High redox potential laccases from the ligninolytic fungi Pycnoporus coccineus and Pycnoporus sanguineus suitable for white biotechnology: from gene cloning to enzyme characterization and applications.

AIMS: Exploitation of natural biodiversity in species Pycnoporus coccineus and Pycnoporus sanguineus to screen for a new generation of laccases with properties suitable for the lignin-processing sector. METHODS AND RESULTS: Thirty strains originating from subtropical and tropical environments, mainly isolated from fresh specimens collected in situ, were screened for laccase activity. On the basis of levels of enzyme activity and percentage of similarity between protein sequences, the laccases from strains BRFM 938, BRFM 66 and BRFM 902 were selected for purification and characterization. Each BRFM 938, BRFM 66 and BRFM 902 laccase gene encoded a predicted protein of 518 amino acids; the three deduced proteins showed 68.7-97.5% similarity with other Polyporale laccases. The three laccases (59.5-62.9 kDa with 7-10% carbohydrate content) had high redox potentials (0.72-0.75 V vs normal hydrogen electrode at pH 6), remained highly stable up to 75-78 degrees C and at pH 5-7 mixtures, and were resistant to methyl and ethyl alcohols, acetonitrile and dimethylsulfoxide at concentrations as high as 50% (v/v). The best laccase-1-hydroxybenzotriazole systems permitted almost 100% of various polyphenolic dye decolourization and oxidation of adlerol and veratryl alcohol. CONCLUSIONS: The three laccases showed complementary biochemical features. BRFM 938 laccase had the highest thermo- and pH stability, catalytic efficiency towards 2,2'-azino-bis-[3-ethylthiazoline-6-sulfonate] and resistance to alcoholic solvents. BRFM 66 laccase had the highest rates of dye decolourization and oxidation of nonphenolic compounds. SIGNIFICANCE AND IMPACT OF THE STUDY: This study identified P. coccineus and P. sanguineus as outstanding producers of high redox potential laccases, easy to purify and scale-up for industrial production. Three new laccases proved to be suitable models for white biotechnology processes and for further molecular breeding to create a new generation of tailor-made enzymes.

The pre-immune variable kappa repertoire of swine is selectively generated from certain subfamilies of Vkappa2 and one Jkappa gene.

Combinatorial diversity is highly restricted during formation of the pre-immune heavy chain repertoire of swine, raising the question of whether the same is true for the pre-immune light chain repertoire. Before addressing this question, we first used competitive PCR to show that kappa and lambda light chains in swine are equally expressed in mature B cells similar to the situation in humans but alike that in other studied Ungulates. This justified efforts to examine the repertoire of both light chain types. These studies also revealed that lambda is preferentially expressed at sites of B cells lymphogenesis, perhaps because of the use of a surrogate light chain containing lambda5. Data are presented here on >100 VkappaJkappa-containing transcripts and approximately 180 genomic Vkappa genes to show that >90% of the pre-immune repertoire is generated from three subfamilies of IGKV2 genes and one of five Jkappa segments. The kappa locus contains >or=50 IGKV2 genes belonging to at least five subfamilies and an undetermined but perhaps equal number of IGKV1 genes. The porcine IGKV1 and IGKV2 genes share 87% sequence similarity with their human counterparts and Jkappa1 through Jkappa5 share sequence and organizational homology with those in sheep, horse, human and mouse. Swine have a single Ckappa gene. These findings contrast with those from rodents and primates but are reminiscent of those on the pre-immune heavy chain repertoire of swine in that it is generated using a relatively restricted number of gene segments. These restricted pre-immune repertoires may reflect the minimal exposure of the fetus to maternal factors and environmental antigens. The significance for swine immunology of characterizing the pre-immune repertoire is discussed.