Reversibly soluble biocatalyst: optimization of trypsin coupling to Eudragit S-100 and biocatalyst activity in soluble and precipitated forms

Eudragit S-100, a copolymer of methacrylic acid and methyl methacrylate is soluble at pH above 5 and insoluble at pH below 4.5. pH-dependent solubility of the polymer is used for the development of reversibly soluble biocatalyst, which combines the advantages of both soluble and immobilized biocatalysts. Activity of trypsin, covalently coupled to Eudragit S-100, was improved by protecting the active site of the enzyme with benzamidine and removing the noncovalently bound proteins with Triton X-100 in 0.15 M Tris buffer (pH 7.6). Accurate choice of coupling conditions combined with proper washing protocol produced highly active enzyme-polymer conjugate with no noncovalently bound protein. Two conjugates with 100-fold difference in the content of trypsin coupled to Eudragit S-100 were studied when the preparations were in soluble and precipitated forms. The K(m)values of the soluble enzyme to the lower molecular weight substrate was less than that of the free enzyme, whereas that to the higher molecular weight substrate was closer to that of the free enzyme. Activities of the soluble and precipitated immobilized trypsin with higher molecular weight substrate were completely inhibited by soy bean trypsin inhibitor, whereas complete inhibition with soy bean trypsin inhibitor was never achieved with lower molecular weight substrate, indicating reduced access of high-molecular weight substrate/inhibitor to some of the catalytically active enzyme molecules in trypsin-Eudragit conjugate.     

Activity and stability of laccase in conjugation with chitosan

Laccase is one of a few enzymes that can directly reduce oxygen into water under ambient conditions, while oxidizing a variety of aromatic compounds. Its conjugation with chitosan generates a pH-sensitive functional biomaterial that changes its solubility in response to pH variation. The molecular conjugation between laccase and chitosan of different molecular mass was investigated with a carbodiimide reaction to understand the mechanism of the enzyme's activity loss during conjugation. With 81-93% laccase being conjugated, a moderate activity loss (16-28% less than the initial activity) was observed in conjugation solution. A second severe activity loss (63-78% less than the conjugated activity) occurred during a cycle of phase change consisting of precipitation, centrifugation and re-dissolution of the enzyme-chitosan conjugates. The chitosan molecular size has little effect on the first moderate activity loss in the conjugation reaction, but visible effect on the substantial activity loss associated with phase change. Small chitosan molecules gave high residual activity. The conjugated laccase exhibited a high stability in the following repeated phase changes and had the same temperature and pH profile as those of free laccase. Compared to free laccase, the conjugated laccase had a similar affinity (Km), but reduced turnover (kcat) that was adversely affected with increase of molecular mass of chitosan.     

Coupling of aromatic amines onto syringylglycerol β-guaiacylether using Bacillus SF spore laccase: A model for functionalization of lignin-based materials

The potential of Bacillus SF spore laccase for coupling aromatic amines to lignin model molecules as a way of creating a stable reactive surface was investigated. The Bacillus spore laccase was shown to be active within the neutral to alkaline conditions (pH 7–8.5) and was more resistant to common laccase inhibitors than fungal laccases. Using this enzyme, tyramine was successfully covalently coupled onto syringylglycerol β-guaiacylether via a 4-O-5 bond, leaving the –NH₂ group free for further attachment of functional molecules. This study demonstrates the potential of Bacillus SF spore laccase for application in lignocellulose surface functionalization and other coupling reactions which can be carried out at neutral to alkaline pH under extreme conditions which normally inhibit fungal laccases.     

Engineering bifunctional laccase-xylanase chimeras for improved catalytic performance

Two bifunctional enzymes exhibiting combined xylanase and laccase activities were designed, constructed, and characterized by biochemical and biophysical methods. The Bacillus subtilis cotA and xynA genes were used as templates for gene fusion, and the xynA coding sequence was inserted into a surface loop of the cotA. A second chimera was built replacing the wild-type xynA gene by a thermostable variant (xynAG3) previously obtained by in vitro molecular evolution. Kinetic measurements demonstrated that the pH and temperature optima of the catalytic domains in the chimeras were altered by less than 0.5 pH units and 5 °C, respectively, when compared with the parental enzymes. In contrast, the catalytic efficiency (k(cat)/K(m)) of the laccase activity in both chimeras was 2-fold higher than for the parental laccase. Molecular dynamics simulations of the CotA-XynA chimera indicated that the two domains are in close contact, which was confirmed by the low resolution structure obtained by small angle x-ray scattering. The simulation also indicates that the formation of the inter-domain interface causes the dislocation of the loop comprising residues Leu-558 to Lys-573 in the laccase domain, resulting in a more accessible active site and exposing the type I Cu(2+) ion to the solvent. These structural changes are consistent with the results from UV-visible electronic and EPR spectroscopy experiments of the type I copper between the native and chimeric enzymes and are likely to contribute to the observed increase in catalytic turnover number.     

Immobilization of functionally unstable catechol-2,3-dioxygenase greatly improves operational stability

Thermophilic catechol 2,3-dioxygenase (EC 1.13.11.2) from Bacillus stearothermophilus has been immobilized on highly activated glyoxyl agarose beads. The enzyme could be fully immobilized at 4 degrees C and pH 10.05 with a high retention of activity (around 80%). Enzyme immobilized under these conditions showed little increase in thermostability compared with the soluble enzyme, but further incubation of immobilized enzyme at 25 degrees C and pH 10.05 for 3 h before borohydride reduction resulted in conjugates exhibiting a 100-fold increase in stability (c.f. the free enzyme). The stability of catechol 2,3-dioxygenase immobilized under these conditions was essentially independent of protein concentration whereas free enzyme was rapidly inactivated at low protein concentrations. An apparent stabilization factor of over 700-fold was recorded in the comparison of free and immobilized catechol 2,3-dioxygenases at protein concentrations of 10 μg/ml. Immobilization increased the 'optimum temperature' for activity by 20 degrees C, retained activity at substrate concentrations where the soluble enzyme was fully inactivated and enhanced the resistance to inactivation during catalysis. These results suggest that the immobilization of the enzyme under controlled conditions with the generation of multiple covalent links between the enzyme and matrix both stabilized the quaternary structure of the protein and increased the rigidity of the subunit structures.     

Advantages in using non-isothermal bioreactors in bioremediation of water polluted by phenol by means of immobilized laccase from Rhus vernicifera

Laccase from Rhus vernicifera was immobilized on a polypropylene membrane chemically modified with chromic acid. Ethylenediamine and glutaraldehyde were used as spacer and bifunctional coupling agent, respectively. Phenol was used as substrate.To know how the immobilization procedures affected the enzyme reaction rate the catalytic behavior of soluble and insoluble laccase was studied under isothermal conditions as a function of pH, temperature and substrate concentration. From these studies, two main singularities emerged: (i) the narrower pH–activity profile of the soluble enzyme in comparison to that of the insoluble counterpart and (ii) the increase in pH and thermal stability of the insoluble enzyme.The laccase catalytic behavior was also studied in a non-isothermal bioreactor as a function of substrate concentration and size of the applied transmembrane temperature difference. It was found that, under non-isothermal conditions and keeping constant the average temperature of the bioreactor, the enzyme reaction rate linearly increased with the increase of the temperature difference.     

The effect of macromolecular conjugates of daunorubicin on nuclear ultrastructure in Trypanosoma brucei rhodesiense

The effects of free and conjugated daunorubicin on T.b. rhodesiense in vitro are described. Free drug caused nucleolar lesions ranging from segregation to complete fragmentation. At equimolar concentrations a soluble bovine serum albumin conjugate with a stable succinyl linkage (D-BSAS) produced no ultrastructural lesions whereas a conjugate with a labile glutaraldehyde linkage (D-BSAG) and a conjugate linked to large agarose beads (D-ag) produced similar though less severe lesions than free drug. Polyisobutylcyanoacrylate nanoparticles caused trypanosomal lysis both with (D-PICA) and without adsorbed daunorubicin.     

Laccase detoxification of steam-exploded wheat straw for second generation bioethanol

In this work we compared the efficiency of a laccase treatment performed on steam-exploded wheat straw pretreated under soft conditions (water impregnation) or harsh conditions (impregnation with diluted acid). The effect of several enzymatic treatment parameters (pH, time of incubation, laccase origin and loading) was analysed. The results obtained indicated that severity conditions applied during steam explosion have an influence on the efficiency of detoxification. A reduction of the toxic effect of phenolic compounds by laccase polymerization of free phenols was demonstrated. Laccase treatment of steam-exploded wheat straw reduced sugar recovery after enzymatic hydrolysis, and it should be better performed after hydrolysis with cellulases. The fermentability of hydrolysates was greatly improved by the laccase treatment in all the samples. Our results demonstrate the action of phenolic compounds as fermentation inhibitors, and the advantages of a laccase treatment to increase the ethanol production from steam-exploded wheat straw.     

A mediated glucose/oxygen enzymatic fuel cell based on printed carbon inks containing aldose dehydrogenase and laccase as anode and cathode

Enzyme electrodes show great potential for many applications, as biosensors and more recently as anodes and cathodes in biocatalytic fuel cells for power generation. Enzymes have advantages over metal catalysts, as they provide high specificity and reaction rates, while operating under mild conditions. Here we report on studies related to development of mass-producible, completely enzymatic printed glucose/oxygen biofuel cells. The cells are based on filter paper coated with conducting carbon inks containing mediators and laccase, for reduction of oxygen, or aldose dehydrogenase, for oxidation of glucose. Mediator performance in these printed formats is compared to relative rate constants for the enzyme-mediator reaction in solution, for a range of anode and cathode mediators. The power output and stability of fuels cells using an acidophilic laccase isolated from Trametes hirsuta is greater, at pH 5, than that for cells based on Melanocarpus albomyces laccase, that shows optimal activity closer to neutral pH, at pH 6. Highest power output, although of limited stability, was observed for ThL/ABTS cathodes, providing a maximum power density of 3.5 μWcm(-2) at 0.34 V, when coupled to an ALDH glucose anode mediated by an osmium complex. The stability of cell voltage above a threshold of 200 mV under a moderate 75 kΩ load is used to benchmark printed fuel cell performance. Highest stability was obtained for a printed fuel cell using osmium complexes as mediators of glucose oxidation by aldose dehydrogenase, and oxygen reduction by T. hirsuta laccase, maintaining cell voltage above 200 mV for 137 h at pH 5. These results provide promising directions for further development of mass-producible, completely enzymatic, printed biofuel cells.     

Production of Laccase and Optimization of Its Production by Bacillus sp. Using Distillery Spent Wash as Inducer

A bacillus sp. isolated from the sediments of a distillery mill was used for laccase production under optimized culture conditions. The distillery effluent was used as an inducer for overproduction of laccase by employing the Taguchi approach. Screening of different medium components and their effect on laccase production was studied using an M-16 orthogonal array. The formation of laccase was considerably increased by addition of 1 mM copper sulfate (51.95 U/ml), which was further enhanced by the use of different inducers. The usefulness of the Taguchi method for optimization of culture conditions was investigated with five selected factors at four levels, and it was observed that the optimized medium resulted in a 9-fold increase in extracellular laccase production compared with the control. The optimized medium composition for laccase production was dextrose (1%), tryptone (0.1%), CuSO₄ (1 mM), and an inducer (distillery effluent 10% [v/v]) at pH 7, which altogether resulted in 107.32 U/ml extracellular laccase activity. Hence, the Taguchi approach proved to be a reliable tool in optimizing culture conditions and achieving the best possible combination for enhanced laccase production.     

Antitumour benzothiazoles. Part 15: The synthesis and physico-chemical properties of 2-(4-aminophenyl)benzothiazole sulfamate salt derivatives

A series of sulfamate salt derivatives of the potent and selective 2-(4-aminophenyl)benzothiazole antitumour agents has been prepared and their evaluation as potential prodrugs for parenteral administration carried out. The salts were sparingly soluble under aqueous conditions (pH 4-9), and degradation to the active free amine was shown to occur under strongly acidic conditions. The salts were found to be markedly less active than their parent amines against sensitive human tumour cell lines in vitro.     

Engineering platforms for directed evolution of Laccase from Pycnoporus cinnabarinus

While the Pycnoporus cinnabarinus laccase (PcL) is one of the most promising high-redox-potential enzymes for environmental biocatalysis, its practical use has to date remained limited due to the lack of directed evolution platforms with which to improve its features. Here, we describe the construction of a PcL fusion gene and the optimization of conditions to induce its functional expression in Saccharomyces cerevisiae, facilitating its directed evolution and semirational engineering. The native PcL signal peptide was replaced by the α-factor preproleader, and this construct was subjected to six rounds of evolution coupled to a multiscreening assay based on the oxidation of natural and synthetic redox mediators at more neutral pHs. The laccase total activity was enhanced 8,000-fold: the evolved α-factor preproleader improved secretion levels 40-fold, and several mutations in mature laccase provided a 13.7-fold increase in k(cat). While the pH activity profile was shifted to more neutral values, the thermostability and the broad substrate specificity of PcL were retained. Evolved variants were highly secreted by Aspergillus niger (～23 mg/liter), which addresses the potential use of this combined-expression system for protein engineering. The mapping of mutations onto the PcL crystal structure shed new light on the oxidation of phenolic and nonphenolic substrates. Furthermore, some mutations arising in the evolved preproleader highlighted its potential for heterologous expression of fungal laccases in yeast (S. cerevisiae).     

Polyester dendritic systems for drug delivery applications: in vitro and in vivo evaluation

High molecular weight polymers (> 20 000 Da) have been widely used as soluble drug carriers to improve drug targeting and therapeutic efficacy. Dendritic polymers are exceptional candidates for the preparation of near monodisperse drug carriers due to their well-defined structure, multivalency, and flexibility for tailored functionalization. We evaluated various dendritic architectures composed of a polyester dendritic scaffold based on the monomer unit 2,2-bis(hydroxymethyl)propanoic acid for their suitability as drug carriers both in vitro and in vivo. These systems are both water soluble and nontoxic. In addition, the potent anticancer drug, doxorubicin, was covalently bound via a hydrazone linkage to a high molecular weight 3-arm poly(ethylene oxide)-dendrimer hybrid. Drug release was a function of pH, and the release rate was more rapid at pH < 6. The cytotoxicity of the DOX-polymer conjugate measured on multiple cancer lines in vitro was reduced but not eliminated, indicating that some active doxorubicin was released from the drug polymer conjugate under physiological conditions. Furthermore, biodistribution experiments show little accumulation of the DOX-polymer conjugate in vital organs, and the serum half-life of doxorubicin attached to an appropriate high molecular weight polymer has been significantly increased when compared to the free drug. Thus, this new macromolecular system exhibits promising characteristics for the development of new polymeric drug carriers.     

p-Benzylthiocarbamoyl-aspartyl-daunorubicin-substituted polytrisacryl. A new drug acid-labile arm-carrier conjugate

A new type of drug acid-labile arm-carrier is described. A hydrophilic polymer [polytrisacryl, or poly(acryloyl 2-amido-2-hydroxymethyl-1,3-propanediol)], used as a model, is substituted with p-nitrobenzyl groups. The nitrobenzyl groups are reduced to aminobenzyl and transformed into benzylisothiocyanate groups which are allowed to react with aminoacyl daunorubicin. The excess of benzylisothiocyanate is transformed into benzylthiocarbamoyl N-methyl glucamine. A conjugate containing benzylthiocarbamoyl-aspartyl daunorubicin is stable at neutral pH, and releases free daunorubicin when it is exposed to pH 5 or below. This conjugate is about 200-fold less toxic than free daunorubicin, on a molecular basis, when it is added to the medium of Lewis lung carcinoma (3LL) cells in culture.     

Protease produced by Pseudomonas aeruginosa K-187 and its application in the deproteinization of shrimp and crab shell wastes

In addition to chitinase/lysozyme, Pseudomonas aeruginosa K-187 also produced a protease useful for the deproteinization of shrimp and crab shell wastes. The optimal culture conditions for P. aeruginosa K-187 to attain the highest protease activity were investigated and discussed. The highest protease activity was as high as 21.2 U/ml, 10-fold that (2.2 U/ml) obtained prior to optimization. The protease of P. aeruginosa K-187, produced under the optimal culture conditions, was tested for crustacean waste deproteinization. The percent of protein removal for shrimp and crab shell powder (SCSP) after 7-day incubation was 72%, while that of natural shrimp shell (NSS) and acid-treated SCSP was 78% and 45%, respectively. In contrast, with the protease produced under pre-optimization conditions, the percent of protein removal for SCSP, NSS, and acid-treated SCSP was 48%, 55%, and 40%, respectively. For comparison, three other protease-producing microbes were tested for crustacean waste deproteinization. However, they were shown to be less efficient in deproteinization than P. aeruginosa K-187. The crude protease produced by P. aeruginosa K-187 can be covalently immobilized on a reversibly soluble polymeric support (hydroxypropyl methycellulose acetate succinate). The immobilized enzyme was soluble above pH 5.5 but insoluble below pH 4.5. Immobilization efficiency was 82%. The immobilized enzyme was stable between pH 6 and 9 and at temperatures below 60 degrees C. The optimum pH and temperature for the immobilized enzyme was pH 8 and 50 degrees C. The half-life of the immobilized enzyme was 12 days, longer than that of free protease (8 days). The utilization of the immobilized enzyme for the deproteinization of SCSP has resulted in a 67% protein removal. By contrast, SCSP protein removal by using free enzymes was 72%. The protease was further purified and characterized. The purification steps included ammonium sulfate precipitation, DEAE-Sepharose CL-6B ion-exchange chromatography, and Sephacryl S-200 gel-permeation chromatography. The enzyme had a molecular weight estimated to be 58.8 kDa by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme was active from pH 7 to 9 and its optimal pH was 8.     

Decolourization of synthetic dyes by a newly isolated strain of Serratia marcescens

A novel dye-decolourizing strain of the bacterium Serratia marcescens efficiently decolourized two chemically different dyes Ranocid Fast Blue (RFB) and Procion Brilliant Blue-H-GR (PBB-HGR) belonging respectively to the azo and anthraquinone groups. Extracellular laccase and manganese peroxidase (MnP) activity were detected during dye decolourization. The involvement of MnP activity was found in the decolourization of both dyes. More than 90% decolourization of PBB-HGR and RFB was obtained on days 8 and 5, respectively at 26 °C under static conditions at pH 7.0. MnP activity was increased by the addition of Mn²⁺ · At 50 M Mn²⁺, high MnP (55.3 U/ml) but low laccase activity (8.3 U/ml) was observed. Influence of oxalic acid on MnP activity was also observed.     

Thiols as myeloperoxidase-oxidase substrates.

Nine low-Mr thiols were compared with regard to their ability to function as myeloperoxidase-oxidase substrates under conditions where no auto-oxidation of the thiols could be observed. The methyl and ethyl esters of cysteine were found to be about twice as active as cysteamine at pH 7.0, in terms of increased O2 consumption. Cysteine itself was poorly active, whereas glutathione, N-acetylcysteine and penicillamine were completely inactive as myeloperoxidase-oxidase substrates under these conditions. The structure-activity relationships indicated that both a free thiol and free amino group were required for peroxidase-oxidase activity, and also that a free carboxy group abolished activity. In analogy with cysteamine, the activities of both cysteine esters were inhibited by superoxide dismutase (less than 5 micrograms/ml) and by catalase and not by the hydroxyl-radical scavenger mannitol. In contrast with cysteamine, the activities of both cysteine esters were stimulated more than 2-fold by high concentrations (greater than 5 micrograms/ml) of superoxide dismutase. The activities of both cysteine esters exhibited broad pH optima at pH 7. A mechanism for the myeloperoxidase-oxidase oxidation of the cysteine esters is proposed, which is partly different from that previously proposed for cysteamine.     

Characterization of Trametes versicolor laccase for the transformation of aqueous phenol

Laccase (oxygen oxidoreductase, EC 1.10.3.2) from Trametes versicolor was thoroughly characterized in terms of its catalytic stability and its effectiveness as a biocatalyst under various reaction conditions when using phenol as a model substrate. This enzyme demonstrated high or moderate degrees of stability at pHs from 5 to 8 at 25 degrees C and at temperatures from 10 to 30 degrees C at pH 6. Exponential decay expressions were successfully used to model laccase inactivation when incubated under various conditions of pH and temperature. Phenol transformation was optimum at pH 6, but significant transformation was observed over a pH range of 4-7, provided that sufficient laccase was present in the reacting solution. Partial inactivation of laccase was observed during the oxidation of phenol, even under conditions of optimal stability (pH 6 and 25 degrees C).     

蜜环菌胞外漆酶的合成、纯化及性质研究

研究了蜜环菌胞外漆酶合成条件和酶学性质。实验表明,培养基初始pH5.5、培养温度25℃有利于菌株产酶;与麦芽糖、山梨糖和半乳糖相比,纤维二糖和棉子糖作为碳源时漆酶产量更高;有机氮源比无机氮源有利于漆酶合成。泥炭提取液可显著诱导漆酶生成,当其含量为50%时,菌株漆酶最高产量是对照组的7倍。在蜜环菌发酵上清液中检测到3个漆酶同功酶组分,其主要活性（约占75%）组份漆酶A经 (NH₄)₂SO₄沉淀、制备级PAGE电泳和阴离子交换柱层析被分离纯化至电泳均一,SDSPAGE法测得酶亚基分子量59kD,凝胶过滤色谱法测定活性酶分子量58kD。纯化的漆酶A等电点pI为4.0,氧化愈创木酚的最适反应pH为5.6,最适温度为60℃,在60℃和65℃时半衰期分别为45min和36.8min,在pH5.2～7.2范围内稳定性较好。100mmol/L Cl^-对该酶有显著抑制作用,1mmol/L SO^2-₄ 对漆酶有激活作用,1mmol/L NaN₃可完全抑制酶活性,10 mmol/L EDTA对漆酶活没有明显影响,1mmol/L Cu²⁺对漆酶有激活作用。以愈创木酚为底物时,测得酶的K_m=1.026mmol/L,V_max=5μmol/(min·mg);以ABTS为底物时,测得其K_m=0.22mmol/L,V_max=69μmol/(min·mg)。     

Effect of Nutritional Parameters on Laccase Production by the Culinary and Medicinal Mushroom, <Emphasis Type="Italic">Grifola frondosa</Emphasis>

Extracellular laccase in cultures of Grifola frondosa grown in liquid culture on a defined medium was first detectable in the early/middle stages of primary growth, and enzyme activity continued to increase even after fungal biomass production had peaked. Laccase production was significantly increased by supplementing cultures with 100–500 μM Cu over the basal level (1.6 μM Cu) and peak levels observed at 300 μM Cu were 7-fold higher than in unsupplemented controls. Decreased laccase activity similar to levels detected in unsupplemented controls, as well as an adverse effect on fungal growth, occurred with further supplementation up to and including 0.9 mM Cu, but higher enzyme titres (2- to 16-fold compared with controls) were induced in cultures supplemented with 1–2 mM Cu²⁺. SDS-PAGE combined with activity staining revealed the presence of a single protein band (M _r 70 kDa) exhibiting laccase activity in control culture fluids, whereas an additional distinct laccase protein band (M _r 45 kDa) was observed in cultures supplemented with 1–2 mM Cu. Increased levels of extracellular laccase activity, and both laccase isozymes, were also detected in cultures of G. frondosa supplemented with ferulic, vanillic, veratric and 4-hydroxybenzoic acids, and 4-hydroxybenzaldehyde. Using 2,2′-azino-bis(ethylbenzothiazoline-6-sulfonate) (ABTS) as substrate, the optimal temperature and pH values for laccase activity were 65°C and pH 2.2, respectively, and the enzyme was relatively heat stable. In solid-state cultures of G. frondosa grown under conditions adopted for industrial-scale mushroom production, extracellular laccase levels increased during the substrate colonization phase, peaked when the substrate was fully colonized, and then decreased sharply during fruit body development.