Novel cold-adapted raw-starch digesting α-amylases from Eisenia fetida: Gene cloning, expression, and characterization.

We identified the raw-starch-digesting α-amylase genes a earthworm Eisenia fetid α amylase I and II (Ef-Amy I and Ef-Amy II). Each gene consists of 1,530 base pairs (bp) that encode proteins of 510 amino acids, as indicated by the corresponding mRNA sequences. Ef-Amy I and II showed an 89% amino acid identity. The amino acid sequences of Ef-Amy I and II were similar to those of the α-amylases from porcine pancreas, human pancreas, Tenebrio molitor, Oryctolagus cuniculus, and Xenopus (Silurana) tropicalis. Each gene encoding mature Ef-Amy I and II was expressed in the GS115 strain of Pichia pastoris. The molecular masses of the recombinant Ef-Amy I and II were 57 kDa each, and catalytically important residues of α-amylases of the GH family 13 were conserved in both proteins. These amylases exhibited raw-starch-digesting activity at 4 °C. The substrate specificities of rEf-Amy I and II were dissimilar. rEf-Amy I and II were shown to be active even in 40% ethanol, 4 M NaCl, and 4 M KCl.

X-ray crystallographic structural studies of α-amylase I from Eisenia fetida.

The earthworm Eisenia fetida possesses several cold-active enzymes, including α-amylase, ß-glucanase and ß-mannanase. E. fetida possesses two isoforms of α-amylase (Ef-Amy I and II) to digest raw starch. Ef-Amy I retains its catalytic activity at temperatures below 10°C. To identify the molecular properties of Ef-Amy I, X-ray crystal structures were determined of the wild type and of the inactive E249Q mutant. Ef-Amy I has structural similarities to mammalian α-amylases, including the porcine pancreatic and human pancreatic α-amylases. Structural comparisons of the overall structures as well as of the Ca2+-binding sites of Ef-Amy I and the mammalian α-amylases indicate that Ef-Amy I has increased structural flexibility and more solvent-exposed acidic residues. These structural features of Ef-Amy I may contribute to its observed catalytic activity at low temperatures, as many cold-adapted enzymes have similar structural properties. The structure of the substrate complex of the inactive mutant of Ef-Amy I shows that a maltohexaose molecule is bound in the active site and a maltotetraose molecule is bound in the cleft between the N- and C-terminal domains. The recognition of substrate molecules by Ef-Amy I exhibits some differences from that observed in structures of human pancreatic α-amylase. This result provides insights into the structural modulation of the recognition of substrates and inhibitors.

Enzymatic characterization of germination-specific cysteine protease-1 expressed transiently in cotyledons during the early phase of germination.

Papain-like cysteine protease activity that shows a unique transient expression profile in cotyledons of daikon radish during germination was detected. The enzyme showed a distinct elution pattern on DEAE-cellulose compared with cathepsin B-like and Responsive to dessication-21 cysteine protease. Although this activity was not detected in seed prior to imbibition, the activity increased markedly and reached a maximum at 2 days after imbibition and then decreased rapidly and completely disappeared after 5 days. Using cystatin-Sepharose, the 26 kDa cysteine protease (DRCP26) was isolated from cotyledons at 2 days after imbibition. The deduced amino acid sequence from the cDNA nucleotide sequence indicated that DRCP26 is an orthologue of Arabidopsis unidentified protein, germination-specific cysteine protease-1, belonging to the C1 family of cysteine protease predicted from genetic information. In an effort to characterize the enzymatic properties of DRCP26, the enzyme was purified to homogeneity from cotyledons at 48 h after imbibition. The best synthetic substrate for the enzyme was carbobenzoxy-Phe-Arg-4-methylcoumaryl-7-amide. All model peptides were digested to small peptides by the enzyme, suggesting that DRCP26 possesses broad cleavage specificity. These results indicated that DRCP26 plays a role in the mobilization of storage proteins in the early phase of seed germination.

Interleukin-10 promoter gene polymorphisms have no clear influence on interleukin-10 protein secretion in AIDS-associated B-cell lines.

Interleukin-10 (IL-10) is a pleiotropic cytokine involved in several immune responses and expressed by a variety of cell types. IL-10 interacts with at least two subunits of the IL-10 receptors (IL-10R1 and IL-10R2), which are members of the interferon receptor family, and play important roles in ligand binding and signaling. Using reverse transcriptase-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) methods, the mRNA expression and secretion patterns of IL-10 were studied. IL-10R1 and IL-10R2 mRNA expression patterns were also studied in the tumor-derived human B-cell lines. IL-10 protein is expressed and predominantly secreted by AIDS-associated B-cell lines (AABCL). However, IL-10R1 and IL-10R2 are constitutively and ubiquitously expressed in all the B-cell lines included in our study. These results suggest that B-cell IL-10 functions as an autocrine growth factor, in AABCL. Furthermore, we report that higher secretion of IL-10 observed in AABCL could be due to the specific GCC haplotype of IL-10 promoter polymorphisms, although no specific correlation was observed between IL-10 promoter polymorphisms and IL-10 protein secretion as analyzed by PCR-sequence specific primers methodology and ELISA, respectively.