Purification and Characterization of Natural Solid-Substrate Degrading and Alcohol Producing Hyperthermostable Alkaline Amylase from Bacillus cereus (sm-sr14)

Objective: Amylases enzymes hydrolyze starch molecules to produce diverse products including dextrins, and progressively smaller polymers. These include glucose units linked through α-1- 1, α-1-4, α-1-6, glycosidic bonds. Methods: This enzyme carrying an (α /β) 8 or TIM barrel structure is also produced containing the catalytic site residues. These groups of enzymes possess four conserved regions in their primary sequence. In the Carbohydrate-Degrading Enzyme (CAZy) database, α-amylases are classified into different Glycoside Hydrolase Families (GHF) based on their amino acid sequence. The present objective was to study one such enzyme based on its molecular characterization after purification in our laboratory. Its main property of solid-natural starch degradation was extensively investigated for its pharmaceutical/ industrial applications. Results: Amylase producing bacteria Bacillus cereus sm-sr14 (Accession no. KM251578.1) was purified to homogeneity on a Seralose 6B-150 gel-matrix and gave a single peak during HPLC. MALDITOF mass-spectrometry with bioinformatics studies revealed its significant similarity to α/β hydrolase family. The enzyme showed an efficient application; favourable Km, Vmax and Kcat during the catalysis of different natural solid starch materials. Analysis for hydrolytic product showed that this enzyme can be classified as the exo-amylase asit produced a significant amount of glucose. Conclusion: Besides the purified enzyme, the present organism Bacillus cereus sm-sr14 could degrade natural solid starch materials like potato and rice up to the application level in the pharmaceutical/ industrial field for alcohol production.

Screening and Characterization of Halophilic Bacteria With Industrial Enzymes from Salt Lake Razazah, Karbala, Iraq

ABSTRACT: A total of 218 halophilic bacterial isolates were obtained from Lake Razazah, west of Karbala, Iraq. Optimum pH and temperature were 7.5 and 37 °C, respectively. According to optimal growth at different salt concentration, the slightly halophilic bacteria were the most abundant isolates with the frequency of 68%. The isolated bacteria were screened for the production of extracellular amylase, alkaline amylase, protease, alkaline protease, lipase, alkaline lipase, pectinase and cellulase. The production of pectinase (55.8%), amylase (52.6%) and lipase (50.0%) were observed in almost half of the halophilic bacteria. Alkaline amylase and alkaline lipase production were reported in less than one third (30%) of isolates. Phylogenetic analysis16S rRNA sequences indicated that all isolates were members of eight genera of the domain Bacteria, including Bacillus, Halobacillus, Virgibacillus, Oceanobacillus, Staphylococcus, Pseudomonas, Idiomarina and Halomonas. The predominant commercial enzymes producers in current study were Halobacillus sp. K51 and Halomonas sp. K46 with the ability to produce 7 out of 8 exoenzymes. The presented data shows that despite drought, dehydration, increased concentrations of salt and contaminants, Lake Razazah represents an untapped source of halophilic bacteria biodiversity.

Taxonomy and Polyphasic Characterization of Alkaline Amylase Producing Marine ActinomyceteStreptomyces rocheiBTSS 1001

Actinomycetes isolated from marine sediments along the southeast coast of Bay of Bengal were investigated for amylolytic activity. Marine actinomycete BTSS 1001 producing an alkaline amylase was identified from marine sediment of Diviseema coast, Bay of Bengal. The isolate produced alkaline amylase with maximum amylolytic activity at pH 9.5 at 50°C. The organism produced white to pale grey substrate mycelium and grayish aerial mycelium with pinkish brown pigmentation. A comprehensive study of morphological, physiological parameters, cultural characteristics, and biochemical studies was performed. The presence of iso-C15 : 0, anteiso-C15 : 0, iso-C16 : 0, and anteiso-C17 : 0as the major cellular fatty acids, LL-diaminopimelic acid as the characteristic cell wall component, and menaquinones MK-9H(6)and MK-9H(8)as the major isoprenoid quinones is attributed to the strain BTSS 1001 belonging to the genusStreptomyces. Comparison of 16S rRNA gene sequences showed that strain BTSS 1001 exhibited the highest similarities to the type strains ofStreptomyces rochei(99%),Streptomyces plicatus(99%), andStreptomyces enissocaesilis(99%). Using the polyphasic taxonomical approach and phenotypic characteristic studies, the isolate BTSS 1001 was characterized as marine actinomyceteStreptomyces rochei.

Georgenia satyanarayanai sp. nov., an alkaliphilic and thermotolerant amylase-producing actinobacterium isolated from a soda lake

A Gram-stain-positive, oxidase-negative, starch-hydrolysing, actinobacterium (strain JC82T) was isolated from a soda lake in Lonar, India. Based on 16S rRNA gene sequence similarity studies, strain JC82T belonged to the genus Georgenia and was most closely related to Georgenia muralis 1A-CT (96.8 %) and other members of the genus Georgenia (<96.5 %). The DNA G+C content of strain JC82T was 73.4 mol%. The cell-wall amino acids were alanine, glutamic acid and lysine with peptidoglycan type A4α. Polar lipids included diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannosides, an unidentified lipid (L1) and an unidentified glycolipid (GL3). The predominant isoprenoid quinone was menaquinone MK-8(H4). Anteiso-C15 : 0 was the predominant fatty acid and significant proportions of iso-C14 : 0, C14 : 0, C16 : 0, iso-C15 : 0 and iso-C16 : 0 were also detected. Strain JC82T produced thermostable alkaline amylase. The results of physiological and biochemical tests allowed a clear phenotypic differentiation of strain JC82T from all other members of the genus Georgenia . Based on these data, strain JC82T represents a novel species of the genus Georgenia , for which the name Georgenia satyanarayanai sp. nov. is proposed. The type strain is JC82T ( = KCTC 19802T = NBRC 107612T).