Sodium azide induced mutation of Actinomycetes I (original) (raw)
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Sodium Azide Induced Mutation in Actinomycetes
Tribhuvan University Journal of Microbiology
Objectives: The study was done with an aim to determine the gain and loss of functions among the actinomycetes mutants induced by sodium azide. Methods: The study was carried out in the laboratory of the Sainik Awasiya Mahavidhayala, Bhaktapur, Nepal from 2016 December to 2017 March. A total of 30 soil samples were collected from Tokha, Bhaktapur area and Godawari area. Actinomycetes were isolated from the soil sample using pour plate technique on selective media; starch casein agar. The isolates were identifi ed by using standard microbiological methods and each isolate was exposed to different concentration of sodium azide to generate mutants. The wild type and mutants were compared in morphology, biochemical reactions and antibiotic susceptibility to test organism to determine the gain and loss of functions. Results: Among 30 samples processed, 20(67%) actinomycetes were isolated, in which 6 (20%) were identifi ed as the Streptomyces spp. A total of 28 mutants were isolated from 6 wild types by exposed at 10ppm, 20ppm, 40ppm, 50ppm, 100ppm concentration of sodium azide. Out of 28 mutants formed, only 10 mutants from sample showed same pigmentation as its wild type while other 18 mutants showed change in their pigmentation. In sugar utilization test, 8 different sugars for 28 mutants each, 56 cases showed Gain of Function (GOF), similarly 44 cases showed Loss of function (LOF). Antibiosis remained unaffected against Pseudomonas i.e. no GOF or LOF was seen. Only 2 cases of LOF against Staphylococcus aureus were seen while there were no cases of LOF in other pathogens. 3 cases of GOF against E. coli, 4 against S Typhi and 4 against S. aureus were observed. Conclusion: The potential of mutant actinomycetes has been realized, and hence opens exciting avenues in the fi eld of biotechnology and biomedical research.
Sodium azide induced mutaion of Actinomycetes II: Biochemical and Genetic Characterization
Sodium azide was employed to generate mutants of an actinomycete isolate (Snp1e) from Sagarmatha National Park of Mount Everest region to establish biochemical and genetic changes among them. Nine mutants were selected from Starch Casein Agar plates containing 50, 75 and 100 ppm sodium azide based on observed morphological differences of individual colonies and purified subsequently. Individual isolates were tested for carbon assimilation tests, Nitrate reduction, Catalase production and hydrolysis tests. Five decamer primers were used to establish genomic polymorphisms among the isolates by Randomly Amplified Polymorphic DNA – PCR method. Most of the mutants grew faster than the wild type isolate in Starch-Casein broth. Many of the mutants were able to hydrolyze urea and Tween-20 and produce Nitrate reductase. All mutants were able to show one or more of either GOF mutation or LOF mutation or both. The primers allowed scoring of total 173 bands of which 53 (30.64%) were polymorphic. Primer P1 demonstrated DNA polymorphisms above 50% among the isolates and large numbers of bands were observed with primers P3 and P5. Ample biochemical and genetic variations were observed among mutants derived from an actinomycete using Sodium azide. Key words: Actinomycetes, biochemical and genetic polymorphisms, Mt. Everest, mutation, sodium azide
Cellular and Molecular Biology, 2018
L-Asparaginase is an enzyme that hydrolyses the amino acid L-Asparagine into aspartic acid and ammonia. As a medication, L-Asparaginase is used in chemotherapy to treat acute lymphoblastic leukaemia by depleting circulating Asparagine and depriving tumor cells. Interest in Actinomycetes as potential producers of antibiotics and enzymes encouraged us to investigate an isolated strain (CA01) from soft wheat bran.The Actinomycete strain was characterized based on its morphological and biochemical characteristics and selected due to a proved promising ability to produce L-Asparaginase optimized in both solid and liquid media cultures.The conditions of enzyme production were standardized according to a one-factor-at-a-time (OFAT) experimental design.To obtain optimal medium combination, a Box-Behnken Response Surface Methodology (RSM) has been adopted by choosing the most influential factors. The optimal conditions for the enzyme production were (g/l): L-Asparagine 10.7; Glucose 2.7; starch 7, in based medium containing (g/l): K 2 HPO 4 0.5; MgSO 4 , 7H 2 O 0.1, corresponding to an optimal enzymatic activity of 8.03 IU/ml at 27.83°C. The maximum production of enzyme was reached on the sixth day of experiment. The ANOVA test (P value ˂ 0.05) and adjusted R 2 values close to the experimental R 2 show that the obtained model of the active L-Asparaginase of CA01 strain production is significant with the following linear terms: temperature, substrate concentration, Glucose concentration and there squared.
APPLIED GENETICS AND MOLECULAR BIOTECHNOLOGY
The Saccharopolyspora erythraea mutB knockout strain, FL2281, having a block in the methylmalonyl-CoA mutase reaction, was found to carry a diethyl methylmalonateresponsive (Dmr) phenotype in an oil-based fermentation medium. The Dmr phenotype confers the ability to increase erythromycin A (erythromycin) production from 250-300% when the oil-based medium is supplemented with 15 mM levels of this solvent. Lower concentrations of the solvent stimulated proportionately less erythromycin production, while higher concentrations had no additional benefit. Although the mutB strain is phenotypically a low-level erythromycin producer, diethyl methylmalonate supplementation allowed it to produce up to 30% more erythromycin than the wild-type (control) strain-a strain that does not show the Dmr phenotype. The Dmr phenotype represents a new class of strain improvement phenotype. A theory to explain the biochemical mechanism for the Dmr phenotype is proposed. Other phenotypes found to be associated with the mutB knockout were a growth defect and hyper-pigmentation, both of which were restored to normal by exposure to diethyl methylmalonate. Furthermore, mutB fermentations did not significantly metabolize soybean oil in the presence of diethyl methylmalonate. Finally, a novel method is proposed for the isolation of additional mutants with the Dmr phenotype.
Applied Microbiology and Biotechnology, 2010
Microbial transglutaminase (MTG) has been used extensively in academic research and the food industries through its cross-linking or posttranslational modification of proteins. To improve MTG, a novel method of rational mutagenesis, called WASH-ROM (Water Accessible Surface Hot-space Region Oriented Mutagenesis), was first attempted. Based on the three-dimensional structure of MTG, 151 point mutations were selected at 40 different residues bearing high solvent accessibility surface area, within a 15 Å of the active center site nucleophile, Cys64. Among them, 32 mutants showed higher specific activity than the wild type enzyme. We found that beneficial mutations are distributed in two regions and with distinctive amino acid substitutions. Next, random mutagenesis was applied to the entire MTG region by developing a new plate assay-based screening system, using Corynebacterium glutamicum as the secretion host strain. This in vivo screening system allowed us to readily distinguish the change in enzymatic activity upon mutation by monitoring the intensity of enzymatic reaction-derived color zones which appeared around the recombinant cell colonies on the plate. From the library of 24,000 clones, 10 mutants were finally selected as beneficial enzymes exhibiting higher specific activity than wild type. Notably, most of the mutations differed from those obtained by WASH-ROM, except for H289Y. Beneficial mutations were distributed in two other regions as well. Furthermore, we found that the FRAP-S199A mutant (FRAP: N-terminal four amino acid residues extension) showed the highest specific activity (45 U/mg: 1.7 times higher than the wild type enzyme). Through these different mutation approaches, various beneficial positions leading to increased specific activity of MTG were surveyed. MATERIALS AND METHODS Bacterial strains, culture medium, and plasmids. E.coli JM109, E.coli DH5α and Corynebacterium glutamicum YDK010 (Kikuchi et al. 2002) were used in this study. E. coli JM109 were grown in Luria broth and used as an intermediate host for various plasmid constructions. E. coli DH5α were used as a host for a random mutagenesis library. C. glutamicum was grown in CM2G medium [5 g of glucose, 10 g of tryptone, 10 g of yeast extract, 5 g of NaCl, 0.2 g of DL-methionine per liter of distilled water, adjusted to pH 7.2] at 30 ºC. A MTG-production medium for C. glutamicum was used with MMTG medium [60 g of glucose, 1 g of MgSO4, 30 g of (NH4)2SO4, 1.5 g of KH2PO4, 0.01 g of FeSO4•7H2O, 0.01 g of MnSO4•4H2O, 450 µg of thiamine hydrochloride, 450 µg of biotin, 0.15 g of DL-methionine, 50 g of CaCO3 per liter of distilled water, adjusted to pH 7.5] at 30 ºC. C. glutamicum was transformed by electroporation as described previously (Lieble et al. 1989). Antibiotics were added to final concentrations of 25 mg/l for kanamycin (C. glutamicum and E. coli), 5 mg/l (C. glutamicum) or 30 mg/l (E. coli) for chloramphenicol, and 50 mg/l for ampicillin (E. coli). DNA manipulations. All DNA manipulations followed standard procedures (Sambrook et al. 1989). PCR with Pyrobest DNA polymerase (Takara Shuzo, Kyoto, Japan) was performed in 50 µl reaction mixtures for 5 min at 94 ºC, and for 25 cycles of 10 sec. at 98 ºC, and 30 sec. at 55 ºC, and 3 min at 72 ºC. Nucleotide sequences were determined by using the BigDye terminator cycle sequencing FS ready reaction kit (Applied Biosystems) and DNA sequencer (model 377; Applied Biosystems).
Journal of Biotechnology Research Center (Special edition) Vol. 8 No.1
2014
This study was designed to evaluate the ability of Saccharomyce buolardi as effective probiotic against Shiegella flexneri. Mice treated with S. boulardii and infected with Sh. flexneri, then serum levels of Aspartate aminotransferase (AST) and Alanine aminotransferase (ALT) of treated mice were measured and histological sections were made from liver to evaluate protective effect. Results showed that mice treated with S. boulardii exhibited no significant p≤0.05 differences in serum level of AST and ALT 131,67 respectively U/L in comparison with their levels in serum of control group 113.2, 72.86 U/L. Mice infected with Sh. flexneri showed a significant increase in serum level of AST and ALT 198, 101 U/L in comparison with their levels 113,72 U/L in control group. Mice treated with S. boulardii and infected with Sh. flexneri showed a significant decrease in serum level of AST and ALT in comparison with their levels in mice infected with Sh. flexneri 80.13,78.26 U/L vs. 198 and 101 U...