Production, characterization, and antitumor efficiency of l-glutaminase from halophilic bacteria

Background Halophiles are an excellent source of enzymes that are not only salt stable, but also can withstand and carry out reaction efficiently under extreme conditions. l-glutaminase has attracted much attention with respect to proposed applications in several fields such as pharmaceuticals and food industries. The aim of the present study was to investigate the anticancer activity of l-glutaminase produced by halophilic bacteria. Various halophilic bacterial strains were screened for extracellular l-glutaminase production. An attempt was made to study the optimization, purification, and characterization of l-glutaminase from Bacillus sp. DV2-37. The antitumor activity of the produced enzyme was also investigated. Results The potentiality of 15 halophilic bacterial strains isolated from the marine environment that produced extracellular l-glutaminase was investigated. Bacillus sp. DV2-37 was selected as the most potent strain and optimized for enzyme production. The optimization of fermentation process revealed that the highest enzyme activity (47.12 U/ml) was observed in a medium supplemented with 1% (w/v) glucose as a carbon source, 1% (w/v) peptone as a nitrogen source, 5% (w/v) NaCl, the initial pH was 7.0, at 37 °C, using 20% (v/v) inoculum size after 96 h of incubation. The produced crude enzyme was partially purified by ammonium sulfate precipitation and dialysis. Of the various parameters tested, pH 7, 40 °C, and 5% NaCl were found to be the best for l-glutaminase activity. The enzyme also exhibited high salt and temperature stability. The antitumor effect against human breast (MCF-7), hepatocellular (HepG-2), and colon (HCT-116) carcinoma cell lines revealed that l-glutaminase produced by Bacillus sp. DV2-37 showed potent cytotoxic activity of all the tested cell lines in a dose-dependent manner with an IC50 value of 3.5, 3.4, and 3.8 µg/ml, respectively. Conclusions The present study proved that l-glutaminase produced by marine bacteria holds proper features and it has a high potential to be useful for many therapeutic applications.

Screening of Lactic Acid Fermentation by Rhizopus oryzae on Banana Peel Using Two-Level Factorial Analysis

For ages, pure sugars or edible crops have produced lactic acid. However, a major concern on lactic acid production lies in the cost of the raw materials used. Thus, an alternative to overcome this situation is urgently needed. Characterization of banana peels shows that it contains promising sugar that can be utilized for lactic acid production, which are xylose (0.774 g/L), glucose (0.756 g/L) and fructose (0.532 g/L). Thus, this study aims to screen the potential of banana peel as a substrate by using Rhizopus oryzae through batch fermentation for lactic acid production, as R. oryzae can synthesize lactic acid in low nutrient requirements. Two-level factorial analysis was designed to screen the effects of moisture content (60% and 80%), temperature (27 °C and 40 °C), pH (4.5 and 6.5) and inoculum size (1x104 spores/mL and 1x108 spores/g) on the lactic acid production. Based on the Two-level factorial (2LF) analysis, the highest lactic acid production of 0.0813 g/L was detected at 80 % moisture content, pH 4.5, the temperature of 27 °C and inoculum size of 1x104 spores/g. The findings show that most of the conditions have a significant difference between each other (p<0.05). Therefore, the fermentation of banana peels by R. oryzae could be a promising method to produce a lactic acid concentration.

Rosmarinic acid production in cell suspension cultures of Ehretia asperula Zollinger & Moritz

Plant cell cultures provide an alternative means for producing secondary compounds in food, cosmetic and pharmaceutical industries. Ehretia asperula Zollinger & Moritzi is used as a traditional medicine for the treatment of liver detoxification, ulcers, tumors, inflammation and enhancing the body's resistance in Vietnam. The study was carried out to select suitable callus line for cell suspension cultures of E. asperula Zollinger & Moritzi and investigate the effects of inoculum size, rotation speed and naphthalene acetic acid (NAA) on the proliferation of cell suspension cultures. In addition, the influence of light intensity on the growth and rosmarinic acid (RA) biosynthesis of cell suspension was also surveyed. After 4 weeks of culture, the white to pale yellow friable callus expanded significantly with a fresh weight (FW) of 0.788 g and a high RA content of 2.062 mg/g FW. An appropriate medium for cell proliferation was the liquid B5 medium, which contained 30 g/l glucose, 0.1 mg/l benzyl adenine (BA) and 0.4 mg/l NAA. The results also demonstrated that a 1:20 ratio (w/v) inoculum size, darkness and rotation speed of 90 rpm were the optimal conditions for the proliferation and RA accumulation to 188.217 mg/l in 4 weeks of culture. These findings showed that E. asperula Zollinger & Moritzi cell suspension cultures could be a potential alternative approach for RA production in vitro.

Characterization of Morganella sp. for its Paraquat Degradation Potential

One of the beneficial roles of the microbial population is their ability to convert toxic herbicides to lesser toxic compounds such as water and carbon (IV) oxide. Paraquat which is an acutely toxic herbicide is used on farmlands and has been found to affect human health. This study was aimed at characterizing bacteria with the potential to degrade paraquat. Previously isolated bacteria from culture collection labelled A-F were screened for their potential to degrade and utilized paraquat as the sole carbon source in Bushnell Hass agar media. Of the six isolates, isolate E (Morganella sp.) was observed to have the highest growth and tolerance to paraquat after 72 h of incubation at 37 ºC. Characterization study revealed that Morganella sp. can utilize and grow with optimum conditions of pH 6.5, the temperature of 30 ºC and can tolerate up to 400 mg/L paraquat concentration with an increase in growth as inoculum size increases. Thus, these findings showed that Morganella sp. can degrade toxic paraquat to a less toxic form and therefore can be a good isolate for the future bioremediation process of the pollutant.

Characterization of Cypermethrin-degradation by a Novel Molybdenum-reducing Morganella sp. Isolated from Active Agricultural Land in Northwestern Nigeria

The increasing use of cypermethrin in agricultural fields, household and industrial applications for effective pest control had increased the global burden of the pollutant over the years. Consequently, there is an urgent need to devise techniques to eliminate this pollutant from the environment. A bacterium capable of degrading cypermethrin has been successfully screened and characterized. The bacterium was grown in a mineral salt medium (MSM) supplemented with cypermethrin as its sole carbon and energy source at an optimum pH 7.5, temperature 40 ºC, a carbon source concentration of 4 g/L, optimum incubation time of 24 h and an inoculum size of 400 µL. The potential of Morganella sp. to degrade cypermethrin makes it an important instrument for the degradation of cypermethrin. This knowledge may be useful for the optimization of environmental conditions for cypermethrin bioremediation and important for detoxification of cypermethrin polluted sites.

Characterization of Butachlor Degradation by A Molybdenum-Reducing and Aniline-degrading Pseudomonas sp.

Butachlor is a chloroacetanilide herbicide that is selective in action and commonly used for pre-emergence control of weeds. It is believed to have range of toxicity from acute to chronic and also can cause DNA strand breaks and chromosomal aberrations in humans. This study was aimed at characterizing the potential of previously isolated bacteria for butachlor degradation. The isolates from culture collection, labelled A-F were screened for butachlor degradation on Bushnell Hass agar media with butachlor as a sole carbon source. Isolate A (molybdenum-reducing and aniline-degrading Pseudomonas sp.) was observed to grow best and tolerated the highest concentration of butachlor supplemented in the media after 72 h of incubation at 37 ℃. Characterization study revealed that the Pseudomonas sp. can utilize and grow with butachlor at optimum pH between 6.0 - 6.5, temperature between 30 – 37 ℃ and can tolerate up to 600 mg/L butachlor concentration with increase in growth as inoculum size increases. Additionally, this bacterial strain shows no lag phase regardless of the concentration of the herbicide used and reach its maximum growth after 24 h of incubation. The ability of this isolate to tolerate and utilize butachlor as sole carbon source makes it suitable for future bioremediation of this toxicant.

Optimization of Small Peptide Feed from Milk Thistle Residue by Synergistic Fermentation of Multiple Strains and Proteases

In order to improve the utilization rate of the milk thistle residue, this study used the synergistic fermentation of multiple strains and proteases to increase the small peptide content of the fermented feed produced by the milk thistle residue. Taking the small peptide content of the milk thistle residue fermented feed as an indicator, the optimal fermentation process was obtained by single-factor optimization experiments and the response surface methodology. The optimal fermentation process was as follows: fermentation time of 7 days, inoculum size of 15%, inoculation ratio of aerobic strains: anaerobic strains = 1: 2, solid-state fermentation water content of 66%, fermentation temperature of 36℃, and amount of protease was 0.25% acid protease+0.25% bromelain. Under the above process, the small peptide content of the fermented feed from milk thistle residue was greatly improved to 57.86%. These results inferred that the added proteases were beneficial to the growth of fermentative microorganisms, the secretion of protease and the increase of the small peptide content.

Applicability of Saccharomyces cerevisiae Strains for the Production of Fruit Wines Using Cocoa Honey Complemented with Cocoa Pulp

Research background. Cocoa honey (CH) and cocoa pulp (CP) are both fruit pulps highly appreciated but, until now, CH is less processed than CP. In this work, it was investigated the applicability of strains of S. cerevisiae to ferment CH complemented with CP, to obtain fruit wines and improve CH commercialization. Experimental approach. The selection of a strain, previously isolated from cachaçaria distilleries in Brazil, took place based on its fermentation performance. The conditions for fermentation with S. cerevisiae L63 were then studied in relation to: volumetric proportion (φCH) of CH (complemented with CP), sucrose addition (γsuc), temperature (T) and inoculum size (No). The best conditions were applied in order to obtain fermentation profiles. Results and conclusions. S. cerevisiae L63 (No=107–108 cell/mL) is capable to ferment φCH of 90 and 80 % (V/V) for 24 or 48 h with γsuc of 50 and 100 g/L at T=28–30 °C resulting in wines with ethanol contents from 8 to 14 % (V/V). Additionally, the φCH=90 % (V/V) wine resulted in the lowest residual sugar concentration (<35 g/L) than the φCH=80 % (V/V) wine (~79 g/L) which could be classified as a sweet wine. In general, S. cerevisiae L63 resulted in a similar fermentation performance than a commercial strain tested, indicating its potential for fruit pulp fermentation. Novelty and scientific contribution. Therefore, S. cerevisiae L63 is capable to ferment CH complemented with CP to produce fruit wines with good commercial potentials that may also benefit small cocoa producers by presenting a product with greater added value.

Biodegradation of Basic Yellow Auramine- O Dye using Staphylococcus sp. Isolated from Textile Industry Effluent

Due to the increased use of synthetic dyes in various industries, there is an increased disposal of wastewater containing harmful dyes. These, in turn, have affected plants, animals, and humans. The physical and chemical methods of dye decolorization have failed to degrade the synthetic dyes in industrial effluents completely. The microbial decolorization is better due to its versatility, dynamic metabolism, and potential machinery of enzymes. This study aimed to degrade basic yellow dye auramine O by bacteria isolated from textile industry effluent. In this regard, five bacterial strains were isolated and screened from a soil sample taken from textile industry effluent. The initial physical and biochemical characterization of the bacterial isolates 1 and 2 indicated catalase test-positive, starch test-negative, motility agar test-negative, gram staining test-positive, and morphology-bacillus. The bacterial isolates 3, 4, and 5 indicated oxidase test-negative, urease test-positive, gram staining test-negative, and morphology-staphylococcus. All the isolates were further subjected to a screening test, where isolate 5 showed maximum dye decolorization of 98.9% in 96 h. The biodegradation of dye was optimized for different values of initial pH (4-10), inoculum size (2% -10%), initial dye concentration (50 mgL-1 to400 mgL-1), carbon source (glucose, fructose, xylose, starch and lactose) and nitrogen source (peptone, ammonium sulphate, yeast extract, ammonium nitrate and urea). Maximum dye decolorization was observed for initial dye concentration of 200 mgL-1, initial pH of 6, inoculum size of 10%, yeast extract as nitrogen source, and glucose as carbon source. Therefore, dye degradation by bacteria can be used as a potential method for auramine O dye treatment.

Production of bacterial cellulose films by Gluconoacetobacter xylinus for lipase immobilization

Bacterial cellulose (BC), a microbial polysaccharide, has chemically equivalent structure to plant cellulose with unbranched pellicle structure of only glucose monomers. Due to the unique nanostructure, BC has great potential in enzyme immobilization. In this study, the effects of different cultivation conditions including rotational speed, initial inoculum concentration and medium pH on the film-like cellulosic biomass formation of Gluconacetobacter xylinus JCM 9730 were examined. The resultant BC films were then studied for its feasibility in the immobilization of lipase, a widely used enzyme in biotechnological and industrial processes including food, pharmaceutical, chemical and paper industries. Results showed that increasing in rotational speed from 0 rpm to 200 rpm converted cellulose-producing cells to non-cellulose-producing ones, leading to a significant decline in BC film formation. The increase in initial inoculum size from 0.01 g/L to 0.1 g/L reduced sugar concentration and surface area of the medium, and therefore inhibiting the formation of film-like cellulosic biomass. In addition, the optimum pH range of Acetobacter species from 5.4 – 6.3 was found not optimal for BC film formation. The highest amount of film-like cellulosic biomass of 19.01 g/L was obtained under static condition (0 rpm) with initial cell concentration of 0.04 g/L and initial pH of 4.0. The BC film samples were then acetylated with acetic anhydride/iodine system to convert the hydroxyl groups to less hydrophilic acetyl groups and were used for lipase immobilization. Results showed that lipase immobilized on acetylated BC still maintained its lipid hydrolytic activity. It can be hence concluded that BC films produced by G. xylinus JCM 9730 were potential for lipase immobilization.