枯草芽胞杆菌L-天冬氨酸a脱羧酶的酶学性质

b-丙氨酸是一种重要的医药化工原料,目前主要依靠化学法进行生产。探寻更为环保和高效的生物生产法是未来研究的一个方向。L-天冬氨酸a脱羧酶 (PanD) 能特异地脱去L-天冬氨酸的a羧基,生成b-丙氨酸。本文比较了3种分别来源于大肠杆菌、谷氨酸棒状杆菌及枯草芽胞杆菌的PanD比酶活 (分别为0.98、7.52和8.4 U/mg)。后两者的最适pH均为6.5,最适反应温度分别为65 ℃及60 ℃。与目前研究最多的来源于大肠杆菌和谷氨酸棒状杆菌的PanD相比,来源于枯草芽胞杆菌的PanD具有更好的活性和热稳定性,具有更强的工业应用潜力。同时,本文对该酶特有的翻译后自剪切及机理性失活现象进行了分析和讨论。     

Characterization of an lrp-like (IrpC ) gene from Bacillus subtilis

In the course of the Bacillus subtilis genome sequencing project, we identified an open reading frame encoding a putative 16.4?kDa protein. This protein shows, respectively, 34% and 25% identity with the Escherichia coli regulatory proteins Lrp and AsnC. Phylogenetic analysis suggests that it represents a new group in the AsnC-Lrp family. Sequence comparisons, as well as immunodetection experiments, lead to the conclusion that the product of this B. subtilislrp-likegene is a bona fide Lrp protein – the first one to be detected in gram-positive bacteria. When expressed in E.?coli, the B. subtilis Lrp-like protein is able to repress, by about two-fold, the expression of the ilvIH operon which is normally regulated by E. coli Lrp, indicating functional similarity in their regulatory targets. Vegetative growth of a B. subtilis lrp-like mutant is not affected in rich medium. However, the lrp-like mutation causes a transitory inhibition of growth in minimal medium in the presence of valine and isoleucine, which is relieved by leucine. This points to a possible role in regulation of amino acid metabolism. In addition, sporogenesis occurs earlier in the lrp-like mutant than in the reference strain, implying that the B. subtilis Lrp-like protein plays a role in the growth phase transition.     

Enzymatic characterization of 5-methylthioribose 1-phosphate isomerase from Bacillus subtilis

The product of the mtnA gene of Bacillus subtilis catalyzes the isomerization of 5-methylthioribose 1-phosphate (MTR-1-P) to 5-methylthioribulose 1-phosphate (MTRu-1-P). The catalysis of MtnA is a novel isomerization of an aldose phosphate harboring a phosphate group on the hemiacetal group. This enzyme is distributed widely among bacteria through higher eukaryotes. The isomerase reaction analyzed using the recombinant B. subtilis enzyme showed a Michaelis constant for MTR-1-P of 138 microM, and showed that the maximum velocity of the reaction was 20.4 micromol min(-1) (mg protein)(-1). The optimum reaction temperature and reaction pH were 35 degrees C and 8.1. The activation energy of the reaction was calculated to be 68.7 kJ mol(-1). The enzyme, with a molecular mass of 76 kDa, was composed of two subunits. The equilibrium constant in the reversible isomerase reaction [MTRu-1-P]/[MTR-1-P] was 6. We discuss the possible reaction mechanism.     

Isolation of constitutive mutants for L-arabinose utilization in Bacillus subtilis.

Constitutive mutants for L-arabinose utilization were isolated from Bacillus subtilis 168T+ and showed resistance to D-fucose, a nonmetabolizable analog of L-arabinose. The mutations that conferred the constitutive phenotype (Arac) were mapped between cysB and hisA. All the mutants showed an isomerase activity which was reduced to 50 to 70% in the presence of L-arabinose and to 10% in the presence of glucose.     

Characterization of a recombinant l-rhamnose isomerase from Bacillus subtilis and its application on production of l-lyxose and l-mannose

The gene of an l-rhamnose isomerase (RhaA) from Bacillus subtilis was cloned to the pET28a(+) and then expressed in the E. coli ER2566. The expressed enzyme was purified with a specific activity of 3.58 U/mg by His-Trap affinity chromatography. The recombinant enzyme existed as a 194 kDa tetramer and the maximal activity was observed at pH 8.0 and 60°C. The RhaA displayed activity for l-rhamnose, l-lyxose, l-mannose, d-allose, d-gulose, d-ribose, and l-talose, among all aldopentoses and aldohexoses and it showed enzyme activity for l-form monosaccharides such as l-rhamnose, l-lyxose, l-mannose, and l-talose. The catalytic efficiency (k _cat/K _m) of the recombinant enzyme for l-rhamnose, l-lyxose, and l-mannose were 7,460, 1,013, and 258 M/sec. When l-xylulose 100 g/L and l-fructose 100 g/L were used as substrates, the optimum concentrations of RpiB were determined with 6 and 15 U/mL, respectively. The l-lyxose 40 g/L was produced from l-xylulose 100 g/L by the enzyme during 60 min, while l-mannose 25 g/L was produced from l-fructose 100 g/L for 80 min. The results suggest that RhaA from B. subtilis is a potential producer of l-form monosaccharides.     

Characterization of an inhibitor of the intracellular protease from Bacillus subtilis.

A specific inhibitor of intracellular serylprotease from Bacillus subtilis has been isolated from both growing and sporulating cells. Like other protease inhibitors isolated from eukaryotic cells, the inhibitor from B. subtilis is a thermostable protein. A purification method is described. The molecular weight estimated by Biogel filtration and SDS gel electrophoresis is about 15,500. Both proteolytic and esterolytic activities of intracellular protease are equally sensitive to inhibition. With azocoll or Z-tyrosine p-nitrophenylester as substrates, noncompetitive inhibition patterns are observed. The inhibitor has no effect on the proteolytic or esterolytic activities of the extracellular serylprotease. A similar thermostable inhibitor is also present in Bacillus megaterium.     

Characterization of a thermostable L-arabinose (D-galactose) isomerase from the hyperthermophilic eubacterium Thermotoga maritima

The araA gene encoding L-arabinose isomerase (AI) from the hyperthermophilic bacterium Thermotoga maritima was cloned and overexpressed in Escherichia coli as a fusion protein containing a C-terminal hexahistidine sequence. This gene encodes a 497-amino-acid protein with a calculated molecular weight of 56,658. The recombinant enzyme was purified to homogeneity by heat precipitation followed by Ni(2+) affinity chromatography. The native enzyme was estimated by gel filtration chromatography to be a homotetramer with a molecular mass of 232 kDa. The purified recombinant enzyme had an isoelectric point of 5.7 and exhibited maximal activity at 90 degrees C and pH 7.5 under the assay conditions used. Its apparent K(m) values for L-arabinose and D-galactose were 31 and 60 mM, respectively; the apparent V(max) values (at 90 degrees C) were 41.3 U/mg (L-arabinose) and 8.9 U/mg (D-galactose), and the catalytic efficiencies (k(cat)/K(m)) of the enzyme were 74.8 mM(-1).min(-1) (L-arabinose) and 8.5 mM(-1).min(-1) (D-galactose). Although the T. maritima AI exhibited high levels of amino acid sequence similarity (>70%) to other heat-labile mesophilic AIs, it had greater thermostability and higher catalytic efficiency than its mesophilic counterparts at elevated temperatures. In addition, it was more thermostable in the presence of Mn(2+) and/or Co(2+) than in the absence of these ions. The enzyme carried out the isomerization of D-galactose to D-tagatose with a conversion yield of 56% for 6 h at 80 degrees C.     

The shape of L-arabinose isomerase from Escherichia coli.

L-Arabinose isomerase, EC 5.3.1.4, catalyzes the conversion of L-arabinose to L-ribulose, the first step in the catabolism of L-arabinose by Escherichia coli B/r. Patrick and Lee (1969) J. Biol. Chem. 244, 4277--4283) demonstrated that native L-arabinose isomerase is composed of six identical subunits of approximately Mr = 60,000. In this paper we describe an electron microscopy study of the arrangement of the six identical subunits. The isomerase is seen in two distinctly different orientations. The first has three subunits visible, with a 3-fold axis of symmetry, corresponding to a face-on view of two stacked, eclipsed trimers. The second orientation is rectangular in shape with 2-fold symmetry; suggesting a side-on view of the stacked trimers. The six identical subunits are thus arranged with D3 symmetry as in a trigonal prism. Measurements were made on the maximum profile of the three 2-fold axes of symmetry of the face-on orientations, and of both the long and short dimensions of the side-on orientation. The best estimate for the maximum profile of the 2-fold axes of symmetry of the face-on view is 106 +/- 8 A, using glutamine synthetase as an internal size standard. Measurements from micrographs of the isomerase alone, using an external magnification calibration, give the following results: for the maximum profile of the three 2-fold axes of symmetry of the face-on view, 132 +/- 7 A; for the long axis of the side-on view, 136 +/- 10 A; and for the short axis, 105 +/- 6 A. These measurements are consisting with the interpretation of the profiles as representing two different orientations of the L-arabinose isomerase.     

Characterization of Bacillus subtilis bacteriophages

Brodetsky, Anna M. (University of California, Los Angeles), and W. R. Romig. Characterization of Bacillus subtilis bacteriophages. J. Bacteriol. 90:1655-1663. 1965.-A group of six phages, SP5, SP6, SP7, SP8, SP9, and SP13, which use the Marburg strain of Bacillus subtilis as host was characterized. These phages, referred to as group 1, were examined for the following properties: host range, plaque morphology, stability, adsorption kinetics, one-step growth characteristics, calcium requirements, serum neutralization, thermal inactivation, and inactivation by ultraviolet irradiation. Five unrelated B. subtilis phages, SP3, SP10, PBS1, SP alpha, and SP beta, were included in the studies. When first isolated, none of the group 1 phages was able to replicate efficiently on B. subtilis SB19, a mutant of the "transforming" B. subtilis 168. Host range mutants capable of growth in SB19 were isolated for all of the group 1 phages except SP13, and are designated the "star" phages (SP5* through SP9*). For characterization, SB19 was used as host for the star phages, and another B. subtilis mutant, 168B, was host for SP13.     

Purification and characterization of an L-arabinose isomerase from an isolated strain of Geobacillus thermodenitrificans producing D-tagatose

The araA gene, encoding l-arabinose isomerase (AI), from the thermophilic bacterium Geobacillus thermodenitrificans was cloned and expressed in Escherichia coli. Recombinant AI was isolated with a final purity of about 97% and a final specific activity of 2.10 U/mg. The molecular mass of the purified AI was estimated to be about 230 kDa to be a tetramer composed of identical subunits. The AI exhibited maximum activity at 70 degrees C and pH 8.5 in the presence of Mn2+. The enzyme was stable at temperatures below 60 degrees C and within the pH range 7.5-8.0. d-Galactose and l-arabinose as substrate were isomerized with high activities. Ribitol was the strongest competitive inhibitor of AI with a Ki of 5.5mM. The apparent Km and Vmax for L-arabinose were 142 mM and 86 U/mg, respectively, whereas those for d-galactose were 408 mM and 6.9 U/mg, respectively. The catalytic efficiency (kcat/Km) was 48 mM(-1)min(-1) for L-arabinose and 0.5mM(-1)min(-1) for D-galactose. Mn2+ was a competitive activator and increased the thermal stability of the AI. The D-tagatose yield produced by AI from d-galactose was 46% without the addition of Mn2+ and 48% with Mn2+ after 300 min at 65 degrees C.     

Isolation and Characterization of a Xylanase from Bacillus subtilis

Partial characterization of an extracellular xylanase isolated by chromatography from Bacillus subtilis gave a molecular weight of 32,000 and optimum pH and temperature of 5.0 and 50°C, respectively. K_m and V_max values, determined with a soluble larchwood xylan, were 0.16% and 7.0 × 10³ μmol min⁻¹ mg⁻¹ of enzyme respectively. The amino acid composition showed more basic amino acid residues than in a previously characterized xylanase from a white-rot fungus.     

枯草芽孢杆菌基因启动子的分离与鉴定

利用启动子探针型载体pSUPV4直接在大肠杆菌 (Escherichiacoli)中分离枯草芽孢杆菌 (Bacillussubtilis)WB6 0 0的基因启动子片段 ,获得 5 5个具有卡那霉素抗性的重组子。对 3个抗性最高的重组子pSU -Bs2 ,pSU -Bs4 ,pSU -Bs8进行序列测定和同源性分析发现 ,所克隆到的基因启动子片段均来自于枯草芽孢杆菌的基因组 ,并且具有枯草杆菌基因启动子的保守序列。对抗性最高的Bs2片段进一步研究表明 ,它可以在大肠杆菌中高效地启动来自于短小芽孢杆菌的碱性蛋白酶基因的表达 ,也能在枯草芽孢杆菌中启动卡那霉素抗性基因的表达。     

Characterization of a thermoacidophilic L-arabinose isomerase from Alicyclobacillus acidocaldarius: role of Lys-269 in pH optimum

The araA gene encoding L-arabinose isomerase (AI) from the thermoacidophilic bacterium Alicyclobacillus acidocaldarius was cloned, sequenced, and expressed in Escherichia coli. Analysis of the sequence revealed that the open reading frame of the araA gene consists of 1,491 bp that encodes a protein of 497 amino acid residues with a calculated molecular mass of 56,043 Da. Comparison of the deduced amino acid sequence of A. acidocaldarius AI (AAAI) with other AIs demonstrated that AAAI has 97% and 66% identities (99% and 83% similarities) to Geobacillus stearothermophilus AI (GSAI) and Bacillus halodurans AI (BHAI), respectively. The recombinant AAAI was purified to homogeneity by heat treatment, ion-exchange chromatography, and gel filtration. The purified enzyme showed maximal activity at pH 6.0 to 6.5 and 65 degrees C under the assay conditions used, and it required divalent cations such as Mn2+, Co2+, and Mg2+ for its activity. The isoelectric point (pI) of the enzyme was about 5.0 (calculated pI of 5.5). The apparent Km values of the recombinant AAAI for L-arabinose and D-galactose were 48.0 mM (Vmax, 35.5 U/mg) and 129 mM (Vmax, 7.5 U/mg), respectively, at pH 6 and 65 degrees C. Interestingly, although the biochemical properties of AAAI are quite similar to those of GSAI and BHAI, the three AIs from A. acidocaldarius (pH 6), G. stearothermophilus (pH 7), and B. halodurans (pH 8) exhibited different pH activity profiles. Based on alignment of the amino acid sequences of these homologous AIs, we propose that the Lys-269 residue of AAAI may be responsible for the ability of the enzyme to act at low pH. To verify the role of Lys-269, we prepared the mutants AAAI-K269E and BHAI-E268K by site-directed mutagenesis and compared their kinetic parameters with those of wild-type AIs at various pHs. The pH optima of both AAAI-K269E and BHAI-E268K were rendered by 1.0 units (pH 6 to 7 and 8 to 7, respectively) compared to the wild-type enzymes. In addition, the catalytic efficiency (kcat/Km) of each mutant at different pHs was significantly affected by an increase or decrease in Vmax. From these results, we propose that the position corresponding to the Lys-269 residue of AAAI could play an important role in the determination of the pH optima of homologous AIs.     

Characterization of Bacillus subtilis recombinational pathways

Recombination in Bacillus subtilis requires the products of numerous rec loci. To dissect the various mechanisms which may be involved in genetic recombination, we constructed a series of isogenic strains containing more than one mutant rec allele. On the basis of their impairment in genetic exchange, the various loci (represented by specific rec alleles) were classified into different epistatic groups. Group alpha consists of rec genes represented by recB, recD, recF, recG, recL, and recR mutations, while group beta comprises the addA and addB mutations. Group gamma consists of the recH and recP mutations. These results suggest that B. subtilis has multiple pathways for genetic recombination and that the products of the genes within the alpha, beta, and gamma epistatic groups are involved in these alternative recombination pathways. The RecA protein is required in all three pathways of intermolecular recombination.