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Pullulanase with high catalytic efficiency has attracted great attention from both the academic and industrial communities for its wide application in cold starch hydrolysis. A novel pullulanase gene pul703 was cloned from a mesophilic bacteria Bacillus pseudofirmus 703. Pul703 was characterized to be a type I pullulanase with maximal activity at 45 °C and good low-temperature stability, more than 70% of activity was detected after incubation at 25–35 °C for 72 h. Pul703 obtained the maximal activity around pH 7.0–8.0, and was highly active and stable over a wide pH range of 5.5–9.5, more than 80% of activity was retained after 12 h incubation in these pHs. Pul703 was EDTA-resistant and detergent-tolerant, with a relative activity of 100, 99, and 114.8% at the presence of 10 mM EDTA, 10% of Triton X-100 and Tween 20, respectively. Pul703 can efficiently hydrolyze pullulan with a specific activity of 270 U/mg, which was higher than all reported type I pullulanases. In addition, Pul703 can act synergistically with α-amylase BLA to efficiently hydrolyze amylopectin. These results suggested that Pul703 was a good candidate for cold starch hydrolysis. [ABSTRACT FROM AUTHOR]

A multiple protein sequence alignment of l -alanine dehydrogenases from different bacterial species revealed that five highly conserved amino acid residues Arg-15, Lys-73, Lys-75, His-96 and Asp-269 are potential catalytic residues of l -alanine dehydrogenase from Bacillus pseudofirmus OF4. In this study, recombinant OF4Ald and its mutants of five conserved residues were constructed, expressed in Escherichia coli , purified by His 6 -tag affinity column and gel filtration chromatography, structure homology modeling, and characterized. The purified protein OF4Ald displayed high specificity to l -alanine (15 U mg −1 ) with an optimal temperature and pH of 40 °C and 10.5, respectively. Enzymatic assay and activity staining in native gels showed that mutations at four conserved residue Arg-15, Lys-75, His-96 and Asp-269 (except residue Lys-73) resulted in a complete loss in enzymatic activity, which signified that these predicted active sites are indispensable for OF4Ald activity. In contrast, the mutant K73A resulted in 6-fold improvement in k cat / K m towards l -alanine as compared to the wild type protein. Further research of the residue Lys-73 substituted by various amino acids and structural modeling revealed that residue Lys-73 might be involved in the catalytic reaction of the enzyme by influencing the enzyme-substrate binding through the hydrogen-bonding interaction with conserved residue Lys-75. [ABSTRACT FROM AUTHOR]

Debranching of (1, 6)-α-linkages in starch is of great significance as it is widely used in different industries. In recent years, identifying a single potential enzyme that could debranch (1, 6)-α-linkages and cleave (1, 4)-α-linkages brought great interest as amylopectin possessed both (1, 4)-α- and (1, 6)-α-linkages. In the present study, a 64\,kDa exo-α-l, 4-glucosidase from Bacillus pseudofirmus 703, annotated as Amy112, was cloned and characterized. Biochemical analysis indicated that heterologous Amy112 expressed in Escherichia coli exhibited a high activity against amylopectin, with the optimal temperature and pH of 40 °C and pH 7.0, respectively. Addition of K + ions improved the amy112 activity by 12%, but Li + , Ca 2+ and Mg 2+ ions showed no significant effect. Amy112 sequence homology revealed that it belonged to glycoside hydrolase family 13, showing 65% identity with α-glucosidase GSJ from Geobacillus sp. HTA-462. This is the first report indicating that Amy112 from B. pseudofirmus 703 belongs to GH13 enzyme family, having both exo-α-1, 4-glucosidase and oligo-l, 6-glucosidase activities. However, no transglycosylation activity was detected in LC–MS analysis. Amy112 would be of great significance of being utilized to debranch starch in different industries in a cost effective manner. [ABSTRACT FROM AUTHOR]

Cracks remaining after autogenous healing of mortar can be further healed when subjected to a bacterial suspension of Bacillus pseudofirmus if it contains organic calcium compounds. The consumption of organic calcium compounds, such as calcium lactate, by bacteria is providing the necessary calcium to precipitate calcite. The addition of inorganic calcium salts is inefficient to promote the formation of calcite during bacterial growth experiments, but can be efficient during biohealing experiments if the anion of the inorganic calcium salt can react with the cement paste. The addition of both organic and inorganic calcium compounds improves the robustness of the biohealing. [ABSTRACT FROM AUTHOR]

Cardiolipin (CL), a membrane phospholipid in bacteria and mitochondria, has been hypothesized to facilitate movement of protons on the outer surface of membranes in support of respiration- dependent ATP synthesis, oxidative phosphorylation (OXPHOS). If so, the high levels of membrane CL found in alkaliphilic bacteria, such as Bacillus pseudofirmus OF4, might facilitate its robust OXPHOS at pH 10.5, where the bulk protonmotive (PMF) force is low. To address the role of CL in Bacillus pseudofirmus OF4, we studied strains in which genes (cls) potentially encoding a CL synthase (CLs) were deleted: three single (ΔclsA, ΔclsB, and ΔclsC), one double (ΔclsA/B), and one triple (ΔclsA/B/C) mutant. Two-dimensional thin layer chromatography analyses of lipid extracts from 32P-labeled strains showed that the wild-type CL content was 15% of total phospholipids at pH 10.5 versus 3% at pH7.5duringlogphase. The % CL was higher (28-33%) at both pH values during stationary phase. The clsA gene plays a major role in CL biosynthesis as no detectable CL was found in ΔclsA-containing mutants, whereas the CL precursor phosphatidylglycerol was elevated. The ΔclsB mutant exhibited no significant reduction in CL, but clsB expression was up-regulated and appeared to support growth at pH 7.5. In the absence of detectable CL, the alkaliphile showed no significant deficits in non-fermentative growth, respiration- dependent ATP synthesis, or salt tolerance. Minor deficits in respiration and ATP synthase assembly were noted in individual mutants. In long term survival experiments, significant growth defects were found in ΔclsA strains and the ΔclsC strain at pH 10.5. [ABSTRACT FROM AUTHOR]

Abstract: An open reading frame of 1100 bp in the partially sequenced genome sequence of alkaliphilic Bacillus pseudofirmus OF4 was identified as a putative alanine racemase gene (dadX OF4 ), which was cloned and expressed in Escherichia coli BL21 (DE3). The encoded protein DadXOF4 was purified to homogeneity by His6-tag affinity column, gel filtration and ion-exchange chromatography. The amino acid sequence has highest identity with the known alanine racemase from Oceanobacillus iheyensis HTE831 (48%). The protein was a dimeric, endogenous PLP-dependent enzyme, which was demonstrated by absorption spectra and enzyme activity with or without PLP. The racemization temperature optimum was 40 °C and the optimal pH was 10.5. The kinetic parameters K m and V max at 40 °C of alanine racemase, determined by HPLC analysis, were 41.79 mM, 10,500 units/mg for L-alanine and 14.91 mM, 3708 units/mg for D-alanine, respectively. [Copyright &y& Elsevier]