RESUMO
BACKGROUND: Polygalacturonases are a group of enzymes under pectinolytic enzymes related to enzymes that hydrolyse pectic substances. Polygalacturonases have been used in various industrial applications such as fruit juice clarification, retting of plant fibers, wastewater treatment drinks fermentation, and oil extraction. OBJECTIVES: The study was evaluated at the heterologous expression, purification, biochemical characterization, computational modeling, and performance in apple juice clarification of a new exo-polygalacturonase from Sporothrix schenckii 1099-18 (SsExo-PG) in Pichia pastoris. METHODS: Recombinant DNA technology was used in this study. Two different pPIC9K plasmids were constructed with native signal sequence-ssexo-pg and alpha signal sequence-ssexo-pg separately. Protein expression and purification performed after plasmids transformed into the Pichia pastoris. Biochemical and structural analyses were performed by using pure SsExo-PG. RESULTS: The purification of SsExo-PG was achieved using a Ni-NTA chromatography system. The enzyme was found to have a molecular mass of approximately 52 kDa. SsExo-PG presented as stable at a wide range of temperature and pH values, and to be more storage stable than other commercial pectinolytic enzyme mixtures. Structural analysis revealed that the catalytic residues of SsExo- PG are somewhat similar to other Exo-PGs. The KM and kcat values for the degradation of polygalacturonic acid (PGA) by the purified enzyme were found to be 0.5868 µM and 179 s-1, respectively. Cu2+ was found to enhance SsExo-PG activity while Ag2+ and Fe2+ almost completely inhibited enzyme activity. The enzyme reduced turbidity up to 80% thus enhanced the clarification of apple juice. SsExo-PG showed promising performance when compared with other commercial pectinolytic enzyme mixtures. CONCLUSION: The clarification potential of SsExo-PG was revealed by comparing it with commercial pectinolytic enzymes. The following parameters of the process of apple juice clarification processes showed that SsExo-PG is highly stable and has a novel performance.
Assuntos
Sucos de Frutas e Vegetais/análise , Proteínas Fúngicas/química , Malus/química , Pectinas/química , Poligalacturonase/química , Sporothrix/química , Cátions Bivalentes , Clonagem Molecular , Cobre/química , Estabilidade Enzimática , Tecnologia de Alimentos/métodos , Proteínas Fúngicas/isolamento & purificação , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Ferro/química , Cinética , Peso Molecular , Pichia/genética , Pichia/metabolismo , Poligalacturonase/isolamento & purificação , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Prata/química , Sporothrix/enzimologia , TemperaturaRESUMO
Laccases are polyphenol oxidoreductases used in a number of industrial applications. Due to the increasing demand for these "green catalysis" enzymes, the identification and biochemical characterisation of their novel properties is essential. In our study, cloned Madurella mycetomatis laccase (mmlac) genes were heterologously expressed in the methylotrophic yeast host Pichia pastoris. The high yield of the active recombinant protein in P. pastoris demonstrates the efficiency of a reliably constructed plasmid to express the laccase gene. The optimal biochemical conditions for the successfully expressed MmLac enzyme were identified. Detailed structural properties of the recombinant laccase were determined, and its utility in decolourisation and textile bleaching applications was examined. MmLac demonstrates good activity in an acidic pH range (4.0-6.0); is stable in the presence of cationic metals, organic solvents and under high temperatures (50-60 °C); and is stable for long-term storage at - 20 °C and - 80 °C for up to eight weeks. The structural analysis revealed that the catalytic residues are partially similar to other laccases. MmLac resulted in an increase in whiteness, whilst demonstrating high efficiency and stability and requiring the input of fewer chemicals. The performance of this enzyme makes it worthy of investigation for use in textile biotechnology applications, as well as within environmental and food technologies.
Assuntos
Biotecnologia/métodos , Lacase/química , Lacase/genética , Madurella/genética , Saccharomycetales/metabolismo , Clareadores/química , Catálise , Clonagem Molecular , Estabilidade Enzimática , Expressão Gênica , Concentração de Íons de Hidrogênio , Cinética , Lacase/isolamento & purificação , Madurella/enzimologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , TemperaturaRESUMO
Staphylococcus aureus, among other staphylococcal species, developed multidrug resistance and causes serious health risks that require complex treatments. Therefore, the development of novel and effective strategies to combat these bacteria has been gaining importance. Since Staphylococcus simulans lysostaphin is a peptidoglycan hydrolase effective against staphylococcal species, the enzyme has a significant potential for biotechnological applications. Despite promising results of lysostaphin as a bacteriocin capable of killing staphylococcal pathogens, it is still not widely used in healthcare settings due to its high production cost. In this study, medium engineering techniques were applied to improve the expression yield of recombinant lysostaphin in E. coli. A new effective inducible araBAD promoter system and different mediums were used to enhance lysostaphin production. Our results showed that the composition of autoinduction media enhanced the amount of lysostaphin production 5-fold with the highest level of active lysostaphin at 30 °C. The production cost of 1000 U of lysostaphin was determined as 4-fold lower than the previously proposed technologies. Therefore, the currently developed bench scale study has a great potential as a large-scale fermentation procedure to produce lysostaphin efficiently.
