RESUMO
In this study, we designed and evaluated a microalgal pretreatment method using cellulolytic bacteria that naturally degrades microalgae in their native habitat. Bacterial strains were isolated from each of two mollusk species in a medium containing 1% carboxymethyl cellulose agar. We selected nine bacterial strains that had endoglucanase activity: five strains from Mytilus chilensis, a Chilean mussel, and four strains from Mesodesma donacium, a clam found in the Southern Pacific. These strains were identified phylogenetically as belonging to the genera Aeromonas, Pseudomonas, Chryseobacterium, and Raoultella. The cellulase-producing capacities of these strains were characterized, and the degradation of cell walls in Botryococcus braunii and Nannochloropsis gaditana was tested with "whole-cell" cellulolytic experiments. Aeromonas bivalvium MA2, Raoultella ornithinolytica MA5, and Aeromonas salmonicida MC25 degraded B. braunii, and R. ornithinolytica MC3 and MA5 degraded N. gaditana. In addition, N. gaditana was pretreated with R. ornithinolytica strains MC3 and MA5 and was then subjected to an anaerobic digestion process, which increased the yield of methane by 140.32% and 158.68%, respectively, over that from nonpretreated microalgae. Therefore, a "whole-cell" cellulolytic pretreatment can increase the performance and efficiency of biogas production.
Assuntos
Biocombustíveis , Microalgas/metabolismo , Microalgas/microbiologia , Aeromonas/classificação , Aeromonas/enzimologia , Aeromonas/isolamento & purificação , Biodegradação Ambiental , Celulase/metabolismo , Celulose/metabolismo , Clorófitas/metabolismo , Clorófitas/microbiologia , Chryseobacterium/classificação , Chryseobacterium/enzimologia , Chryseobacterium/isolamento & purificação , DNA Bacteriano/genética , Enterobacteriaceae/classificação , Enterobacteriaceae/enzimologia , Enterobacteriaceae/isolamento & purificação , Metano/metabolismo , Filogenia , Pseudomonas/classificação , Pseudomonas/enzimologia , Pseudomonas/isolamento & purificação , RNA Ribossômico 16S/genéticaRESUMO
Keratinases are exciting keratin-degrading enzymes; however, there have been relatively few studies on their immobilization. A keratinolytic protease from Chryseobacterium sp. kr6 was purified and its partial sequence determined using mass spectrometry. No significant homology to other microbial peptides in the NCBI database was observed. Certain parameters for immobilization of the purified keratinase on chitosan beads were investigated. The production of the chitosan beads was optimized using factorial design and surface response techniques. The optimum chitosan bead production for protease immobilization was a 20 g/l chitosan solution in acetic acid [1.5% (v/v)], glutaraldehyde ranging from 34 g to 56 g/l, and an activation time between 6 and 10 h. Under these conditions, above 80% of the enzyme was immobilized on the support. The behavior of the keratinase loading on the chitosan beads surface was well described using the Langmuir model. The maximum capacity of the support (qm) and dissociation constant (Kd) were estimated as 58.8 U/g and 0.245 U/ml, respectively. The thermal stability of the immobilized enzyme was also improved around 2-fold, when compared with that of the free enzyme, after 30 min at 65 degrees C. In addition, the activity of the immobilized enzyme remained at 63.4% after it was reused five times. Thus, the immobilized enzyme exhibited an improved thermal stability and remained active after several uses.
Assuntos
Quitosana/metabolismo , Chryseobacterium/enzimologia , Enzimas Imobilizadas/metabolismo , Queratinas/metabolismo , Metaloproteases/metabolismo , Sequência de Aminoácidos , Biotecnologia/métodos , Estabilidade Enzimática , Enzimas Imobilizadas/química , Enzimas Imobilizadas/isolamento & purificação , Glutaral/metabolismo , Espectrometria de Massas , Metaloproteases/química , Metaloproteases/isolamento & purificação , Ligação Proteica , Homologia de Sequência de Aminoácidos , TemperaturaRESUMO
The keratinolytic potential and protease properties of three novel Gram-negative feather-degrading bacteria isolated from Brazilian soils was described. Aeromonas hydrophila K12, Chryseobacterium indologenes A22 and Serratia marcescens P3 were able to degrade feather meal, producing high amounts of soluble proteins and forming thiol groups. The proteases of strains K12, A22 and P3 had optimal pH of 8.0, 7.5 and 6.0, respectively; this last is an uncommon feature for bacterial keratinases. The optimal temperature was in the range 45-55°C. All three proteases were active towards azokeratin and were inhibited by EDTA, suggesting that they are keratinolytic metalloproteases. The proteolytic activity of K12 was stimulated by organic solvents and the detergent SDS, suggesting its potential application for detergent formulations and peptide synthesis. Strains A22, K12 and P3 have great potential for use in biotechnological processes involving hydrolysis of keratinous byproducts.
Assuntos
Aeromonas hydrophila/enzimologia , Proteínas de Bactérias/metabolismo , Biodegradação Ambiental , Chryseobacterium/enzimologia , Plumas/metabolismo , Microbiologia Industrial/métodos , Queratinas/metabolismo , Peptídeo Hidrolases/metabolismo , Serratia marcescens/enzimologia , Animais , Brasil , Meios de Cultura , Ácido Edético/farmacologia , Plumas/química , Concentração de Íons de Hidrogênio , Hidrólise/efeitos dos fármacos , Aves Domésticas , Dodecilsulfato de Sódio/farmacologia , Solo , Microbiologia do Solo , Especificidade por Substrato , Temperatura , ResíduosRESUMO
The effects of nutritional conditions on growth and protease production by the feather-degrading Chryseobacterium sp. kr6 were investigated. Higher growth was observed on feather-containing or tryptone (TR) medium when compared to casein (CA) or glucose-nitrogen (GN) base medium. Protease production occurred during growth on feather-containing and TR media, whereas no protease activity was detected on CA or GN medium, indicating that protease production is not constitutive, depending on the presence of specific complex nitrogen sources. Supplementation of whole feathers (WF) medium with glucose (WFG) or NH(4)Cl (WFN) did not result in major differences in growth and protease production, whereas soluble protein was lower in supplemented media. Glucose consumption and growth were higher on WFG than on GN medium, suggesting that the absence of a specific complex nitrogen source limited bacterial growth. On WF medium, this strain grew closely attached to the feather structures, initially on the barbules and subsequently on the feather rachis. It was observed, through zymogram analysis, that strain kr6 produced diverse proteolytic enzymes in response to different growth substrates. These results were confirmed by the differential behaviors of crude proteases towards protease inhibitors.
Assuntos
Chryseobacterium/enzimologia , Chryseobacterium/crescimento & desenvolvimento , Endopeptidases/biossíntese , Plumas/microbiologia , Plumas/patologia , Animais , Proteínas de Bactérias/biossíntese , Meios de Cultura/química , Inibidores Enzimáticos/metabolismo , Plumas/ultraestruturaRESUMO
A novel keratinase from Chryseobacterium sp. strain kr6 was purified to homogeneity by (NH(4))(2)SO(4) precipitation, gel permeation on Sephadex G-100, and Q-Sepharose Fast Flow anion-exchange chromatography. The molecular weight of the purified enzyme was around 20 kDa. Kinetic and thermodynamic parameters for thermal inactivation were determined. The influence of Ca(2+) and Mg(2+) ions and purification degree on the enzyme stability was evaluated in the range of 50 to 60 degrees C. The results showed that first-order kinetics explained well the thermal denaturation of the keratinase in this temperature interval. The presence of Ca(2+) increases significantly the enzyme stability. Compared with the controls, the half-life of the purified enzyme after two purification steps in the presence of Ca(2+) increased 7.3, 20.2, and 9.8 fold at 50, 55, and 60 degrees C, respectively. Thermodynamics parameters for thermal inactivation were also determined.
Assuntos
Chryseobacterium/enzimologia , Peptídeo Hidrolases/química , Termodinâmica , Cálcio/metabolismo , Estabilidade Enzimática , Temperatura Alta , Cinética , Peptídeo Hidrolases/isolamento & purificação , Peptídeo Hidrolases/metabolismo , Desnaturação ProteicaRESUMO
The Chryseobacterium sp. kr6 strain has been described as a highly keratinolytic bacterium showing effective feather-degrading and de-hairing activities. A keratinase Q1 enzyme was purified from Chryseobacterium sp. kr6 culture by Phenyl Sepharose and Superose 12HR chromatography. This enzyme showed a specific activity of 967U/mg for keratin azure. Electrophoresis under denaturing conditions showed a monomeric protein with approximately 64kDa. The enzyme showed pH and temperature optima of 8.5 and 50 degrees C, respectively. The inhibitory effect of EDTA, EGTA and 1,10-phenanthroline characterized Q1 enzyme as a Zn-metalloprotease. Its activity was increased by three-fold in the presence of Ca(2+). ESI-MS/MS analysis of peptides generated from a tryptic digestion revealed sequence homology which may characterize the Q1 keratinase as a member of the M14 metalloprotease family, with a consensus glycosylation region similar to proteins from Chryseobacerium meningosepticum.
Assuntos
Chryseobacterium/enzimologia , Chryseobacterium/genética , Metaloproteases/isolamento & purificação , Peptídeo Hidrolases/isolamento & purificação , Sequência de Aminoácidos , Animais , Clonagem Molecular , Plumas/metabolismo , Concentração de Íons de Hidrogênio , Hidrólise , Metaloproteases/genética , Dados de Sequência Molecular , Peso Molecular , Peptídeo Hidrolases/genética , Aves Domésticas , Homologia de Sequência de Aminoácidos , TemperaturaRESUMO
The strain Chryseobacterium sp. kr6 shown to be useful for biotechnological purposes such as hydrolysis of poultry feathers and de-hairing of bovine pelts. The effect of temperature, initial pH and media composition on protease production by this keratinolytic strain was studied. The enzyme was produced between 25 and 37ºC, with maximum activity and yield at 30ºC. When protease production was tested in media with different initial pH, maximum activity was observed when cultivation was carried out at 30ºC and initial pH ranging from 6.0 to 8.0. Higher activity was observed when feathers or feather meal were used as growth substrates, followed by soybean meal. The addition of carbohydrates or surfactants to feather broth resulted in decrease in keratinolytic activity.