Abstract
Laccases are glycoprotein polyphenol oxidases which are involved in fungal pathogenicity and they are also useful for biotechnological applications. The ligninolytic ascomycete, Botryosphaeria rhodina, has been studied as producer of exopolysaccharide and PPO-I and PPO-II laccases induced by veratryl alcohol. However, as the induced laccases have not been isolated, the aim of this study was to purify the enzyme and to identify the carbohydrates constituents of the glycosidic moiety. The fungus was cultivated on broth Vogel, 1% glucose and 30.4mM veratryl alcohol during 4.5 days at 28°C/180 rpm. The extracellular fluid showed high carbohydrate concentration and the stability of PPO-I laccase under conditions of refrigeration and freezing at 4ºC-18ºC over 40 days. The purification was developed by ultrafiltration using a NMWL 100 and 30 kDa membrane, gelfiltration on Sephadex G-100, and ion-exchange chromatography on DEAE-cellulose. The purified laccase was identified as a glycoprotein, weight molecular 113 kDa, consisting of 40% protein and 60% carbohydrate identified by HPAEC-PAD as fucose, galactose, mannose, glucose and glucosamine
Highlights
As lacases ρ-difenol: oxigênio oxidoredutase, EC 1.10.3.2 são glicoproteínas polifenoloxidases envolvidas na degradação de lignina
Leonowicz et al (2001) revisaram as propriedades e a atividade de lacases fúngicas e concluíram que polímeros de celulose e de lignina são degradados simultaneamente por sistemas enzimáticos envolvendo reações de desmetilação catalisadas por lacases induzidas por radicais e mediadores de baixo peso molecular resultantes da ação inicial de lignina (LiP) e manganês peroxidases (MnP)
Todas as etapas do isolamento das lacases foram realizadas a 4oC e acompanhadas por determinações de atividade de PPO-I, proteínas e carboidratos
Summary
O fungo Botryosphaeria rhodina MAMB-5 foi cultivado a 28 ± 2°C, sob agitação a 180 rpm durante 4,5 dias em meio líquido de Vogel (1956), contendo glucose 1% (p/v) e álcool veratrílico na concentração de 30,4 mM (veratryl [3,4dimethoxybenzyl] alcohol – Aldrich) como agente indutor (Vasconcelos et al, 2000). O cultivo foi interrompido através de centrifugação a 2000xg por 15 minutos. Após filtração em lã de vidro, o sobrenadante foi dialisado exaustivamente (membrana de porosidade controlada NMWL 12 kDa – Sigma) contra água deionizada por 24 horas a 4oC. O extrato livre de células (ELC) foi analisado quanto à atividade de lacase PPO-I, proteínas e carboidratos, e utilizado para purificação das lacases
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
