Effective Purification of Cerrena unicolor Laccase Using Microfiltration, Ultrafiltration and Acetone Precipitation

Abstract

Microfiltration followed by concentration and diafiltration on ultrafiltration membranes (Biomax-100, Biomax-10 and Ultracel-10) was used to recover extracellular laccase (EC 1.10.3.2) from culture broth of wood-rotting fungus Cerrena unicolor. Feed, permeate, retentate and membrane wash-out solutions were analysed for the presence of laccase, proteases, protein and brown impurities. An easy, cheap and short-term procedure was proposed to obtain retentates with low yields of total proteins (less than 14%), proteases activity (less than 15%) and brown impurities (from 2% to 29%) with a simultaneous laccase recovery above 73%. The degree of laccase purification varied from 6.7 to 11.0 and depended on the type of membrane used and content of brown pigments in the feed. Subsequent protein precipitation with cold acetone increased the degree of purification about twice and reduced proteases and brown impurities to some extent. Ultracel-10 membrane was recommended as the best solution to prevent fouling of membranes and to obtain laccase-enriched fraction with a very low content of proteases and brown pigments.

Partitioning of cerrena unicolor laccase activity in an aqueous two-phase system

“…According to Kula et al (1982), about 60-80% of all expenses in biotechnological processes are spent on downstream processing. Various conventional methods, such as micro-and ultrafiltration (Bryjak and Rekuć, 2009), chromatography (Yang et al, 2013), or precipitation (Rajeeva and Lele, 2010), have been employed for downstream processing of laccases. Unfortunately, those methods are highly inefficient in terms of yields and energy consumption.…”

Effectivity of tyrosinase purification by membrane techniques versus fractionation by salting out

“…Activity of proteolytic enzymes was assessed by the non-specific caseinolytic test as in previous paper (Bryjak and Rekuć 2010). One unit of proteolytic activity (U) was defined as the amount of enzyme that induced absorbance (280 nm) increase of 1.0 per 10 min at 37 °C.…”

“…Microfiltration was carried out using an Amicon (USA) stirred cell with 400 mL working volume as presented previously (Bryjak and Rekuć, 2010). Disc membranes were of 41.8 cm 2 effective membrane filtration area.…”

Laccase activity and stability in the presence of menthol-based ionic liquids.

“…Microorganism cultivation and laccase production was performed according to the method described by Al-Adhami et al (2002). The laccase containing culture fluid was purified according to procedure proposed by Bryjak & Rekuć (2010). The enzyme activity was determined from the change of optical density versus time and calculated from the initial reaction rate region, using 0.2 mM ABTS in 0.025 M citrate-phosphate buffer, pH 5.3, as a substrate (Childs & Bardsley, 1975).…”