Purification of an alpha amylase from Aspergillus flavus NSH9 and molecular characterization of its nucleotide gene sequence

In this study, an alpha-amylase enzyme from a locally isolated Aspergillus flavus NSH9 was purified and characterized. The extracellular α-amylase was purified by ammonium sulfate precipitation and anion-exchange chromatography at a final yield of 2.55-fold and recovery of 11.73%. The molecular mass of the purified α-amylase was estimated to be 54 kDa using SDS-PAGE and the enzyme exhibited optimal catalytic activity at pH 5.0 and temperature of 50 °C. The enzyme was also thermally stable at 50 °C, with 87% residual activity after 60 min. As a metalloenzymes containing calcium, the purified α-amylase showed significantly increased enzyme activity in the presence of Ca2+ ions. Further gene isolation and characterization shows that the α-amylase gene of A. flavus NSH9 contained eight introns and an open reading frame that encodes for 499 amino acids with the first 21 amino acids presumed to be a signal peptide. Analysis of the deduced peptide sequence showed the presence of three conserved catalytic residues of α-amylase, two Ca2+-binding sites, seven conserved peptide sequences, and several other properties that indicates the protein belongs to glycosyl hydrolase family 13 capable of acting on α-1,4-bonds only. Based on sequence similarity, the deduced peptide sequence of A. flavus NSH9 α-amylase was also found to carry two potential surface/secondary-binding site (SBS) residues (Trp 237 and Tyr 409) that might be playing crucial roles in both the enzyme activity and also the binding of starch granules. © 2018, Springer-Verlag GmbH Germany, part of Springer Nature

Bradyrhizobium forestalis sp. nov., an efficient nitrogen-fixing bacterium isolated from nodules of forest legume species in the Amazon

Three strains of nitrogen-fixing bacteria isolated from nodules of Inga sp. (INPA54B(T)) and Swartzia sp. (INPA86A and INPA01-91A) in soils under native forest in the Brazilian Amazon were previously identified as belonging to the Bradyrhizobium genus. In this study, these strains were characterized using a polyphasic approach to establish their taxonomic position. The three strains shared more than 99.5% sequence similarity of the 16S rRNA gene with the type strains of five Bradyrhizobium species (B. japonicum USDA 6(T), B. liaoningense LMG 18230(T), B. ottawaense OO99(T), B. subterraneum 58 2-1(T) and B. yuanmingense LMG 21827(T)). However, multilocus sequence analysis of two (recA and glnII) or three (atpD, gyrB, and recA) housekeeping genes indicated that these three strains represent a new Bradyrhizobium species, which is closely related to B. subterraneum 58 2-1(T) and B. yuanmingense LMG 21827(T). DNA-DNA hybridization values between INPA54B(T) and B. subterraneum 58 2-1(T) and B. yuanmingense LMG 21827(T) were only 41.5 and 30.9%, respectively. Phenotypic characterization also allowed the differentiation of the novel species from B. subterraneum 58 2-1(T) and B. yuanmingense LMG 21827(T). In the phylogenetic analysis of the nodC and nifH genes, the three strains showed similar sequences that were divergent from those of type strains of all Bradyrhizobium species. We concluded that these strains represent a novel species, for which the name Bradyrhizobium forestalis is proposed, with INPA54B(T) (= LMG 10044(T)) as type strain. The G+C content in the DNA of INPA54B(T) is 63.7 mol%