Lactobacillus reuteri 121 uses the glucosyltransferase A (GTFA) enzyme to convert sucrose into large amounts of the ␣-D-glucan reuteran, an exopolysaccharide. Upstream of gtfA lies another putative glucansucrase gene, designated gtfB. Previously, we have shown that the purified recombinant GTFB protein/enzyme is inactive with sucrose. Various homologs of gtfB are present in other Lactobacillus strains, including the L. reuteri type strain, DSM 20016, the genome sequence of which is available. Here we report that GTFB is a novel ␣-glucanotransferase enzyme with disproportionating (cleaving ␣134 and synthesizing ␣136 and ␣134 glycosidic linkages) and ␣136 polymerizing types of activity on maltotetraose and larger maltooligosaccharide substrates (in short, it is a 4,6-␣-glucanotransferase). Characterization of the types of compounds synthesized from maltoheptaose by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), methylation analysis, and 1-dimensional 1 H nuclear magnetic resonance (NMR) spectroscopy revealed that only linear products were made and that with increasing degrees of polymerization (DP), more ␣136 glycosidic linkages were introduced into the final products, ranging from 18% in the incubation mixture to 33% in an enriched fraction. In view of its primary structure, GTFB clearly is a member of the glycoside hydrolase 70 (GH70) family, comprising enzymes with a permuted (␤/␣) 8 barrel that use sucrose to synthesize ␣-D-glucan polymers. The GTFB enzyme reaction and product specificities, however, are novel for the GH70 family, resembling those of the GH13 ␣-amylase type of enzymes in using maltooligosaccharides as substrates but differing in introducing a series of ␣136 glycosidic linkages into linear oligosaccharide products. We conclude that GTFB represents a novel evolutionary intermediate between the GH13 and GH70 enzyme families, and we speculate about its origin.Glucansucrase (GS) (or glucosyltransferase [GTF]) enzymes (EC 2.4.1.5) of lactic acid bacteria (LAB) use sucrose to synthesize a diversity of ␣-glucans with ␣136 (dextran; found mainly in Leuconostoc), ␣133 (mutan; found mainly in Streptococcus), alternating ␣133 and ␣136 (alternan; reported only in Leuconostoc mesenteroides), and ␣134 (reuteran; synthesized by GTFA and GTFO from Lactobacillus reuteri strains) glycosidic bonds (1,14,16,23,34).The first glycoside hydrolase 70 (GH70) family 3-dimensional (3D) structures, recently elucidated (9, 38), showed that the catalytic domains of GS enzymes possess a (␤/␣) 8 barrel structure similar to that of members of the GH13 family, confirming earlier secondary-structure predictions (4, 21). The core of the proteins belonging to the GH13 family comprises 8 ␤-sheets alternated with 8 ␣-helices. In GS enzymes, however, this (␤/␣) 8 barrel structure is circularly permuted (21). Also, the four conserved regions (regions I to IV) identified in members of the ␣-amylase family GH13 (31) are present in glucansucrases. However, as a consequence of the circular per...

Homopolysaccharide (glucan and fructan) synthesis from sucrose by sucrase enzymes in lactic acid bacteria (LAB) has been well studied in the genera Leuconostoc, Streptococcus and Lactobacillus. This study aimed to identify and characterize genes encoding glucansucrase/glucosyltransferase (GTF) and fructansucrases/fructosyltransferase (FTF) enzymes from genomic DNA of 'rare' Indonesian exopolysaccharide-producing LAB. From a total of 63 exopolysaccharide-producing LAB isolates obtained from foods, beverages and environmental samples, 18 isolates showing the most slimy and mucoid colony morphologies on sucrose were chosen for further study. By comparing bacterial growth on De Man, Rogosa and Sharpe (MRS)-sucrose with that on MRS-raffinose, and using the results of a previous PCR screening study with degenerate primer pairs targeting the conserved catalytic domain of GTFs, various strains were identified as producers of fructan (13), of glucan only (five) or as potential producers of both glucan and fructan (nine). Here, we report the characteristics of three gtf genes and one ftf gene obtained from Weissella confusa strains MBF8-1 and MBF8-2. Strain MBF8-1 harbored two putative gtf genes with high sequence similarity to GTFB of Lactobacillus reuteri 121 and GTF180 of L. reuteri 180, respectively. Strain MBF8-2 possessed single gtf and ftf genes with high sequence similarity to GTFKg3 of Lactobacillus fermentum Kg3 and DSRWC of Weissella cibaria, and FTF levansucrase of L. reuteri 121, respectively.

The purpose of this study is to isolate G. latissima Miq. leaves ethyl acetate extract and to understand its activity against B. subtilis. The isolation was performed by open column chromatography, recrystallization, preparative thin layer chromatography and the isolate antibacterial assay by microdilution method. Compound characterization using spectroscopy. The isolated compound was white crystal, soluble in chloroform and insoluble in n-hexane, ethyl acetate. The melting point of the isolated compound was 262-264oC. IR spectrum showed strong signals for C-H stretching of methyl groups and carbonyl oxygen (C=O). The based on NMR data, the chemical shifts of chemical compounds from the isolate was compared with the literature showed that the isolate was friedelin. The MIC of friedelin against B. subtilis was 2,500 ppm.

Members of the Gram-positive lactic acid bacteria (LAB) are well-known for their beneficial properties as starter cultures and probiotics. Many LAB species produce ribosomally synthesized proteinaceous antibiotics (bacteriocins). Weissella confusa MBF8-1 is a strain isolated from a fermented soybean product that not only produces useful exopolysaccharides but also exhibits bacteriocin activity, which we call weissellicin MBF. Here, we show that bacteriocin production by W. confusa MBF8-1 is specified by a large plasmid, pWcMBF8-1. Plasmid pWcMBF8-1 (GenBank accession number KR350502), which was identified from the W. confusa MBF8-1 draft genome sequence, is 17 643 bp in length with a G + C content of 34.8% and contains 25 open reading frames (ORFs). Six ORFs constitute the weissellicin MBF locus, encoding three putative double-glycine-motif peptides (Bac1, Bac2, Bac3), an ABC transporter complex (BacTE) and a putative immunity protein (BacI). Two ORFs encode plasmid partitioning and mobilization proteins, suggesting that pWcMBF8-1 is transferable to other hosts. To the best of our knowledge, plasmid pWcMBF8-1 not only represents the first large Weissella plasmid to be sequenced but also the first to be associated with bacteriocin production in W. confusa.

ObjectiveThe genetic involvement in unconjugated neonatal hyperbilirubinemia has been extensively studied. Despite the high incidence of hyperbilirubinemia in Indonesia, studies are lacking. The objective of this study is to elucidate the role of polymorphism in the UGT1A1 in Neonatal Hyperbilirubinemia in Bengkulu, Indonesia.ResultsThere were 41 neonates enrolled in the study; 30 had a total serum bilirubin level ≥ 15 mg/dL (hyperbilirubinemia neonates) while 11 has < 15 mg/dL (control neonates). Genetic mutations in Exon 1, UGT1A1*6 (c211g > a) and one in promoter region, UGT1A1*60 (c3279t > g) were determined by polymerase chain reaction–restriction fragment length polymorphism. We found 18 (60%) mutation in exon 1 in hyperbilirubinemia group and 7 (64%) in the control group with an identical allele frequency of 0.3 in both groups. We found heterozygous UGT1A1*60 4 times (13.3%) and homozygous 26 times (86.7%) in the hyperbilirubinemia group, with an identical allele frequency of 0.935 in hyperbilirubinemia and 1 in control group. This study supports the involvement of genetic factors in the development of unconjugated hyperbilirubinemia in Bengkulu population.