scholarly journals Protease Production from Bacillus sp. Isolated from Gastrointestinal Tract of Catfish (Clarias sp.) with Different Medium

2021 ◽  
Vol 10 (2) ◽  
pp. 186
Author(s):  
Asep Awaludin Prihanto ◽  
Kartika D. Aninta ◽  
Soffi Trisnaningrum
Keyword(s):  
Gastrointestinal Tract ◽  
Specific Activity ◽  
Skim Milk ◽  
Luria Bertani ◽  
Protease Production ◽  
Growth Media ◽  
Bacillus Sp ◽  
Screening Process ◽  
Luria Bertani Broth ◽  
The Media

The need for protease in the industrial field has been increasing. Candidates for producing these enzymes can be isolated from the digestive tract of catfish (Clarias sp.). The purpose of this study was to obtain bacterial isolates that produce proteolytic from the gastrointestinal tract of catfish and determine the effect of different production media on the activity of proteolytic. The first step of this study was isolation, screening, and identification of bacteria. The second step was to test the effect of the media Luria Bertani, trypticase soy broth, and skim milk broth on proteolytic activity. Nineteen isolates were obtained from the screening process of proteolytic bacteria. Isolate no 1, was known as the best isolate in producing enzymes and was known as Bacillus sp. Tests with different growth media gave results that semi-quantitative, nutrient growth media produced the highest activity with a proteolytic index value of 2.09 ± 0.41. In addition, based on quantitative tests, the media Luria Bertani Broth produced the highest specific activity with a value of 36.479 U/mg. The conclusion of this study, Bacillus sp. from the gastrointestinal tract of catfish that cultured on the Luria Bertani Broth medium produced the best activity.

Statistical Optimization of the production of protease from Bacillus sp. MSK-01 in submerged fermentation

2018 ◽  
Vol 23 (4) ◽  
Author(s):  
Kulwant Kaur ◽  
Sonica Sondhi ◽  
Palki Sahib Kaur
Keyword(s):  
Submerged Fermentation ◽  
Milk Powder ◽  
Skim Milk ◽  
Fold Increase ◽  
Skim Milk Powder ◽  
Protease Production ◽  
High Yield ◽  
Bacillus Sp ◽  
Increasing Demand ◽  
Optimized Conditions

Protease has been in increasing demand in industries due to its hydrolytic nature. In industries, high yield of enzyme is required to meet the industrial need at a relatively cheaper cost. In the present study, the protease from Bacillus sp. MSK-01 was produced in large quantity by submerged fermentation. Statistical techniques including Plackett-Burman and Response surface methodology are useful tools for optimizing many parameters at a time and are used for increasing the protease production from Bacillus sp. MSK-01. 19 different parameters were chosen, out of which 15 factors had positive effect on protease yield. Four maximum influencing factors were peptone, magnesium sulphate, skim milk powder and casein were chosen to further increase the protease yield. 397.3 IU ml-1 of enzyme yield was obtained under optimized conditions which lead to 198 fold increase in the yield of protease from unoptimized condition. 

Real-Time Monitoring of Luciferase-Tagged Cronobacter sakazakii in Reconstituted Infant Milk Formula

2011 ◽  
Vol 74 (4) ◽  
pp. 573-579 ◽  
Author(s):  
RUTH MORRISSEY ◽  
MÁIRE BEGLEY ◽  
SATORU OSHIMA ◽  
MARY REA ◽  
R. PAUL ROSS ◽  
...  
Keyword(s):  
Real Time ◽  
High Throughput ◽  
Skim Milk ◽  
Luria Bertani ◽  
Milk Formula ◽  
Cronobacter Sakazakii ◽  
Real Time Monitoring ◽  
Luria Bertani Broth ◽  
Infant Milk Formula

The aim of this study was to examine the potential of using a lux-tagged Cronobacter sakazakii strain to monitor growth of the bacterium in various liquids. C. sakazakii was transformed with plasmid p16Slux, and integration of the plasmid at the desired site on the chromosome was confirmed by PCR. The growth of the lux-tagged strain was similar to that of the non–lux-tagged strain, and the integrated plasmid was stable when cells were cultured in the absence of antibiotic. Growth of the lux-tagged strain was monitored in real time in Luria-Bertani broth, skim milk, and infant milk formula by using both the Luminoskan luminometer and the Xenogen IVIS imager. Bioluminescence could be detected when the lux-tagged strain was cocultured with other bacteria. The effect of monocaprylin and nisin on the growth of C. sakazakii in milk was monitored by measuring bioluminescence. In conclusion, growth of a lux-tagged C. sakazakii can be monitored in real time in both clear and opaque liquids by measuring bioluminescence. lux-tagged C. sakazakii strains could be potentially used in high-throughput assays to monitor the effects of various infant milk formula compositions on growth of the bacterium.

scholarly journals Optimization of Culture Conditions for Protease Production using Three Strains of Bacillus

2021 ◽  
Author(s):  
Cyr Jonas Morabandza ◽  
Valentin Dibangou ◽  
Faly Armel Soloka Mabika ◽  
Elgie Viennechie Gatse ◽  
Tarcisse Baloki Ngoulou ◽  
...  
Keyword(s):  
Enzyme Production ◽  
Skim Milk ◽  
Nitrogen Sources ◽  
Petri Dish ◽  
Culture Conditions ◽  
Luria Bertani ◽  
Protease Production ◽  
Carbon And Nitrogen ◽  
Cassava Leaves ◽  
Milk Casein

The aim of this work was to determine the effect of a few external factors on bacterial growth and the production of enzymes with a proteolytic effect in three strains of Bacillus: CMS5 (Bacillus subtilis), CMS4 (Bacillus sp.) and SPo5 14′ (Bacillus velenzensis) isolated from squashes packed in traditionally prepared cassava leaves, but also to determine the best source of carbon and nitrogen. All three strains have the ability to actively degrade milk casein. The strains were grown in Luria Bertani medium and the suspension from the cell culture was used to measure optical density and demonstrate enzyme activity on a petri dish containing skim milk. Several parameters were verified including the influence of temperature, pH, and carbon and nitrogen source on growth and enzyme production. Growth was possible from 25 to 60°C with an optimal temperature of 30°C after 24 hours. Enzyme production was observed from 25 to 55°C with an optimum at 37°C. For pH, growth and enzyme production was possible from pH 5.7 and 9 with an optimum of 7 in all three strains. Among the sources of carbons used, galactose is the best source for growth after 24 h in all three strains, and starch for production. Among nitrogen sources, Bacto-peptone is best for growth as well as production.

scholarly journals Isolation of Shewanella putrefaciens GRD 03 from Fish and Explication of Biofilm Adherence Potency on Different Substrates

2022 ◽  
Author(s):  
Swathy Krishna Jayalekshmi ◽  
Arya Radhakrishnan Krishna ◽  
Trisha Mary Pandipilly Antony ◽  
Suganthi Ramasamy
Keyword(s):  
Foodborne Pathogens ◽  
Biofilm Formation ◽  
Food Poisoning ◽  
Brain Heart Infusion ◽  
Microtiter Plate ◽  
Shewanella Putrefaciens ◽  
Luria Bertani ◽  
Growth Media ◽  
Luria Bertani Broth ◽  
Strong Attachment

Foodborne pathogens are the main threat and cause of food poisoning. The majority of food infections have been related to the biofilm formation of foodborne pathogens in the food industry. Shewanella putrefaciens (KX355803, GRD 03), a Gram-negative pathogen isolated from mackerel fish, was identified and recognized as a food spoilage bacterium and a strong biofilm producer. The adhesion or attachment ability of Shewanella putrefaciens was determined on steel, plastic, glass, PVC and wood. NB (Nutrient broth), LB (Luria-Bertani broth), TSB (Tryptic soy broth) and BHI (Brain heart infusion broth) were enriched with glucose and shows optimum for bacterial adhesion. In the microtiter plate method (MTP), the strong attachment was observed at 48 and 72 hours of incubation and significant differences were obtained at p < 0.05. As the incubation period increases, the OD value (Optical density) of samples also increase. Biofilm formation is the major cause cross-contamination, and shows resistance to certain disinfectants, which leads to environmental stress tolerance. This study suggested with optimum biofilm production of isolate from fish by using glucose enriched media on different substrates, also comparing different growth media provide a detailed idea about biofilm-forming ability at different incubation time intervals.

scholarly journals Production of 7-methylxanthine from Theobromine by Metabolically Engineered E. coli

2020 ◽  
Vol 21 (3) ◽  
pp. 19-27
Author(s):  
Khalid Hussein Rheima Algharrawi ◽  
Mani Subramanian
Keyword(s):  
Potassium Phosphate ◽  
Reaction Medium ◽  
Luria Bertani ◽  
Preparative Chromatography ◽  
Growth Media ◽  
Scale Production ◽  
E Coli ◽  
Luria Bertani Broth ◽  
Product Solution ◽  
Biocatalytic Process

In this work, a novel biocatalytic process for the production of 7-methylxanthines from theobromine, an economic feedstock has been developed. Bench scale production of 7-methlxanthine has been demonstrated. The biocatalytic process used in this work operates at 30 OC and atmospheric pressure, and is environmentally friendly. The biocatalyst was E. coli BL21(DE3) engineered with ndmB/D genes combinations. These modifications enabled specific N7- demethylation of theobromine to 7-methylxanthine. This production process consists of uniform fermentation conditions with a specific metabolically engineered strain, uniform induction of specific enzymes for 7-methylxanthine production, uniform recovery and preparation of biocatalyst for reaction and uniform recovery of pure 7-methylxanthine.    Many E. coli BL21(DE3) strains metabolically engineered with single and/or multiple ndmB/D genes were tested for catalytic activity, and the best strains which had the higher activity were chosen to carry out the N-demethylation reaction of theobromine. Strain pBD2dDB had the highest activity for the production of 7-methylxanthine from theobromine. That strain was used to find the optimum amount of cells required to achieve complete conversion of theobromine to 7-methylxanthine within two hours. It was found that the optimum concentration of pBD2dDB strain to achieve 100% conversion of 0.5 mM theobromine to 7-methylxanthine was 5 mg/mL. The cell growth of pBD2dDB strain was studied using two different growth media, (Luria-Bertani Broth and Super Broth). Super broth was found to be the best medium to produce the highest amount of cell paste (1.5 g). Subsequently, the process was scaled up in which 2 L reaction volume was used to produce 7-methylxanthine (100% conversion) from 0.5 mM theobromine catalyzed by pBD2dDB strain. The reactions was carried out at 30 oC and 250 rpm shaker speed, and the reaction medium was 50 mM potassium phosphate buffer (pH=7). 7-methylxanthines was separated by preparative chromatography with high recovery, and the product solution was collected, purified by drying at 120-140 oC for 4 hours and, recovered (127 mg). Purity of the isolated 7-methylxanthine was comparable to authentic standards with no contaminant peaks, as observed by HPLC, LC-MS, and NMR. 

Effect of Growth Media and Extended Incubation on the Appearance of Lactose - Nonfermenting Variants in Lactococci

1990 ◽  
Vol 53 (7) ◽  
pp. 583-587 ◽  
Author(s):  
R. P. SINHA
Keyword(s):  
Cell Survival ◽  
Room Temperature ◽  
Simple Technique ◽  
Skim Milk ◽  
Extended Period ◽  
Growth Media ◽  
Genetic Traits ◽  
Stabilizing Effect ◽  
The Media ◽  
And Storage

Growth and storage were investigated for the development of Lac− (lactose-nonfermenting) variants of Lactococcus lactis subsp. lactis C2, ML3 and L. lactis subsp. cremoris ML1, SC607 under different conditions, in an unbuffered medium (M17−), and in media buffered with inorganic or organic phosphates. Strains were grown overnight (16–18 h) at 32°C and subsequently held at 32 and 22°C. The cell survival was much higher at 22°C than at 32°C after storage for 96 h. Most of the survivors in M17− broth were of the Lac− phenotype. Lac− variants were also observed when the cultures were grown in skim milk at 32°C and then held at that temperature for 96 h. These results showed that the lactose-fermenting ability of lactococcus in general is lost when overnight cultures in M17− broth are kept either at room temperature (22°C) or at 32°C for an extended period. However, the cultures in buffered media under similar conditions showed little or no loss of lactose-fermenting ability, suggesting that phosphate in the media had a stabilizing effect on plasmid-encoded lactose-fermenting gene(s). These observations indicate the possibility of utilizing this method as a simple technique for isolating mutants deficient in plasmid-linked genetic traits in lactococci.

Growth History Influences Starvation-Induced Expression of uspA, grpE, and rpoS and Subsequent Cryotolerance in Escherichia coli O157:H7

2005 ◽  
Vol 68 (6) ◽  
pp. 1154-1158 ◽  
Author(s):  
PURUSHOTTAM V. GAWANDE ◽  
MANSEL W. GRIFFITHS
Keyword(s):  
Escherichia Coli ◽  
Foodborne Pathogens ◽  
Molecular Mechanisms ◽  
Frozen Storage ◽  
Luria Bertani ◽  
Growth Media ◽  
Escherichia Coli O157 ◽  
E Coli ◽  
Luria Bertani Broth ◽  
And Control

In this study, we investigated the effect of starvation on cryotolerance of Escherichia coli O157:H7 grown in tryptic soy broth (TSB) and Luria-Bertani broth (LB). Starved cells (cells suspended in water at 37°C for 6 h) and control cells (cells in TSB or LB) were frozen at −18°C for up to 240 h in their respective growth media. The E. coli grown in TSB showed a greater starvation effect (the difference in percent survival of starved and control cells) and cryotolerance. The starved E. coli grown in TSB showed a 30% increase in their ability to survive frozen storage for 24 h at −18°C. The corresponding increase in survival for LB-grown E. coli was only 3.8%. Cryotolerance induced by starvation of TSB- and LB-grown E. coli was correlated with the expression of genes involved in general stress response pathways, such as uspA, grpE, and rpoS. The expression of uspA, grpE, and rpoS was quantified by measuring the green fluorescence generated from autofluorescent E. coli harboring puspA::gfp, pgrpE::gfp, and prpoS::gfp gene fusions. The results obtained in this study indicate that uspA, grpE, and rpoS were induced on starvation when E. coli was grown in TSB, and their expression correlated well with subsequent induction of cryotolerance developed at −18°C. In contrast, cells grown in LB and subsequently exposed to starvation conditions showed no increase in expression of uspA, grpE, or rpoS, and, as expected, these cells did not exhibit increased cryotolerance at −18°C. Knowledge of molecular mechanisms involved in cross-protection might make it possible to devise strategies to limit their effects and lead to ways to predict the survival of foodborne pathogens in stressful environments.

scholarly journals Construction and Evaluation of Self-Directing Expression System Using Regulatory Elements of Cry Gene of Bacillus Thuringiensis

2021 ◽  
Author(s):  
Drishtant Singh ◽  
Samiksha . ◽  
Seema Madhumal Thayil ◽  
Satwinder Kaur Sohal ◽  
Anup Kumar Kesavan
Keyword(s):  
Promoter Region ◽  
Specific Activity ◽  
Expression System ◽  
Skim Milk ◽  
Regulatory Elements ◽  
Time Interval ◽  
Bacillus Sp ◽  
Fluorescent Intensity ◽  
Cry Gene ◽  
Gene Regulatory Elements

Abstract An expression system based on the cry gene regulatory elements was constructed. The Terminator region of cry gene from B. thuringiensis subsp. kurstaki HD-1 was cloned in pSG1151 plasmid downstream to gfpmut1. The promoter region of the cry gene was amplified to give three different reading frames. The Promoter region of cry gene was cloned in pSG1151T plasmid upstream to gfpmut1. The expression of GFP under the promoter/terminator expression system was evaluated by checking the expression of gfpmut1 under the same promoter. The GFP content of pSG1151 and three constructs; pDSA1, pDSA2 and pDSA3 were compared by fluorescence spectroscopy. The fluorescent intensity of pSG1151 and pDSA1 were compared at time interval of 6 hours upto 72 hours. Both the samples showed detectable fluorescence that increased with time up to 12 hours, but the increase in the fluorescence of pDSA1 was 3 times higher as compared to pSG1151. A cold peptidase gene was cloned under the control of the cry promoter. The transformed E.coli DH5α colonies were patched on skim milk agar plates and the clones of pSG1151CP and pDSA1CP were compared on the basis of zone of clearance. The zone of clearance of pDSA1CP was much higher as compared to that of pSG1151CP. The cell-free supernatant of Bacillus sp. S1DI 10 and recombinant pDSA1CP collected at different time points was assayed for the specific activity of the extracellular protease. At 72 hours the protease activity in pDSA1CP was 2.7 fold higher compared to that of wild Bacillus sp. S1DI 10.

scholarly journals Comparable Expressions of Bacillus species on Different Growth Media

2021 ◽  
Vol 9 (9) ◽  
pp. 573-577
Author(s):  
Emoleila Itoandon
Keyword(s):  
Bacillus Subtilis ◽  
Luria Bertani ◽  
Bacillus Species ◽  
Growth Media ◽  
Growth Promoters ◽  
Media Formulation ◽  
Commercial Strain ◽  
Luria Bertani Broth ◽  
Peptone Water ◽  
Metabolic Activities

Abstract: An investigation was carried out using Pikovskaya Broth (PKB), Luria Bertani Broth (LBB), and Peptone Water (PW) to analyse growth expressions of constructed Bacillus subtilis sub sp and compared to a commercial Bacillus subtilis RIK 1285. The aim was to determine the effect of carbon, nitrogen and other elements at different variations on the metabolic activities under different conditions. The results obtained showed growth density of 4.1 g/ml at 70oC and 3.1 g/ml at pH 6.0; 3.3 g/ml at 70oC and 2.8 g/ml at pH 6.0; 3.8 g/ml at 60oC and 2.6 g/ml at pH 7.0 from PKB, LBB and PW respectively. The growth density of the commercial strain recorded 3.8 g/ml at 50oC and 2.8 g/ml at pH 7.0; 3.1 g/ml at 50oC and 2.3 g/ml; 3.0 at 50oC and 2.3 at pH respectively. The investigation showed importance and relevance of gene metabolic upgrade on the utilization of multiple nutrients present from one media to another. Keywords: media formulation, microbial reaction, growth promoters, growth density

Cytotoxic Effect of Pseudomonas aurogenosa Protease on Cancer Cells

2019 ◽  
Vol 10 (03) ◽  
Author(s):  
Ghanyia J. Shanyoor ◽  
Fatima R. Abdul ◽  
Nehad A. Taher ◽  
Ihsan A. Raheem
Keyword(s):  
Cell Line ◽  
Normal Cell ◽  
Cytotoxic Effect ◽  
Human Cancer ◽  
Skim Milk ◽  
Exchange Chromatography ◽  
Protease Production ◽  
Normal Cell Line ◽  
Protease Enzyme ◽  
Embryonic Fibroblast

About (20) Pseudomonas rogenosa isolate were experienced for their ability of protease production by calculating the diameter of lysis area after developing on skim milk agar medium (qualitatively ) and the results exhibited that only isolate no (5), was higher isolate for protease making of (26mm) of lysis area. Then, the protein concentration also identified by Bradford method and it was found of 0.16 mg/ ml , then purification was done by using an ion- exchange chromatography with DEAE sephadex G- 100 column and the results showed the presence of 1 peak of enzyme with 50 Kd of molecular weight 2 peaks of other proteins . we tried to investigate the invitro Cytotoxic effect of purified enzyme against two human cancer lines, HeP2 (Human larynx epidermed carcinoma ) , RD ( Rabdo- Sarcoma ) , and one normal cell line Ref ( Rat embryonic fibroblast ) . The cancer and normal cells were treated with different concentrations of protease enzyme ranging from ( 0.05, 0.1, 0.2, 0.4,0.8and 0.16 mg/ml) then incubated for additional 48h at 37C0 and the results showed highest toxicity ( 80.28%) of protease enzyme on RD , moderate cytotoxicity (45.52%) on Hep andslight toxicity ( 37.12% ) on normal cell line (Ref) in a concentration (0.8mg/ml).

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