scholarly journals Co-Cultivation of Two Bacillus Strains for Improved Cell Growth and Enzyme Production to Enhance the Degradation of Aflatoxin B1

Toxins ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 435
Author(s):  
Le Wang ◽  
Wei Huang ◽  
Yu Sha ◽  
Haicheng Yin ◽  
Ying Liang ◽  
...  

Bacillus sp. H16v8 and Bacillus sp. HGD9229 were identified as Aflatoxin B1 (AFB1) degrader in nutrient broth after a 12 h incubation at 37 °C. The degradation efficiency of the two-strain supernatant on 100 μg/L AFB1 was higher than the bacterial cells and cell lysate. Moreover, degradations of AFB1 were strongly affected by the metal ions in which Cu2+ stimulated the degradation and Zn2+ inhibited the degradation. The extracellular detoxifying enzymes produced by co-cultivation of two strains were isolated and purified by ultrafiltration. The molecular weight range of the detoxifying enzymes was 20–25 kDa by SDS-PAGE. The co-culture of two strains improved the total cell growth with the enhancement of the total protein content and detoxifying enzyme production. The degradation efficiency of the supernatant from mixed cultures increased by 87.7% and 55.3% compared to Bacillus sp. H16v8 and HGD9229, individually. Moreover, after the degradation of AFB1, the four products of the lower toxicity were identified by LC-Triple TOF-MS with the two proposed hypothetical degradation pathways.

2012 ◽  
Vol 59 (1) ◽  
Author(s):  
Nurulhanis Ahmad Sanusi ◽  
Haryati Jamaluddin

Bacillus sp. strain B1 producing wild type fibrinolytic enzyme was isolated from Budu. The fibrinolytic enzyme was collected from the supernatant of Bacillus sp. strain B1 culture broth and purified to electrophoretic homogeneity through a combination of various purification schemes, which include ammonium sulphate precipitation, followed by anion exchange chromatography using DEAE–Sepharose Fast Flow and gel filtration chromatography on Sephadex G–75 column. During ammonium sulphate precipitation screening, it was observed that the crude enzyme from Bacillus sp. strain B1 precipitated at 40% and 50% of ammonium sulphate saturation respectively. The fibrinolytic enzyme was purified 58.5–fold with a final yield of 0.51%. The specific activity was determined to be 1.17 Units/mg using plasmin as standard and the final total protein content was 8.58 mg/ml. After the successive purification steps, the estimated molecular mass of fibrinolytic enzymes from strain B1 was estimated via sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS–PAGE). SDS–PAGE analysis showed a single band at 45 kDa corresponding to the purified fraction with fibrinolytic activity.


2019 ◽  
Vol 17 ◽  
Author(s):  
Farzane Kargar ◽  
Mojtaba Mortazavi ◽  
Mahmood Maleki ◽  
Masoud Torkzadeh Mahani ◽  
Younes Ghasemi ◽  
...  

Aims: The purpose of this study was to screen the bacteria producing cellulase enzymes and their bioinformatics studies. Background: Cellulose is a long-chain polymer of glucose that hydrolyzes by cellulases to glucose molecules. In order to design the new biotechnological applications, some strategies have been used as increasing the efficiency of enzyme production, generating cost-effective enzymes, producing stable enzymes and identification of new strains. Objective: On the other hand, some bacteria special features have made them suitable candidates for the identification of the new source of enzymes. In this regard, some native strains of bacteria were screened. Method: These bacteria were grown on a culture containing the liquid M9 media containing CMC to ensure the synthesis of cellulase. The formation of a clear area in the culture medium indicated decomposition of cellulose. In the following, the DNA of these bacteria were extracted and their 16S rDNA genes were amplified. Result: The results show that nine samples were able to synthesize cellulase. In following, these strains were identified using 16S rDNA. The results show that these screened bacteria belonged to the Bacillus sp., Alcaligenes sp., Alcaligenes sp., and Enterobacter sp.conclusionThe enzyme activity analysis shows that the Bacillus toyonensis, Bacillus sp. strain XA15-411 Bacillus cereus have produced the maximum yield of cellulases. However, these amounts of enzyme production in these samples are not proportional to their growth rate. As the bacterial growth chart within 4 consecutive days shows that the Alcaligenes sp. Bacillus cereus, Bacillus toyonensis, Bacillus sp. strain XA15-411 have a maximum growth rate. The study of the phylogenetic tree also shows that Bacillus species are more abundant in the production of cellulase enzyme. These bioinformatics analyses show that the Bacillus species have different evolutionary relationships and evolved in different evolutionary time. Other: However, for maximum cellulase production by this bacteria, some information as optimum temperature, optimum pH, carbon and nitrogen sources are needed for the ideal formulation of media composition. The cellulase production is closely controlled in microorganisms and the cellulase yields appear to depend on a variety of factors. However, the further studies are needed for cloning, purification and application of these new microbial cellulases in the different commercial fields as in food, detergent, and pharmaceutical, paper, textile industries and also various chemical industries. However, these novel enzymes can be further engineered through rational design or using random mutagenesis techniques.


2004 ◽  
Vol 70 (8) ◽  
pp. 4582-4587 ◽  
Author(s):  
Jan Kostal ◽  
Rosanna Yang ◽  
Cindy H. Wu ◽  
Ashok Mulchandani ◽  
Wilfred Chen

ABSTRACT The metalloregulatory protein ArsR, which offers high affinity and selectivity toward arsenite, was overexpressed in Escherichia coli in an attempt to increase the bioaccumulation of arsenic. Overproduction of ArsR resulted in elevated levels of arsenite bioaccumulation but also a severe reduction in cell growth. Incorporation of an elastin-like polypeptide as the fusion partner to ArsR (ELP153AR) improved cell growth by twofold without compromising the ability to accumulate arsenite. Resting cells overexpressing ELP153AR accumulated 5- and 60-fold-higher levels of arsenate and arsenite than control cells without ArsR overexpression. Conversely, no significant improvement in Cd2+ or Zn2+ accumulation was observed, validating the specificity of ArsR. The high affinity of ArsR allowed 100% removal of 50 ppb of arsenite from contaminated water with these engineered cells, providing a technology useful to comply with the newly approved U.S. Environmental Protection Agency limit of 10 ppb. These results open up the possibility of using cells overexpressing ArsR as an inexpensive, high-affinity ligand for arsenic removal from contaminated drinking and ground water.


2001 ◽  
Vol 56 (11-12) ◽  
pp. 1022-1028 ◽  
Author(s):  
Kristina Uzunova ◽  
Anna Vassileva ◽  
Margarita Kambourova ◽  
Viara Ivanova ◽  
Dimitrina Spasova ◽  
...  

Abstract Enzyme production of newly isolated thermophilic inulin-degrading Bacillus sp. 11 strain was studied by batch cultivation in a fermentor. The achieved inulinase and invertase activi­ ties after a short growth time (4.25 h) were similar or higher compared to those reported for other mesophilic aerobic or anaerobic thermophilic bacterial producers and yeasts. The investigated enzyme belonged to the exo-type inulinases and splitted-off inulin, sucrose and raffinose. It could be used at temperatures above 65 °C and pH range 5.5-7.5. The obtained crude enzyme preparation possessed high thermostability. The residual inulinase and inver­ tase activities were 92-98% after pretreatment at 65 °C for 60 min in the presence of substrate inulin.


Nutrients ◽  
2018 ◽  
Vol 10 (11) ◽  
pp. 1679 ◽  
Author(s):  
Elisabetta De Angelis ◽  
Simona Bavaro ◽  
Graziana Forte ◽  
Rosa Pilolli ◽  
Linda Monaci

Almond is consumed worldwide and renowned as a valuable healthy food. Despite this, it is also a potent source of allergenic proteins that can trigger several mild to life-threatening immunoreactions. Food processing proved to alter biochemical characteristics of proteins, thus affecting the respective allergenicity. In this paper, we investigated the effect of autoclaving, preceded or not by a hydration step, on the biochemical and immunological properties of almond proteins. Any variation in the stability and immunoreactivity of almond proteins extracted from the treated materials were evaluated by total protein quantification, Enzyme Linked Immunosorbent Assay (ELISA), and protein profiling by electrophoresis-based separation (SDS-PAGE). The sole autoclaving applied was found to weakly affect almond protein stability, despite what was observed when hydration preceded autoclaving, which resulted in a loss of approximately 70% of total protein content compared to untreated samples, and a remarkable reduction of the final immunoreactivity. The final SDS-PAGE protein pattern recorded for hydrated and autoclaved almonds disclosed significant changes. In addition, the same samples were further submitted to human-simulated gastro-intestinal (GI) digestion to evaluate potential changes induced by these processing methods on allergen digestibility. Digestion products were identified by High Pressure Liquid Chromatography-High Resolution Tandem Mass Spectrometry (HPLC-HRMS/MS) analysis followed by software-based data mining, and complementary information was provided by analyzing the proteolytic fragments lower than 6 kDa in size. The autoclave-based treatment was found not to alter the allergen digestibility, whereas an increased susceptibility to proteolytic action of digestive enzymes was observed in almonds subjected to autoclaving of prehydrated almond kernels. Finally, the residual immunoreactivity of the GI-resistant peptides was in-silico investigated by bioinformatic tools. Results obtained confirm that by adopting both approaches, no epitopes associated with known allergens survived, thus demonstrating the potential effectiveness of these treatments to reduce almond allergenicity.


2020 ◽  
pp. 1-4
Author(s):  
Opara C N ◽  
◽  
Anumudu C K ◽  

Lipases form an important group of relevant enzymes which have applications in various fields including; food, pharmaceutical, detergent, textile and cosmetic industries. Lipases can be produced from diverse sources including microorganisms. This study evaluated the potential of bacteria isolates from fresh-water clam Mercenaria Mercenaria to produce lipolytic enzymes. Ten samples of Clam (Mercenaria Mercenaria) were screened for the presence of lipase producing bacteria using classical culture methods. Eleven bacteria species were obtained, of which six (Actinomyces sp., E. coli, Bacillus sp., Pseudomonas sp., Clostridium sp. and Klebsiella sp.) produced lipases that had lipolytic activity in breaking down olive oil used in media supplementation. The best culture media and conditions for optimal production of lipases was studied and it was shown that supplementation of growth media with 2% dextrose at neutral pH gave the greatest yield of lipases when lipase producing isolates were grown in shake flasks. Measurement of biomass by culture and turbidimetric methods indicates that the highest cell mass was recorded by Pseudomonas sp at 7.8 x 105 CFU/ml, closely followed by Actinomyces sp. and Bacillus sp., at 6.2 x 105 CFU/ml and 5.3 x 105 respectively. The produced lipases were partially purified by precipitating with ammonium sulphate followed by dialysis. The total protein content of produced lipases was evaluated by the Lowry’s method, showing that estimated protein content followed the same trend as cell biomass with the highest recorded by Pseudomonas sp. at 1.53mg/ml, followed by Actinomyces sp. and Bacillus sp. at 1.47mg/ml and 1.32mg/ml respectively. The results obtained in this study shows that isolates obtained from freshwater clam can produce potent lipases which can be employed for industrial, food and other diverse uses


2010 ◽  
Vol 10 (2) ◽  
pp. 256-260 ◽  
Author(s):  
Hasnah Natsir ◽  
Abd. Rauf Patong ◽  
Maggy Thenawidjaja Suhartono ◽  
Ahyar Ahmad

Chitinase is an extracellular enzyme which is capable in hydrolyzing insoluble chitin to its oligomeric and monomeric components. The enzyme produced by thermophilic bacteria was screened and isolated from Sulili hot spring in Pinrang, South Sulawesi, Indonesia. The gram positive spore forming rod shape bacteria was identified as Bacillus sp. HSA,3-1a through morphological and physiological analysis. The production of chitinase enzyme was conducted at various concentration of colloidal chitin at a pH of 7.0 and a temperature of 55 °C. The bacteria optimally was produced the enzyme at a colloidal chitin concentration of 0.5% after 72 h of incubation. The optimum temperature, pH and substrate concentration of chitinase were 60 °C, 7.0 and 0.3%, respectively. The enzyme was stable at a pH of 7.0 and a temperature of 60 °C after 2 h of incubation. The chitinase activities was increased by addition of 1 mM Mg2+, Ca2+ and Mn2+ ions, whereas the activities were  decreased by 1 mM Co2+, Fe2+ and Zn2 ions. The molecular weight of the crude enzyme was determined using SDS-PAGE analysis. Five protein fractions were obtained from SDS-PAGE, with MWs of 79, 71, 48, 43 and 22 kDa.   Keywords: colloidal chitin, thermophilic bacteria, chitinase


2017 ◽  
Vol 18 (2) ◽  
pp. 1-10 ◽  
Author(s):  
Dzun Noraini Jimat ◽  
Intan Baizura Firda Mohamed ◽  
Azlin Suhaida Azmi ◽  
Parveen Jamal

A newly bacterial producing L-asparaginase was successful isolated from Sungai Klah Hot Spring, Perak, Malaysia and identified as Bacillus sp. It was the best L-asparaginase producer as compared to other isolates. Production of L-asparaginase from the microbial strain was carried out under liquid fermentation. The crude enzyme was then centrifuged and precipitated with ammonium sulfate before further purified with chromatographic method. The ion exchange chromatography HiTrap DEAE-Sepharose Fast Flow column followed by separation on Superose 12 gel filtration were used to obtain pure enzyme. The purified enzyme showed 10.11 U/mg of specific activity, 50.07% yield with 2.21 fold purification. The purified enzyme was found to be dimer in form, with a molecular weight of 65 kDa as estimated by SDS-PAGE. The maximum activity of the purified L-asparaginase was observed at pH 9 and temperature of 60°C.


2020 ◽  
Vol 2 (1) ◽  
pp. 52-68
Author(s):  
Mircea BOLOGA ◽  
Elvira VRABIE ◽  
Irina PALADII ◽  
Olga ILIASENCO ◽  
Tatiana STEPURINA ◽  
...  

Introduction. Whey is a by-product and an excellent source of proteins that is rather aggressive due to a large amount of organic substances it contains. The electro-activation of whey applied in the experiments is a wasteless method that allows the va-lorification of all whey components. β-lactoglobulin (β-Lg) makes up 50% of the whey proteins and 12% of the total protein content in milk. Material and methods. The recovery of β-Lg in protein-mineral concentrates (PMC) by electro-activation processing of different types of whey with different initial protein content was investigated in seven configurations. The recovery of protein fractions in the PMCs were analyzed via electrophoresis with sodium dodecyl sulfate (SDS-PAGE) and 15% non-denaturing polyacrylamide gel (PAAG).      Results. Whey electro-fractionation and the obtaining of PMCs with predetermined protein content, namely of β-Lg, were studied on three whey types, processed at different treatment regimens and in seven configurations. The proper management of electroactivation by varying the treatment regimens will allow the electro-fractionation of different types of dairy by-products. Conclusions. The maximum amount of β-Lg recovered in PMCs on electroactivation is  66-71% depending on the processed whey and on the treatment regimens. Obviously, the extraction of β-Lg from initially lower protein content shows a higher recovery degree of β-Lg. The registered temperatures allows formation of PMCs without thermal denaturation.


2018 ◽  
Vol 84 (8) ◽  
pp. e02508-17 ◽  
Author(s):  
Xiaofei Yuan ◽  
Yanqing Song ◽  
Yizhi Song ◽  
Jiabao Xu ◽  
Yinhu Wu ◽  
...  

ABSTRACTLasers are instrumental in advanced bioimaging and Raman spectroscopy. However, they are also well known for their destructive effects on living organisms, leading to concerns about the adverse effects of laser technologies. To implement Raman spectroscopy for cell analysis and manipulation, such as Raman-activated cell sorting, it is crucial to identify nondestructive conditions for living cells. Here, we evaluated quantitatively the effect of 532-nm laser irradiation on bacterial cell fate and growth at the single-cell level. Using a purpose-built microfluidic platform, we were able to quantify the growth characteristics, i.e., specific growth rates and lag times of individual cells, as well as the survival rate of a population in conjunction with Raman spectroscopy. Representative Gram-negative and Gram-positive species show similar trends in response to a laser irradiation dose. Laser irradiation could compromise the physiological function of cells, and the degree of destruction is both dose and strain dependent, ranging from reduced cell growth to a complete loss of cell metabolic activity and finally to physical disintegration. Gram-positive bacterial cells are more susceptible than Gram-negative bacterial strains to irradiation-induced damage. By directly correlating Raman acquisition with single-cell growth characteristics, we provide evidence of nondestructive characteristics of Raman spectroscopy on individual bacterial cells. However, while strong Raman signals can be obtained without causing cell death, the variety of responses from different strains and from individual cells justifies careful evaluation of Raman acquisition conditions if cell viability is critical.IMPORTANCEIn Raman spectroscopy, the use of powerful monochromatic light in laser-based systems facilitates the detection of inherently weak signals. This allows environmentally and clinically relevant microorganisms to be measured at the single-cell level. The significance of being able to perform Raman measurement is that, unlike label-based fluorescence techniques, it provides a “fingerprint” that is specific to the identity and state of any (unlabeled) sample. Thus, it has emerged as a powerful method for studying living cells under physiological and environmental conditions. However, the laser's high power also has the potential to kill bacteria, which leads to concerns. The research presented here is a quantitative evaluation that provides a generic platform and methodology to evaluate the effects of laser irradiation on individual bacterial cells. Furthermore, it illustrates this by determining the conditions required to nondestructively measure the spectra of representative bacteria from several different groups.


Sign in / Sign up

Export Citation Format

Share Document