A protein showing endoglucanase-peptidase activity was prepared from a newly isolated bacterium (ST15c10). We identified ST15c10 as Brevibacillus agri based on electron-microscopic images and its 16S-rDNA sequence (GenBank accession No. HM446043), which exhibits 98.9% sequence identity to B. agri (KZ17)/B. formosus (DSM-9885T)/B. brevis. The enzyme was purified to homogeneity and gave a single peak during high-performance liquid chromatography on a Seralose 6B-150 gel-matrix/C-18 column. MALDI-TOF mass-spectrometry and bioinformatics studies revealed significant similarity to M42-aminopeptidases/endoglucanases of the CelM family. These enzymes are found in all Brevibacillus strains for which the genome sequence is known. ST15c10 grows optimally on carboxymethyl cellulose (CMC)-gelatin (40°C/pH 8-9), and also shows strong growth/carboxymethyl cellulase (CMCase) activity in submerged bagasse fermentation. The purified enzyme also functions as endoglucanase with solid bagasse/rice straw. Its CMCase activity (optimal at pH 5.6 and 60°C/K_m = 35.5 µM/V_max = 1,024U) was visualized by zymography on a CMC-polyacrylamide gel, which provided a strong band of approximately 70 kDa. The purified enzyme also showed strong peptidase (gelatinase) activity (pH 7.2/40°C during zymography on 6-12% gelatin/1% gelatin-PAGE (at approx. 70 kDa). The CMCase activity is inhibited by the metal ions Mn/Cu/Fe/Co (50%), Hg/KMnO₄ (100%), and by glucose or lactose (50-75%; all at 1 mM). The observed dose/time-dependent inhibition by Hg ions could be prevented with 2-mercaptoethanol. A comparison of the B. agri endoglucanase-aminopeptidase (ELK43520; 350 aa) with other members of the M42-family revealed the conservation of active-site residues Cys256/Cys260, which were previously identified as metal-binding sites. Regulation of the endoglucanase activity probably occurs via metal binding-triggered changes in the redox state of the enzyme. Studies on this type of enzyme are of high importance for basic scientific and industrial research.

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Salt-tolerant alkaline amylase producing Bacillus sp. MRS6 was isolated from three municipal waste disposal site of Medinipur town in the Paschim Medinipur district of southern West Bengal, India. The strain had been characterized from a polyphasic approach. Extracellular enzyme production was carried out in mineral salt media at pH-7, 35°C with 2% soluble starch as sole carbon source. It was found that the amylase, produced by the salttolerant strain possessed high activity in a range of alkaline pH (6-9), with pH-8 as optimum. An incubation period of 30 min at 35°C was found as optimum temperature condition of its activity. This alkaliphilic, salt-tolerant enzyme was found to stay stable upto 80°C, 4M NaCl. Mn 2+, Ca 2+ plays critical role in >80% increasing of this enzyme activity. EDTA, β-merkeptoethanol and detergents strongly inhibit enzyme activity. A solid state fermentation for production of this enzyme was performed with 85% moisture content where wheat bran was found as most effective and cheap carbon source. This enzyme was purified partially by cold acetone precipitation followed by gel filtration chromatography (seralose 6B) and the molecular mass was found to be 55 kDa by SDS PAGE. The present findings suggested the enzyme to be halophilic alkaline amylase. The present enzyme is of great significance in present day biotechnology with applications ranging from food, fermentation, textile, detergent to paper industries.

: Amylases enzymes hydrolyze starch molecules to producediverse products including dextrins, and progressively smaller polymers. These include glucose units linked through α-1-1, α-1-4, α-1-6, glycosidic bonds. This enzyme carry an (α /β) 8 or TIM barrel structure ia also produced containing the catalytic site residues.These groups of enzymespossess 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. Present objectives were to study on one such enzyme based on its molecular characterization after it purification in our laboratory. Its main property of solid-natural starch degradation was extensively investigated for its pharmaceutical/ industrialapplications.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. MALDI-TOF mass-spectrometry with bioinformatics studies revealed its significant similarity to α/β hydrolase family. The enzyme showed a very high 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 exoamylaseas because it produced significant amount of glucose.Beside the purified enzyme, the present whole organism Bacillus cereus sm-sr14 could degrade natural solid starch materials like potato, rice up to the application level in pharmaceutical/ industrial field for alcohol production.