桦褶孔菌漆酶固定化及其对染料的降解

采用壳聚糖交联法和海藻酸钠-壳聚糖包埋交联法固定化桦褶孔菌产生的漆酶,探讨最佳固定化条件,固定化漆酶的温度,pH稳定性及操作稳定性,并以两种固定化酶分别对4种染料进行了降解.结果表明:(1)壳聚糖交联法固定化漆酶的最佳条件为:壳聚糖2.5%,戊二醛7%,交联时间2h,固定化时间5h,给酶量1g壳聚糖小球:1mL酶液(1U/mL),固定化效率56%;(2)海藻酸钠-壳聚糖包埋交联法固定化漆酶的最佳条件为:海藻酸钠浓度4%,壳聚糖浓度0.7%,氯化钙浓度5%,戊二醛浓度0.6%,给酶量4mL 4%海藻酸钠:1mL酶液(1U/mL),固定化效率高达86%;(3)固定化的漆酶相比游离漆酶有更好的温度和pH稳定性;(4)比较两种固定化漆酶,海藻酸钠-壳聚糖包埋交联法固定化酶的温度及酸度稳定性要优于壳聚糖固定化酶,但可重复操作性要弱于后者,两者重复使用8次后的剩余酶活比率分别为71%及64%;(5)两种固定化酶对所选的4种不同结构的合成染料均有较好的降解效果,其中壳聚糖固定化酶对茜素红的降解效果及重复使用性极佳,重复降解40mg/L的茜素红10次,降解率仍保持在100%.     

应用Coriolus vericolor 菌丝球脱色染料及印染废水的研究

对白腐真菌（Coriolus vericolor）产生漆酶进行了研究。发现该菌产漆酶的最适初始pH值为4．5。提高微量元素浓度或添加藜芦醇都可使C.versiclor的产酶能力增加,添加Tween80会有一定的抑制作用。采用C.versicolor菌丝球进行重复分批产酶试验,结果表明菌丝球的稳定性很好,同一批菌丝球可连续利用14次,平均每批酶活力可保持在6．72U／mL,产酶能力优于聚氨酯泡沫固定化菌丝。将粗酶液用要料的脱色降解试验,在酶活力为3．3IU／mL,酸性橙浓度为500mg/L条件下,经过24h反应,脱色率达到98．5％;对含弱酸大红和卡布龙红的印染废水进行脱色试验,脱色率也达到了93％。     

Polyphenoloxidases immobilized in organic gels: Properties and applications in the detoxification of aromatic compounds

Gelatine gels originate from water in oil microemulsions in which the ternary system consists of isooctane/ sulfosuccinic acid bis [2-ethyl hexyl] ester/water; the solubilization of gelatin in the water pool of these microemulsions transforms them into viscous gels in which it is possible to cosolubilize various reactive molecules. These gels were used to immobilize two phenoloxidases, a laccase from Trametes versicolor and a tyrosinase from mushroom. The best balance between gel retention and catalytic activity was reached at a gelatine concentration of 2.5% (w/v) in the case of tyrosinase, while laccase immobilization was independent of gelatine concentration. Both enzymes kept the same optimum pH as the corresponding soluble controls, while a partial loss of activity was observed when they were immobilized. Immobilized enzymes showed an increased stability when incubated for several days at 4 degrees C with a very low release from the gels in the incubation solutions. The immobilization of tyrosinase and of laccase enhanced stability to thermal inactivation. Furthermore, gel-entrapped tyrosinase was almost completely preserved from proteolysis: more than 80% of the activity was maintained, while only 25% of the soluble control activity was detected after the same proteolytic treatments. A column packed with gel-immobilized tyrosinase was used to demonstrate that enzymes immobilized with this technique may be reused several times in the same reaction without loosing their efficiency. Finally, gel-entrapped tyrosinase and laccase were capable of removing naturally occurring and xeno-biotic aromatic compounds from aqueous suspensions with different degrees of efficiency. (c) 1995 John Wiley & Sons, Inc.     

Degradation of chlorophenols catalyzed by laccase

The degradations of 2,4-dichlorophenol (2,4-DCP), 4-chlorophenol (4-CP) and 2-chlorophenol (2-CP) catalyzed by laccase were carried out. The optimal condition regarding degradation efficiency was also discussed, which included reaction time, pH value, temperature, concentration series of chlorophenols and laccase. Results showed that the capability of laccase was the best, while to oxidize 2,4-DCP among the above-mentioned chlorophenols. Within 10 h, the removal efficiency of 2,4-DCP, 2-CP and 4-CP could reach 94%, 75% and 69%, respectively. The optimal pH for laccase to degrade chlorophenols was around 5.5. The increase of laccase concentration or temperature might result in the degradation promotion. The trends of degradation percentage were various among these three chlorophenols with the concentration increase of chlorophenols. Degradation of 2,4-DCP is a first-order reaction and the reaction activation energy is about 44.8 kJ mol⁻¹. When laccase was immobilized on chitosan, crosslinked with glutaraldehyde, the activity of immobilized laccase was lower than that of free laccase, but the stability improved significantly. The removal efficiency of immobilized laccase to 2,4-DCP still remained over 65% after six cycles of operation.     

Immobilization of Laccase by Alginate–Chitosan Microcapsules and its Use in Dye Decolorization

Laccase is a ligninolytic enzyme that is widespread in white-rot fungi. Alginate–chitosan microcapsules prepared by an emulsification–internal gelation technique were used to immobilize laccase. Parameters of the immobilization process were optimized. Under the optimal immobilization conditions (2% sodium alginate, 2% CaCl₂, 0.3% chitosan and 1:8 ratio by volume of enzyme to alginate), the loading efficiency and immobilized yield of immobilized laccase were 88.12% and 46.93%, respectively. Laccase stability was increased after immobilization. Both the free and immobilized laccase alone showed a very low decolorization efficiency when Alizarin Red was selected for dye decolorization test. When 0.1 mM 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) was added into the decolorization system, the decolorization efficiency increased significantly. Immobilized laccase retained 35.73% activity after three reaction cycles. The result demonstrated that immobilized laccase has potential application in dyestuff treatment.     

Biodesulfurization using Pseudomonas delafieldii in magnetic polyvinyl alcohol beads

AIMS: To immobilize Pseudomonas delafieldii R-8 cells in magnetic polyvinyl alcohol (PVA) beads for biodesulfurization. METHODS AND RESULTS: Magnetic PVA beads were prepared by a freezing-thawing technique under liquid nitrogen. The beads have distinct super-paramagnetic properties and their saturation magnetization is 8.02 emu g(-1). The desulfurization rate of the immobilized cells could reach 40.2 mmol kg(-1) h(-1). Desulfurization patterns of dibenzothiophene in model oil with the immobilized and free cells were represented by the Michaelis-Menten equation. The Michaelis constant for both immobilized and free cells was 1.3 mmol l(-1). CONCLUSIONS: The cells immobilized in magnetic PVA beads could be stably stored and be repeatedly used over 12 times for biodesulfurization. The immobilized cells could be easily separated by magnetic field. SIGNIFICANCE AND IMPACT OF THE STUDY: Magnetic PVA beads are easy to prepare. The immobilization process in the paper is to increase the efficiency of cells and to decrease the cost of operations.     

Removal of acetaminophen and carbamazepine in single and binary systems with immobilized laccase from Trametes hirsuta

Laccase-based bioprocesses represent a fascinating prospective for the removal of contaminants of emerging concern in wastewaters. In this work, immobilized laccase from Trametes hirsuta was used to transform carbamazepine (CBZ) and acetaminophen (ACE) in spiked single and binary solutions. The effects of pH, time course and reaction pathways on the transformation were studied. T. hirsuta secreted only laccase. The immobilized laccase was able to degrade 40% and 70% of CBZ and ACE, respectively, in the binary system, while only 5% and 25% of transformation was achieved in the single system for ACE and CBZ, respectively. The maximum removal of acetaminophen was found at pH 7. These obtained results confirm that the acetaminophen is a good laccase mediator compound. The most probable pathway in the binary system involved the formation of acetaminophen dimers and ACE-ACE-CBZ oligomers.     

Immobilization of porcine pancreatic lipase on glycidyl methacrylate grafted poly vinyl alcohol

Graft copolymerization of glycidyl methacrylate (GMA) on to polyvinyl alcohol (PVA) using benzophenone (BP) as initiator was carried out. Grafted PVA was used as carrier for pancreatic lipase immobilization. The effects of GMA and BP concentrations as well as grafting reaction times on grafting yields and activities of the immobilized lipase were determined. The influence of enzyme concentrations was also studied. The optimal conditions for the grafting reaction were: 1 h at 15 mM BP and 2.3 M GMA, the optimum enzyme concentration for immobilization was 1 mg/ml. After optimization of the immobilization process a physical and chemical characterization of the immobilized enzyme was performed. Furthermore, the thermal, pH, storage and operational stability of the immobilized enzyme in comparison to the free form was tested.     

The treatment of chlorophenols with laccase immobilized on sol–gel-derived silica

Laccase, a so-called “blue-copper” oxidase, is able to oxidize a variety of organic compounds. Sol–gel derived silica glasses are frequently adopted as an immobilization method to improve the stability of enzymes and make them reusable. In this study, immobilization conditions were optimized to achieve improved embedding results. The thermal stability, reaction stability and storage stability were improved with the immobilized enzyme when compared to the free enzyme. 2,4-Dichlorophenol (DCP) and 2,4,6-trichlorophenol (TCP) were chosen as model compounds. The treatment of chlorophenols (CPs) by immobilized laccase demonstrated excellent removal and response stability. The affinity of TCP for immobilized laccase was higher than that of DCP. This finding leads to different removal efficiencies under variable conditions (reaction time, initial concentration, dosage of immobilized laccase and removal rate in mixed solution). By fitting the experimental data with the diffusion model of the degradation process, the degradation of CPs by immobilized laccase matches an intraparticle diffusion-controlled model.     

Semi-continuous production of laccase byPhlebia radiata in different culture media

The white-rot fungusPhlebia radiata, immobilized on a polypropylene carrier, was cultivated in a laboratory fermentor under semi-continuous conditions on culture media varying in the content of nitrogen, glucose, vitamins and microelements. Moreover, two laccase inducers were used: veratryl alcohol and veratraldehyde. Throughout the cultivation except the growth phase in the first cycle of fermentation, the observed rate of laccase expression reached up to about 2.0 nkat/mL per 1 h of cultivation, as determined by ABTS oxidation. In most experiments, phenol oxidase activity was determined also in the reaction with syringaldazine, giving reaction rates almost two times lower than in the case of ABTS.     

Laccase production using Pleurotus ostreatus 1804 immobilized on PUF cubes in batch and packed bed reactors: influence of culture conditions

The feasibility of laccase production by immobilization of Pleurotus ostreatus 1804 on polyurethane foam (PUF) cubes with respect to media composition was studied in both batch and reactor systems. Enhanced laccase yield was evidenced due to immobilization. A relatively high maximum laccase activity of 312.6 U was observed with immobilized mycelia in shake flasks compared to the maximum laccase activity of free mycelia (272.2 U). It is evident from this study that the culture conditions studied, i.e. biomass level, pH, substrate concentration, yeast extract concentration, Cu2+ concentration, and alcohol nature, showed significant influence on the laccase yield. Gel electrophoretic analysis showed the molecular weight of the laccase produced by immobilized P. ostreatus to be 66 kDa. The laccase yield was significantly higher and more rapid in the packed bed reactor than in the shake flask experiments. A maximum laccase yield of 392.9 U was observed within 144 h of the fermentation period with complete glucose depletion.     

Sequential reactors for the removal of endocrine disrupting chemicals by laccase immobilized onto fumed silica microparticles

Abstract

The main objective of this study is the evaluation of the capability of laccase from Myceliophthora thermophila immobilized on fumed silica microparticles (fsMP) for the removal of endocrine disrupting chemicals (EDCs) in two enzymatic reactor configurations. This type of support can also be magnetized to allow the straightforward separation of the biocatalyst under a magnetic field. The support exhibited excellent biocompatibility with the enzyme, superior tolerance to pH and temperature as well as improved stability in comparison with the free enzyme, even in the presence of organic solvents and enzyme inhibitors. The technical feasibility of the removal of EDCs by immobilized laccase was assessed in two types of enzymatic reactors operated in sequential mode: a membrane reactor using fsMP-laccase and a reactor with magnetic separation using magnetized fsMP-laccase. The extent of transformation for the target compounds: bisphenol A (BPA) and 17β-estradiol (E2) was high and comparable to free laccase in both systems (up to 80%). The possibility of reusing the immobilized enzyme, especially for magnetized supports, offers an interesting approach in the development of enzyme based processes for the biotransformation of emerging pollutants.     

Ready to use p-Benzoquinone activated supports for biochemical coupling,with special applications for laccase immobilization

Summary A new type of p-Benzoquinone activated support (agarose, polyvinyl alcohol, chitosan) which can be stored for more than one year, is described. Coupling yields of 10–95% were obtained for various compounds (propylamine, serum albumin, alpha-amylase). A retained activity of 150% for the immobilized laccase, was obtained. This peculiar result is probably due to the presence of hydroquinone groups on the support, which, as a substrate of laccase, can generate a simultancous affinity retention of the enzyme.     

Immobilized laccase of Cerrena unicolor for elimination of endocrine disruptor micropollutants

The white-rot fungus Cerrena unicolor C-139 produced 450?000 U l⁻¹ of laccase when cultivated in submerged (50 ml) fermentation of wheat bran. Laccase (benzenediol: oxygen oxidoreductase, EC 1.10.3.2.), from C. unicolor C-139 was immobilized covalently on control porosity carrier silica beads. The activity of the immobilized laccase was approximately 15.8 units per gram of silica beads. The pH optimum was between 2.5 and 3.0 for free and immobilized laccase. The immobilization of enzyme appeared to be the main factor for retention of laccase activity at high temperature of 80 °C. The apparent K_m value (100 μmol) of immobilized laccase from C. unicolor C-139 was 6.7 times higher than free laccase (15 μmol) using 2,2-azino-bis-[3-ethylthiazoline-6-sulfonate] (ABTS) as the substrate. Immobilized laccase was able to eliminate 80 % of Bisphenol A, 40 % of Nonylphenol, and 60 % of Triclosan from solutions containing 50 μmol of each micropollutant separately. The experiments were run three times consecutively with the same immobilized laccase without loss of enzyme activity.     

Stability of immobilized laccase on Luffa Cylindrica fibers and assessment of synthetic hormone degradation

In this work were studied the pH, thermal, and storage stability of free and immobilized laccases. Enzymes were produced by Pleurotus ostreatus on potato dextrose (PD) broth and potato dextrose modified (PDM) broth, and immobilized using Luffa cylindrica fibers as support. Both free and immobilized enzymes were assessed on their respective enzymatic activities and for 17α-ethinylestradiol (EE2) degradation. The optimum pH conditions concerning laccase activity ranged from 3.6 to 4.6, while temperature ranged between 30?°C and 50?°C for both free and immobilized enzyme. Laccase produced using PD broth presented greater storage stability and thermal stability than that of PDM. Best EE2 removals were of 79.22% and 75.00% for the free and immobilized enzymes, respectively. Removal rates were assessed during 8?h at pH 5. The removal of 17α-ethinylestradiol was stabilized in the fourth cycle of use. Results imply that immobilization promoted stability towards pH and temperature variations, although media played a decisive role in the enzymatic activity. Both free and immobilized laccases of P. ostreatus were able to degrade EE2, whereas immobilized laccase in PDM medium presented possible reuse applicability, albeit removal was not optimal when compared to other reports.     

A packed-bed reactor utilizing porous resin enables high density culture of hepatocytes

Summary To enable high density culture of hepatocytes for use as a hybrid artificial liver support system or a bioreactor system, a packed-bed reactor using collagen-coated reticulated polyvinyl formal (PVF) resin was applied to a primary culture of hepatocytes. Cubic PVF resins (2×2×2 mm, mean pore size: 100, 250 or 500 m) were used as supporting substrates to immobilize hepatocytes. Two hundred and fifty cubes were packed in a cylindrical column, and 2.6–11.3×10⁷ hepatocytes were seeded in the column by irrigating with 3 ml of the medium containing hepatocytes. Perfusion culture experiments using this packed-bed reactor, as well as monolayer cultures using conventional collagen-coated petri dishes as control experiments, were performed. Sufficient amounts of hepatocytes were found to be immobilized in the reticulated structure of the PVF resins. The highest density of immobilized hepatocytes attained with PVF resin was 1.2×10⁷ cells/cm³ PVF, which showed levels of ammonium removal and urea-N secretion comparable to those in the monolayer culture. It is concluded that the packed-bed reactor system utilizing PVF resin is a promising process for developing a bioreactor or a bioartificial organ using hepatocytes. Correspondence to: N. Ohshima     

Cooperativity of covalent attachment and ion exchange on alcalase immobilization using glutaraldehyde chemistry: Enzyme stabilization and improved proteolytic activity

Alcalase was scarcely immobilized on monoaminoethyl-N-aminoethyl (MANAE)-agarose beads at different pH values (<20% at pH 7). The enzyme did not immobilize on MANAE-agarose activated with glutaraldehyde at high ionic strength, suggesting a low reactivity of the enzyme with the support functionalized in this manner. However, the immobilization is relatively rapid when using low ionic strength and glutaraldehyde activated support. Using these conditions, the enzyme was immobilized at pH 5, 7, and 9, and in all cases, the activity vs. Boc-Ala-ONp decreased to around 50%. However, the activity vs. casein greatly depends on the immobilization pH, while at pH 5 it is also 50%, at pH 7 it is around 200%, and at pH 9 it is around 140%. All immobilized enzymes were significantly stabilized compared to the free enzyme when inactivated at pH 5, 7, or 9. The highest stability was always observed when the enzyme was immobilized at pH 9, and the worst stability occurred when the enzyme was immobilized at pH 5, in agreement with the reactivity of the amino groups of the enzyme. Stabilization was lower for the three preparations when the inactivation was performed at pH 5. Thus, this is a practical example on how the cooperative effect of ion exchange and covalent immobilization may be used to immobilize an enzyme when only one independent cause of immobilization is unable to immobilize the enzyme, while adjusting the immobilization pH leads to very different properties of the final immobilized enzyme preparation. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2768, 2019.     

Amine functional monodisperse microbeads via precipitation polymerization of N-vinyl formamide: immobilized laccase for benzidine based dyes degradation

Densely cross-linked poly(vinylamine) microbeads (∼2 μm) were prepared by precipitation copolymerization of N-vinyl formamide and ethylene glycoldimethacrylate in acetonitrile. The formamido groups of the microbeads were hydrolyzed into amino groups. Then, amino-functionalized microbeads were used for covalent immobilization of laccase via glutaraldehyde coupling. The average amount of immobilized enzyme was 18.7 mg/g microbeads. Kinetic parameters, V_max and K_m values were determined as 20.7 U/mg protein and 2.76 × 10⁻² mmol/L for free enzyme and 15.8 U/mg protein and 4.65 mmol/L for the immobilized laccase, respectively. The immobilized laccase was operated in a batch reactor for the degradation of two different benzidine based dyes (i.e., Direct Blue 1 and Direct Red 128). The laccase immobilized on the microbeads was very effective for removal of these dyes which interfere with the hormonal system.     

Enzymatic removal of flatulence-inducing sugars in chickpea milk using free and polyvinyl alcohol immobilized α-galactosidase from <Emphasis Type="Italic">Aspergillus oryzae</Emphasis>

The treatment of chickpea milk was carried out in batch, repeated batch and continuous reaction by soluble and polyvinyl alcohol (PVA) immobilized Aspergillus oryzae alpha-galactosidase for the removal of raffinose family oligosaccharides (RFOs). In the batch mode of treatment 96 and 92% of RFOs hydrolysis was observed by soluble and immobilized enzyme, respectively. In repeated batch experiments, immobilized enzyme showed 70% RFOs hydrolysis up to sixth cycle. Polyvinyl alcohol immobilized alpha-galactosidase in fluidized bed reactor showed highest reduction of 94% at a flow rate of 30 ml/h. The results obtained from the present study are very interesting for industrial use of PVA-immobilized enzyme.     

Production of NADP by immobilized cells with NAD kinase

By the radiation-copolymerization method with polyethylene glycoldimethacrylate (PGD) as a main polymerizable reagent, microbial cells of Brevibacterium ammoniagenes were immobilized with high specific activity of NAD kinase and high mechanical strength. The reagents used for the immobilization such as PGD, polyvinyl alcohol (PVA), and N,N'-methylenebisacrylamide (Bis) did not inversely affect the enzyme activity. Freezing and irradiation treatment of the cell-reagent solution did not inactivate the enzyme either, but longer freezing time or a lower irradiation dose (less than 400 krad) resulted in the unsatisfactory mechanical strength of the immobilized cells. Almost all of NAD and ATP consumed were converted into NADP within three hours reaction time. The drum reactor was found to be ideal for the reaction of immobilized cells, since it gave little mechanical stress to the immobilized cells for the effective mixing of the cells and the substrates. The immobilized cells were subjected to three hours reaction repeatedly for 30 times without any activity loss.