Expression and characterization of recombinant l -asparaginase from Pseudomonas fluorescens

Sindhu, R.; Manonmani, H. K.

doi:10.1016/j.pep.2017.09.009

Cited by 45 publications

(16 citation statements)

References 36 publications

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“…Another study reported the production of the enzyme in Pseudomonas fluorescens as homotetramero and total molecular weight of 140 KDa and after purified this ASNase had an activity of 26 U/mg protein and half-life higher than commercially available L-asparaginase from E. coli. [27].…”

Section: Discussionmentioning

confidence: 99%

Optimization of aqueous two-phase micellar system for partial purification of L-asparaginase from Penicillium sp. grown in wheat bran as agro-industrial residue

et al. 2020

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L-asparaginase has been used in the remission of malignant neoplasms such as acute lymphoblastic leukemia. The search for new sources of this enzyme has become attractive for therapeutics. Traditional methods for biomolecule purification involve several steps. A two-phase system may be a good strategy to anticipate one of these stages. This study aimed to produce and purify a fungal L-asparaginase through an aqueous two-phase micellar system (ATPMS) using Triton X-114. The fungus Penicillium sp.-encoded 2DSST1 was isolated from Cerrado soil. Plackett-Burman design followed by a 2 4 full factorial design was used to determine the best conditions to produce L-asparaginase. The evaluated variables were L-asparagine, L-proline, wheat bran, potato dextrose broth, ammonium sulfate, yeast extract, sucrose and glucose concentrations, incubation temperature, incubation period, and initial pH of the culture medium. L-asparaginase quantification was valued by the formation of β-aspartyl hydroxamate. The significant positive variables, L-asparagine, L-proline, potato dextrose broth, and sucrose concentrations, were evaluated at 2 levels (+ 1 and − 1) with triplicate of the central point. After 34 runs, maximum activity (2.33 IU/mL) was achieved at the factorial design central point. A central composite design was performed in ATPMS at two levels (+ 1 and − 1) varying Triton X-114 concentration (w/v), separation phase temperature, and crude extract concentration (w/v). The Lasparaginase partition coefficient (K) was considered the experimental design response. Out of the 16 systems that were examined, the most promising presented a purification factor of 1.4 and a yield of 100%.

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Section: Discussionmentioning

confidence: 99%

Optimization of aqueous two-phase micellar system for partial purification of L-asparaginase from Penicillium sp. grown in wheat bran as agro-industrial residue

et al. 2020

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“…Nessler's reagent is widely used to quantify ASNase activity, yet little attention has been paid to the effects of salts present in the analyte that can yield erroneous estimates of enzyme activity 22–28 . Here, we determined that the salts of Co 2+ , Fe 2+ , Mg 2+ , Mn 2+ and Ni 2+ interfered with NH 3 quantification using Nessler's reagent, while the salts of Ca 2+ , Cu 2+ , Mo 2+ , Na + and Zn 2+ did not.…”

Section: Resultsmentioning

confidence: 89%

An improved method for simple and accurate colorimetric determination of l‐asparaginase enzyme activity using Nessler's reagent

Simas

Kleingesinds

Pessoa

et al. 2021

J of Chemical Tech & Biotech

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BACKGROUND Colorimetric determination of l‐asparaginase enzyme activity most commonly uses Nessler's reagent, but often yields unreliable results due to precipitation of tetraiodomercurate salts. Interference caused by some commonly used inorganic and organic compounds of biochemical buffers, reagents and microbiology fermentation media was determined. The stabilising effects of tartrate and polyvinyl alcohol upon the impact of these salts was next evaluated using the following two‐step assay in 96‐well microtitre plates: 160 μL of 50 mmol L−1 Tris–HCl pH 8.6, 32 μL of 100 mmol L−1 l‐asparagine, 16 μL analyte and 128 μL water were added to each well. After incubation at 37 °C for 1 h, 16 μL of 1.5 mol L−1 trichloroacetic acid was added to stop the reaction. A 35 μL volume was next transferred to a fresh well containing 35 μL Nessler's reagent, 35 μL stabiliser solution (4 mmol L−1 disodium tartrate and 10 mg L−1 polyvinyl alcohol) and 245 μL water. After incubation at room temperature for 10 min, absorbance was recorded at 436 nm and the enzyme activity calculated by extrapolation of a (NH4)2SO4 calibrated standard curve. RESULTS Addition of tartrate and polyvinyl alcohol proved to be effective in reducing interference caused by precipitation of salts, allowing determination of l‐asparaginase activity in a range from 0.050 to 0.796 IU mL−1, ideal for monitoring enzyme production in fermentations. CONCLUSION A method is offered for rapid and reliable determination of l‐asparaginase activity, particularly in complex samples such as microbial fermentation broths where existing methodologies do not account for interferences that frequently yield overvalues. © 2020 Society of Chemical Industry

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“…Moreover, their general occurrence in foods along with widespread use means that they are Generally Recognized as Safe for human consumption [25]. [29] which showed no activity following incubation at 45°C and P. fluorescens, which was stable at 37°C [30].…”

Section: Discussionmentioning

confidence: 99%

Comparative Biochemical Characterization of L-Asparaginases from Four Species of Lactic Acid Bacteria

Phetsri¹,

Furukawa²,

Yamashiro³

et al. 2019

J Biotechnol Biomed

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L-Asparaginase (ASNase; EC 3.5.1.1) is an enzyme that catalyzes the hydrolysis of L-asparagine to L-aspartic acid and ammonia. Generally, ASNases from Escherichia coli and Erwinia chrysanthemi are used for the treatment of acute lymphoblastic leukemia. However, few studies focusing on ASNase from lactic acid bacteria (LAB) have been reported. The aim of this study is to characterize ASNase genes from four LAB strains: Streptococcus thermophiles, Lactobacillus plantarum, L. acidophilus, and L. sakei. ASNase genes from each strain amplified by polymerase chain reaction PCR were inserted into NdeI and XhoI sites of pET28a-(+) and cloned in E. coli BL21(DE3). Recombinant ASNases were purified using nickel-nitrilotriacetic acid column chromatography. Among the four strains, the purified recombinant ASNase from S. thermophilus exhibited the highest specific activity of 113.0 U/mg and specificity for L-asparagine. The pH and temperature ranges for S. thermophilus ASNase were pH 8.0-9.0 and 30°C-50°C, respectively. The activity of the enzyme was significantly inhibited by Ni 2+ . K m and k cat values were 2.91 mM and 1.53 × 10 2 s -1 , respectively. In this study, we described the biochemical properties of ASNases from four LAB and demonstrated that ASNase from S. thermophilus has potential applications in food processing.

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Expression and characterization of recombinant l -asparaginase from Pseudomonas fluorescens

Cited by 45 publications

References 36 publications

Optimization of aqueous two-phase micellar system for partial purification of L-asparaginase from Penicillium sp. grown in wheat bran as agro-industrial residue

Optimization of aqueous two-phase micellar system for partial purification of L-asparaginase from Penicillium sp. grown in wheat bran as agro-industrial residue

An improved method for simple and accurate colorimetric determination of l‐asparaginase enzyme activity using Nessler's reagent

Comparative Biochemical Characterization of L-Asparaginases from Four Species of Lactic Acid Bacteria

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