Purification of a Thermostable β-mannanase from Paenibacillus Thiaminolyticus - characterization and its Potential Use as a Detergent Additive

Endo-1, 4- β- D-mannanase (EC 3.2.1.78) is a glycoside hydrolase involved in random cleavage of β-1, 4- D-manno-pyranosyl linkages within mannans and heteromannans and generates branched and linear oligosaccharides. A β-mannanase was purified from a thermotolerant bacterium Paenibacillus thiaminolyticus isolated from a soil sample. Enzyme was purified to homogeneity with specific activity of 8812 U/mg protein. Sodium dodecyl sulfate (SDS) and native poly-acryl amide gel electrophoresis indicated that the purified mannanase is a monomeric protein with a molecular mass of 38 kDa. The purified enzyme was found to be maximally active at temperature and pH of 60°C and 7.0, respectively. It was stable at 55°C for 24 h and maintained more than 50 % activity up to 3 h at 60°C. The enzyme was very stable in the pH range of 5.0-9.0. Purified β-mannanase demonstrated high stability after 1 h of pre-incubation with most of the tested organic solvents. Enzyme retained significant stability in the presence of various detergent additives, commercially available detergents and dish washing liquids. The high compatibility and substantial stability in the presence of nonionic detergents and dishwashing liquids confirmed its utility as an additive to dish washing liquids and laundry detergents. Enzyme exhibited efficacious de-staining of heteromannan based stains of chocolate ice cream and salad dressing in the wash performance test for detergent application. It also exhibited anti-soil redeposition effect on cotton swatches treated with tennis court clay and heteromannans.

The architecture of the diaminobutyrate acetyltransferase active site provides mechanistic insight into the biosynthesis of the chemical chaperone ectoine

Ectoine is a solute compatible with the physiologies of both prokaryotic and eukaryotic cells and is widely synthesized by bacteria as an osmotic stress protectant. Because it preserves functional attributes of proteins and macromolecular complexes, it is considered a chemical chaperone and has found numerous practical applications. However, the mechanism of its biosynthesis is incompletely understood. The second step in ectoine biosynthesis is catalyzed by l-2,4-diaminobutyrate acetyltransferase (EctA; EC 2.3.1.178), which transfers the acetyl group from acetyl-CoA to EctB-formed l-2,4-diaminobutyrate (DAB), yielding N-γ-acetyl-l-2,4-diaminobutyrate (N-γ-ADABA), the substrate of ectoine synthase (EctC). Here, we report the biochemical and structural characterization of the EctA enzyme from the thermotolerant bacterium Paenibacillus lautus (Pl). We found that (Pl)EctA forms a homodimer whose enzyme activity is highly regiospecific by producing N-γ-ADABA but not the ectoine catabolic intermediate N-α-acetyl-l-2,4-diaminobutyric acid. High-resolution crystal structures of (Pl)EctA (at 1.2–2.2 Å resolution) (i) for its apo-form, (ii) in complex with CoA, (iii) in complex with DAB, (iv) in complex with both CoA and DAB, and (v) in the presence of the product N-γ-ADABA were obtained. To pinpoint residues involved in DAB binding, we probed the structure-function relationship of (Pl)EctA by site-directed mutagenesis. Phylogenomics shows that EctA-type proteins from both Bacteria and Archaea are evolutionarily highly conserved, including catalytically important residues. Collectively, our biochemical and structural findings yielded detailed insights into the catalytic core of the EctA enzyme that laid the foundation for unraveling its reaction mechanism.

Prevalence and Diversity of the Thermotolerant Bacterium Bacillus cytotoxicus among Dried Food Products

ABSTRACT Bacillus cytotoxicus, a member of the Bacillus cereus group, is a thermotolerant species originally reported from a lethal foodborne infection in France in 1998. The strain NVH391-98, isolated from this outbreak, produces cytotoxin K1, a potential cytotoxic enterotoxin. However, the habitat and diversity of B. cytotoxicus isolates so far have been poorly explored. The objective of this study was to assess the prevalence of this bacterium in different food products (mainly dried) and to estimate its diversity. Among the 210 samples analyzed, all potato flakes contained the bacterium at low concentrations (≤102 CFU/g). However, prepared and kept at room temperature for 2 days, the puree contained ca. 105 CFU/g B. cytotoxicus. Besides potato flakes, some samples of millet flour, salted potato chips, and soups also contained B. cytotoxicus. From these samples, 55 thermotolerant B. cytotoxicus isolates were obtained. When classified into six distinct random amplified polymorphism DNA patterns, they showed the existence of 11 distinct plasmid profiles. Although most isolates (including the reference strains NVH391-98 and NVH883-00) contained no detectable plasmid, some displayed one to three plasmids with sizes from ca. 8 to 90 kb. It also emerged from this study that a single food sample could contain B. cytotoxicus isolates with different genetic profiles. HIGHLIGHTS

Draft Genome Sequence of Natranaerobius trueperi DSM 18760T, an Anaerobic, Halophilic, Alkaliphilic, Thermotolerant Bacterium Isolated from a Soda Lake

ABSTRACT The anaerobic, halophilic, alkaliphilic, thermotolerant bacterium Natranaerobius trueperi was isolated from a soda lake in Wadi An Natrun, Egypt. It grows optimally at 3.7 M Na+, pH 9.5, and 43°C. The draft genome consists of 2.63 Mb and is composed of 2,681 predicted genes. Genomic analysis showed that various genes are potentially involved in the adaptation mechanisms for osmotic stress, pH homeostasis, and high temperatures.

First Insights into the Genome Sequence of the Alkaliphilic Thermotolerant Bacterium Clostridium thermoalcaliphilum JW/YL23-2T

ABSTRACT Clostridium thermoalcaliphilum is an obligate anaerobic and rod-shaped bacterium isolated from sewage sludge. It is an alkaliphilic thermotolerant organism and utilizes sucrose, glucose, fructose, maltose, cellobiose, amino acids, and Casamino Acids as substrates. The draft genome comprises 2.031 Mbp and 2,027 predicted protein-coding genes.

Bacillus caseinilyticus sp. nov., an alkali- and thermotolerant bacterium isolated from a soda lake

A novel Gram-stain-positive, rod-shaped, motile, endospore-forming and proteolytic bacterial strain, SPT, was isolated from Lonar soda lake, in India. On the basis of 16S rRNA gene sequence analysis it was identified as belonging to the class Firmibacteria and was most closely related to Bacillus cellulosilyticus DSM 2522T (96.7 %) and other members of the genus Bacillus ( < 95.9 %). Strain SPT was catalase- and oxidase-positive. The cell-wall peptidoglycan of strain SPT contained meso-diaminopimelic acid. Polar lipids included diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, three phospholipids, two aminolipids and two unknown lipids. The predominant isoprenoid quinone was MK-7. Anteiso-C15 : 0 (26.8 %) was the predominant fatty acid and significant proportions (>5 %) of iso-C15 : 0 (20.9 %), C16 : 1ω7c alcohol (6.3 %), iso-C16 : 0 (6.3 %) and anteiso-C17 : 0 (5.3 %) were also detected in strain SPT. The DNA G+C content of strain SPT was 38.9 mol%. The results of phylogenetic, chemotaxonomic and biochemical tests allowed a clear differentiation of strain SPT from all other members of the genus Bacillus. Strain SPT represents a novel member of the genus Bacillus, for which the name Bacillus caseinilyticus sp. nov. is proposed. The type strain is SPT ( = MCC 2612T = JCM 30246T).

Thermotolerant Bacillus kokeshiiformis sp. nov. isolated from marine animal resources compost

A novel Gram-staining-positive, endospore-forming, rod-shaped, facultatively anaerobic, thermotolerant bacterium, designated strain MO-04T, was isolated from a marine animal resources (MAR) compost. The 16S rRNA gene sequence of strain MO-04T showed 99.4 % similarity with Bacillus thermolactis R-6488T, 94.1 % similarity with Bacillus thermoamylovorans CNCM I-1378T, 93.3 % similarity with Bacillus humi LMG 22167T, 93.2 % similarity with Bacillus niacini IFO 15566T and the similarities with other species were less than 93 %. DNA–DNA relatedness between strain MO-04T and B. thermolactis DSM 23332T was 45 %. The DNA G+C content of strain MO-04T was 33.4 mol%, comparatively lower than that of B. thermolactis R-6488T (35.0 mol%). Strain MO-04T grew at 35–61 °C (optimum 50 °C), pH 4.5–9.0 (optimum pH 7.2) and tolerated up to 8.0 % (w/v) NaCl (optimum 2 %). The MO-04T cell wall peptidoglycan type was meso-2,6-diaminopimelic acid, and the major fatty acids were C16 : 1, C14 : 1, C17 : 0 and C17 : 1. The major polar lipids were represented by diphosphatidylglycerol and phosphatidylglycerol and two unidentified phospholipids. The analysed polyphasic data presented here clearly indicate that the isolate MO-04T is considered to represent a novel species within the genus Bacillus for which the name Bacillus kokeshiiformis sp. nov. is proposed. The type strain of B. kokeshiiformis is MO-04T ( = JCM 19325T = KCTC 33163T).