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1.
A new epoxide hydrolase with high enantioselectivity toward (R)-glycidyl phenyl ether (R-GPE) was partially purified from Bacillus megaterium strain ECU1001. The maximum activity of the isolated enzyme was observed at 30 degrees C and pH 6.5 in a buffer system with 5% (v/v) of DMSO as a cosolvent. The enzyme was quite stable at pH 7.5 and retained full activity after incubation at 40 degrees C for 6 h. Interestingly, when the cosolvent DMSO was replaced by an emulsifier (Tween-80, 0.5% w/v) as an alternative additive to help disperse the water-insoluble substrate, the apparent activity of the epoxide hydrolase significantly increased by about 1.8-fold, while the temperature optimum shifted from 30 to 40 degrees C and the half-life of the enzyme at 50 degrees C increased by 2.5 times. The enzymatic hydrolysis of rac-GPE was highly enantioselective, with an E-value (enantiomeric ratio) of 69.3 in the Tween-80 emulsion system, which is obviously higher than that (41.2) observed in the DMSO-containing system. 相似文献
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It is shown that the Bacillus megaterium population has two types of qualitatively differing cells: exo- and endotrophic ones, i.e. the cells which assimilate in a given moment of time the source of carbon and energy from the medium or only intracellular sources of carbon and energy. Trophic structure of the population has been analyzed in different phases of the batch culture as well as in case of cell starvation. In all cases the content of exotrophic cells in the bacilli population considerably decreases with exhaustion of the nutrient medium. The obtained results are hypothetically explained by the alteration of the exo- and endotrophic processes in a cell cycle of bacteria. 相似文献
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Burger S Tatge H Hofmann F Genth H Just I Gerhard R 《Biochemical and biophysical research communications》2003,307(3):584-588
Pathogenic Clostridium difficile produces two major protein toxins, toxin A and toxin B. We used the Bacillus megaterium expression system for expression of recombinant toxin A. The construct for the toxin A gene was obtained by the following cloning strategy: the gene for toxin A was generated in three parts, each of them ligated into a cloning vector. The three parts were sequentially fused to the complete gene. The holotoxin gene was ligated into the expression vector pWH1520. This vector was modified to generate a toxin with a C-terminally located His-tag. Gene expression in the B. megaterium system resulted in an approximate 300 kDa protein, which was identified by specific antibody as toxin A. Recombinant, His-tagged toxin A was purified by Ni(2+) as well as thyroglobulin affinity chromatography. Characterization of the recombinant toxin A showed identical cytotoxicity and in vitro-glucosyltransferase activity as the native toxin A from C. difficile. 相似文献
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Kyu-Min Kim Jin-Soo Park HaeRi Choi Min-Seon Kim Joo-Hyun Seo 《Biocatalysis and Biotransformation》2013,31(6):469-475
AbstractBiotransformation of daidzein was performed by using Bacillus amyloliquefaciens KCTC 13588, Lactococcus lactis subsp. lactis KCTC 3769, Leuconostoc citreum KCTC 13186, Kluyveromyces lactis var. lactis KCTC 17704, Pediococcus pentosaceus KCTC 3116, and Lactobacillus sakei KCTC 13416 cells as a biocatalyst. Four derivatives of daidzein such as daidzein-7-O-phosphate, daidzein-7-O-β-D-glucoside, daidzein-7-O-β-(6′′-O-succinyl)-D-glucoside, and 4′-Ethoxy-daidzein-7-O-β-(6′′-O-succinyl)-D-glucoside were isolated from the biotransformation reaction mixture. The structures of the molecules were elucidated by HPLC, HR-QTOF-ESI/MS and 1H-NMR analyses. Among them 4′-Ethoxy-daidzein-7-O-β-(6′′-O-succinyl)-D-glucoside derivative is novel compound and not reported elsewhere till now. 相似文献
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In chiral separation, enantioseparation factor is an important parameter which influences the resolution of enantiomers. In this current overview, a biphasic chiral recognition method is introduced to the readers. This method can significantly improve the enantioseparation factor in two‐phase solvent through adding lipophilic and hydrophilic chiral selectors which have opposite chiral recognition ability to organic and aqueous phases, respectively. This overview presents the development and applications of biphasic chiral recognition in liquid‐liquid extraction and counter current chromatography. It mainly focuses on the topics of mechanism, advantages and limitations, applications, and key factors of biphasic chiral recognition. In addition, the future outlook on development of biphasic chiral recognition also has been discussed in this overview. 相似文献
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1. The lipids of Bacillus megaterium were extracted and three lipids containing glucosamine were identified. One of these is not a phospholipid, but the other two, which differ in their chromatographic behaviour, contain phosphorus, glycerol, fatty acid and d-glucosamine in the molar proportions 1:2:2:1. 2. In both phosphoglycolipids, the fatty acids are bound in ester linkage, and both yield 2,5-anhydromannose and 3-sn-phosphatidyl-1'-sn-glycerol on treatment with sodium nitrite. 3. Both phosphoglycolipids were N-acetylated and, after removal of fatty acids by mild alkaline hydrolysis, in both cases N-acetylglucosamine was quantitatively released by beta-N-acetylhexosaminidase. 4. The glucosaminylglycerols derived from the two phosphoglycolipids by partial acid hydrolysis differ in their behaviour towards periodate. In one case 1 mole of periodate is rapidly consumed/mole of glucosaminylglycerol, but in the other case under identical conditions the consumption of periodate is negligible. 5. The phosphoglycolipids were identified as 1'-(1,2-diacyl-sn-glycero-3-phosphoryl)-3'-O-beta-(2-amino-2-deoxy-d-glucopyranosyl)-sn-glycerol and as 1'-(1,2-diacyl-sn-glycero-3-phosphoryl)-2'-O-beta-(2-amino-2-deoxy-d-glucopyranosyl)-sn-glycerol. 6. Both phosphoglycolipids are good substrates for phospholipase A: neither is a substrate for phospholipase C from Clostridium perfringens, and only the 3'-glucosaminide is a substrate for phospholipase D. 相似文献
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Das-Bradoo S Svensson I Santos J Plieva F Mattiasson B Hatti-Kaul R 《Journal of biotechnology》2004,110(3):273-285
Whole cells of alkaliphilic Bacillus pseudofirmus AR-199, induced for beta-galactosidase activity, were used for the synthesis of 1-hexyl-beta-d-galactoside and 1-octyl-beta-d-galactoside, respectively, by transglycosylation reaction between lactose and the corresponding alcohol acceptor. The product yield was strongly influenced by the initial water content in the reaction mixture. Water content of 10% (v/v) was optimal providing 3.6-36 mM hexyl galactoside from 10 to 150 mM lactose, and no secondary product hydrolysis. Product yield could be enhanced by supplementing the reaction mixture with more cells or partly replacing the product with fresh substrate, but was decreased with time to the initial equilibrium level. Cell permeabilisation or disruption resulted in increased reaction rate and higher product yield but was followed by product hydrolysis. Octyl galactoside synthesis using whole cells was optimal at water content of 2% (v/v) with a yield of 26%. The cells were immobilised in cryogels of polyvinyl alcohol for use in continuous process, where hexyl galactoside was produced with a constant yield of 50% from 50mM lactose for at least a week. 相似文献
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K B Shark F D Smith P R Harpending J L Rasmussen J C Sanford 《Applied and environmental microbiology》1991,57(2):480-485
We present a simple and rapid method for introducing exogenous DNA into a bacterium, Bacillus megaterium, utilizing the recently developed biolistic process. A suspension of B. megaterium was spread onto the surface of nonselective medium. Plasmid pUB110 DNA, which contains a gene that confers kanamycin resistance, was precipitated onto tungsten particles. Using a biolistic propulsion system, the coated particles were accelerated at high velocities into the B. megaterium recipient cells. Selection was done by use of an agar overlay containing 50 micrograms of kanamycin per ml. Antibiotic-resistant transformants were recovered from the medium interface after 72 h of incubation, and the recipient strain was shown to contain the delivered plasmid by agarose gel electrophoresis of isolated plasmid DNA. All strains of B. megaterium tested were successfully transformed by this method, although transformation efficiency varied among strains. Physical variables of the biolistic process and biological variables associated with the target cells were optimized, yielding greater than 10(4) transformants per treated plate. This is the first report of the biolistic transformation of a procaryote. 相似文献
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Morphogenesis of the membrane-bound electron-transport system in sporulating Bacillus megaterium KM.
The properties of electron transport systems present in soluble and particulate fractions of spores of Bacillus megaterium KM?HAVE BEEN COMPARED WIth those of similar fractions prepared from exponential-phase vegetative cells of this organism. The timing and localization of modifications of the electron transport system occurring during sporulation have been investigated by using a system for separating forespores from mother cells at all stages during development [8]. Spore membranes contained cytochromes a + a3, and o at lower concentrations than in vegetative membranes, and in addition cytochrome c, which was not found in exponential-phase vegetative membranes. An NADH oxidase activity of similar specific activity was found in both spore and vegetative membranes but DL-glycerol 3-phosphate and L-malate oxidase activities were found only in vegetative membranes. A soluble NADH oxidase of low specific activity was found in spores and vegetative cells which probably involves a flavoprotein reaction with oxygen because the activity was stimulated by FAD or FMN and difference spectra of concentrated soluble fractions showed spectra typical of a flavoprotein. Particulate NADH oxidase was sensitive to all classical inhibitors of electron transport tested whereas soluble NADH oxidase was insensitive to many of these inhibitors. Cytochrome c was formed between stage I and II of sporulation and this coincided with a five-fold increase in NADH-cytochrome c reductase activity. Forespore membranes had lower contents of cytochromes than sporangial cell membranes but similar levels of NADH and L-malate oxidases; DL-glycerol 3-phosphate oxidase activity could not be detected in either membranes by stage III of sporulation. This characterization of spore electron transport systems provides a basis for suggestions concerning initial metabolic events during spore germination and the effect of a number of germination inhibitors. 相似文献
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Passage of a membrane protein through the walls of toluene-treated Bacillus megaterium cells. 总被引:2,自引:1,他引:1
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Based on autoradiographic and microscopic evidence, it seems likely that a membrane protein essential for peptidoglycan synthesis can be extracted from uhlysed toluene-treated Bacillus megaterium cells. Furthermore, this protein can be added back to the membrane through the wall to reconstitute peptidoglycan synthesis. Autoradiograms also show that peptidoglycan is synthesized from externally added nucleotide precursors over the entire length of the toluene-treated bacterial. The amounts of peptidoglycan made is to small to be visible by thin section electron microscopy. 相似文献
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A Berg J A Gustafsson M Ingelman-Sundberg 《The Journal of biological chemistry》1976,251(9):2831-2838
Cell-free extracts from sonically disrupted Bacillus megaterium ATCC 13368 hydroxylated a variety of 3-oxo-delta4-steroids in position 15beta in the presence of NADPH and O2. Ring A-reduced, aromatic and 3beta-hydroxy-delta5-steroids did not serve as substrates for the 15beta-hydroxylase system. Using ion exchange chromatography on DEAE-cellulose and gel filtration on Ultrogel ACA-54 it was possible to resolve the hydroxylase system into three proteins: a strictly NADPH-dependent FMN-containing (megaredoxin reductase), an iron-sulfur protein (megaredoxin), and cytochrome P-450 (P-450meg). The activity of the 15beta-hydroxylase system was fully reconstituted upon combination of these three proteins and addition of NADPH. Megaredoxin had an apparent sulfur to iron ration of 0.98 and showed g-signals at 1.90, 1.93, and 2.06 when analyzed by electron paramagnetic reso0 times and the preparation contained 1 to 2 nmol of cytochrome P-450 per mg of protein. This preparation of cytochrome P-450meg sedimented as a homogeneous zone on sucrose gradients with a sedimentation coefficient of 3.3 S and contained 0.94 nmol of heme per nmol of cytochrome P-450. The oxidized form of cytochrome P-450meg showed absolute absorption maxima at 416, 528, and 565 nm whereas the reduced form showed maxima at 411 and 542 nm. The following scheme is suggested for the electron transport in the 15beta-hydroxylase system in B. megaterium: NADPH leads to megaredoxin reductase leads to megaredoxin leads to cytochrome P-450meg. 相似文献
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《Journal of Fermentation and Bioengineering》1989,67(6):374-379
A new type of glucose dehydrogenase was purified from vegetative cells of Bacillus megaterium IAM1030. The characteristics of the vegetative-cell enzyme were investigated and compared with the enzyme from sporulating cells of B. megaterium IWG3. They are very similar in the following points: molecular size (Mr 120,000), subunit composition (homo tetramer), pH-activity profile with an optimum pH at around 8, pH-stability profile with a stable pH range of 6.0–7.5 (at 25°C, for 30 min), substrate specificity (specific for d-glucose and 2-deoxy-d-glucose), and the affinity for glucose (a Km value of 11–12 mM at pH 8.0, 2.5 mM NAD). They are a little different in the following points: slower mobility for the vegetative-cell enzyme in polyacrylamide-gel electrophoresis at pH 8, immunological determinants (some of them are common), and higher heat resistance for the vegetative-cell enzyme at pH 6.5. They are quite different in their affinity for NAD and NADP. The Km values for NAD are 0.1 mM for the vegetative-cell enzyme and 1.0 mM for the spore enzyme, while the values for NADP are 7.1 mM for the vegetative-cell enzyme and 0.09 mM for the spore enzyme, at pH 8.0, 0.1 M d-glucose. These results suggest that B. megaterium has at least two types of glucose dehydrogenase. 相似文献
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Expression of a Bacillus megaterium sporulation-specific gene during sporulation of Bacillus subtilis 总被引:34,自引:28,他引:6
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The gene for the Bacillus megaterium spore C protein, a sporulation-specific gene, has been transferred into Bacillus subtilis. The B. megaterium gene was expressed little, if at all, during log-phase and early-stationary-phase growth, but was expressed during sporulation with the same kinetics as and at a level similar to that of the analogous B. subtilis genes. This finding is most consistent with the regulation of this class of genes by a mechanism of positive control. 相似文献
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Production and characterization of a glycolipid biosurfactant from Bacillus megaterium using economically cheaper sources 总被引:1,自引:0,他引:1
R. Thavasi S. Jayalakshmi T. Balasubramanian Ibrahim M. Banat 《World journal of microbiology & biotechnology》2008,24(7):917-925
Criteria selected for screening of biosurfactant production by Bacillus megaterium were hemolytic assay, bacterial cell hydrophobicity and the drop-collapse test. The data on hemolytic activity, bacterial
cell adherence with crude oil and the drop-collapse test confirmed the biosurfactant-producing ability of the strain. Accordingly,
the strain was cultured at different temperatures, pH values, salinity and substrate (crude oil) concentration in mineral
salt medium to establish the optimum culture conditions, and it was shown that 38°C, 2.0% of substrate concentration, pH 8.0
and 30‰ of salt concentration were optimal for maximum growth and biosurfactant production. Laboratory scale biosurfactant
production in a fermentor was done with crude oil and cheaper carbon sources like waste motor lubricant oil and peanut oil
cake, and the highest biosurfactant production was found with peanut oil cake. Characterization of partially purified biosurfactant
inferred that it was a glycolipid with emulsification potential of waste motor lubricant oil, crude oil, peanut oil, diesel,
kerosene, naphthalene, anthracene and xylene. 相似文献