Chemical Modifications of Laccase from White-Rot Basidiomycete Cerrena unicolor

K. H. Kucharzyk G. Janusz I. Karczmarczyk J. Rogalski Laccases belong to the group of phenol oxidizes and constitute one of the most promising classes of enzymes for future use in various fields. For industrial and biotechnological purposes, laccases were among the first enzymes providing larger-scale applications such as removal of polyphenols or conversion of toxic compounds. The wood-degrading basidiomycete Cerrena unicolor C-139, reported in this study, is one of the high-laccase producers. In order to facilitate novel and more efficient biocatalytic process applications, there is a need for laccases with improved biochemical properties, such as thermostability or stability in broad ranges of pH. In this work, modifications of laccase isoforms by hydrophobization, hydrophilization, and polymerization were performed. The hydrophobized and hydrophilized enzyme showed enhanced surface activity and higher ranges of pH and temperatures in comparison to its native form. However, performed modifications did not appear to noticeably alter enzyme's native structure possibly due to the formation of coating by particles of saccharides around the molecule. Additionally, surface charge of modified laccase shifted towards the negative charge for the hydrophobized laccase forms. In all tested modifications, the size exclusion method led to average 80 % inhibition removal for hydrophilized samples after an hour of incubation with fluoride ions. Samples that were hydrophilized with lactose and cellobiose showed an additional 90 % reversibility of inhibition by fluoride ions after an hour of concluding the reaction and 40 % after 24 h. The hydrophobized laccase showed higher level of the reversibility after 1 h (above 80 %) and 24 h (above 70 %) incubation with fluoride ions. The addition of ascorbate to laccase solution before a fluoride spike resulted in more efficient reversibility of fluoride inhibitory effect in comparison to the treatments with reagents used in the reversed sequence. - Laccases (EC 1.10.3.2, p-diphenol: oxygen oxidoreductase) are blue copper-containing oxidases, catalyzing the oxidation of ortho- and para- diphenols, polyphenols, arylamines, aminophenols, and some inorganic ions, while simultaneously reducing molecular dioxygen to water [50, 51, 53]. Laccases are classified into two groups depending on their source: plant and fungal. However, diphenol oxidases have also been identified in bacteria [12] and insects [20]. Three forms of laccase (so called blue, yellow, and white) were discovered in fungi, the most effective producers of this biocatalyst. Laccase is a copper protein containing four metal ions classified as a T1 (one copper), T2 (one copper), and T3 (two coppers) sites according to their spectroscopic characteristics [51]. The T1 copper is responsible for the blue color of the enzyme [48]. An electron from the substrate is transferred to the T1 site (the primary electron acceptor), and then through an intermolecular electron transfer (IET) mechanism via a His-Cys-His bridge to the T2/T3 cluster, where O2 is reduced to H2O [15, 50]. Recently, Cerrena unicolor, was determined as a new fungal source of extracellular laccase, excreting the enzyme under non-induced conditions with the highest activities of 60,000 nkat/l on the 6th day of its cultivation as in Janusz et al. [17]. Several attempts to increase its production including optimization of medium composition and physical parameters of the culture [17, 25, 45] were undertaken. Moreover, laccase from C. unicolor was recently purified and characterized as a glycoprotein with a molecular weight of 45 to 75 kDa, depending on the isoforms composition [2, 33, 45]. Up to date, laccase from C. unicolor was used in biodegradation and bioremediation [4, 5], delignification [27], and decolorization [32, 34]. The fact that laccase has a broad specificity for the phenolic substrates makes it an attractive candidate as a component of biosensor [19], for the determination of total phenols [44] and biofuel cell cathodes [20, 23, 38]. It is known that the substrates attach to the binding site of laccase by hydrophobic interactions [44]. Thus, the effectiveness of electrode constructed of laccase would depend on the quality and quantity of the enzyme, its physical and chemical activity parameters and its ability of surface attachment [9]. Since the new applications of laccases expose it to environments of suboptimal pH and temperature, modifications to develop more resistant enzyme must be found [54]. In the present study, we investigated several techniques that potentially may have altered laccase stability in broad ranges of pH and temperatures, obtained by chemical modifications of enzyme molecule through cross linking, deglicosylation, hydrophobization, or hydrophilization [13, 30, 41, 54]. We have also determined laccases resistance to halides as those factors remain a bottleneck for many new industrial applications of enzymes. Materials and Methods Medium and Growth Conditions C. unicolor C-139 was obtained from the culture collection of the Regensburg University and deposited in the fungal collection at the Department of Biochemistry (Maria CurieSklodowska University, Poland) under the strain number 139. The crude laccase was obtained by fermentor scale cultivation in optimized Lindenberg and Holm medium [17]. The after-culture liquid was centrifuged at 10,000g for 15 min, concentrated 10 times on the ultrafiltration system Pellicon 2 Mini holder (Millipore, Bedford, MA) with an Biomax 10 membrane (10 kDa cut off) and used as the source of crude enzyme. The purification procedure was performed on a chromatographic EconoSystem (Bio-Rad, Richmond, VA). The semi-pure laccase was obtained after the chromatography on a DEAESepharose (fast flow). The purification of laccase isoforms to homogeneity was performed using DEAE-Sepharose ion exchange, vanillyl-CPG (affinity chromatography), and chromatofocusing [45]. Determination of Carbohydrate Content The hydrolysis of laccase carbohydrate compounds was performed according to NikuPaavola et al. with some modifications [39]. The 450 l of samples (0.2 mg protein) was mixed with 50 l 10 % SDS at 100 C for 5 min. Then, Triton X-100 (50 l) and Nglucosidase F (10 l) (Calbiochem, San Diego, CA, USA) were added and incubated for 48 h at 37 C. The obtained hydrolysates were next purified from the residual protein by ultrafiltration on Amicon Ultra-2 filter (3 kDa cut off membrane) using 10,000g and analyzed by HPLC method on a VP chromatographic system (Shimadzu, Tokio, Japan) composed of a LC-10AD pump, a RID-10A refractive index detector, a SCL-10A controller, a CTO 10-AS oven (all of which were controlled by Class VP 5.03 Workstation Software; Shimadzu, 1999) and sampling valve Model 7725 (Rheodyne, Berkeley, USA) with a 20-l loop. The mobile phase (a mixture of acetonitrile and water in the ratio 72: 28 v/v) was run at a flow rate of 1 ml/min through KromosilNH (...truncated)