Engineered dextranase from Streptococcus mutans enhances the production of longer isomaltooligosaccharides

Herein, we investigated enzymatic properties and reaction specificities of Streptococcus mutans dextranase, which hydrolyzes α-(1→6)-glucosidic linkages in dextran to produce isomaltooligosaccharides. Reaction specificities of wild-type dextranase and its mutant derivatives were examined using dextran and a series of enzymatically prepared p-nitrophenyl α-isomaltooligosaccharides. In experiments with 4-mg·mL⁻¹ dextran, isomaltooligosaccharides with degrees of polymerization (DP) of 3 and 4 were present at the beginning of the reaction, and glucose and isomaltose were produced by the end of the reaction. Increased concentrations of the substrate dextran (40 mg·mL⁻¹) yielded isomaltooligosaccharides with higher DP, and the mutations T558H, W279A/T563N, and W279F/T563N at the -3 and -4 subsites affected hydrolytic activities of the enzyme, likely reflecting decreases in substrate affinity at the -4 subsite. In particular, T558H increased the proportion of isomaltooligosaccharide with DP of 5 in hydrolysates following reactions with 4-mg·mL⁻¹ dextran.Abbreviations CI: cycloisomaltooligosaccharide; CITase: CI glucanotransferase; CITase-Bc: CITase from Bacillus circulans T-3040; DP: degree of polymerization of glucose unit; GH: glycoside hydrolase family; GTF: glucansucrase; HPAEC-PAD: high performance anion-exchange chromatography-pulsed amperometric detection; IG: isomaltooligosaccharide; IGn: IG with DP of n (n, 2‒5); PNP: p-nitrophenol; PNP-Glc: p-nitrophenyl α-glucoside; PNP-IG: p-nitrophenyl isomaltooligosaccharide; PNP-IGn: PNP-IG with DP of n (n, 2‒6); SmDex: dextranase from Streptococcus mutans; SmDexTM: S. mutans ATCC25175 SmDex bearing Gln100‒Ile732

Intron sequences from the CCT7 gene exhibit diverse evolutionary histories among the four lineages within the Babesia microti-group, a genetically related species complex that includes human pathogens

Babesia microti, the primary causal agent of human babesiosis in North America, was thought to distribute in Europe in association with ixodid ticks and rodents. Recent analyses of β-tubulin and the eta subunit of the chaperonin-containing t-complex protein 1 (CCT7) genes revealed discrete clusters (a species-complex comprised of at least 4 taxa for the U.S., Kobe, Munich, and Hobetsu). To further assess the micro-evolutionary history and genetic variability within the taxon, we combined a set of 6 introns from the CCT7 gene to use as a rapidly evolving DNA marker. Phylogenetic and comparative sequence analyses subdivided the U.S. taxon into 3 geographic subclades-North America, western to central Eurasia, and northeastern Eurasia (≥98% bootstrap supports for each node). The Kobe taxon, which occurs only in a few geographic foci of Japan, could further be subdivided into 2 subgroups (100% support). The Munich and Hobetsu taxa, common to Europe and Japan, respectively, exhibited little or no pairwise sequence divergence among geographically diverse samples, suggesting an extreme population bottleneck during recent history. Despite the small sample size, this study provides a better understanding of the micro-evolutionary relationships and the genetic variability present within each lineage of the B. microti-group

Short report : Identification and phylogenetic analysis of Japanese Macaque Babesia-1 (JM-1) detected from a Japanese Macaque (Macaca fuscata fuscata)

We demonstrate here the identification and phylogenetic characterization of Babesia microti (B. microti)-like parasite detected from a splenectomized Japanese macaque (Macaca fuscata fuscata) at a facility for laboratory animal science. On Day 133 after splenectomy, intra-erythrocytic parasites were found on light microscopic examination, and the level of parasitemia reached 0.3% on blood smear. Molecular characterization of the parasite using nested-polymerization chain reactions targeting the 18S rRNA, β-tubulin, and subunit 7 (eta) of the chaperonin-containing t-complex polypeptide 1 (CCT7) genes were identified as a B. microti-like parasite, designated the Japanese Macaque Babesia-1 (JM-1)

Molecular evidence of the multiple genotype infection of a wild Hokkaido brown bear (Ursus arctos yesoensis) by Babesia sp. UR1

A frozen-stored blood clot of a wild brown bear cub Ursus arctos yesoensis that had been captured in Hokkaido, Japan was examined for piroplasma infection using polymerase chain reaction (PCR). Two 18S ribosomal RNA gene (SSU rDNA) sequences were generated. One 1565-bp sequence showed the highest similarity with B. gibsoni (95.9% identity) but, phylogenetically, was found to belong to a distinct lineage. The other sequence (1709-bp) could not be definitively assigned to a described taxon, sharing only limited homology to the closest named species (90.1% identity with C. felis). In order to enhance information obtained from the SSU rDNA sequence, further detection and sequence analysis of the CCTη gene sequence were done revealing the simultaneous presence of three closely related genotypes (all in a monophyletic lineage) within a single bear host. This finding suggested the possibility that a new Babesia species (Babesia sp. UR1) might have been maintained in nature in wild brown bears. While the parasite\u27s biology is yet unknown, to our knowledge, this is, excepting the single case documentation in 1910 of a hemoparasite in a bear at Russian zoo, the first reported case of piroplasms inhabiting a bear species