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Microbiology (Reading, England) Aug 1997A method was established to measure the cytoplasmic pH of the facultative alkaliphilic strain, . The bacterium was loaded with a pH-sensitive fluorescent probe,...
A method was established to measure the cytoplasmic pH of the facultative alkaliphilic strain, . The bacterium was loaded with a pH-sensitive fluorescent probe, 2',7'-bis-(2-carboxyethyl)-5 (and -6)-carboxyfluorescein (BCECF), and cytoplasmic pH was determined from the intensity of fluorescence of the intracellular BCECF. The activity of the organism to maintain neutral cytoplasmic pH was assessed by measuring the cytoplasmic pH of the cells exposed to various pH conditions. The cytoplasmic pH maintenance activity of C-125 increased with increasing culture pH, indicating that the activity was regulated in response to the culture pH.
PubMed: 33657729
DOI: 10.1099/00221287-143-8-2531 -
Applied and Environmental Microbiology Mar 1981Anaerobic and facultatively anaerobic bacteria associated with soft rot of yam (Diascorea rotundata) were isolated by the looping-out method and found to consist of...
Anaerobic and facultatively anaerobic bacteria associated with soft rot of yam (Diascorea rotundata) were isolated by the looping-out method and found to consist of Clostridium (three isolates), Corynebacterium (three isolates), Vibrio (one isolate), and Bacillus lentus (one isolate). Enzyme assay for hydrolase, lyase, and pectinesterase activities by the cup-plate method showed that except for Vibrio sp., B. lentus, and two isolates of Corynebacterium no pectinase activity could be detected for organisms cultured on pectin medium. Most of the cultures on yam tissue, however, showed activities for the three enzymes. The viscometric assay for hydrolase and lyase enzymes indicated a significant level of hydrolase activity (a 40.90% decrease in viscosity for Vibrio sp. and Corynebacterium spp.), but no lyase activity for most of the isolates. Two isolates of Corynebacterium and B. lentus caused changes in fresh yams suggestive of soft rot.
PubMed: 16345726
DOI: 10.1128/aem.41.3.563-567.1981 -
Bioscience, Biotechnology, and... Jan 2002Cytoplasmic pH homeostatic activities of cell wall-defective derivatives of the alkaliphile Bacillus lentus C-125 were assessed using a pH-sensitive fluorescent probe,...
Cytoplasmic pH homeostatic activities of cell wall-defective derivatives of the alkaliphile Bacillus lentus C-125 were assessed using a pH-sensitive fluorescent probe, BCECF. It was shown that the acidic cell wall components took part in maintenance of the cytoplasmic pH neutrality at alkaline pH.
Topics: Alkalies; Bacillus; Cell Wall; Cytoplasm; Fluoresceins; Fluorescent Dyes; Homeostasis; Hydrogen-Ion Concentration; Intracellular Fluid
PubMed: 11866113
DOI: 10.1271/bbb.66.218 -
Applied Microbiology Mar 1972The spores of Fusarium solani reduced the C(2)-carbonyl group, 1-dehydrogenated ring "A" and cleaved the side chain of 16alpha, 17alpha-oxidopregn-4-ene-3, 20-dione...
Reduction of the 20-carbonyl group of C-21 steroids by spores of Fusarium solani and other microorganisms. I. Side-chain degradation, epoxide cleavage, and substrate specificity.
The spores of Fusarium solani reduced the C(2)-carbonyl group, 1-dehydrogenated ring "A" and cleaved the side chain of 16alpha, 17alpha-oxidopregn-4-ene-3, 20-dione (16alpha, 17alpha-oxidoprogesterone)(I) to give the following products: 20alpha-hydroxy-16alpha, 17alpha-oxidopregn-4-en-3-one(II); 20alpha-hydroxy-16alpha, 17alpha-oxidopregna-1, 4-dien-3-one(III); 16alpha-hydroxy-17a-oxa-androsta-1, 4-diene-3, 17-dione (16alpha-hydroxy-1-dehydrotestololactone)(IV); and 16alpha, 17beta-dihydroxy-androsta-1, 4-dien-3-one (16alpha-hydroxy-1-dehydrotestosterone)(V). When II was used as a substrate, it was metabolized into III, IV, and V at a slower rate than I. Furthermore, 16alpha-hydroxy-androst-4-ene-3, 17-dione (16alpha-hydroxyandrostenedione)(X) was transformed into IV and V. Pregn-4-ene-3, 20-dione (progesterone)(XII) was transformed into androsta-1, 4-diene-3, 17-dione (androstadienedione)(VIII) and 17a-oxa-androsta-1, 4-diene-3, 17-dione (1-dehydrotestololactone)(IX), while 17alpha-hydroxy-pregn-4-ene-3, 20-dione (17alpha-hydroxyprogesterone)(VI) was converted into its 1-dehydro analogue (VII) without accumulation of any 20-dihydro compounds. Substrate specificity in the 20-reductase system of F. solani, Cylindrocarpon radicicola, Septomyxa affinis, Bacillus lentus, and three strains of B. sphaericus are demonstrated. The 20-reductase is active only on steroids having the 16alpha, 17alpha-oxido, and Delta(4)-3-keto functions. Evidence of competition between side-chain degrading enzymes and the 20-reductase for the steroid molecule and evidence of side-chain degradation followed by epoxide cleavage (and not the reverse) are presented. A mechanism for the epoxide opening by nongerminating spores of F. solani is postulated.
Topics: 17-Ketosteroids; Androstanes; Bacillus; Ethers, Cyclic; Fusarium; Hydroxyprogesterones; Hydroxysteroid Dehydrogenases; Hydroxysteroids; Ketosteroids; Oxidoreductases; Pregnanes; Progesterone; Species Specificity; Spores; Spores, Fungal; Structure-Activity Relationship
PubMed: 5021973
DOI: 10.1128/am.23.3.601-612.1972 -
European Journal of Biochemistry Dec 1990The subtilisins are known to be susceptible to chemical oxidation due to the conversion of Met222 into the corresponding sulfoxide. A number of derivatives with...
The subtilisins are known to be susceptible to chemical oxidation due to the conversion of Met222 into the corresponding sulfoxide. A number of derivatives with resistance towards oxidation have previously been prepared by replacement of this group with the other 19 amino acid residues. Unfortunately, the activities of these enzymes were of the order of 1-10% of that obtained with the wild-type enzyme. In contrast, the oxidation-labile cysteine mutant exhibited much higher activity, suggesting that this is associated with the presence of a sulphur atom in the amino acid at position 222. It is shown here that it is possible to maintain a sulphur atom in the amino acid at position 222 without the enzyme becoming labile towards oxidation. A subtilisin from Bacillus lentus, subtilisin 309, in which Met222 was replaced with a cysteinyl residue by site-directed mutagenesis was modified with thioalkylating reagents. Treatment of such enzyme derivatives with H2O2 revealed that their stabilities towards oxidation had increased significantly compared to both wild-type and unmodified [Cys222]subtilisin. One of the chemically modified enzyme derivatives, [Me-S-Cys222]subtilisin, exhibited a kcat/Km value of 56% of that obtained with the wild-type enzyme when assayed against the substrate Suc-Ala-Ala-Pro-Phe-NH-Ph-NO2 (Suc, succinyl) and it exhibited 89% activity when tested in an assay with dimethyl casein as a substrate. The corresponding values obtained for unmodified [Cys222]subtilisin were lower, i.e. 39% for the dimethyl casein activity and 46% for the kcat/Km for the hydrolysis of Suc-Ala-Ala-Pro-Phe-NH-Ph-NO2. This demonstrates the feasibility of replacing the oxidation-labile methionyl residue group in a subtilisin enzyme with a group stable towards oxidation without substantially reducing the activity.
Topics: Bacillus; Cysteine; Kinetics; Methionine; Mutagenesis, Site-Directed; Oxidation-Reduction; Protein Conformation; Subtilisins
PubMed: 2269308
DOI: 10.1111/j.1432-1033.1990.tb19484.x -
FEBS Letters Feb 1992Two subfamilies of the subtilisins, distinguished by the presence or absence of a free cysteinyl residue near the essential histidyl residue of the catalytic triad, are...
Two subfamilies of the subtilisins, distinguished by the presence or absence of a free cysteinyl residue near the essential histidyl residue of the catalytic triad, are known. In order to evaluate the significance of the presence of this -SH group a cysteinyl residue has been introduced by site-directed mutagenesis into the cysteine-free subtilisin-like enzyme from Bacillus lentus, i.e. Savinase. The free cysteine affects the enzyme activity only slightly but renders it sensitive to mercurials presumably due to an indirect effect. The results indicate that the -SH group is not involved in catalysis.
Topics: Cysteine; Detergents; Endopeptidase K; Hydrolysis; Kinetics; Sequence Homology, Nucleic Acid; Serine Endopeptidases; Serine Proteinase Inhibitors
PubMed: 1551423
DOI: 10.1016/0014-5793(92)80351-g