Amarila Malik

4,6-α-Glucanotransferase, a Novel Enzyme That Structurally and Functionally Provides an Evolutionary Link between Glycoside Hydrolase Enzyme Families 13 and 70

ABSTRACT Lactobacillus reuteri 121 uses the glucosyltransferase A (GTFA) enzyme to convert sucrose into large amounts of the α-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 α-glucanotransferase enzyme with disproportionating (cleaving α1→4 and synthesizing α1→6 and α1→4 glycosidic linkages) and α1→6 polymerizing types of activity on maltotetraose and larger maltooligosaccharide substrates (in short, it is a 4,6-α-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 1H nuclear magnetic resonance (NMR) spectroscopy revealed that only linear products were made and that with increasing degrees of polymerization (DP), more α1→6 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 (β/α)8 barrel that use sucrose to synthesize α-d-glucan polymers. The GTFB enzyme reaction and product specificities, however, are novel for the GH70 family, resembling those of the GH13 α-amylase type of enzymes in using maltooligosaccharides as substrates but differing in introducing a series of α1→6 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.

Cloning, DNA sequence, and expression of Aeromonas caviae WS7b chitinase gene

A chitinase-producing bacterium, designated WS7b, was isolated from a soil sample obtained from a black-pepper plantation on Bangka Island, Indonesia. Fatty-acid methyl-ester analysis indicated that the isolate was Aeromonas caviae. A chitinase gene from WS7b was cloned in a pUC19-based plasmid vector, but without its natural promoter. The complete nucleotide sequence of the gene was determined, and the structural gene consisted of a 2748-bp region encoding 864 amino acids. DNA sequence analysis indicated that the gene had been cloned without its promoter, and this was confirmed by chitinase-plate assay of the truncated version of the gene in Escherichia coli. The chitinase gene product showed amino-acid sequence similarity to chiA from A. caviae. Chitinase enzyme activity was determined spectrophotometrically, using colloidal chitin azure as substrate for extracellular and intracellular fractions. The ability of the chitinase cloned in E. coli to hydrolyze chitin was less than that of the enzyme in its indigenous host.

Identification and sequence analysis of pWcMBF8-1, a bacteriocin-encoding plasmid from the lactic acid bacterium Weissella confusa

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.

Cloning and heterologous expression of the ftfCNC-2(1) gene from Weissella confusa MBFCNC-2(1) as an extracellular active fructansucrase in Bacillus subtilis.

Fructan-exopolysaccharides (fructan-EPS) (inulin and levan) and their oligosaccharides (fructooligosaccharides, FOS) have drawn considerable interest in the food and pharmaceutical industries. EPS-producing lactic acid bacteria have been reported to produce β-fructans (inulin and levan), as well as α-glucans, by the function of sucrase enzymes, i.e., fructansucrase and glucansucrase. A fructansucrase ftfCNC-2(1) gene from Weissella confusa strain MBFCNC-2(1) was previously cloned in Escherichia coli. In this study, we aimed to express the ftf[CNC-2(1)] gene in Bacillus subtilis to obtain the active form of the extracellular recombinant protein FTF[CNC-2(1)]. This cloning was achieved by inserting the gene in-fusion with the signal sequence of the B. subtilis subtilisin E. SDS-polyacrylamide gel electrophoresis analysis and in situ activity assay with Periodic Acid-Schiff staining revealed that the recombinant FTF[CNC-2(1)] was successfully expressed as an extracellular protein from B. subtilis DB403 in its active form, which was confirmed using sucrose and raffinose.

Draft Genome Sequence of Weissella confusa MBF8-1, a Glucansucrase- and Bacteriocin-Producing Strain Isolated from a Homemade Soy Product

ABSTRACT We report here the draft genome sequence of Weissella confusa MBF8-1, an isolate from a homemade fermented soybean product that produces sucrases and exhibits antibacterial (bacteriocin) activity. The draft genome of W. confusa MBF8-1 comprises a 2.2-Mbp chromosome and a 17.8-kbp bacteriocin-encoding plasmid. Two putative glucansucrase genes were also identified.

Multidrug resistance gene 1 polymorphisms in pediatric patients with leukemia at a national referral hospital in Indonesia

Abstract Background Acute lymphoblastic leukemia (ALL) is the most prevalent cancer in the pediatric population. From 25% to 30% of patients with ALL will have a relapse that leads to death when they are teenagers. At Cipto Mangunkusumo Hospital, 40% of 126 pediatric patients with ALL relapsed from 2005 to 2011. A multiple variant of multidrug resistance gene 1 (MDR1) is C3435T, which can be used to understand the genetic basis of susceptibility to relapse. Objectives To identify the profile of MDR1 polymorphism in pediatric Indonesian patients with ALL. Methods We collected data from 44 patients with ALL who attended Cipto Mangunkusumo Hospital between January and June 2014. We investigated a silent C3435T polymorphism in MDR1 exon 26 with polymerase chain reaction- restriction fragment length polymorphism using MboI. Results There were 32 male and 12 female patient participants in this study. Eighteen patients were 1–3 years old and 26 were over 3 years. The mean age at 1–3 years was 2.4 ± 0.86, and over 3 years it was 6.3 ± 2.67 years. There were 27 patients with ALL in the standard risk group and 17 in the high risk group. We determined that the 25 samples from patients with ALL in the standard risk group were not digestible (allele T) and the 6 samples from patients with ALL in the high risk group were digestible (allele C). Conclusions The prevalence of the T allele was higher than that of the C allele in pediatric Indonesian patients with ALL.

Pharmacognostic and Antimicrobial Studies of Garcinia latissima Miq. Leaves (Clusiaceae)

Introduction: Garcinia latissima Miq known as Dolo magota (Maluku), is a medicinal plant belonging to the family Clusiaceae. The purpose of the research was to explore the phyto constituents present, pharmacognostic details, and their antimicrobial efficacy. Methods: The preliminary phytochemical components were qualitatively examined using the standard method systems. The antimicrobial screening was carried out using the good diffusion method and the minimum inhibitory concentration (MIC) using dilution method. Results: The phytochemical screening of different extract of G. latissima Miq leaves revealed the presence of tannins, saponins, and alkaloids and the results were tabulated. The ethyl acetate and methanolic extracts from its leaves showed antimicrobial activity especially for Bacillus subtilis, a positive bacteria; the hexane extract did not show any activity against the selected microba. Conclusion: The results of the phytochemical and bio-efficacy study revealed most valuable information and also support the continued sustainable use of this leaves in the traditional system of medicine.

Evaluation of Antimicrobial Activities of Garcinia latissima Miq. Stem Bark Extract

Introduction: To evaluate the antimicrobial activities of hexane, ethyl acetate, and methanol extracts from the stem bark of Garcinia latissima Miq. Method: The G. latissima Miq. was collected from Bogor. This study uses multilevel maceration extraction methods. The antimicrobial activity was determined by the good diffusion method and the broth dilution method. Result: The ethyl acetate extracts of stem bark were active against Bacillus subtilis, aeruginosa Staphylococcus aureus, and Ps aeruginosa, and virtually inactive against Escherichia coli, Candida albicans, and Trichophyton mentagrophytes. The methanol extracts of stem bark were active against B. subtilis and S. aureus, and virtually inactive against E. coli, P. aeroginosa, C. albicans, and T. mentagrophytes. The hexane extracts of stem bark were inactive against B. subtilis, S. aureus, E. coli, P. aeroginosa, C. albicans, and T. mentagrophytes. The 2% methanol extracts of G. latissima Miq. stem bark showed the maximum zone of inhibition against B. subtilis (10.70±0.638 mm), followed by the methanol extracts of G. latissima Miq. stem bark against S. aureus (10.38±0.653 mm). 2% Ethyl acetate extracts of G. latissima Miq. stem bark exhibited a maximum zone of inhibition against B. subtilis (10.35±0.867 mm) followed by P. aeruginosa (10.07±0.971 mm). The results of the antibacterial activity showed that the MeOH extract of G. latissima Miq. stem bark exhibited activities against B. subtilis (MIC/MBC = 625 ppm/2500 ppm)and S. aureus (MIC/MBC = 2500 ppm/5000 ppm). The ethyl acetate extract of G. latissima Miq. stem bark exhibited activities against P. aeruginosa (MIC/ MBC = 5000 ppm/5000 ppm) and B. subtilis (MIC/MBC > 5000 ppm/5000 ppm). Conclusion: The results of the present study revealed most valuable information and also support the continued sustainable use of G. latissima Miq. stem barks in a traditional system of medicine. Key words : Garcinia latissima, Antimicrobial activities, Stem bark extract, Hexane, Ethyl acetate, Methanol.