a 13 × 4.1 mm ID column to create an immobilized enzyme reactor (HLADH‐IMER). The reactor was connected through a switching valve to a column containing a chiral stationary phase (CSP) based upon p‐methylphenylcarbamate derivatized cellulose (Chiralcel OJR‐CSP). The results from the combined HLADH‐IMER/CSP and chromatographic system demonstrate that the enzyme retained its activity and stereoselectivity after immobilization in the column and that the substrate and products from the enzymatic reduction could be transferred to a second column for analytical or preparative separation. The combined HLADH‐IMER/CSP system is a prototype for the preparative on‐line use of cofactor‐dependent enzymes in large‐scale chiral syntheses. Chirality 11:39–45, 1999. © 1999 Wiley‐Liss, Inc.     

Assessing the effectiveness of low‐pressure ultraviolet light for inactivating Mycobacterium avium complex (MAC) micro‐organisms

Aims: To assess low‐pressure ultraviolet light (LP‐UV) inactivation kinetics of Mycobacterium avium complex (MAC) strains in a water matrix using collimated beam apparatus. Methods and Results: Strains of M. avium (n = 3) and Mycobacterium intracellulare (n = 2) were exposed to LP‐UV, and log₁₀ inactivation and inactivation kinetics were evaluated. All strains exhibited greater than 4 log₁₀ inactivation at fluences of less than 20 mJ cm^?2. Repair potential was evaluated using one M. avium strain. Light repair was evaluated by simultaneous exposure using visible and LP‐UV irradiation. Dark repair was evaluated by incubating UV‐exposed organisms in the dark for 4 h. The isolate did not exhibit light or dark repair activity. Conclusions: Results indicate that MAC organisms are readily inactivated at UV fluences typically used in drinking water treatment. Differences in activation kinetics were small but statistically significant between some tested isolates. Significance and Impact of the Study: Results provide LP‐UV inactivation kinetics for isolates from the relatively resistant MAC. Although UV inactivation of Mycobaterium species have been reported previously, data collected in this effort are comparable with recent UV inactivation research efforts performed in a similar manner. Data were assessed using a rigorous statistical approach and were useful towards modelling efforts.     

Structure of 6‐diazo‐5‐oxo‐norleucine‐bound human gamma‐glutamyl transpeptidase 1, a novel mechanism of inactivation

Intense efforts are underway to identify inhibitors of the enzyme gamma‐glutamyl transpeptidase 1 (GGT1) which cleaves extracellular gamma‐glutamyl compounds and contributes to the pathology of asthma, reperfusion injury and cancer. The glutamate analog, 6‐diazo‐5‐oxo‐norleucine (DON), inhibits GGT1. DON also inhibits many essential glutamine metabolizing enzymes rendering it too toxic for use in the clinic as a GGT1 inhibitor. We investigated the molecular mechanism of human GGT1 (hGGT1) inhibition by DON to determine possible strategies for increasing its specificity for hGGT1. DON is an irreversible inhibitor of hGGT1. The second order rate constant of inactivation was 0.052 mM ^?1 min^?1 and the K _i was 2.7 ± 0.7 mM . The crystal structure of DON‐inactivated hGGT1 contained a molecule of DON without the diazo‐nitrogen atoms in the active site. The overall structure of the hGGT1‐DON complex resembled the structure of the apo‐enzyme; however, shifts were detected in the loop forming the oxyanion hole and elements of the main chain that form the entrance to the active site. The structure of hGGT1‐DON complex revealed two covalent bonds between the enzyme and inhibitor which were part of a six membered ring. The ring included the OG atom of Thr381, the reactive nucleophile of hGGT1 and the α‐amine of Thr381. The structure of DON‐bound hGGT1 has led to the discovery of a new mechanism of inactivation by DON that differs from its inactivation of other glutamine metabolizing enzymes, and insight into the activation of the catalytic nucleophile that initiates the hGGT1 reaction.     

A comprehensive kinetic model of laccase‐catalyzed oxidation of aqueous phenol

A comprehensive model was developed to describe the kinetics of the laccase‐catalyzed oxidation of phenol that incorporates enzyme kinetics, enzyme inactivation, variable reaction stoichiometry between substrate and oxygen, and oxygen mass‐transfer. The model was calibrated and validated against data obtained from experiments conducted in an open system, which allowed oxygen to transfer from air to the reacting mixture and phenol conversion to approach completion. Inactivation of laccase was observed over the course of the reaction and was found to be dependent on the rate of substrate transformation. A single kinetic expression was sufficient to describe laccase inactivation arising from interaction with reacting species over time. Excellent agreement was found between model predictions of phenol and oxygen concentrations and experimental data over time for a wide range of initial substrate concentrations and enzyme activities. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009