Background: Laccases are a class of multi-copper oxidases that can catalyze the oxidation of various phenolic substrates while reduacing molecular oxygen to water. Although only a few bacterial laccases have been studied to date, recent advances in genome research suggest that these enzymes are widespread in bacteria. Due to their ability to oxidize a broad range of phenolic compounds, laccases have numerous biotechnological applications. The aim of this study was to isolate the gene encoding laccase (CotA) from recombinant Escherichia coli BL21 (DE3) containing the Bacillus licheniformis LS04 CotA-laccase gene and investigate its properties.Methods: The bacterial strains, vectors, and growth conditions were used in the study, and also the recombinant and expression host strain construction was described. Plasmid isolation, PCR amplification, gel electrophoresis, and protein purification were also carried out. SDS-PAGE was used to visualize the protein bands and plasmid stability was analyzed. In addition, this study characterized the CotA laccase by evaluating its optimum temperature, pH, thermal stability, and activity after bathing at 50 °C for 10 min.Results: The results showed that the CotA laccase produced a protein with a molecular weight of 65 kDa, and the plasmid was stable in the absence of antibiotic pressure for 200 generations. The pH profile for laccase activity showed a peak at pH 7.4, and the optimal temperature was found to be 45 °C. However, the pH and temperature stability of the CotA laccase was lower than that of the spore laccase.Conclusion: The purified recombinant CotA-laccase showed high stability towards alkaline pH, high temperatures, and a broad pH range for catalyzing substrates. Nevertheless, the study demonstrates that CotA-laccase has the potential for industrial use due to its high stability and broad substrate range.

To clarify taxonomic complexity within the Anthemis species, pollen grains of 19 species belonging to three sections of the genus Anthemis sensu stricto and two species of the genus Cota from Iran were examined by using scanning electron microscopy. The pollen grains morphological characteristics are provided for this genus in detail. Pollen grains were trizonocolporate and echinate. The pollen grains shape was prolate and spheroidal with the mean polar axes 20– 35 μ m and the mean equatorial diameter 20. 3– 26. 5 μ m. Among the studied taxa, A. microcephala showed the smallest pollen grains, and A. odontostephana possessed the largest ones. The spines were commonly conical with a broad basis, ornamentation within spines was generally rugulate-perforate. Thirteen pollen quantitative and qualitative characters have been analysed statistically by employed UPGMA, PCA and MDS in PAST software. Numerical analysis showed that, features as pollen shape, exine ornamentation, spine length and density were valuable characters for separating the examined taxa. In the resulting clusters, three groups within the investigated species have been recognized. The results derived from the present study, therefore, proved to be concomitant with the previous study on taxonomic infrageneric classification. The palynological evidences confirmed the sectional divisions, and revealed that, A. brachystephana and A. lorestanica in A. sect. Anthemis, have close relationship with A. odontostephana. The palynological characters of sect. Anthemis were similar to Tripleurospermum and Cota. The analysis of the palynological data confirmed the conventional taxonomic classification of the genus Anthemis rather than the phylogenetic classification of the genus.

The pollen grains of 32 populations from 13 genera belonging to the tribe Anthemideae - Asteraceae (one to three populations of each species) from Iran were studied by light and scanning electron microscopes. Pollen grains of all the studied samples were tricolporate. Based on pollen shape, 55, 22, 15 and 8 % were oblate- sp heroidal, suboblate, spheroidal and prolate - spheroidal, respectively. The pollen grains with oblate- spheroidal and spheroidal shapes were common among the genera studied.The pollen grains of all taxa were operculate and tricolporate. The pollen ornamentat ions were echinate perforate except Artemisia that was spinulose non- perforate in SEM.The dendrogram obtained from UPGMA method based on four quantitative and qualitative characters was classified into two main clusters. The first cluster (A) includes pop ulations of Artemisia which show "Artemisia - type" pollen, while the second cluster (B) comprises of the rest of the taxa which have pollen grains of "Anthemis - type". The subcluster B-1 possesses samples with bigger (polar axis 28.64- 32.5 mm and equatorial axis 30.65- 33.2 mm ) pollen grains and subcluster B - 2 comprised of populations with smaller (polar axis 21.69 - 30.38 mm and equatorial axis 23.29-32.4 mm ) pollen grains. The results showed that pollen micromorphological characters could be utilized in inter pretation of intergeneric relationship of tribe Anthemideae. Moreover, pollen size and shape were determined as taxonomic diagnostic characteristics.

Laccases have many biotechnological applications due to their oxidation ability towards a wide range of phenolic compounds. In this study, the strain QZA2 isolated from Iran soils was used. This strain showed high similarity to Bacillus anthracis strain ATCC 14578 according to 16S rRNA gene sequence. Laccase gene was amplified by cloning primers. then PCR product cloned in the expression vector (pET21a) and transferred to BL21(DE3) strain of E. coli under the control phage T7 and sequence analysis carried out. The gene of the QZA2 has an open reading frame composed of 1656 bases, which encodes 551 amino acid residues and contain four histidine rich copper-binding domains. The laccase gene from QZA2 showed 16% similarity with CotA from B. subtilis and 57% similarity with multicopper oxidase B. halodurans. The expression was performed under microaerobic condition in order to obtain high amounts of soluble protein. Biochemical properties were investigated using common laccase substrates ABTS.

Purpose: Robust pharmaceutical and industrial enzymes from extremophile microorganisms are main source of enzymes with tremendous stability under harsh conditions which make them potential tools for commercial and biotechnological applications.Methods: The genome of a Gram-positive halo-thermotolerant Bacillus sp. SL1, new isolate from Saline Lake, was investigated for the presence of genes coding for potentially pharmaceutical enzymes. We determined gene sequences for the enzymes laccase (CotA), l-asparaginase (ansA3, ansA1), glutamate-specific endopeptidase (blaSE), l-arabinose isomerase (araA2), endo-1, 4-b mannosidase (gmuG), glutaminase (glsA), pectate lyase (pelA), cellulase (bglC1), aldehyde dehydrogenase (ycbD) and allantoinases (pucH) in the genome of Bacillus sp. SL1.Results: Based on the DNA sequence alignment results, six of the studied enzymes of Bacillus sp. SL-1 showed 100% similarity at the nucleotide level to the same genes of B. licheniformis 14580 demonstrating extensive organizational relationship between these two strains. Despite high similarities between the B. licheniformis and Bacillus sp. SL-1 genomes, there are minor differences in the sequences of some enzyme. Approximately 30% of the enzyme sequences revealed more than 99% identity with some variations in nucleotides leading to amino acid substitution in protein sequences.Conclusion: Molecular characterization of this new isolate provides useful information regarding evolutionary relationship between B. subtilis and B. licheniformis species. Since, the most industrial processes are often performed in harsh conditions, enzymes from such halo-thermotolerant bacteria may provide economically and industrially appealing biocatalysts to be used under specific physicochemical situations in medical, pharmaceutical, chemical and other industries.