RESUMEN
Bacterial cellulose (BC) has numerous excellent properties but the absence of antibacterial activity restricts its applications in biomedical field. Therefore, in order to introduce the antibacterial characteristics into BC; herein, a facile method for incorporation of ZnO nanoparticles (ZnO-NPs) is presented. BC films were first immersed in zinc nitrate solution, followed by treating with NaOH solution, the BC loaded ZnO nanocomposite films were dried by a sheet former instrument at 80⯰C for 20â¯min. The obtained BC/ZnO nanocomposites were characterized by different techniques. XRD results showed the hexagonal wurtzite structure of ZnO-NPs while FE-SEM results displayed the particle size of ZnO-NPs was ranging from 70 to 100â¯nm. Thermogravimetric study revealed the thermal stability of nanocomposite films. The nanocomposite exhibited photocatalytic activity and revealed 91% degradation of methyl orange (MO) under UV-irradiation within 2â¯h. Moreover, the nanocomposites demonstrated significant UV-blocking properties and showed antibacterial activities against tested Gram-positive and Gram-negative bacterial strains. This work provides a simple and novel method for the synthesis of BC/ZnO nanocomposite as a functional biomaterial.
Asunto(s)
Antibacterianos/química , Antibacterianos/farmacología , Celulosa/química , Gluconacetobacter xylinus/química , Nanocompuestos/química , Procesos Fotoquímicos , Óxido de Zinc/química , Compuestos Azo/química , Catálisis , Nanopartículas/química , Tamaño de la PartículaRESUMEN
Cross-linked enzyme aggregate (CLEA) technology has been regarded as an effective carrier-free immobilization method. This method is very attractive due to its simplicity and robustness, as well as for the possibility of using the crude enzyme extract and the opportunity to co-immobilize multiple different enzymes. The resulting CLEAs generally exhibit high catalyst productivities, improved storage and operational stability and are easy to recycle. Nowadays, although the technology has been applied to various enzymes, some undesirable properties have limited its further application. To overcome these limitations, novel strategies have been developing in recent years. This mini-review focuses on process optimization, new improved strategies and the latest advances on CLEAs technology.
Asunto(s)
Biotecnología/métodos , Enzimas Inmovilizadas/química , Biocatálisis , Reactivos de Enlaces Cruzados/químicaRESUMEN
Cordyceps militaris is a potential harborer of biometabolites for herbal drugs. For a long time, C. militaris has gained considerable significance in several clinical and biotechnological applications. Much knowledge has been gathered with regard to the C. militaris's importance in the genetic resources, nutritional and environmental requirements, mating behavior and biochemical pharmacological properties. The complete genome of C. militaris has recently been sequenced. This fungus has been the subject of many reviews, but few have focused on its biotechnological production of bioactive constituents. This mini-review focuses on the recent advances in the biotechnological production of bioactive compositions of C. militaris and the latest advances on novel applications from this laboratory and many others.
Asunto(s)
Biotecnología , Cordyceps/genética , Medicina Tradicional China , Cordyceps/química , Cordyceps/metabolismo , HumanosRESUMEN
Novel hybrid magnetic cross-linked enzyme aggregates of phenylalanine ammonia lyase (HM-PAL-CLEAs) were developed by co-aggregation of enzyme aggregates with magnetite nanoparticles and subsequent crosslinking with glutaraldehyde. The HM-PAL-CLEAs can be easily separated from the reaction mixture by using an external magnetic field. Analysis by scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) indicated that PAL-CLEAs were inlayed in nanoparticle aggregates. The HM-PAL-CLEAs revealed a broader limit in optimal pH compared to free enzyme and PAL-CLEAs. Although there is no big difference in Km of enzyme in CLEAs and HM-PAL-CLEAs, Vmax of HM-PAL-CLEAs is about 1.75 times higher than that of CLEAs. Compared with free enzyme and PAL-CLEAs, the HM-PAL-CLEAs also exhibited the highest thermal stability, denaturant stability and storage stability. The HM-PAL-CLEAs retained 30% initial activity even after 11 cycles of reuse, whereas PAL-CLEAs retained 35% of its initial activity only after 7 cycles. These results indicated that hybrid magnetic CLEAs technology might be used as a feasible and efficient solution for improving properties of immobilized enzyme in industrial application.
Asunto(s)
Biotecnología/métodos , Nanopartículas de Magnetita/química , Fenilanina Amoníaco-Liasa/metabolismo , Agregado de Proteínas/fisiología , Rhodotorula/enzimología , Estudios Cruzados , Glutaral/metabolismo , Concentración de Iones de Hidrógeno , Cinética , Magnetismo , Microscopía Confocal , Microscopía Electrónica de Rastreo , Rhodotorula/fisiologíaRESUMEN
Phenylalanine ammonia lyase (PAL) catalyzes the nonoxidative deamination of l-phenylalanine to form trans-cinnamic acid and a free ammonium ion. It plays a major role in the catabolism of l-phenylalanine. The presence of PAL has been reported in diverse plants, some fungi, Streptomyces and few Cyanobacteria. In the past two decades, PAL has gained considerable significance in several clinical, industrial and biotechnological applications. Since its discovery, much knowledge has been gathered with reference to the enzyme's importance in phenyl propanoid pathway of plants. In contrast, there is little knowledge about microbial PAL. Furthermore, the commercial source of the enzyme has been mainly obtained from the fungi. This study focuses on the recent advances on the physiological role of microbial PAL and the improvements of PAL biotechnological production both from our laboratory and many others as well as the latest advances on the new applications of microbial PAL.
Asunto(s)
Fenilanina Amoníaco-Liasa , Bacterias/metabolismo , Hongos/metabolismo , Microbiología Industrial/métodos , Fenilanina Amoníaco-Liasa/aislamiento & purificación , Fenilanina Amoníaco-Liasa/metabolismoRESUMEN
A separable and highly-stable enzyme system was developed by adsorption of phenylalanine ammonia lyase (PAL) from Rhodotorula glutinis in amino-functionalized macroporous silica gel and subsequent enzyme crosslinking. This resulted in the formation of cross-linked enzyme aggregates (PAL-CLEAs) into macroporous silica gel (MSG-CLEAs). The effect of adsorptive conditions, type of aggregating agent, its concentration as well as that of cross-linking agent was studied. MSG-CLEAs production was most effective using ammonium sulfate (40%-saturation), followed by cross-linking for 1 h with 1.5% (v/v) glutaraldehyde. The resulting MSG-CLEAs extended the optimal temperature and pH range compared to free PAL and PAL-CLEAs. Moreover, MSG-CLEAs exhibited the excellent stability of the enzyme against various deactivating conditions such as temperature and denaturants, and showed higher storage stability compared to the free PAL and the conventional PAL-CLEAs. Such as, after 6 h incubation at 60°C, the MSG-CLEAs still retained more than 47% of the initial activity whereas PAL-CLEAs only retained 7% of the initial activity. Especially, the MSG-CLEAs exhibited good reusability due to its suitable size and active properties. These results indicated that PAL-CLEAs on MSG might be used as a feasible and efficient solution for improving properties of immobilized enzyme in industrial application.
Asunto(s)
Enzimas Inmovilizadas/química , Fenilanina Amoníaco-Liasa/química , Gel de Sílice/química , Adsorción , Reactivos de Enlaces Cruzados/química , Activación Enzimática , Concentración de Iones de Hidrógeno , Cinética , Porosidad , Rhodotorula/enzimología , Espectroscopía Infrarroja por Transformada de Fourier , Propiedades de Superficie , Temperatura , TermodinámicaRESUMEN
Cross-linked enzyme aggregates (CLEAs) have been recently proposed as an alternative to conventional immobilization methods on solid carriers. However, the low cross-linking efficiency causes the major activity loss and instability in the conventional protocol for CLEA preparation. Herein, the effects of bovine serum albumin and starch addition on the cross-linking efficiency of CLEAs of phenylalanine ammonia lyase (PAL) from Rhodotorula glutinis were evaluated. A co-aggregation strategy was developed to improve cross-linking efficiency by adding starch and bovine serum albumin (BSA). CLEAs of PAL prepared in the presence of BSA and starch (PSB-CLEAs) retained 36 % activity, whereas CLEAs prepared without BSA and starch (PAL-CLEAs) retained only 8 % activity of the starting enzyme preparation. Compared with PAL-CLEAs, the thermal stability of PSB-CLEAs has improved considerably, maintaining 30 % residual activity after 4 h of incubation at 70 °C, whereas the PAL-CLEAs have only 13 % residual activity. PSB-CLEAs also exhibited the expected increased stability of PAL against hydrophilic organic solvents, superior operability, and higher storage stability. The proposed technique of preparing CLEAs using co-aggregation with starch and BSA would rank among the potential strategies for efficiently preparing robust and highly stable enzyme aggregates.
Asunto(s)
Reactivos de Enlaces Cruzados/química , Fenilanina Amoníaco-Liasa/química , Rhodotorula/enzimología , Albúmina Sérica Bovina/química , Almidón/química , Activación Enzimática , Estabilidad de Enzimas , Enzimas Inmovilizadas/químicaRESUMEN
During the two-stage submerged fermentation of medicinal mushroom Cordyceps militaris, it was found that K(+), Ca(2+), Mg(2+), and Mn(2+) were favorable to the mycelial growth. The EPS production reached the highest levels in the media containing Mg(2+) and Mn(2+). However, Ca(2+) and K(+) almost failed to increase significantly exopolysaccharides (EPS) production. Sodium dodecyl sulfate (SDS) significantly enhanced EPS production compared with that of without adding SDS when SDS was added on static culture stage of two-stage cultivation process. The presence of Tween 80 in the medium not only simulated mycelial growth but also increased EPS production. By response surface methods (RSM), EPS production reached its peak value of 3.28 g/L under optimal combination of 27.6 mM Mg(2+), 11.1 mM Mn(2+), and 0.05 mM SDS, which was 3.76-fold compared with that of without metal ion and surfactant. The results obtained were useful in better understanding the regulation for efficient production of EPS of C. militaris in the two-stage submerged culture.
Asunto(s)
Cordyceps/crecimiento & desarrollo , Cordyceps/metabolismo , Medios de Cultivo/metabolismo , Técnicas de Cultivo/métodos , Metales/metabolismo , Polisacáridos/biosíntesis , Tensoactivos/metabolismo , Biomasa , Concentración de Iones de HidrógenoRESUMEN
Cross-linked enzyme aggregates of phenylalanine ammonia lyase (PAL-CLEAs) from Rhodotorula glutinis were prepared. The effects of the type of aggregating agent, its concentration, and that of cross-linking agent were studied. PAL-CLEAs production was most effective using ammonium sulfate (40 % saturation), followed by cross-linking for 1 h with 0.2 % (v/v) glutaraldehyde. Moreover, the storage and operational stability of the resulting PAL-CLEAs were also investigated. Compared to the free enzyme, the PAL-CLEAs exhibited the expected increased stability of the enzyme against various deactivating conditions such as pH, temperature, denaturants, and organic solvents and showed higher storage stability than its soluble counterpart. Additionally, the reusability of PAL-CLEAs with respect to the biotransformation of L-phenylalanine was evaluated. PAL-CLEAs could be recycled at least for 12 consecutive batch reactions without dramatic activity loss, which should dramatically increase the commercial potential of PAL for synthesis of L: -phenylalanine. To the best of our knowledge, this is the first report of immobilization of PAL as cross-linked enzyme aggregates.
Asunto(s)
Biocatálisis , Reactivos de Enlaces Cruzados/farmacología , Fenilanina Amoníaco-Liasa/química , Fenilanina Amoníaco-Liasa/metabolismo , Fenilalanina/biosíntesis , Estabilidad de Enzimas/efectos de los fármacos , Enzimas Inmovilizadas/química , Enzimas Inmovilizadas/metabolismo , Concentración de Iones de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Compuestos Orgánicos/química , Compuestos Orgánicos/farmacología , Desnaturalización Proteica/efectos de los fármacos , Rhodotorula/enzimología , Solventes/química , Solventes/farmacología , TemperaturaRESUMEN
Curdlan is a microbial polysaccharide composed exclusively of ß-(1,3)-linked glucose residues. Until now only bacteria belonging to the Alcaligenes and Agrobacterium species have been reported to produce Curdlan. In this study, a bacterium capable of producing extracellular Curdlan, identified as Pseudomonas sp. on the basis of 16S rDNA gene sequencing, was isolated from soil samples. From the HPLC, permethylation linkage analysis, (13)C NMR, and FT-IR analytical data, the polysaccharide consisted exclusively of glucose; the most prominent sugar was 1,3-linked glucose, and most glycosidic bonds joining these sugar residues were of the ß-type. This also supported that the exopolysaccharide produced by Pseudomonas sp. was actually Curdlan. In addition, the Pseudomonas sp. was studied for the production of Curdlan by conventional "one-factor-at-a-time technique" and response surface methodology (RSM). It was observed that glucose and yeast extract were the most suitable carbon source and nitrogen source for Curdlan production, respectively. By using RSM, Curdlan production was increased significantly by 188%, from 1.25 to 2.35 g/L, when the strain was cultivated in the optimal condition developed by RSM, and the highest Curdlan production rate of 0.81 g/(L h) was obtained. To the best of the authors' knowledge, this is the first report on Curdlan production by Pseudomonas sp.
Asunto(s)
Espacio Extracelular/metabolismo , Pseudomonas/metabolismo , beta-Glucanos/metabolismo , Espacio Extracelular/química , Datos de Secuencia Molecular , Estructura Molecular , Filogenia , Pseudomonas/clasificación , Pseudomonas/genética , Pseudomonas/aislamiento & purificación , Microbiología del Suelo , Solubilidad , beta-Glucanos/química , beta-Glucanos/aislamiento & purificaciónRESUMEN
Some approaches for improving recombinant phenylalanine ammonia lyase (PAL) stability in Escherichia coli during the enzymatic methods of l-phenylalanine (l-Phe) production were developed following preliminary studies by means of statistical-based experiment designs (response surface method). The traditional non-statistical technology was used to screen four critical factors for PAL stability during the bioconversion process, viz., glycerin, sucrose, 1,4-dithiothreitol (DTT), and MgSO(4). The central composite design (CCD) was applied to optimize the combined effect of critical factors for recombinant PAL stability and understand the relationship between the factors and PAL stability. The optimum values for testing variables were 13.04 mM glycerin, 1.87 mM sucrose, 4.09 mM DTT, and 69 mM Mg(2+). A second-order model equation was suggested and then validated experimentally. The model adequacy was very satisfactory because the coefficient of determination was 0.88. The maximum PAL activity was retained as 67.73 units/g after three successive cycles of bioconversion. In comparison to initial PAL activity, the loss of PAL activity was only 22%. PAL activity was enhanced about 23% in comparison to the control (without any stabilizer additives). PAL stability was significantly improved during successive bioconversion. The results obtained here verified the effectiveness of the applied methodology and may be helpful for l-Phe production on an industrial scale.