Bilirubin Oxidase Activity of Bacillus subtilis CotA

The spore coat protein CotA from Bacillus subtilis was previously identified as a laccase. We have now found that CotA also shows strong bilirubin oxidase activity and markedly higher affinity for bilirubin than conventional bilirubin oxidase. This is the first characterization of bilirubin oxidase activity in a bacterial protein.     

A Novel Extracellular Multicopper Oxidase from Phanerochaete chrysosporium with Ferroxidase Activity

Lignin degradation by the white rot basidiomycete Phanerochaete chrysosporium involves various extracellular oxidative enzymes, including lignin peroxidase, manganese peroxidase, and a peroxide-generating enzyme, glyoxal oxidase. Recent studies have suggested that laccases also may be produced by this fungus, but these conclusions have been controversial. We identified four sequences related to laccases and ferroxidases (Fet3) in a search of the publicly available P. chrysosporium database. One gene, designated mco1, has a typical eukaryotic secretion signal and is transcribed in defined media and in colonized wood. Structural analysis and multiple alignments identified residues common to laccase and Fet3 sequences. A recombinant MCO1 (rMCO1) protein expressed in Aspergillus nidulans had a molecular mass of 78 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the copper I-type center was confirmed by the UV-visible spectrum. rMCO1 oxidized various compounds, including 2,2′-azino(bis-3-ethylbenzthiazoline-6-sulfonate) (ABTS) and aromatic amines, although phenolic compounds were poor substrates. The best substrate was Fe²⁺, with a K_m close to 2 μM. Collectively, these results suggest that the P. chrysosporium genome does not encode a typical laccase but rather encodes a unique extracellular multicopper oxidase with strong ferroxidase activity.     

Direct Identification of a Bacterial Manganese(II) Oxidase, the Multicopper Oxidase MnxG, from Spores of Several Different Marine Bacillus Species

Microorganisms catalyze the formation of naturally occurring Mn oxides, but little is known about the biochemical mechanisms of this important biogeochemical process. We used tandem mass spectrometry to directly analyze the Mn(II)-oxidizing enzyme from marine Bacillus spores, identified as an Mn oxide band with an in-gel activity assay. Nine distinct peptides recovered from the Mn oxide band of two Bacillus species were unique to the multicopper oxidase MnxG, and one peptide was from the small hydrophobic protein MnxF. No other proteins were detected in the Mn oxide band, indicating that MnxG (or a MnxF/G complex) directly catalyzes biogenic Mn oxide formation. The Mn(II) oxidase was partially purified and found to be resistant to many proteases and active even at high concentrations of sodium dodecyl sulfate. Comparative analysis of the genes involved in Mn(II) oxidation from three diverse Bacillus species revealed a complement of conserved Cu-binding regions not present in well-characterized multicopper oxidases. Our results provide the first direct identification of a bacterial enzyme that catalyzes Mn(II) oxidation and suggest that MnxG catalyzes two sequential one-electron oxidations from Mn(II) to Mn(III) and from Mn(III) to Mn(IV), a novel type of reaction for a multicopper oxidase.     

Mannanase Components from Bacillus pumilus

beta-d-Mannanase from Bacillus pumilus was purified into two components, A and B, which exhibited homogeneity on polyacrylamide gel electrophoresis. These components had molecular weights of 55,000 (A) and 37,000 (B); carbohydrate contents of 15.3% (A) and 7.2% (B); specific activities of 78 (A) and 1,616 (B) U/mg; pH optima of 5.5 to 6.9 (A) and 6.0 (B); and half-lives of 60 (A) and 21 (B) min at 70 degrees C.     

Extracellular ribonuclease from Bacillus pumilus]

The extracellular ribonuclease (RNAse Bp) was isolated from the cultural medium filtrate of Bacillus pumilus by ammonium sulfate precipitation and two stages of ion-exchange chromatography on carboxymethyl- and phospho-cellulose columns. The amino acid composition and N-terminal amino acid residue have been determined. The kinetic parameters of cleavage reaction of synthetic polynucleotides have been measured. According to their structural homology RNAse Bp has been shown to be similar to RNAses Ba and Bi. Catalytic properties of the enzyme are very close to RNAse Bi.     

CotA of Bacillus subtilis is a copper-dependent laccase

The spore coat protein CotA of Bacillus subtilis displays similarities with multicopper oxidases, including manganese oxidases and laccases. B. subtilis is able to oxidize manganese, but neither CotA nor other sporulation proteins are involved. We demonstrate that CotA is a laccase. Syringaldazine, a specific substrate of laccases, reacted with wild-type spores but not with DeltacotA spores. CotA may participate in the biosynthesis of the brown spore pigment, which appears to be a melanin-like product and to protect against UV light.     

Cloning, expression and characterization of a novel esterase from Bacillus pumilus

The gene encoding esterase (CE1) from Bacillus pumilus ARA with a calculated molecular weight of 28.4 kDa was cloned, sequenced and efficiently expressed in Escherichia coli. The open reading frame of 747 nucleotides encoded a protein, which was classified as a carboxylesterase with an identity of 87 % to esterase from Bacillus subtilis 168. Recombinant CE1 was purified in a single step to electrophoretic homogeneity by IMAC (Ni²⁺). The enzyme displayed maximum activity toward p-nitrophenyl (pNP) acetate at 37–40 °C and pH?6.5–7.0. It was stable in the pH range from 6.5 to 8.0, and at temperature from 25 to 40 °C. Among four p-nitrophenyl esters tested, the best substrate was pNP acetate with K _m and k _cat values of 0.33 mM and 4.07 s^?1, respectively. Amounts of 2 mM Ca²⁺ and Co²⁺ significantly increased the esterase activity to 190 and 121 %, respectively. These results suggest that CE1 has very attractive applications of increasing feed digestibility in animal nutrition in this moderate temperature range.     

Soybean-milk-coagulating activity of Bacillus pumilus derives from a serine proteinase

A proteolytic enzyme from Bacillus pumilus strain TYO-67, which was able to coagulate the protein in soybean milk, was characterized enzymologically. The optimum pH and temperature for its activities were 9.0 and 50 °C, respectively. The enzyme was strongly believed to be a serine proteinase because it was completely inhibited by the addition of diisopropyl fluorophosphate or phenylmethanesulfonyl fluoride. Hammerstein milk casein, cytochrome c and soybean protein were good substrates for the enzyme. Seven cleavages were detected using the oxidized insulin B-chain as peptide substrate for the proteolytic specificity test of the serine proteinase from B. pumilus. The bonds most susceptible to the action of the serine proteinase from B. pumilus were Leu-15–Tyr-16. The mode of action on soybean milk protein by the enzyme from B. pumilus was also investigated. The acidic subunit in glycinin and the α′-, α- and β-subunits in β-conglycinin were degraded during the enzyme reaction. However, the basic subunit in glycinin could not be degraded by the enzyme. The formation of coagula in soybean milk caused by the serine proteinase from B. pumilus was mainly due to the hydrophobic interaction. Received: 9 July 1999 / Received last revision: 22 October 1999 / Accepted: 5 November 1999     

一株高效羽毛降解菌株的分离与鉴定

采用以羽毛粉为唯一碳源和氮源的培养基,从自然界中分离到一株能够高效降解羽毛角蛋白的细菌,经形态学观察,生理生化实验和16SrRNA基因鉴定,初步确定该菌株为短小芽孢杆菌(Bacillus pumilus),且命名为短小芽孢杆菌WHK4。发酵48 h时,羽毛粉降解率达到85.76%。本研究为微生物降解羽毛角蛋白提供了优良的菌株,在蛋白饲料生产中具有潜在的广泛的应用前景。     

Purification and mode of action of an alkali-resistant endo-1, 4-beta-glucanase from Bacillus pumilus

Alkaline endo-1,4-beta-d-glucanase was secreted by Bacillus pumilus grown in submerged culture on a combination of oat spelt xylan and corn starch as carbon sources. The enzyme was purified to homogeneity by Sephacryl S-200 and Q-Sepharose column chromatography. The protein corresponded to molecular mass and pI values of 67 kDa and 3.7, respectively. The enzyme was optimally active at pH 7.0-8.0 and 60 degrees C and retained 50% of its optimum activity at pH 12. The most notable characteristic of the endoglucanase was its high stability up to pH 12 for 20 h at 30 degrees C. The enzyme hydrolyzed carboxymethylcellulose (CMC) and cello-oligosaccharides but was inactive on cellobiose, cellotriose, Avicel, xylan, 4-nitrophenyl-beta-d-glucoside, 4-nitrophenyl-beta-d-cellobioside, and 4-nitrophenyl-beta-d-xyloside. Analysis of reaction mixtures by HPLC revealed that the enzyme produced almost exclusively cellotriose when acted on CMC and appeared to hydrolyze cello-oligosaccharides by successively releasing cellotriose. The use of 4-methylumbelliferyl cello-oligosaccharides and the determination of bond cleavage frequency revealed that the enzyme preferentially hydrolyzed the third glycosidic bond adjacent to the glycon. The enzyme mediated a decrease in the viscosity of CMC associated with a release of only small amounts of reducing sugar. The enzyme activity was not inhibited by metal ions, surfactants, and chelating agents used as components of laundry detergents.     

Regulation of a glutamine amidotransferase subunit from Bacillus subtilis by a cloned trpE gene from Bacillus pumilus.

The influence of a cloned trpE (trpE+p) gene from Bacillus pumilus on the expression of the gat locus in Bacillus subtilis was examined. The trpE gene was regulated by tryptophan and the mtr locus, which specifies the presumed aporepressor. The specific activity of subunit G varied directly with the level of subunit Ep, and the heterologous EpG complex that was formed was stable to gel filtration.     

Improving Glyphosate Oxidation Activity of Glycine Oxidase from Bacillus cereus by Directed Evolution

Glyphosate, a broad spectrum herbicide widely used in agriculture all over the world, inhibits 5-enolpyruvylshikimate-3-phosphate synthase in the shikimate pathway, and glycine oxidase (GO) has been reported to be able to catalyze the oxidative deamination of various amines and cleave the C-N bond in glyphosate. Here, in an effort to improve the catalytic activity of the glycine oxidase that was cloned from a glyphosate-degrading marine strain of Bacillus cereus (BceGO), we used a bacteriophage T7 lysis-based method for high-throughput screening of oxidase activity and engineered the gene encoding BceGO by directed evolution. Six mutants exhibiting enhanced activity toward glyphosate were screened from two rounds of error-prone PCR combined with site directed mutagenesis, and the beneficial mutations of the six evolved variants were recombined by DNA shuffling. Four recombinants were generated and, when compared with the wild-type BceGO, the most active mutant B3S1 showed the highest activity, exhibiting a 160-fold increase in substrate affinity, a 326-fold enhancement in catalytic efficiency against glyphosate, with little difference between their pH and temperature stabilities. The role of these mutations was explored through structure modeling and molecular docking, revealing that the Arg⁵¹ mutation is near the active site and could be an important residue contributing to the stabilization of glyphosate binding, while the role of the remaining mutations is unclear. These results provide insight into the application of directed evolution in optimizing glycine oxidase function and have laid a foundation for the development of glyphosate-tolerant crops.     

Purification and characterization of a soybean-milk-coagulating enzyme from Bacillus pumilus TYO-67

Bacillus pumilus TYO-67 was isolated from tofu (soybean curd) as the best producer of a soybean-milk-coagulating enzyme, induced by the addition of soybean protein to the growth medium. The enzyme was purified approximately 30-fold with an 11% yield. The homogeneous preparation of the enzyme showed that it is a monomer with a molecular mass of about 30 kDa and has an isoelectric point at pH 9.75. The results of amino acid composition analyses showed that the enzyme is rich in alanine, aspartic acid, glycine, serine and valine. Although the amino-terminal amino acid (alanine) was identical with that of subtilisins, the amino-terminal sequence was different from those of subtilisins. The α-helix content of the enzyme was calculated to be 28.2%. The optimum pH and temperature were observed at 6.0–6.1 and 65 °C respectively. The enzyme was significantly activated by the addition of 1 mM Mn²⁺, Ca²⁺, Mg²⁺, and Sr²⁺ ions in the reaction mixture, and its thermal stability was significantly increased by Ca²⁺ ion. Received: 31 August 1998 / Received last revision: 1 December 1998 / Accepted: 20 December 1998     

Purification and characterization of a thermostable alkaline protease from alkalophilic Bacillus pumilus

AIMS: An investigation was carried out on the purification and characterization of an alkaline protease from Bacillus pumilus MK6-5. METHODS AND RESULTS: An alkalophilic Bacillus pumilus MK6-5 was grown in a laboratory fermenter containing 1% reverse osmosis concentrated cheese whey powder, 0.25% corn steep liquor, 1% glucose, 0.5% tryptone, 1% sodium citrate, 0.02% MgSO4.7H2O and 0.65% Na2CO3 at 35 degrees C and pH 9.6, agitation at 250 rev min(-1) and aeration of 1 vvm for 60 h. When the enzyme was purified using ammonium sulphate precipitation, ion exchange and gel filtration chromatographies, a 26.2% recovery of enzyme with 36.6-fold purification was recorded. The purified protease was found to be homogenous by SDS-PAGE with molecular mass estimate of 28 kDa. The enzyme was optimally active at pH 11.5 and temperature of 55-60 degrees C. The Km and kcat values observed with synthetic substrates at 37 degrees C and pH 8.0 were 1.1 mmol l(-1) and 624 s(-1) for Glu-Gly-Ala-Phe-pNA and 3.7 mmol l(-1) and 826 s(-1) for Glu-Ala-Ala-Ala-pNA, respectively. The kinetic data revealed that small aliphatic and aromatic residues were the preferred residues at the P1 position. Inhibition profile exhibited by PMSF suggested the B. pumilus protease to be an alkaline serine protease. CONCLUSIONS: Bacillus pumilus MK6-5 produced a calcium-dependent, thermostable alkaline serine protease. SIGNIFICANCE AND IMPACT OF THE STUDY: The thermostable alkaline protease from Bacillus pumilus MK6-5 will be extremely useful in ultrafiltration membrane cleaning due to its ability to work in broad pH and temperature ranges, and tolerance to detergents, unlike the mesophilic proteases which face these limitations.     

短小芽孢杆菌degQ基因的克隆与鉴定

degQ基因编码一个由46个氨基酸组成的多肽,能增强许多芽孢杆菌胞外酶基因的表达．以pMK4作克隆载体构建短小芽孢杆菌基因文库,并用DNA探针原位杂交法从中钓出degQ基因．对克隆基因的DNA序列进行了分析并证明克隆的短小芽孢杆菌degQ基因具有增强枯草杆菌蛋白酶和果聚糖蔗糖酶基因表达的能力．degQ基因克隆有助于研究芽孢杆菌的正调控机理并可望提高外源基因在芽孢杆菌中表达．     

Identification and Characterization of a Gamma-Glutamyl Transpeptidase from a Thermo-Alcalophile Strain of Bacillus pumilus

A γ-glutamyltranspeptidase (GGT, E.C. 2.3.2.2) was isolated from a strain (A8) originating from Lake Bogoria (Kenya) and homologous with Bacillus pumilus. This GGT shows an optimal activity at pH 8.9 and 62°C. The enzyme is thermostable up to 43°C. The best reagent among the potential inhibitors was shown to be DON, which is an inhibitor highly specific for GGTs. Gly-Gly-Ala, Gly-Gly-Gly and Gly-Gly were identified as the best acceptors for the transpeptidation reactions catalyzed by the enzyme. The SDS-PAGE study revealed that the enzyme consists of two non-identical subunits (38,000 and 23,000). Only the large subunit was active when the enzyme was dissociated under denaturing conditions. The behavior of the native enzyme suggests that the active site of the large subunit is masked by the small subunit.     

Characterization and a point mutational approach of a psychrophilic lipase from an arctic bacterium,Bacillus pumilus

A bacterium with lipolytic activity was isolated from the Chukchi Sea within the Arctic Ocean. The lipase BpL5 from the isolate, Bacillus pumilus ArcL5, belongs to subfamily 4 of lipase family I. The optimum pH and temperature of the recombinant enzyme BpL5, as expressed in Escherichia coli, were 9.0 and 20 °C, respectively. The enzyme retained 85 % of its activity at 5 °C. There was a significant difference between temperatures for maximal activity (20 °C) and for protein denaturation (approx. 45 °C). The enzyme preferred middle-chain (C8) p-nitrophenyl substrates. Two mutants, S139A and S139Y, were rationally designed based on the 3D-structure model, and their activities were compared with that of the wild type. The both mutants showed significantly improved activity against tricaprylin.