Identification of Catalytically Active Groups in Inulinase from Bacillus polymyxa 722

Inulinase from Bacillus polymyxa 722, hydrolyzing a polyfructosan inulin, was studied. The dependence of inulinase activity on pH, measurements of pK value, calculation of the ionization heat, photoinactivation with methylene blue, and inhibition with p-chloromercuribenzoate suggest that the active center of this enzyme contains imidazole and sulfhydryl groups. A possible mechanism underlying the cleavage of -2,1-fructoside bonds in the inulin molecule by inulinase is considered.     

Identification of catalytically active groups of lipoxygenase from wheat germ

Catalytically active groups of lipoxygenase in wheat germs have been identified. Blocking of catalytically active groups by specific reagents, dependence of the enzyme activity on pH, enthalpy of ionization of pK1 and pK2, the enzyme photoinactivation allowed to make a conclusion that imidasole and hydroxyl groups take part in lipoxygenase catalytic cycle.     

Identification of catalytically active groups of pea (Pisum sativum L.) beta-glucosidase

Functional groups ofcytoplasmic pea beta-glucosidase pretreated to an electrophoretically homogeneous state were identified. Data on the pH dependence of the enzyme activity, calculated heat of ionization, photoinactivation of the enzyme in the presence of methylene blue, and inactivation of the enzyme with diethyl pyrocarbonate suggest that the catalytic site of beta-glucosidase contains the carboxyl group of glutamic or aspartic acids and the imidazole group of histidine.     

Identification of catalytically active groups of wheat (Triticum aestivum L.) germ lipase

The active site of wheat germ lipase was studied by the Dixon method and chemical modification. The profile of curve logV = f(pH), pK and ionization heat values, lipase photoinactivation, and lipase inactivation with diethylpyrocarbonate and dicyclohexylcarbodiimide led us to assume that the active site of the enzyme comprises the carboxylic group of aspartic or glutamic acid and the imidazole group of histidine. Apparently, the OH-group of serine plays a key role in catalysis: as a result of incubation for 1 h in the presence of phenylmethylsulfonyl fluoride, the enzyme activity decreased by more than 70%. It is shown that ethylenediamine tetraacetate is a noncompetitive inhibitor of lipase. Wheat germs are very healthful because they are rich in vitamins, essential amino acids, and proteins. For this reason, wheat germs are widely used in food, medical, and feed mill industries [1-3]. However, their use is limited by instability during storage, which is largely determined by the effect of hydrolytic and redox enzymes. Representative enzymes of this group are lipase (glycerol ester hydrolase, EC 3.1.1.3), which hydrolyzes triglycerides of higher fatty acids, and lipoxygenase (EC 1.13.11.13), which oxidizes polyunsaturated higher fatty acids.     

Production of optically active 2,3-butanediol by Bacillus polymyxa

Bacillus polymyxa produces (R, R)-2,3-butanediol from a variety of carbohydrates. Other metabolites are also produced including acetoin, acetate, lactate, and ethanol. The excretion of each metabolite was found to depend on the relative availability of oxygen to the culture. When the relative oxygen uptake rate was high, enhanced yields of acetate and acetoin were noted. At an intermediate oxygen availability, the butanediol yield was maximal. When the availability of oxygen was more restricted, higher yields of lactate and ethanol occurred. The cells appeared to regulate themselves such that energy generation is optimal subject to the constraint that the cells do not produce more reducing equivalents than can be oxidized by the electron transport system. The dependence of each product yield on the relative oxygen availability was determined, and this knowledge was used to carry out a fed-batch fermentation that attained a final butanediol concentration of over 40 g/L in 50 h.     

Bacillus polymyxa bacteriophages from Brazilian soils

Ten phages of Bacillus polymyxa were isolated from four different Brazilian soils. All were dsDNA-containing phages belonging to Bradley types A and B. Data obtained from electron microscopy and tests of resistance against physical and chemical agents showed that the isolates could be distributed among six different groups. Host range data were in agreement with this classification. When tested against 88 strains of 18 Bacillus species, these phages only infected B. polymyxa strains, thus revealing specificity for this species. Three phage groups lysed all 42 available B. polymyxa strains and are suggested for use in rapid identification of this species.This work was sponsored by the National Research Council of Brasil (CNPq) and CAPES.     

Identification of a second catalytically active trans-sialidase in Trypanosoma brucei

The procyclic stage of Trypanosoma brucei is covered by glycosylphosphatidylinositol (GPI)-anchored surface proteins called procyclins. The procyclin GPI anchor contains a side chain of N-acetyllactosamine repeats terminated by sialic acids. Sialic acid modification is mediated by trans-sialidases expressed on the parasite’s cell surface. Previous studies suggested the presence of more than one active trans-sialidases, but only one has so far been reported. Here we cloned and examined enzyme activities of four additional trans-sialidase homologs, and show that one of them, Tb927.8.7350, encodes another active trans-sialidase, designated as TbSA C2. In an in vitro assay, TbSA C2 utilized α2-3 sialyllactose as a donor, and produced an α2-3-sialylated product, suggesting that it is an α2-3 trans-sialidase. We suggest that TbSA C2 plays a role in the sialic acid modification of the trypanosome cell surface.     

The propeptide is not required to produce catalytically active neutral protease from Bacillus stearothermophilus

The thermolysin-like neutral protease from Bacillus stearothermophilus (TLP-ste) is usually produced extracellularly in Bacillus subtilis, where it is expressed as preproenzyme and subsequently processed in an autocatalytic, intramolecular process. To create the basis for the production of inactive mutants of TLP-ste, which cannot be processed in B. subtilis, we studied the expression of TLP-ste and its propeptide in cis and in trans in Escherichia coli. In contrast to thermolysin, subtilisin and alpha-lytic protease, which could be obtained only in the presence of the corresponding propeptides, TLP-ste could be produced as an active mature enzyme in E. coli in the absence of its prosequence. Surprisingly, however, a much more effective access to active mature protease was found when TLP-ste (devoid of its prosequence) was expressed as protein with an N-terminal His6 tag which accumulated in the form of inclusion bodies. Completely unexpected, the protein could be renatured from the inclusion bodies after solubilization in guanidine hydrochloride solutions in high yields. Purification to homogeneity was possible by affinity chromatography on Bacitracin silica as well as by immobilized metal ion affinity chromatography. By addition of separately expressed propeptide to the renaturation mixture yields of renaturation could not be increased significantly, confirming that the propeptide is not essential for proper folding of the enzyme or its stabilization during the folding process. Also in vivo, the expression levels of active mature TLP-ste in Escherichia coli did not significantly differ when the mature sequence was expressed alone or coexpressed with the prosequence in cis or in trans.     

Bacteriophages of Bacillus polymyxa

Virulent bacteriophages of colistin--producing Bacillus polymyxa strains were studied. The phages were found to differ in lytic spectrum and were active only against strains of B. polymyxa. They did not attack other strains of the genus Bacillus. The virulent bacteriophages belong to two morphological groups differing in size. The size of the DNA of the bacteriophages of both groups is similar and ranges from 74.9 X 10(6) to 87.8 X 10(6) daltons. The cells of different B. polymyxa strains were also found to carry various defective phages which could be shown after mitomycin C induction of cell cultures. The antibacterial activity of mitomycin C induced cell lysates was not detected. Strains of B. polymyxa most probably devoid of defective bacteriophages (delysogenized) were isolated.     

Purification and characterization of an arabinofuranosidase from Bacillus polymyxa expressed in Bacillus subtilis

Two polypeptides showing -l-arabinofuranosidase activity have been purified to homogeneity from culture supernatants of a Bacillus subtilis clone harbouring the xynD gene [Gosalbes et al. (1991) J Bacteriol 173: 7705–7710] from Bacillus polymyxa. Both polypeptides, with determined molecular masses of 64 kDa and 53 kDa, share the same sequence at their N termini, which also coincides with the sequence deduced for the mature protein from the previously determined sequence of nucleotides (Gosalbes et al. 1991). The two polypeptides have been biochemically characterized. Arabinose is the unique product released from arabinose-containing xylans which are substrates for both enzyme forms. Other natural arabinose-containing polysaccharides, such as arabinogalactans, are not attacked by them but some artificial arabinose derivatives are good substrates for both polypeptides. Their arabinose-releasing activity on arabinoxylans facilitates the hydrolysis of the xylan backbone by some endoxylanases from Bacillus polymyxa.     

Purification and properties of glutamine synthetase from Bacillus polymyxa

Glutamine synthetase (EC 6.3.1.2) was purified to homogeneity from a free-living nitrogen fixing bacteria, Bacillus polymyxa. The holoenzyme, relative molecular mass (M_r) of 600 000 is composed of monomeric sub-units of 60 000 (M_r). The isoelectric point of the sub-units was 5.2. The pH optimum for the biosynthetic and transferase enzyme activity was 8.2 and 7.8, respectively. The apparent K _m values (K _m ^app ) in the biosynthetic reaction for glutamate, NH₄Cl and ATP were 3.2, 0.22 and 1 mM, respectively. In the transferase reaction the K _m values for glutamine, hydroxylamine and ADP were 6.5, 3.5 and 8×10^-4 mM respectively. L-Methionine-D-L-sulfoximine was a very potent inhibitor in both biosynthetic and transferase reactions. Similar to most Gram positive bacteria there was no evidence of in vivo adenylylation and the enzyme seemed to be mainly regulated by feed-back mechanism.Abbreviations PMSF phenylmethylsulfonylfluoride - TCA trichloroacetic acid - GS glutamine synthetase - MSO L-Methionine-D-L-sulfoximine - SDS-PAGE sodium dodecyl sulfatepolyacrylamide gel electrophoresis - SVPDE snake venum phosphodiesterase     

Four-Iron (Sulfide) Ferredoxin from Bacillus polymyxa

Ferredoxin from Bacillus polymyxa contains (per mole) four non-heme iron residues, four acid-labile sulfide residues, and four cysteine residues. Its molecular weight is approximately 8,800, and it has an oxidation-reduction potential (E(m)) of -390 mv. It is active as an electron carrier in several ferredoxin-linked enzyme systems.     

Biotin-deficient growth of Bacillus polymyxa

When Bacillus polymyxa, a wild-type biotin auxotroph, is grown in biotin-deficient medium, a retardation of cell division and consequential cell elongation are the initial detectable consequences of limited biotin. Subsequent events in biotin-deficient cells include, in chronological order: inhibition of net ribonucleic acid (RNA) synthesis and a simultaneous arithmetical accumulation of protein; loss of net RNA, deoxyribonucleic acid, and protein synthesis; morphological aberration, death, and lysis. Incorporation studies employing ³²P-phosphate and ¹⁴CO₂ demonstrate an initial selective inhibition of net ribosomal RNA synthesis over that of ribosomal protein or total protein. Biotin could not be replaced by various extracts from which biotin had been removed, nor could osmotic stabilizers be found which could prevent lysis of the culture.     

Cloning and characterization of an exoinulinase from Bacillus polymyxa

A gene encoding an exoinulinase (inu) from Bacillus polymyxa MGL21 was cloned and sequenced. It is composed of 1455 nucleotides, encoding a protein (485 amino acids) with a molecular mass of 55522 Da. Inu was expressed in Escherichia coli and the His-tagged exoinulinase was purified. The purified enzyme hydrolyzed sucrose, levan and raffinose, in addition to inulin, with a sucrose/inulin ratio of 2. Inulinase activity was optimal at 35°C and pH 7, was completely inactivated by 1 mM Ag⁺ or Hg²⁺. The K _m and V _max values for inulin hydrolysis were 0.7 mM and 2500 M min^–1 mg^–1 protein. The enzyme acted on inulin via an exo-attack to produce fructose mainly.     

Purification and Characterization of Alkaline Xylanases from Bacillus polymyxa

By applying different classical and fast protein liquid chromatographic techniques, three xylanases (beta-1,4-d-xylan xylanhydrolase) were purified to homogeneity from the extracellular enzymatic complex of Bacillus polymyxa. The three enzymes (X(34)C, X(34)E, and X(22)) were small proteins of 34, 34, and 22 kDa and basic pIs 9.3, >9.3, and 9.0, respectively. X(34)C and X(34)E are closely related and seem to be isoforms of the same enzyme. However, they differ in some characteristics. The three enzymes had different pH and temperature optima. One of them, X(34)E, showed a high thermal stability. The V(max) values determined for X(34)C, X(34)E, and X(22) enzymes on oat spelts xylan were 14.9, 85.5, and 64.0 U mg, respectively, and 16.1, 62.0, and 150.6 U mg on birchwood xylan. When oat spelts xylan was the substrate used, K(m) values of 3.4, 2.4, and 1.9 mg ml were obtained for X(34)C, X(34)E, and X(22) enzymes, respectively, and 0.65, 6.3, and 0.32 mg ml were the respective K(m) values determined with birchwood xylan as the substrate. The enzymes were nondebranching endo-beta-xylanases. Xylose was one of the products of xylan hydrolysis by xylanases X(34)C and X(34)E, but this monosaccharide was not released by X(22) enzyme. However, neither of the enzymes was able to degrade xylobiose.