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Abstract: Bacillus sp. KR-8104 was selected from a set of 18 bacteria strains isolated from soil samples and screened for production of amylase. The maximum productivity obtained at pH 5–6 and 60–65h after cultivation in production medium. New extracellular Ca-independent α-amylase was highly purified using ion exchange and hydrophobic interaction chromatography, which showed a single band with an apparent molecular weight of 59kDa by SDS-PAGE. This enzyme is active in a wide pH range with its maximum activity at low pH values (4.0–6.0) and has the 90% of its maximum activity at pH 3.5. The α-amylase is optimally active at 75–80°C. The presence or absence of Ca2+ and EDTA did not affect enzyme activity and thermal stability. [Copyright &y& Elsevier]

Abstract: A native α-amylase capable of activity at low pH was isolated from Bacillus sp. KR-8104 (KRA) naturally occurred in rhizoplane zone of potato cultured soils. An investigation was set out to account some evidence for its specific properties. Kinetic parameters of KRA with respect to Bacillus licheniformis α-amylase (BLA) and B. amyloliquefaciens α-amylase (BAA) showed a shift in acidic limb for KRA pH-profile. After isolation of KRA gene and determination of its nucleotide and protein sequences, the three-dimensional structure of KRA was simulated based on BLA as template. Using PROPKA program the pK a values of titrable groups in KRA model together with other few members of this family were predicted either from simulated model or available structure at protein data bank. A correlation between catalytic residue pK a values and the optimal pH-activity profile was seen. Both our experimental data and prediction studies indicated a shift in pK a value of catalytic residues to acidic pH. Moreover alignment studies raised amino acid substitutions in KRA compare to BLA which may affect the KRA putative active site leading to the formation of an extra hydrogen bond between Glu261 and Arg229 (regarding to BLA numbering) and in turn shifts the pH-activity profile to lower amount. Also a common intermediate with characteristics of molten globular state was detected at a pH value lower than other members of α-amylase family using far UV circular dichroism, intrinsic and ANS fluorescence and quenching experiments. [Copyright &y& Elsevier]

Abstract: Bacterial polyphenol oxidases (PPOs) with a high oxidizing capacity for a wide range of substrates could make their applications in phenolic biotransformation, food processing, cosmetics, and textile industry. We have isolated a melanogenic soil bacterium by differential screening of a number of strains which were isolated from the Iranian microflora. The taxonomic characterization of this strain indicates that it belongs to the genus Bacillus (HR03), and has the ability to produce all types of PPOs; cresolase (EC 1.14.18.1), cathecolase (EC.1.10.3.1), and laccase (EC 1.10.3.2). We studied the tyrosinase activity using l-tyrosine and l-dopa as substrates and the laccase activity with specific substrates such as syringaldazine and 2,6-dimethoxyphenol. The optimum pH and temperature, obtained for all types of polyphenol oxidases, were at about pH 5.5 and 55°C, respectively. Tyrosinase-like enzyme of this strain shows a lag period in its tyrosine hydroxylase activity that could be avoided by the addition of small amounts of l-dopa and sodium dodecyl sulfate (SDS). In addition, tyrosinase and laccase were activated by SDS below the critical micelle concentration and were inhibited by 1mM EDTA. We tested the resistance of melanized-cells against UVA, UVC and H2O2. Results show that melanin protects strain HR03 against UV lights and the oxidant. [Copyright &y& Elsevier]

Abstract: An investigation was carried out to compare the proteolytic resistance and acidic digestion of the mesophilic α-amylase from Bacillus amyloliquefaciens (BAA) and its thermophilic counterpart from Bacillus licheniformis (BLA). Correlation between sites of proteolytic cleavage and the three-dimensional structure of the α-amylases, with the application of theoretical modeling of BAA, allowed discussion of the flexibility and the stability of both enzymes. The thermophilic enzyme shows higher resistance to trypsin, papain and thermolysin but is sensitive to pronase and acidic digestion. Proteolytic digestion of the thermophilic enzyme leads to an increased activity of the enzyme at room temperature whereas results of SDS-PAGE indicate proteolytic cleavage. Furthermore, thermal stability and resistance to proteolysis for BLA and BAA in the presence of additives such as sorbitol, trehalose and glycerol were also investigated. In addition to thermal stabilization of the two enzymes, these additives also augmented the resistance of the enzymes to proteolysis. [Copyright &y& Elsevier]

Abstract: Horseradish peroxidase is an important heme-containing plant enzyme with enormous medical diagnostic, biosensing, bioremediation and biotechnological applications. Any improvement in the stability of the enzyme will greatly enhance its application in mentioned areas. In the present study, the stabilizing effects of certain additives and chemical modification by citraconic anhydride on the thermal behavior of HRP were investigated. Both strategies brought about dramatic enhancement of the thermostability of the enzyme. Results obtained on T m, changes in the circular dichroism (CD) spectra and kinetic parameters of HRP and its modified form are discussed in terms of contributions to the mechanism of the thermal stability and the activity enhancement. Polyols were very effective in providing protection against the irreversible thermoinactivation of the native and the modified forms of the enzyme. Our results reveal that a combination of medium change and surface modification may provide an effective strategy for the enhancement of the thermodynamic and the kinetic stability of the enzyme. [Copyright &y& Elsevier]

Bacillus amyloliquefaciens α-amylase (BAA) is an industrially important extra cellular enzyme with a number of applications. The acid-induced unfolding of BAA was studied by far- and near-UV circular dichroism (CD), intrinsic fluorescent emission spectroscopy, 1-anilino naphthalene-8-sulfonate (ANS) binding, light scattering and fluorescence quenching by acrylamide. At pH 3.0, BAA acquires partially folded state, which shows characteristics of molten globules, i.e., reduction of defined tertiary structure and almost no change in the secondary structure. ANS binding and light scattering experiments show that at acidic pH, BAA unfolds in such a way that its hydrophobic surface is exposed to a greater extent compared to the native form. In addition, acrylamide quenching of the intrinsic tryptophan residues in the protein molecules indicate that at pH 3.0, the protein is in a partially unfolded conformation with more tryptophan residues exposed to the solvent as compared to the native conformation in the neutral pH. Calcium ion is required for the refolding of the molten globule state to the native form. Enzymatic activity is recovered in the presence of Ca2+ but no reactivation of the enzyme can be observed in the presence of Mg2+, Mn2+ and EDTA. Based on several lines of evidence, it is proposed that such acid-denatured state may be the competent state involved in the secretion process of α-amylase. [Copyright &y& Elsevier]