Physical crosslinking of lipase from Rhizomucor miehei immobilized on octyl agarose via coating with ionic polymers: Avoiding enzyme release from the support

Lipase from Rhizomucor miehei (RML) was immobilized on octyl-agarose (OC) at different loadings. Using low enzyme loadings (1/7 of the maximum loading), the incubation of the enzyme with polyethylenimine (PEI) increased the resistance to enzyme desorption in the presence of Triton X-100. However, more than 10% of the enzyme activity could be released from the OC-RML-PEI. The same treatment using fully loaded biocatalyst reduced the enzyme desorption to less than 5%. Further treatment with dextran sulfate (DS) of the PEI treaded immobilized enzyme fully avoids the enzyme desorption even in presence of a Triton X-100 concentration higher than that required for the complete enzyme release from OC-RML. This treatment produced a high stabilization of OC-RML in thermal or organic solvent inactivations, reducing the enzyme release under these drastic conditions. Nevertheless, the support could be recovered by incubation under adequate conditions, and reused in several adsorption/desorption cycles. Thus, the strategy permitted to avoid enzyme desorption, very likely by physical intermolecular crosslinking improving enzyme stability, while still maintaining the reversibility of the immobilization.     

Enzymatic enrichment of polyunsaturated fatty acids using novel lipase preparations modified by combination of immobilization and fish oil treatment

Novel modification methods for lipase biocatalysts effective in hydrolysis of fish oil for enrichment of polyunsaturated fatty acids (PUFAs) were described. Based on conventional immobilization in single aqueous medium, immobilization of lipase in two phase medium composed of buffer and octane was employed. Furthermore, immobilization (in single aqueous or in two phase medium) coupled to fish oil treatment was integrated. Among these, lipase immobilized in two phase medium coupled to fish oil treatment (IMLAOF) had advantages over other modified lipases in initial reaction rate and hydrolysis degree. The hydrolysis degree increased from 12% with the free lipase to 40% with IMLAOF. Strong polar and hydrophobic solvents had negative impact on immobilization-fish oil treatment lipases, while low polar solvents were helpful to maintain the modification effect of immobilization-fish oil treatment. After five cycles of usage, the immobilization-fish oil treatment lipases still maintained more than 80% of relative hydrolysis degree.     

Production of omega-3 polyunsaturated fatty acids through hydrolysis of fish oil by Candida rugosa lipase immobilized and stabilized on different supports

This paper describes the fish oil hydrolysis performed to obtain Omega-3 fatty acids using Candida rugosa lipase (CRL) immobilized and stabilized on different supports. The enzyme was successfully immobilized, presenting higher thermal stability than the free enzyme. Besides, the cationic derivatives were more stable than the others derivatives and free enzyme in methanol, propanol and cyclohexane. Reactions of fish oil hydrolysis were carried out in organic aqueous medium using 10?U of biocatalyst per gram of oil, at 37?°C. After 96?h, the CRL immobilized on cyanogen bromide agarose rendered the lower fish oil hydrolysis, producing 218?μM of Omega-3, which was 1.1-fold more than the hydrolysis catalyzed by free enzyme, while the ionic derivatives rendered the highest fish oil hydrolysis producing 582 and 577?μM of Omega-3 using the carboxymethyl and sulfopropyl derivatives, respectively. The carboxymethyl and the sulfopropyl derivatives resulted in a 2.9-fold increase in the hydrolysis of fish oil, making these derivatives attractive for industrial applications.     

固定化脂肪酶选择性水解废弃鱼油富集DHA和EPA甘油酯

以鱼油下脚料为原料．以固定化Geotrichum sp．脂肪酶为催化剂,选择性水解粗鱼油,使目标脂肪酸EPA和DHA富集于甘油骨架上,显著提高了甘油酯中EPA和DHA的质量分数。通过单因素试验和响应面分析得出最佳水解条件：2g粗鱼油,2．5g水,221．3U脂肪酶,30℃,反应11．7h。最佳条件下的水解度为42．1％。EPA和DHA质量分数分别为7．8％和41．1％。经过二次水解后,水解度提高到45．9％,EPA和DHA质量分数分别提高到8．5％和42％。与初始鱼油相比,分别提高了2倍和2．2倍。     

Preparation of regenerable magnetic nanoparticles for cellulase immobilization: Improvement of enzymatic activity and stability

To obtain regenerable magnetic nanoparticles, triethoxy(3-isocyanatopropyl)silane and iminodiacetic acid (IZ) were used as the starting material and immobilized on Fe₃O₄ nanoparticles. Copper ions (Cu²⁺ ions) were loaded on the Fe-IZ nanoparticles and used for cellulase immobilization. The support was characterized by spectroscopic methods (FTIR, NMR) and thermogravimetric analysis, transmission electron microscopy, scanning electron microscope, X-ray diffraction, energy dispersive X-ray analysis, and vibrating sample magnetometer techniques. As a result of experiments, the amount of protein bound to immobilized cellulase (Fe-IZ-Cu-E) and cellulase activity was found to be 33.1 mg/g and 154 U/g at pH 5, 50°C, for 3 h. The results indicated that the free cellulase had kept only 50% of its activity after 2 h, while the Fe-IZ-Cu-E was observed to be around 77%, at 60°C. It was found that the immobilized cellulase maintained 93% of its initial catalytic activity after its sixth use. Furthermore, the Fe-IZ-Cu-E retained about 75% of its initial activity after 28 days of storage. To reuse the support material (Fe-IZ-Cu), it was regenerated by thorough washing with ammonia or imidazole.     

A novel nanobiosensor for the detection of paraoxon using chitosan-embedded organophosphorus hydrolase immobilized on Au nanoparticles

Organophosphorus (OP) compounds are one of the most hazardous chemicals used as insecticides/pesticide in agricultural practices. A large variety of OP compounds are hydrolyzed by organophosphorus hydrolases (OPH; EC 3.1.8.1). Therefore, OPHs are among the most suitable candidates that could be used in designing enzyme-based sensors for detecting OP compounds. In this work, a novel nanobiosensor for the detection of paraoxon was designed and fabricated. More specifically, OPH was covalently embedded onto chitosan and the enzyme–chitosan bioconjugate was then immobilized on negatively charged gold nanoparticles (AuNPs) electrostatically. The enzyme was immobilized on AuNPs without chitosan as well, to compare the two systems in terms of detection limit and enzyme stability under different pH and temperature conditions. Coumarin 1, a competitive inhibitor of the enzyme, was used as a fluorogenic probe. The emission of coumarin 1 was effectively quenched by the immobilized Au-NPs when bound to the developed nanobioconjugates. However, in the presence of paraoxon, coumarin 1 left the nanobioconjugate, leading to enhanced fluorescence intensity. Moreover, compared to the immobilized enzyme without chitosan, the chitosan-immobilized enzyme was found to possess decreased K_m value by more than 50%, and increased V_max and K_cat values by around 15% and 74%, respectively. Higher stability within a wider range of pH (2–12) and temperature (25–90°C) was also achieved. The method worked in the 0 to 1050?nM concentration ranges, and had a detection limit as low as 5?×?10^?11 M.     

Improvement of activity and stability of Rhizomucor miehei lipase by immobilization on nanoporous aluminium oxide and potassium sulfate microcrystals and their applications in the synthesis of aroma esters

In this study, Rhizomucor miehei lipase (RML) was immobilized on the hexagonally-ordered nanoporous aluminium oxide membranes (RML-Al₂O₃-NP) by adsorption and as protein-coated microcrystals (RML-PCMCs) by simultaneously precipitating RML on micron-sized potassium sulfate crystals (K₂SO₄) in pre-chilled acetone. The hydrolytic activities of immobilized lipase preparations were investigated in terms of p-nitrophenyl palmitate hydrolysis and their esterification activities were examined for the synthesis of some aroma esters such as butyl acetate, isoamyl acetate, hexyl acetate, heptyl acetate, and geranyl acetate. The immobilization yields were 33.8 and 25.1%, respectively for RML immobilized on Al₂O₃-NP membranes and potassium sulfate crystals. The catalytic efficiency ratios of RML-Al₂O₃-NP and RML-PCMCs were 2.3- and 3.9-fold higher than that of the free lipase, respectively in terms of hydrolytic activity. The free lipase was stabilized as 4.1- and 10.5-fold, respectively at 40 and 50?°C when immobilized on Al₂O₃-NP. The corresponding stabilization factors were 4.6- and 12.8-fold higher for RML-PCMCs. RML-Al₂O₃-NP and RML-PCMCs maintained 84 and 86% of their initial hydrolytic activities, respectively after 10 reuses. Of the synthesized aroma esters, the highest yield was obtained for the geranyl acetate. After 4?h reaction time, no geraniol was detected in the preparative-scale (196?g/L) synthesis of geranyl acetate for both the immobilized lipases when the initial geraniol amount, vinyl acetate amount, RML-PCMCs amount, and reaction temperature values were 1?mmol, 3?mmol, 100?mg (or 300?mg RML-Al₂O₃-NP), and 50?°C, respectively. These results show that the immobilization of R. miehei lipase by adsorption on nanoporous aluminium oxide and as protein-coated microcrystals leads to the obtention of highly stable, catalytically more active, and reusable lipase preparations.     

鱼油对断奶大鼠脏器指数及肠道菌群影响的研究

目的探讨鱼油对断奶大鼠脏器指数及肠道菌群的影响。方法选取（21±3）d日龄断奶SD大鼠96只,雌雄各半,随机分为2组,实验组饲喂添加0．5％鱼油的饲料,对照组饲喂正常饲料。分别于第7、14、21、28天处死各组大鼠12只,分别测定大鼠肝脏指数、胸腺指数及脾脏指数;应用梯度稀释法和培养法测定大鼠4种肠道正常菌群,即肠杆菌、双歧杆菌、乳酸杆菌、葡萄球菌。结果实验组第7天脾脏指数与对照组相比差异有显著性（P〈0．05）,第28天肝脏指数和胸腺指数与对照组相比差异有显著性（P〈0．05）;实验组第7、14和21天肠道内肠杆菌数量与对照组相比差异有显著性（P〈0．05）,第7天大鼠肠道内葡萄球菌数量与对照组相比差异有显著性（P〈0．05）,第14天大鼠肠道内葡萄球菌数量与对照组相比差异有非常显著性（P〈0．01）,实验组大鼠肠道内乳酸杆菌和双歧杆菌数量与对照组相比虽有所增高,但差异无显著性（P〉0．05）。结论鱼油可以明显提高断奶大鼠的脏器指数,并且可通过增加断奶大鼠肠道内双歧杆菌和乳酸杆菌数量,降低肠道内肠杆菌和葡萄球菌数量,进而调节断奶大鼠的肠内菌群,改善肠道内环境。     

Partial replacement of dietary fish oil with blends of vegetable oils (rapeseed, linseed and palm oils) in diets for European sea bass (Dicentrarchus labrax L.) over a long term growth study: effects on muscle and liver fatty acid composition and effectiveness of a fish oil finishing diet

Triplicate groups of European sea bass (Dicentrarchus labrax L.), of initial mass 5 g, were fed one of three practical type diets for 64 weeks. The three diets differed only in the added oil and were 100% fish oil (FO; diet A), 40% FO/60% vegetable oil blend (VO; diet B) where the VO blend was rapeseed oil, linseed oil and palm oil in the ratio 10/35/15 by weight and 40% FO/60% VO blend (diet C) where the ratio was 24/24/12 by weight. After final sample collection the remaining fish were switched to a 100% FO finishing diet for a further 20 weeks. After 64 weeks fish fed 60% VO diet B had significantly lower live mass and liver mass than fish fed diets A and C although SGR, FCR and length were not different between groups. There were no differences in any of the above parameters after either 14 or 20 weeks on the FO finishing diet. Fatty acid compositions of flesh were correlated to dietary fatty acids although there was selective retention of docosahexaenoic acid (22:6n-3; DHA) regardless of dietary input. Inclusion of dietary VO resulted in significantly reduced flesh levels of DHA and eicosapentaenoic acid (20:5n-3; EPA) while 18:1n-9, 18:2n-6 and 18:3n-3 were all significantly increased in fish fed the 60% VO diets. Fatty acid compositions of liver showed broadly similar changes, as a result of dietary fatty acid composition, as was seen in flesh. However, the response of flesh and liver to feeding a FO finishing diet was different. In flesh, DHA and EPA values were not restored after 14 or 20 weeks of feeding a FO finishing diet with the values in fish fed the two 60% VO diets being around 70% of the values seen in fish fed FO throughout. Conversely, and despite liver DHA and EPA levels being reduced to only 40% of the value seen in fish fed 100% FO after 64 weeks, the levels of liver DHA and EPA were not significantly different between treatments after feeding the FO finishing diet for 14 weeks. However, a 200 g portion of sea bass flesh, after feeding the experimental diets for 64 weeks followed by a FO diet for 14 weeks, contained 1.22 and 0.95 g of EPA + DHA for fish fed FO or 60% VO, respectively. Therefore, sea bass grown for most of the production cycle using diets containing 60% VO can still contribute a significant quantity of healthy n-3 HUFA to the human consumer.     

Replacement of fish oil with sunflower oil in feeds for Atlantic salmon (Salmo salar L.): effect on growth performance,tissue fatty acid composition and disease resistance

Dietary sunflower oil (SFO) was used to gradually replace fish oil (FO) in six diets (which also contained fish meal) for Atlantic salmon parr (initial mass: 21.7 g). The effect on growth performance, tissue fatty acid profiles and disease resistance was monitored after 63 days. At the conclusion of the trial, no significant differences were detected in growth between any of the feeds. Fatty acid composition of whole carcass, dorsal muscle and liver generally reflected that of the diets. Forty percent of the FO could be replaced by SFO before tissue 22:6n-3 was significantly reduced, although other essential and non-essential fatty acids were more susceptible to change. Significant differences were detected in cumulative mortality of Atlantic salmon challenged with Vibrio anguillarum at the trials conclusion, although this was not correlated to the inclusion level of SFO. Despite the changes observed to the tissue fatty acid profile, there was no significant effect on growth suggesting that SFO is a suitable alternative to FO in diets for Atlantic salmon parr when fish meal is also included.     

Comparison of hydrolysis and esterification behavior of Humicola lanuginosa and Rhizomucor miehei lipases in AOT-stabilized water-in-oil microemulsions: II. Effect of temperature on reaction kinetics and general considerations of stability and productivity

Humicola lanuginosa lipase (HIL) and Rhizomucor miehei lipase (RrnL), isolated from commercial preparations of Lipolase and Lipozyme, respectively, were solubilized in AOT-stabilized water-in-oil (w/o) microemulsions in n-heptane and aspects of their hydrolysis and condensation activity examined. The temperature dependence of HIL hydrolysis activity in unbuffered R = 10 microemulsions matched very closely that for tributyrin hydrolysis by Lipolase in an aqueous emulsion assay. Apparent activation energies were measured as 13 +/- 2 and 15 +/- 2 kJ mol / respectively. Condensation activity, however, was essentially independent of temperature over the range 5 degrees to 37 degrees C. The stability of HIL over a 30-day period was very good at all pH levels (6.1, 7.2, 9.3) and R values studied (5, 7.5, 10, 20), except when high pHs and low R values were combined. The excellent stability was reflected by the linearity of the productivity profiles which facilitate system optimization. The temperature dependence of RmL hydrolysis activity toward pNPC(4) showed a maximum at 40 degrees C and an apparent E(act) = 20 +/- 2 kJ mol(-1) was calculated based on the linear region of the profile (5 degrees to 40 degrees C). RmL esterification activity showed only a slight dependence on temperature over the studied range (0 degrees to 40 degrees C) and an apparent E(act) = 5 +/- 1 kJ mol(-1) was measured for octyl decanoate synthesis. Both RmL and HIL, therefore, have potential for application in low temperature biotransformations in microemulsion-based media. The stability of RmL over a 30-day period was good in R = 7.5 and R = 10 microemulsions containing pH 6.1 buffer, and this was reflected in the linearity of their respective productivity profiles. RmL stability was markedly poorer at more alkaline pH, however, and proved to be sensitive to relatively small changes in the R value. (c) 1995 John Wiley & Sons, Inc.     

Laccase from Pleurotus sajor-caju on functionalised SBA-15 mesoporous silica: Immobilisation and use for the oxidation of phenolic compounds

Laccase from Pleurotus sajor-caju was immobilised on functionalised SBA-15 mesoporous silica. The immobilisation process reached the equilibrium after about 100 min. In order to study the effect of loading (L) on activity of the immobilised laccase, the adsorption isotherm was built and the activity of the corresponding immobilised biocatalysts was determined. The activity of the immobilised preparations reached a maximum at L = 217 kU g_SBA-15⁻¹, whereas higher loadings gave rise to a less-efficient biocatalyst. The immobilised laccase was used for the oxidation of a mixture of four phenolic compounds (protocatechuic acid, ferulic acid, sinapic acid and caffeic acid) chosen among those present in olive mill wastewaters (OMWs). These compounds determine the phytotoxicity of OMWs. Different reaction rates were observed for the oxidation of the examined phenolic compounds. The biocatalyst was recycled and a conversion of 84 mol% at the 10th reuse and of about 60 mol% after the 14th reuse was obtained. In conclusion, the laccase immobilised on SBA-15 is a potential biocatalyst for bioremediation of OMWs, which is an important environmental problem in the regions around the Mediterranean Sea.     

Effect of dietary fish oil on mouse testosterone level and the distribution of eicosapentaenoic acid-containing phosphatidylcholine in testicular interstitium

Low levels of serum testosterone are characteristically associated with diabetes, coronary atherosclerosis, obstructive sleep apnea, rheumatoid arthritis, and chronic obstructive pulmonary disease. Testosterone replacement therapy is effective against many of these disorders, indicating the importance of maintaining a healthy testosterone level. In this study, we investigated the effects of fish oil on murine testosterone metabolism and analyzed the dynamics of relevant lipids in testes by matrix-assisted laser desorption ionization mass spectrometry imaging. Testosterone was upregulated in mice that received fish oil. In the testicular interstitium, eicosapentaenoic acid-containing phosphatidylcholine was distributed characteristically. These data suggest that eicosapentaenoic acid is involved in testosterone metabolism.     

Biotechnology for the production of nutraceuticals enriched in conjugated linoleic acid: I. Uniresponse kinetics of the hydrolysis of corn oil by a Pseudomonas sp. lipase immobilized in a hollow fiber reactor

The kinetics of the hydrolysis of corn oil in the presence of a lipase from Pseudomonas sp. immobilized within the walls of a hollow fiber reactor can be modeled in terms of a three‐parameter rate expression. This rate expression consists of the product of a two‐parameter rate expression for the hydrolysis reaction itself (which is of the general Michaelis–Menten form) and a first‐order rate expression for deactivation of the enzyme. Optimum operating conditions correspond to 30°C and buffer pH values of 7.0 during both immobilization of the enzyme and the hydrolysis reaction. Under these conditions, the total fatty acid concentration in the effluent oil stream for a fluid residence time of 4 h is approximately 1.6 M. This concentration corresponds to hydrolysis of approximately 50% of the glyceride bonds present in the feedstock corn oil. The fatty acid of primary interest in the effluent stream is linoleic acid. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 64: 568–579, 1999.     

Enzymatic esterification of an acid with an epoxide using an immobilized lipase from Mucor miehei as catalyst: optimization of the yield and isomeric excess of ester by statistical analysis

We have developed the methodology for the esterification of an acid with an epoxide using 2-chlorobutyric acid and 1,2-epoxy-5-hexene catalysed by a Mucor miehei-immobilized lipase. Thus, this methodology could be applied to obtain 2-chloroesters. A factorial design of experiments and a central composite design have been used to optimise the synthesis of these esters. The variables chosen were temperature and initial catalyst concentration, while the responses were yield and isomeric excess of the ester. According to this study, temperature was the most important factor, having a positive influence on the yield and a small negative influence on the isomeric excess of the ester. The yield and isomeric excess of the ester show a greater dependence on temperature compared to the catalyst concentration. Although the effect of the catalyst concentration on both responses is smaller than the temperature effect, the higher selectivity presented by the biocatalyst towards the studied ester considerably decreased the final product distribution. Journal of Industrial Microbiology & Biotechnology (2002) 28, 173–179 DOI: 10.1038/sj/jim/7000218 Received 27 June 2001/ Accepted in revised form 25 October 2001     

Immobilization of glucose oxidase in thin polypyrrole films: influence of polymerization conditions and film thickness on the activity and stability of the immobilized enzyme

Using monomers that polymerize to form electrically conducting polymers, one can control the thickness of the polymer film and the amount of enzyme that can be immobilized in the films. First, an investigation of the major variables that influence the immobilization of glucose oxidase by entrapment in polypyrrole films, prepared by electropolymerization from aqueous solutions containing the enzyme and monomer, was carried out. Then the optimized conditions were used to assess the effects of film thickness on the activity and stability of immobilized enzyme. For the films ranged in thickness from 0.1 mum to 1.6 mum, the resulting apparent activity and stability of the immobilized enzyme were found to be a strong function of the polymer film thickness. Above a thickness of 1.0 mum, the apparent activity of the immobilized enzyme increases linearly with increasing film thickness. The nonlinearity observed for films of thickness less than 1.0 mum can be attributed to the changes observed in the morphology of the resulting polypyrrole films. Furthermore, it was noted that when the glucose oxidase/polypyrrole films are stored in phosphate buffer, at 4 degrees C, the observed rate of loss in apparent activity of the immobilized enzyme is highest for the first few days, also being higher for the thinner films. However, after the loosely entrapped enzyme is leached from the polymer film, the rate of loss in activity is very low indicating that the well-entrapped enzyme, as well as the polypyrrole films, exhibit good stability. Finally, the reproducibility of the immobilization technique is excellent. (c) 1993 John Wiley & Sons, Inc.     

Effects of FADS and ELOVL polymorphisms on indexes of desaturase and elongase activities: results from a pre-post fish oil supplementation

Polymorphisms (SNPs) within the FADS gene cluster and the ELOVL gene family are believed to influence enzyme activities after an omega-3 (n-3) fatty acid (FA) supplementation. The objectives of the study are to test whether an n-3 supplementation is associated with indexes of desaturase and elongase activities in addition to verify whether SNPs in the FADS gene cluster and the ELOVL gene family modulate enzyme activities of desaturases and elongases. A total 208 subjects completed a 6-week supplementation period with 5 g/day of fish oil (1.9–2.2 g/day of EPA + 1.1 g/day of DHA). FA profiles of plasma phospholipids were obtained by gas chromatography (n = 210). Desaturase and elongase indexes were estimated using product-to-precursor ratios. Twenty-eight SNPs from FADS1, FADS2, FADS3, ELOVL2 and ELOVL5 were genotyped using TaqMan technology. Desaturase indexes were significantly different after the 6-week n-3 supplementation. The index of δ-5 desaturase activity increased by 25.7 ± 28.8 % (p < 0.0001), whereas the index of δ-6 desaturase activity decreased by 17.7 ± 18.2 % (p < 0.0001) post-supplementation. Index of elongase activity decreased by 39.5 ± 27.9 % (p < 0.0001). Some gene–diet interactions potentially modulating the enzyme activities of desaturases and elongases involved in the FA metabolism post-supplementation were found. SNPs within the FADS gene cluster and the ELOVL gene family may play an important role in the enzyme activity of desaturases and elongases, suggesting that an n-3 FAs supplementation may affect PUFA metabolism.     

Improvement of the enantioselectivity and activity of lipase from Pseudomonas sp. via adsorption on a hydrophobic support: kinetic resolution of 2-octanol

Pseudomonas sp. lipase (PSL) was successfully immobilized on a novel hydrophobic polymer support through physical adsorption and the immobilized PSL was used for resolution of (R,S)-2-octanol with vinyl acetate as acyl donor. Enhanced activity and enantioselectivity were observed from the immobilized PSL compared with free PSL. The effects of reaction conditions such as temperature, water activity, substrate molar ratio and the amount of immobilized lipase were investigated. Under optimum conditions, the residual (S)-2-octanol was recovered with 99.5% enantiomeric excess at 52.9% conversion. The results also indicated that the immobilized PSL could maintain 94% of its initial activity even after reusing it five times.     

Oriented and selective enzyme immobilization on functionalized silica carrier using the cationic binding module Z basic2: design of a heterogeneous D-amino acid oxidase catalyst on porous glass

D-amino acid oxidase from Trigonopsis variabilis (TvDAO) is applied in industry for the synthesis of pharmaceutical intermediates. Because free TvDAO is extremely sensitive to exposure to gas-liquid interfaces, biocatalytic processing is usually performed with enzyme immobilizates that offer enhanced stability under bubble aeration. We herein present an "Immobilization by Design" approach that exploits engineered charge complementarity between enzyme and carrier to optimize key features of the immobilization of TvDAO. A fusion protein between TvDAO and the positively charged module Z(basic2) was generated, and a corresponding oppositely charged carrier was obtained by derivatization of mesoporous glass with 3-(trihydroxysilyl)-1-propane-sulfonic acid. Using 250 mM NaCl for charge screening at pH 7.0, the Z(basic2) fusion of TvDAO was immobilized directly from E. coli cell extract with almost absolute selectivity and full retention of catalytic effectiveness of the isolated enzyme in solution. Attachment of the homodimeric enzyme to the carrier was quasi-permanent in low-salt buffer but fully reversible upon elution with 5 M NaCl. Immobilized TvDAO was not sensitive to bubble aeration and received substantial (≥ tenfold) stabilization of the activity at 45°C as compared to free enzyme, suggesting immobilization via multisubunit oriented interaction of enzyme with the insoluble carrier. The Z(basic2) enzyme immobilizate was demonstrated to serve as re-usable heterogeneous catalyst for D-amino acid oxidation. Z(basic2) -mediated binding on a sulfonic acid group-containing glass carrier constitutes a generally useful strategy of enzyme immobilization that supports transition from case-specific empirical development to rational design.     

Direct and indirect impacts of predation by fish on the zooplankton community: an experimental analysis using tanks

The impact of Pseudorasbora parva, a common zooplanktivorous fish species in Japan, on a zooplankton community was analyzed in experimental tanks, half of which were stocked with the fish. Different zooplankton species showed different responses to the introduction of the fish. In the presence of the fish, the populations of the large cladoceran Ceriodaphnia and the predatory copepod Mesocyclops were reduced, but the population of the herbivorous copepod Eodiaptomus and the small cladocerans Bosmina fatalis and Bosminopsis deitersi increased relative to the controls. The increase of Mesocyclops seen in the control tanks might have suppressed the populations of the small cladocerans, which are vulnerable to invertebrate predation. The results suggest that the population densities of the large prey items preferred by the fish, Ceriodaphnia and Mesocyclops, were controlled directly by fish predation, but the population densities of the smaller and less preferred zooplankton were controlled indirectly through the food-web cascade.