Mutation of AN39-1 for production and characterization of constitutive, thermostable and pH-resistant dextransucrase / Konstitutif, sıcaklığa ve pH’ya dayanıklı dekstransukrazların üretimi ve karakterizasyonu için AN39-1’in mutasyonu

2016 ◽  
Vol 41 (2) ◽  
Author(s):  
Çiğdem Yamaner ◽  
Murat Kemal Avcı ◽  
Aziz Tanrıseven ◽  
İsmail Yavuz Sezen
Keyword(s):  
Mutant Strain ◽  
Parent Strain ◽  
Leuconostoc Mesenteroides ◽  
Industrial Applications ◽  
Wild Type ◽  
Methane Sulfonate ◽  
Ethyl Methane ◽  
Sucrose Medium ◽  
Higher Temperature

AbstractObjective: Leuconostoc mesenteroides AN39-1 has recently been isolated from Crataegus orientalis var. Orientalis. It produces inducible extracellular dextransucrase (EC 2.4.1.5) forming dextran from sucrose. The aim of this study was (1) to obtain constitutive, pH-resistant and thermostable dextransucrase, (2) to characterization of these dextransucrase.Methods: Mutagenesis was carried out on the parent strain (AN39-1) using UV, ethyl methane sulfonate, and N- methyl- N´-nitro-N-nitrosoguanidine. Dextransucrases from wild type (AN39-1) and the mutant strain (A26-2/11) were purified by polyethylene glycol (PEG) precipitation and characterized.Results: Mutants (A26, A26-2, and A26-2/11) hyper producing and constitutive for dextransucrase were isolated. The mutants (A26, A26-2, A26-2/11) produced 7.2, 8.1, and 2.0 times more dextransucrase activity as compared to parent strain on sucrose medium, respectively. In addition, the mutants produced dextransucrase on glucose medium with higher activities (3.0-5.8 times) than what the parental strain produced on sucrose medium. The mutant enzyme (A26-2/11) was much more thermostable than the native enzyme and resistant to pH more than dextransucrase of AN39-1. The dextransucrase from mutant strain was stable up to 35°C and pH of 7.5 for 3 hr.Conclusion: The structures of dextrans produced by wild type and mutant enzymes were similar to commercially produced B-512 F dextran. Thus, the newly dextransucrases produced by mutant strain could find industrial applications at higher temperature and pH.

scholarly journals Mutagenesis and Genotypic Characterization of Aspergillus niger FCBP-02 for Improvement in Cellulolytic Potential

2009 ◽  
Vol 4 (4) ◽  
pp. 1934578X0900400
Author(s):  
Shazia Shafique ◽  
Rukhsana Bajwa ◽  
Sobiya Shafique
Keyword(s):  
Aspergillus Niger ◽  
Wild Strain ◽  
Cellulase Activity ◽  
Soluble Carbohydrates ◽  
Wild Type ◽  
Methane Sulfonate ◽  
Ethyl Methane ◽  
Rapd Pcr ◽  
Mutant Strains

Cellulase is a collective term that encompasses enzymes which catalyze reactions that participate in the degradation of insoluble cellulose to soluble carbohydrates. In the present study, production of extra cellular cellulases by a filamentous fungus, Aspergillus niger FCBP-02, was studied in solid-state fermentation (SSF) as well as in submerged fermentation (SmF). Trials were conducted to evaluate the effect of mutagenesis by UV irradiation (5–40 min) and ethyl methane sulfonate (EMS) treatment (50–300 μg mL−1) to obtain hyper active cellulase enzyme producers among the potential strains. The enzyme activity assays of parental and mutant strains clearly revealed significantly higher cellulase activity of mutant A-Ch-5.5 (96 Units mL−1), followed by A-UV-5.6 (71 Units mL−1) with respect to the wild strain of A. niger FCBP–02 (53.7 Units mL−1). The profile of genetic variability among wild and mutant derivatives was scrutinized through RAPD–PCR. The expression pattern of mutants exhibited that the mutants were isogenic variants of the wild type and the out performance of the mutants could be attributed to the change in genetic make up.

scholarly journals Curly Stem – an Induced Mutation in Flax (Linum usitatissimum L.)

2012 ◽  
Vol 38 (No. 3-4) ◽  
pp. 125-128 ◽  
Author(s):  
E. Tejklová
Keyword(s):  
Linum Usitatissimum ◽  
Wild Type Allele ◽  
Ethyl Methane Sulfonate ◽  
Wild Type ◽  
Methane Sulfonate ◽  
Ethyl Methane ◽  
Straight Stem ◽  
Linum Usitatissimum L ◽  
Factors Influencing ◽  
Stem Shape

After ethyl methane sulfonate (EMS) treatment of two flax lines, curly stem mutations appeared in both, besides of other mutations. Genetic analysis of one CS mutant line confirmed a monogenic inheritance of the changed stem shape. The curly stem allele is partially dominant over the wild type allele for straight stem. Homozygotic mutants have a curly stem, heterozygotic plants have a flexuous stem, while the stem of homozygotic recessive plants is straight. The expression of the curly stem character is affected by factors influencing plant growth. The utilisation of this mutation for ornamental and other purposes is considered.

scholarly journals Characterization of the Different Dextransucrase Activities Excreted in Glucose, Fructose, or Sucrose Medium byLeuconostoc mesenteroides NRRL B-1299

1998 ◽  
Vol 64 (4) ◽  
pp. 1298-1302 ◽  
Author(s):  
Marguerite Dols ◽  
M. Remaud-Simeon ◽  
R. M. Willemot ◽  
M. Vignon ◽  
P. Monsan
Keyword(s):  
Gel Electrophoresis ◽  
Leuconostoc Mesenteroides ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Traditional Culture ◽  
Culture Conditions ◽  
Acceptor Reaction ◽  
Sucrose Medium ◽  
Molecular Masses

ABSTRACT When grown in glucose or fructose medium in the absence of sucrose,Leuconostoc mesenteroides NRRL B-1299 produces two distinct extracellular dextransucrases named glucose glucosyltransferase (GGT) and fructose glucosyltransferase (FGT). The production level of GGT and FGT is 10 to 20 times lower than that of the extracellular dextransucrase sucrose glucosyltransferase (SGT) produced on sucrose medium (traditional culture conditions). GGT and FGT were concentrated by ultrafiltration before sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Their molecular masses were 183 and 186 kDa, respectively, differing from the 195 kDa of SGT. The structural analysis of the dextran produced from sucrose and of the oligosaccharides synthesized by acceptor reaction in the presence of maltose showed that GGT and FGT are two different enzymes not previously described for this strain. The polymer synthesized by GGT contains 30% α(1→2) linkages, while FGT catalyzes the synthesis of a linear dextran only composed of α(1→6) linkages.

scholarly journals Loss of Catabolite Repression Function of HPr, the Phosphocarrier Protein of the Bacterial Phosphotransferase System, Affects Expression of the cry4A Toxin Gene in Bacillus thuringiensis subsp. israelensis

2002 ◽  
Vol 184 (19) ◽  
pp. 5410-5417 ◽  
Author(s):  
Sharik R. Khan ◽  
Nirupama Banerjee-Bhatnagar
Keyword(s):  
Bacillus Thuringiensis ◽  
Mutant Strain ◽  
Catabolite Repression ◽  
Parent Strain ◽  
Inhibitory Effect ◽  
Toxin Gene ◽  
Wild Type ◽  
Phosphotransferase System ◽  
Gene Transcripts ◽  
Bacterial Phosphotransferase System

ABSTRACT HPr, the phosphocarrier protein of the bacterial phosphotransferase system, mediates catabolite repression of a number of operons in gram-positive bacteria. In order to participate in the regulatory process, HPr is activated by phosphorylation of a conserved serine-46 residue. To study the potential role of HPr in the regulation of Cry4A protoxin synthesis in Bacillus thuringiensis subsp. israelensis, we produced a catabolite repression-negative mutant by replacing the wild-type copy of the ptsH gene with a mutated copy in which the conserved serine residue of HPr was replaced with an alanine. HPr isolated from the mutant strain was not phosphorylated at Ser-45 by HPr kinase, but phosphorylation at His-14 was found to occur normally. The enzyme I and HPr kinase activities of the mutant were not affected. Analysis of the B. thuringiensis subsp. israelensis mutant harboring ptsH-S45A in the chromosome showed that cry4A expression was derepressed from the inhibitory effect of glucose. The mutant strain produced both cry4A and σ35 gene transcripts 4 h ahead of the parent strain, but there was no effect on σ28 synthesis. In wild-type B. thuringiensis subsp. israelensis cells, cry4A mRNA was observed from 12 h onwards, while in the mutant it appeared at 8 h and was produced for a longer period. The total amount of cry4A transcripts produced by the mutant was higher than by the parent strain. There was a 60 to 70% reduction in the sporulation efficiency of the mutant B. thuringiensis subsp. israelensis strain compared to the wild-type strain.

scholarly journals Characterization of Acp, a Peptidoglycan Hydrolase of Clostridium perfringens with N-Acetylglucosaminidase Activity That Is Implicated in Cell Separation and Stress-Induced Autolysis

2010 ◽  
Vol 192 (9) ◽  
pp. 2373-2384 ◽  
Author(s):  
Emilie Camiade ◽  
Johann Peltier ◽  
Ingrid Bourgeois ◽  
Evelyne Couture-Tosi ◽  
Pascal Courtin ◽  
...  
Keyword(s):  
Mutant Strain ◽  
Clostridium Perfringens ◽  
Vegetative Growth ◽  
Cell Separation ◽  
Catalytic Domain ◽  
Peptidoglycan Hydrolase ◽  
Wild Type ◽  
Induced Autolysis

ABSTRACT This work reports the characterization of the first known peptidoglycan hydrolase (Acp) produced mainly during vegetative growth of Clostridium perfringens. Acp has a modular structure with three domains: a signal peptide domain, an N-terminal domain with repeated sequences, and a C-terminal catalytic domain. The purified recombinant catalytic domain of Acp displayed lytic activity on the cell walls of several Gram-positive bacterial species. Its hydrolytic specificity was established by analyzing the Bacillus subtilis peptidoglycan digestion products by coupling reverse phase-high-pressure liquid chromatography (RP-HPLC) and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis, which displayed an N-acetylglucosaminidase activity. The study of acp expression showed a constant expression during growth, which suggested an important role of Acp in growth of C. perfringens. Furthermore, cell fractionation and indirect immunofluorescence staining using anti-Acp antibodies revealed that Acp is located at the septal peptidoglycan of vegetative cells during exponential growth phase, indicating a role in cell separation or division of C. perfringens. A knockout acp mutant strain was obtained by using the insertion of mobile group II intron strategy (ClosTron). The microscopic examination indicated a lack of vegetative cell separation in the acp mutant strain, as well as the wild-type strain incubated with anti-Acp antibodies, demonstrating the critical role of Acp in cell separation. The comparative responses of wild-type and acp mutant strains to stresses induced by Triton X-100, bile salts, and vancomycin revealed an implication of Acp in autolysis induced by these stresses. Overall, Acp appears as a major cell wall N-acetylglucosaminidase implicated in both vegetative growth and stress-induced autolysis.

PHOTOSYNTHETIC CHARACTERIZATION OF A MUTANT OF NANNOCHLOROPSIS DEFICIENT IN THE SYNTHESIS OF EICOSAPENTAENOIC ACID

1998 ◽  
Vol 46 (2) ◽  
pp. 101-108 ◽  
Author(s):  
Assaf Sukenik ◽  
Jane Schneider C. ◽  
Paul Roessler G. ◽  
Alexander Livne ◽  
Tamar Berner ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Eicosapentaenoic Acid ◽  
Photosynthetic Apparatus ◽  
Photosynthetic Electron Transport ◽  
Photosynthetic Performance ◽  
Minor Role ◽  
Wild Type ◽  
Higher Temperature ◽  
A Minor

Photosynthetic performance of an eicosapentaenoic acid (EPA; 20:5ω3) deficient mutant of the eustigmatophyte Nannochloropsis sp. was compared to the wild type (Wt) strain in order to evaluate the effect of fatty acid composition on the function of the photosynthetic apparatus. Cellular photosynthetic capacity and the cellular pool of pigments and of reaction centers were reduced in the mutant concomitant with a reduction in the amount of thylakoid membranes and their volume-specific density. Despite the changes observed in photosynthetic activity, the fluorescence properties of the mutant were virtually the same as those of the wild type, although the phase transition of thylakoid membrane was recorded at higher temperature in the mutant than in the Wt. The results suggest that the change in one double bond in a very long chain fatty acid of the thylakoid lipids plays a minor role in regulating photosynthetic electron transport, but that the mutation modified the ability of the mutant to acclimate to low-irradiance conditions.

scholarly journals Construction and Phenotypic Characterization of an Auxotrophic Mutant of Mycobacterium tuberculosis Defective in l-Arginine Biosynthesis

2002 ◽  
Vol 70 (6) ◽  
pp. 3080-3084 ◽  
Author(s):  
Bhavna G. Gordhan ◽  
Debbie A. Smith ◽  
Heidi Alderton ◽  
Ruth A. McAdam ◽  
Gregory J. Bancroft ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mutant Strain ◽  
Auxotrophic Mutant ◽  
Phenotypic Characterization ◽  
Wild Type ◽  
Arginine Biosynthesis ◽  
High Concentrations

ABSTRACT A mutant of Mycobacterium tuberculosis defective in the metabolism of l-arginine was constructed by allelic exchange mutagenesis. The argF mutant strain required exogenous l-arginine for growth in vitro, and in the presence of 0.96 mM l-arginine, it achieved a growth rate and cell density in stationary phase comparable to those of the wild type. The mutant strain was also able to grow in the presence of high concentrations of argininosuccinate, but its auxotrophic phenotype could not be rescued by l-citrulline, suggesting that the ΔargF::hyg mutation exerted a polar effect on the downstream argG gene but not on argH. The mutant strain displayed reduced virulence in immunodeficient SCID mice and was highly attenuated in immunocompetent DBA/2 mice, suggesting that l-arginine availability is restricted in vivo.

scholarly journals PRODUKSI LIPASE DARI ISOLAT KAPANG HASIL MUTASI UNTUK TRANSESTERIFIKASI

2019 ◽  
Vol 6 (1) ◽  
pp. 20
Author(s):  
Galih Cendana Nabilasani ◽  
Trismilah Siswodarsono ◽  
Dadang Suhendar ◽  
Nisa Rachmania Mubarik
Keyword(s):  
Uv Light ◽  
Palm Kernel ◽  
Lipase Production ◽  
Transesterification Reaction ◽  
Biodiesel Production ◽  
Ethyl Methane Sulfonate ◽  
Wild Type ◽  
Methane Sulfonate ◽  
Ethyl Methane ◽  
Type Mutant

Lipase Production by Mutant Fungal Isolates for Transesterification ABSTRACTLipase is used amongst others in biodiesel production, namely in the transesterification reaction. Kernel B (KB) was a fungus isolated from the waste of palm kernel and seed. The fungus produced lipase that catalysed the transesterification reaction with a lower activity compared to that of AK Amano commercial lipase. The purpose of this study was to obtain mutant fungi with higher transesterification activities than the wild type (KB). The mutation process was carried out using ultraviolet (UV) light, ethyl methane sulfonate (EMS), and N-methyl-N’-nitro-N-nitrosoguanidine (NMNG) on KB fungus. The mutations using UV light produced 11 isolates, of which isolate m4.1KB1 produced a higher transesterification activity (0.172 U·mg-1) compared to the wild type. Mutant m5.7KB, which was generated from mutant m4.1KB1 treated using EMS, had its transesterification activity decreased to only 0.051 U·mg-1. Mutant m6.0,3KB2, which was resulted through NMNG treatment, experienced an increase in transesterification activity which was 91.2% higher than that of KB.Keywords: ethyl methane sulfonate, lipase, mutant fungi, N-methyl-N’-nitro-N-nitrosoguanidine, ultraviolet ABSTRAKLipase dimanfaatkan salah satunya dalam produksi biodiesel, yaitu dalam reaksi transesterifikasi. Kernel B (KB) merupakan kapang yang diisolasi dari limbah inti dan biji kelapa sawit, yang menghasilkan lipase sebagai katalis dalam reaksi transesterifikasi. Namun aktivitas transesterifikasi yang dihasilkan oleh lipase dari KB lebih rendah dibandingkan dengan lipase komersial AK Amano. Tujuan penelitian ini adalah mendapatkan mutan kapang dengan aktivitas transesterifikasi yang lebih tinggi dibandingkan tipe liarnya (KB). Proses mutasi dilakukan dengan menggunakan sinar ultraviolet (UV), ethyl methane sulfonate (EMS), dan N-methyl-N’-nitro-N-nitrosoguanidine (NMNG) terhadap kapang KB. Mutasi KB dengan menggunakan sinar UV menghasilkan 11 isolat, dimana isolat dengan kode m4.1KB1 menghasilkan aktivitas transesterifikasi yang lebih tinggi dibandingkan tipe liar, yaitu 0,172 U·mg-1. Mutan m5.7KB, yang dihasilkan dari mutan m4.1KB1 dengan perlakuan EMS, mengalami penurunan aktivitas transesterifikasi hingga hanya sebesar 0,051 U·mg-1. Mutan m6.0,3KB2 hasil perlakuan NMNG mengalami peningkatan aktivitas transesterifikasi sebesar 91,2% lebih tinggi dari KB.Kata Kunci: ethyl methane sulfonate, kapang mutan, lipase, N-methyl-N’-nitro-N-nitrosoguanidine, ultraviolet

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