Degradation of phytate by the 6-phytase from Hafnia alvei: a combined structural and solution study.

Phytases hydrolyse phytate (myo-inositol hexakisphosphate), the principal form of phosphate stored in plant seeds to produce phosphate and lower phosphorylated myo-inositols. They are used extensively in the feed industry, and have been characterised biochemically and structurally with a number of structures in the PDB. They are divided into four distinct families: histidine acid phosphatases (HAP), ß-propeller phytases, cysteine phosphatases and purple acid phosphatases and also split into three enzyme classes, the 3-, 5- and 6-phytases, depending on the position of the first phosphate in the inositol ring to be removed. We report identification, cloning, purification and 3D structures of 6-phytases from two bacteria, Hafnia alvei and Yersinia kristensenii, together with their pH optima, thermal stability, and degradation profiles for phytate. An important result is the structure of the H. alvei enzyme in complex with the substrate analogue myo-inositol hexakissulphate. In contrast to the only previous structure of a ligand-bound 6-phytase, where the 3-phosphate was unexpectedly in the catalytic site, in the H. alvei complex the expected scissile 6-phosphate (sulphate in the inhibitor) is placed in the catalytic site.

Thermoactinospora rubra gen. nov., sp. nov., a thermophilic actinomycete isolated from Tengchong, Yunnan province, south-west China.

Two novel Gram-positive, spore-forming, thermophilic actinomycetes, designated as strain YIM 77501(T) and YIM 77570, were isolated from a sandy soil sample collected at Tengchong National Volcanic Geological Park, Yunnan province, south-west China. Phylogenetic analysis based on the 16S rRNA gene sequences suggested that the two isolates fell within the family Streptosporangiaceae. The strains formed extensively branched substrate and aerial mycelia which carried masses of long, straight or irregular spore chains composed of warty ornamented spores. Cell walls of the two strains contained meso-diaminopimelic acid and glucose, galactose, mannose and ribose were detected as whole-cell sugars. The predominant menaquinones were MK-9(H(4)) and MK-9(H(6)). The major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, N-acetylglucosamine-containing phospholipids and phosphatidylinositol, phosphatidylinositolmannosides. The major cellular fatty acids were iso-C(16:0) and 10-methyl C(17:0). The DNA G+C content was 74-76 mol%. On the basis of the morphological and chemotaxonomic characteristics as well as the phylogenetic analysis, these strains represents a novel species of a new genus within the family Streptosporangiaceae, for which the name Thermoactinospora rubra gen. nov., sp. nov. is proposed. The type strain of T. rubra is YIM 77501(T) (=DSM 45614(T) = CCTCC AA 2011014(T)).

Anoxybacillus bogrovensis sp. nov., a novel thermophilic bacterium isolated from a hot spring in Dolni Bogrov, Bulgaria.

A novel moderately thermophilic bacterium, designated strain BT 13(T), was isolated from a geothermal water source in Dolni Bogrov, near Sofia, Bulgaria. The isolate was spore-forming, Gram-positive, facultatively anaerobic, alkalitolerant and heterotrophic, and was able to ferment a wide variety of carbon sources including d-glucose, sucrose, l-arabinose, l-rhamnose, starch, sorbitol and glycogen. Strain BT 13(T) grew optimally at pH 8.0 and 65 degrees C. Intracellular amylolytic activity was registered with glucose as the main product of starch hydrolysis. Phylogenetic analysis based on the 16S rRNA gene revealed that the strain belonged to the genus Anoxybacillus, the closest relatives being Anoxybacillus flavithermus and Anoxybacillus kamchatkensis. The DNA G+C content was 44.1 mol%. The fatty acid profile with a content of iso-branched fatty acids of around 80 % of the total fatty acids is similar to that of recognized Anoxybacillus species. On the basis of genotypic differentiation and significant differences in phenotypic characteristics, it was concluded that strain BT 13(T) represents a novel species of the genus Anoxybacillus, for which the name Anoxybacillus bogrovensis sp. nov. is proposed. The type strain is BT 13(T) (=DSM 17956(T)=NBIMCC 8427(T)).

Anoxybacillus rupiensis sp. Nov., a novel thermophilic bacterium isolated from Rupi basin (Bulgaria).

Three strains of a novel thermophilic, strictly aerobic, Gram-positive, spore-forming hemo-organotrophic bacterium were isolated from three hot springs in the region of Rupi basin, Bulgaria as producers of amylolytic enzymes. Their 16S rRNA gene sequences (first 500 nucleotides) were very similar (99.8%). Strains were able to ferment a wide spectrum of carbohydrates such as sugars, polyols, and polysaccharides like xylan, glycogen and starch. Optimal growth was observed at 55-58 degrees C, and pH at 6.0-6.5. Phylogenetic analysis of the whole 16S rRNA gene sequence clustered the strain R270(T) with the representatives of the genus Anoxybacillus and with Geobacillus tepidamans. The G + C content of the genomic DNA was 41.7%. DNA-DNA hybridization analysis revealed low homology with the closest relatives (32.0 mol% homology to Geobacillus tepidamans). Fatty acid profile (major fatty acids iso-C15:0 and iso-C17:0) confirmed the affiliation of the strain to the genus Anoxybacillus. On the basis of the data presented here, we propose that strain R270(T), represents a new species of the genus Anoxybacillus for which, we recommend the name Anoxybacillus rupiensis sp. nov. (=DSM 17127(T) = NBIMCC 8387(T)). The 16S rRNA gene sequence data of a strain R270(T) have been deposited in the EMBL databases under the accession number AJ879076.

Biosynthesis of a thermostable gellan lyase by newly isolated and characterized strain of Geobacillus stearothermophilus 98.

The thermophilic strain able to degrade gellan was isolated from Bulgarian hot spring. According to its morphological and biochemical properties and by partial sequencing of its 16S rDNA, it was classified as Geobacillus stearothermophilus. It grew in a synthetic medium with gellan as the only carbon source with a specific growth rate of 0.69 h(-1) and generation time of 60 min. The strain produced thermostable gellan lyase extracellularly during exponential phase. Its synthesis was inducible; the enzyme was not registered in culture liquid without gellan. The enzyme activity was increased tenfold in conditions of continuous cultivation compared to data from batch fermentations and enzyme productivity was almost sixfold higher. The enzyme showed optimal activity at 75 degrees C in a very large pH area 4-8.5. This enzyme is the first reported thermostable gellan lyase, its residual activity was 100% after 24 h incubation at 60 degrees C and its half-life was 60 min at 70 degrees C.