scholarly journals EXPRESSION OF b-XYLOSIDASE ENCODING GENE IN PHIS/ Bacillus megaterium MS SYSTEM

2015 ◽  
Vol 2 (1) ◽  
pp. 25 ◽  
Author(s):  
Sri Sumarsih
Keyword(s):  
Culture Medium ◽  
Culture Supernatant ◽  
Specific Activity ◽  
Agar Medium ◽  
Nitrilotriacetic Acid ◽  
Protoplast Transformation ◽  
E Coli ◽  
Recombinant Cells ◽  
Relative Molecular Weight ◽  
Encoding Gene

b-Xylosidase encoding gene from G. thermoleovorans IT-08 had been expressed in the pHIS1525/ B. megaterium MS941 system. The b-xylosidase gene (xyl) was inserted into plasmid pHIS1525 and propagated in E. coli DH10b. The recombinant plasmid was transformed into B. megaterium MS941 by protoplast transformation. Transformants were selected by growing the recombinant cells on solid LB medium containing tetracycline (10 µg/ ml). The expression of the b-xylosidase gene was assayed by overlaid the recombinant B. megaterium MS941 cell with agar medium containing 0.2% ethylumbelliferyl-b-D-xyloside (MUX). This research showed that the b-xylosidase gene was succesfully sub-cloned in pHIS1525 system and expressed by the recombinant B. megaterium MS941. Theaddition of 0.5% xylose into the culture medium could increase the activity of recombinantactivity of recombinant of recombinantb-xylosidase by 2.74 fold. The recombinant B. megaterium MS941 secreted 75.56% of the expressed b-xylosidase into culture medium. The crude extract b-xylosidase showed the optimum activity at 50° C and pH 6. The recombinant b-xylosidase was purified from culture supernatant by affinity chromatographic method using agarose containing Ni-NTA (Nickel-Nitrilotriacetic acid). The pure b-xylosidase showed a specific activity of 10.06 Unit/mg protein and relative molecular weight ± 58 kDa.

scholarly journals Overexpression of Lipase Gene from Alcaligenes sp. JG3 and its Activity toward Hydrolysis Reaction

2019 ◽  
Vol 20 (1) ◽  
pp. 200
Author(s):  
Norman Yoshi Haryono ◽  
Winarto Haryadi ◽  
Tri Joko Raharjo
Keyword(s):  
Specific Activity ◽  
Catalytic Triad ◽  
Amino Acid Sequences ◽  
Lipase Gene ◽  
High Quality ◽  
Atp Binding Site ◽  
E Coli ◽  
Transporter Protein ◽  
Encoding Gene ◽  
Hydrolase Family

Bacterial lipase holds an important role as a new source for many industrial catalysts. The investigation and understanding of the lipase-encoding gene become apparent as the key step for generating high-quality lipase as biocatalyst for many chemical reactions. In this study, bacterial lipase from Alcaligenes sp. JG3 was produced via overexpression gene method. This specific lipase was successfully overexpressed using pQE-30 vector and E. coli M15[pREP4] as host, producing His-tagged protein sized 46 kDa and was able to hydrolyze triacylglycerol from olive oil with the calculated unit activity and specific activity of 0.012 U and 1.175 U/mg respectively. The in silico investigation towards lipase JG3 revealed that it was categorized as ABC transporter protein as opposed to the conventional hydrolase family. Lastly, amino acid sequences SGSGKTT from lipase JG3 was highly conserved sequences and was predicted as the ATP-binding site but the catalytic triad of serine, histidine, and aspartate has not been solved yet.

scholarly journals EKSPRESI GEN PENYANDI b-XILOSIDASE DALAM SISTEM pHIS1525/ Bacillus megaterium MS941

2008 ◽  
Vol 13 (2) ◽  
pp. 157-161
Author(s):  
Sri Sumarsih ◽  
Ni Nyoman Tri Puspaningsih ◽  
Sofijan Hadi ◽  
Ami Soewandi J.S.
Keyword(s):  
Bacillus Megaterium ◽  
Recombinant Plasmid ◽  
Culture Medium ◽  
Protein Band ◽  
Extracellular Protein ◽  
Protoplast Transformation ◽  
E Coli ◽  
Sds Page ◽  
Xylosidase Activity

The aim of this research was to express the β-xylosidase gene in the pHIS1525 or Bacillus megaterium MS941 system. The xyl gene was amplified from pTP510 and cloned into pHIS1525 in E. coli DH10b. The recombinant plasmid was transformed into B. megaterium MS941 by protoplast transformation. Transformants were selected by growing the recombinant B. megaterium MS941 on solid LB medium containing tetracycline (10 μg/ml). The expression of β-xylosidase was assayed using 0.2 percent methylumbelliferyl-β-D-xyloside (MUX) and the proteins were analyzed by SDS-PAGE method. The b-xilosidase activity was determined toward p-nitrophenyl-b-Dxylopyranoside (pNPX) as a substrate and p-nitrofenol releasing was measured by UV/Vis spectrophotometer at λ 405 nm. This research showed that recombinant B. megaterium MS941 expressed the β-xylosidase gene (xyl) and secreted it into the culture medium. The SDS-PAGE analysis of extracellular protein (culture medium) showed a 60,0 kD protein band. The recombinant Bacillus megaterium MS941 expressed and secreted the β-xilosidase into culture medium 5 hours after adding 5 percent xylose. The b-xylosidase activity was 0.441 unit/ml toward pNPX as a substrate.

scholarly journals Constitutive High Level Expression of an Endoxylanase Gene from the Newly IsolatedBacillus subtilisAQ1 inEscherichia coli

2010 ◽  
Vol 2010 ◽  
pp. 1-12 ◽  
Author(s):  
Is Helianti ◽  
Niknik Nurhayati ◽  
Maria Ulfah ◽  
Budiasih Wahyuntari ◽  
Siswa Setyahadi
Keyword(s):  
Specific Activity ◽  
Biochemical Properties ◽  
Extracellular Expression ◽  
High Level Expression ◽  
Periplasmic Fraction ◽  
16S Rdna Sequence ◽  
E Coli ◽  
Encoding Gene ◽  
High Level ◽  
Very High

A xylanolytic bacterium was isolated from the sediment of an aquarium. Based on the 16S rDNA sequence as well as morphological and biochemical properties the isolate was identified and denoted asBacillus subtilis(B. subtilis) AQ1 strain. An endoxylanase-encoding gene along with its indigenous promoter was PCR amplified and after cloning expressed inE. coli. InE. colithe recombinant enzyme was found in the extracellular, in the cytoplasmic, and in the periplasmic fraction. The specific activity of the extracellular AQ1 recombinant endoxylanase after 24-hour fermentation was very high, namely,2173.6 ± 51.4and2745.3 ± 11 U/mg in LB and LB-xylan medium, respectively. This activity was clearly exceeding that of the nativeB. subtilisAQ1 endoxylanase and that of 95% homologous recombinant one fromB. subtilisDB104. The result shows that the original AQ1 endoxylanase promoter and the signal peptide gave a very high constitutive extracellular expression inE. coliand hence made the production inE. colifeasible.

scholarly journals Production Optimization of an Active β-Galactosidase of Bifidobacterium animalis in Heterologous Expression Systems

2019 ◽  
Vol 2019 ◽  
pp. 1-10 ◽  
Author(s):  
Xinxin Xu ◽  
Xiaohu Fan ◽  
Chao Fan ◽  
Xing Qin ◽  
Bo Liu ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Culture Medium ◽  
Specific Activity ◽  
Food Additives ◽  
Production Optimization ◽  
E Coli ◽  
Bifidobacterium Animalis ◽  
Wide Ph Range ◽  
Heterologous Expression Systems ◽  
Ph Range

β-Galactosidase (E.C.3.2.1.23) catalyzes the hydrolysis of lactose into glucose and galactose and the synthesis of galacto-oligosaccharides as well. The β-galactosidases from bacteria, especially lactobacilli, and yeast have neutral pH and are much more likely to be developed as food additives. However, the challenges of cumbersome purification, product toxicity, and low yield in protein production have limited the commercialization of many excellent candidates. In this study, we identified a β-galactosidase gene (bg42-106) in Bifidobacterium animalis ACCC05790 and expressed the gene product in Escherichia coli BL21(DE3) and Pichia pastoris GS115, respectively. The recombinant bG42-106 purified from E. coli cells was found to be optimally active at pH 6.0 and 60°C and had excellent stability over a wide pH range (5.0–8.0) and at high temperature (60°C). The specific activity of bG42-106 reached up to 2351 U/mg under optimal conditions. The galacto-oligosaccharide yield was 24.45 g/L after incubation with bG42-106 at 60°C for 2 h. When recombinant bG42-106 was expressed in Pichia pastoris GS115, it was found in the culture medium but only at a concentration of 1.73 U/ml. To increase its production, three strategies were employed, including codon optimization, disulfide formation, and fusion with a Cherry tag, with Cherry-tag fusion being most effective. The culture medium of P. pastoris that expressed Cherry-tagged bG42-106 contained 24.4 U/mL of β-galactosidase activity, which is 14-fold greater than that produced by culture of P. pastoris harboring wild-type bG42-106.

Microbiological performance and salmonella dynamics in a wastewater depuration pond system of southeastern spain

1995 ◽  
Vol 31 (12) ◽  
pp. 239-248 ◽  
Author(s):  
Ana Emparanza-Knörr ◽  
Francisco Torrella
Keyword(s):  
Fecal Coliform ◽  
No Reduction ◽  
Agar Medium ◽  
Microbiological Quality ◽  
Total Coliform ◽  
Fecal Coliforms ◽  
Selective Media ◽  
E Coli ◽  
The Village ◽  
Final Effluent

The Salmonella presence and the microbiological quality indicators, total and fecal coliforms and coliphages of E. coli C, have been studied in a overloaded wastewater lagoon system treating urban wastewatrers of the village of Guardamar del Segura (Alicante, Spain). Classical microbiological technology to detect salmonellae was used, including pre-enrichment, enrichment, selective media plating and biochemical and serological confirmation. Water was physicochemically characterized according to COD, SS, temperature, pH and dissolved oxygen. The selective migration step through Rappaport-Vassiliadis semisolid agar medium was essential for the consistent detection of Salmonella in the different lagoon effluents. Total and fecal coliform levels of up to 105-106 MPN/100 ml were detected in the final effluent. High coliphage concentrations of 103-104 pfu/ml were also found in the effluent waters. Salmonella was always detected in 100 ml samples and eventually reached an order of value of 103 MPN/100 ml. Total coliform reduction was higher in the anaerobic ponds whereas fecal coliforms were more efficiently eliminated in the facultative (mostly “anoxic”) lagoons. Coliphage reduction was higher in the facultative lagoons when compared to the anaerobic ponds. On many occasions, no reduction or eventual increment of the concentration of salmonellae was detected in the effluents from the anaerobic ponds compared to concentrations of the patohogen in the influent raw wasterwaters. The possibility exists for a capacity of Salmonella to multiply in the anoxic phase of the wastewater treatment, but the presence of microorganisms in raw sewage waters which could maskSalmonella detection with the enrichment methodology employed cannot be ruled out.

scholarly journals Enhanced Extracellular Synthesis of Gold Nanoparticles by Soluble Extracts from Escherichia coli Transformed with Rhizobium tropici Phytochelatin Synthase Gene

Metals ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 472
Author(s):  
Qunying Yuan ◽  
Manjula Bomma ◽  
Zhigang Xiao
Keyword(s):  
Gold Nanoparticles ◽  
Gold Nanoparticle ◽  
Synthesis Method ◽  
Nanoparticle Synthesis ◽  
Morphological Properties ◽  
Phytochelatin Synthase ◽  
Rhizobium Tropici ◽  
Whole Cells ◽  
E Coli ◽  
Recombinant Cells

Phytochelatins, the enzymatic products of phytochelatin synthase, play a principal role in protecting the plants from heavy metal and metalloid toxicity due to their ability to scavenge metal ions. In the present study, we investigated the capacity of soluble intracellular extracts from E. coli cells expressing R. tropici phytochelatin synthase to synthesize gold nanoparticle. We discovered that the reaction mediated by soluble extracts from the recombinant E. coli cells had a higher yield of gold nanoparticles, compared to that from the control cells. The compositional and morphological properties of the gold nanoparticles synthesized by the intracellular extracts from recombinant cells and control cells were similar. In addition, this extracellular nanoparticle synthesis method produced purer gold nanoparticles, avoiding the isolation of nanoparticles from cellular debris when whole cells are used to synthesize nanoparticles. Our results suggested that phytochelatins can improve the efficiency of gold nanoparticle synthesis mediated by bacterial soluble intracellular extracts, and the potential of extracellular nanoparticle synthesis platform for the production of nanoparticles in large quantity and pure form is worth further investigation.

Poly(β-L-malate) hydrolase from Plasmodia of Physarum polycephalum

1995 ◽  
Vol 41 (13) ◽  
pp. 192-199 ◽  
Author(s):  
Christian Korherr ◽  
Michael Roth ◽  
Eggehard Holler
Keyword(s):  
Hydrophobic Interaction ◽  
Malic Acid ◽  
Physarum Polycephalum ◽  
Culture Medium ◽  
Specific Activity ◽  
Hydrophobic Interaction Chromatography ◽  
Reduced Form ◽  
Serine Esterase ◽  
Hydroxybutyric Acid ◽  
Ph Optimum

A 68-kDa extracellular glycoprotein from Physarum polycephalum that hydrolyses specifically poly(β-L-malic acid) by removing monomers of L-malic acid in an exolytic manner has been purified and characterized. The enzyme was purified 1740-fold from the culture medium by ammonium sulfate precipitation, hydrophobic interaction chromatography on butyl-Toyopearl, and gel permeation chromatography on Superdex 200 to a specific activity of 9.0 μmol∙min−1∙mg−1. The hydrolase was also purified from the cytosol, which contained 1 mg in 43 g cells in contrast to 1 mg extracellular enzyme in 28 L of culture medium. The pH optimum was pH 3.5 as a result of the effect of an acidic side chain on Vmax and the preferred binding of poly(β-L-malate) in the ionized form. Intracellular hydrolase was only marginally active on [14C]poly(β-L-malate) that had been injected into plasmodia. Poly(L-aspartate), poly(L-glutamate), poly(vinyl sulfate), and poly(acrylate) were neither bound nor degraded by the hydrolase. Poly(β-hydroxybutyric acid), which was considered the reduced form of poly(β-L-malate), was not a substrate. The enzyme is neither a metallo- nor a serine-esterase, and is distinct from poly(3-hydroxybutyric acid) depolymerases. It is related to a glucosidase with respect to hydrophobic interaction chromatography, the pH-activity dependence, and its inhibition with mercuribenzoate, N-bromosuccinimide, and D-gluconolactone, but not the use of the substrates.Key words: poly(β-L-malate), polymalatase, Physarum polycephalum, biodegradative polymer.

scholarly journals Improvement of the Culture Medium for the Dichlorvos-Ammonia (DV-AM) Method to Selectively Detect Aflatoxigenic Fungi in Soil

Toxins ◽  
2018 ◽  
Vol 10 (12) ◽  
pp. 519 ◽  
Author(s):  
Kimiko Yabe ◽  
Haruna Ozaki ◽  
Takuya Maruyama ◽  
Keisuke Hayashi ◽  
Yuki Matto ◽  
...  
Keyword(s):  
Culture Medium ◽  
Agar Medium ◽  
Carbon Sources ◽  
Tween 80 ◽  
Selective Medium ◽  
Selective Detection ◽  
Aflatoxigenic Fungi ◽  
Aspergillus Nomius ◽  
Rhizopus Sp ◽  
Triton X

The dichlorvos-ammonia (DV-AM) method is a simple but sensitive visual method for detecting aflatoxigenic fungi. Here we sought to develop a selective medium that is appropriate for the growth of aflatoxigenic fungi among soil mycoflora. We examined the effects of different concentrations of carbon sources (sucrose and glucose) and detergents (deoxycholate (DOC), Triton X-100, and Tween 80) on microorganisms in soils, using agar medium supplemented with chloramphenicol. The results demonstrated that 5–10% sucrose concentrations and 0.1–0.15% DOC concentrations were appropriate for the selective detection of aflatoxigenic fungi in soil. We also identified the optimal constituents of the medium on which the normal rapid growth of Rhizopus sp. was completely inhibited. By using the new medium along with the DV-AM method, we succeeded in the isolation of aflatoxigenic fungi from non-agricultural fields in Fukui city, Japan. The fungi were identified as Aspergillus nomius based on their calmodulin gene sequences. These results indicate that the new medium will be useful in practice for the detection of aflatoxigenic fungi in soil samples including those from non-agricultural environments.

Screening and characterization of thermostable fibrinolytic enzyme from Bacillus amyloliquefaciens EGE-B-2d.1 / Bacillus amyloliquefaciens EGE-B-2d.1’den termostabil fibrinolitik enzimin taranması ve karakterizasyonu

2016 ◽  
Vol 41 (3) ◽  
Author(s):  
Birkan Slem ◽  
Yüksel Gezgin ◽  
Rengin Eltem
Keyword(s):  
Bacillus Amyloliquefaciens ◽  
Fibrinolytic Activity ◽  
Culture Supernatant ◽  
Specific Activity ◽  
Fibrinolytic Therapy ◽  
Fibrinolytic Enzyme ◽  
Fibrinolytic Enzymes ◽  
Enzyme Thermostability ◽  
Natural Agent

AbstractObjective: To screen fibrinolytic enzyme-producing Bacillus isolates (n=210) and to characterize of thermostable fibrinolytic enzyme from Bacillus amyloliquefaciens EGE-B-2d.1 that had the highest level of fibrinolytic activity together with the highest thermostability.Methods: Firstly, a total of 210 isolates were screened for their fibrinolytic enzyme production. The potent fibrinolytic enzyme producing isolates were evaluated for the thermostability of their fibrinolytic enzymes and one isolate showing prominent fibrinolytic activity was identified as molecular. Fermentation process was carried out on the isolate that had both the highest level of fibrinolytic activity and enzyme thermostability. The thermostable fibrinolytic enzyme from this isolate was then purified and characterized.Results: The fibrinolytic enzyme activities of 21 Bacillus sp. isolates in Nutrient Yeast Salt Medium were found to be in the range of 0.176-1.734 U/ml. The fibrinolytic activity of the enzyme purified from the culture supernatant of Bacillus amyloliquefaciens EGE-B-2d.1 was relatively stable at pH 7.0-11.0 for 24 h and also showed stability at a temperature of 60°C for 60 min. The enzyme degraded the fibrin clots by direct fibrinolysis. The specific activity and the molecular weight of the purified enzyme were estimated to be 44.46 units/mg protein and 30 kD respectively.Conclusion: The thermostable fibrinolytic enzyme from Bacillus amyloliquefaciens EGE-B-2d.1 was purified and characterized. This enzyme might also be used as a natural agent for oral fibrinolytic therapy or thrombosis prevention.

scholarly journals A Novel Gene Encoding Xanthan Lyase of Paenibacillus alginolyticus Strain XL-1

2000 ◽  
Vol 66 (9) ◽  
pp. 3945-3950 ◽  
Author(s):  
Harald J. Ruijssenaars ◽  
Sybe Hartmans ◽  
Jan C. Verdoes
Keyword(s):  
Signal Sequence ◽  
Fold Increase ◽  
Side Chain ◽  
Cloning And Sequencing ◽  
Gene Encoding ◽  
E Coli ◽  
Mature Enzyme ◽  
Locust Bean ◽  
Encoding Gene ◽  
Amino Acid Signal

ABSTRACT Xanthan-modifying enzymes are powerful tools in studying structure-function relationships of this polysaccharide. One of these modifying enzymes is xanthan lyase, which removes the terminal side chain residue of xanthan. In this paper, the cloning and sequencing of the first xanthan lyase-encoding gene is described, i.e., thexalA gene, encoding pyruvated mannose-specific xanthan lyase of Paenibacillus alginolyticus XL-1. ThexalA gene encoded a 100,823-Da protein, including a 36-amino-acid signal sequence. The 96,887-Da mature enzyme could be expressed functionally in Escherichia coli. Like the native enzyme, the recombinant enzyme showed no activity on depyruvated xanthan. Compared to production by P. alginolyticus, a 30-fold increase in volumetric productivity of soluble xanthan lyase was achieved by heterologous production in E. coli. The recombinant xanthan lyase was used to produce modified xanthan, which showed a dramatic loss of the capacity to form gels with locust bean gum.

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