L-asparaginase Activity Research Articles

Improvement of Biocatalytic Properties and Cytotoxic Activity of L-Asparaginase from Rhodospirillum rubrum by Conjugation with Chitosan-Based Cationic Polyelectrolytes.

L-asparaginases (L-ASNases, EC 3.5.1.1) are a family of enzymes that are widely used for the treatment of lymphoblastic leukemias. L-ASNase from Rhodospirillum rubrum (RrA) has a low molecular weight, low glutaminase activity, and low immunogenicity, making it a promising enzyme for antitumor drug development. In our work, the complex formation and covalent conjugation of the enzyme with synthetic or natural polycationic polymers was studied. Among non-covalent polyelectrolyte complexes (PEC), polyethyleneimine (PEI) yielded the highest effect on RrA, increasing its activity by 30%. The RrA-PEI complex had increased stability to trypsinolysis, with an inactivation constant decrease up to 10-fold compared to that of the native enzyme. The covalent conjugation of RrA with chitosan-PEI, chitosan-polyethylene glycol (chitosan-PEG), and chitosan-glycol resulted in an increase in the specific activity of L-asparagine (up to 30%). RrA-chitosan-PEG demonstrated dramatically (by 60%) increased cytotoxic activity for human chronic myeloma leukemia K562 cells in comparison to the native enzyme. The antiproliferative activity of RrA and its conjugates was significantly higher (up to 50%) than for that of the commercially available EcA at the same concentration. The results of this study demonstrated that RrA conjugates with polycations can become a promising strategy for antitumor drug development.

Response surface methodological optimization of l-asparaginase production from the medicinal plant endophyte Acinetobacter baumannii ZAS1

BackgroundThis study targets the enhanced production of l-asparaginase, an antitumor enzyme by Acinetobacter baumannii ZAS1. This organism is an endophyte isolated from the medicinal plant Annona muricata. Plackett–Burman design (PBD) and central composite design (CCD) were used for statistical optimization of media components. ResultsThe organism exhibited 18.85 ± 0.2 U/mL enzyme activities in unoptimized media. Eight variables: l-asparagine, peptone, glucose, lactose, yeast extract, NaCl, MgSO4, and Na2HPO4 were screened by PBD. Among them, only four factors—l-asparagine, peptone, glucose, and Na2HPO4—were found to affect enzyme production significantly (p < 0.05). Furthermore, the best possible concentrations and interactive effects of the components that enhance this enzyme’s output were chosen by using CCD on these selected variables. The results revealed that an optimized medium produces a higher concentration of enzymes than the unoptimized medium. After optimizing media components, the maximum l-asparaginase activity was 45.59 ± 0.36 U/mL, around the anticipated value of 45.04 ± 0.42 U/mL. After optimization of process parameters, it showed a 2.41-fold increase in the production of l-asparaginase by the endophyte Acinetobacter baumannii ZAS1. ConclusionThe findings of this study indicated that an endophyte, Acinetobacter baumannii ZAS1 that produces l-asparaginase could be used to increase enzyme output. However, using the statistical methods Plackett-Burman design and central composite design of response surface methodology is a handy tool for optimizing media components for increased l-asparaginase synthesis.

Isolation of asparaginase-producing microorganisms and evaluation of the enzymatic antitumor activity

Background L-asparaginase is an enzyme with very high biological activity owing to its activity on several tumor cells. It is mainly used to treat acute lymphoblastic leukemia. The complicated immunogenic adverse effects of present microbial sources present a need for switching to natural novel sources that have no immunogenic effect and better activity of L-asparaginase, so screening for other sources of L-asparaginase, like marine bacteria, may result in an enzyme having fewer adverse effects.Objective To screen and identify marine eco-friendly and potent L-asparaginase-producing bacteria, having a novel immunological property that possibly will avoid hypersensitivity.Materials and methods In the present study, bacterial strains were screened for extracellular L-asparaginase production from marine isolates, identified by 16 s rDNA technology, and L-asparaginase productivity was assessed using semiquantitative and quantitative enzymatic assays. The antiproliferative effect of the partially purified enzyme on different tumor human cell lines [HepG-2 (human hepatocellular carcinoma cell line), MCF-7 (breast cancer cell line), and PC-3 (prostate carcinoma cell line)] was assessed by the mitochondrial-dependent reduction of yellow MTT.Results and conclusion Bacillus safensis was established as the bacterial strain (Gene Bank accession number: MK541039). The extracellular enzyme-yielding capacity of the isolate B. safensis (518 IU/ml) was found to be 4.18 times higher than Bacillus pumilus (157.03 IU/ml) and higher than Bacillus circulans species (85 IU/ml). The marine isolate is environmentally friendly and can be used to produce significant quantities of extracellular L-asparaginase for the treatment of a variety of tumors and preparation of acrylamide-free fry food.

Study of L-Asparaginase from Marine Fungi and its Potential as an Anti-cancer Agent: A Preliminary Study using Chorioallantoic Membrane Assay

Introduction: L-asparaginase is a therapeutic enzyme that is used in combination therapy for mainly Acute Lymphocytic Leukemia (ALL) and other cancers. It hydrolyzes L-Asparagine required by neoplastic cells into L-aspartate and ammonia which leads to the death of tumor cells. But commercially available L-asparaginases show fatal side effects because of its prokaryotic source of origin, and associated glutaminase and urease co-activities. Aims: Screening of marine fungi for L- asparaginase activity and study of its potential as an anti – cancer agent. Methodology: This study was designed to isolate fungi from marine water and soil samples collected from regions of Maharashtra and Goa that are capable of producing L-asparaginase enzymes that are free of urease and glutaminase activity, and further study the potential of anticancer activity of these eucaryotic enzymes using Chorioallantoic membrane (CAM) assay. Result: Among all twelve marine fungi isolated, RF2 (identified as Aspergillusaculeatus (Genbank accession no.ON023823)) from Mandvi beach, Ratnagiri MH demonstrated high levels of L-asparaginase activity within 48-96 hrs, while lacking glutaminase and urease activity. Conclusion: The Chorioallantoic membrane (CAM) assay revealed that this fungal isolate could be a promising source of eucaryotic L-asparaginase to treat cancer with lower side effects.

EFFECT OF SUBSTRATE CONCENTRATION AND FeCl2 ADDITION ON L-ASPARAGINASE ACTIVITIES OF BACTERIA SYMBIONTS BROWN ALGAE Sargassum sp. AND ITS POTENTIAL AS A NEW ANTI-DENGUE AGENTS

The disintegration of the amide link, causing L-Asparaginase catalyzes the hydrolysis process of L-Asparagine into ammonia and aspartic acid. The purposes of this study were to determine the optimal conditions for enzyme production and enzymatic activity, including their potential as a new anti-dengue of L-asparaginase from Klebsiella sp. The Nessler method used a UV-Visible spectrophotometer to determine the L-asparaginase activity by measuring ammonia levels produced by L-asparaginase catalysis. The ideal fermentation period was at the 48th hour, and the optimal substrate concentration was 10 g/L with an activity value of 49.572 U/mL, and the optimal FeCl2 concentration was 0.5 g/L with an activity value of 60.1183 U/mL, and protein content of 0.1878 mg/mL. The optimum conditions for L-asparaginase, which has an activity value of 61.1182 U/mL, were observed at pH 8 and 37 °C. The optimum cofactor concentration of FeCl2 was 0.5 mM, which resulted in an activity value of 106.5046 U/mL. Both the inhibition percentage and CC50 value were 80% and 167.15 μg/mL respectively, showing that the L-asparaginase has a high antidengue potential.

Highly Active Thermophilic L-Asparaginase from Melioribacter roseus Represents a Novel Large Group of Type II Bacterial L-Asparaginases from Chlorobi-Ignavibacteriae-Bacteroidetes Clade.

L-asparaginase (L-ASNase) is a biotechnologically relevant enzyme for the pharmaceutical, biosensor and food industries. Efforts to discover new promising L-ASNases for different fields of biotechnology have turned this group of enzymes into a growing family with amazing diversity. Here, we report that thermophile Melioribacter roseus from Ignavibacteriae of the Bacteroidetes/Chlorobi group possesses two L-ASNases—bacterial type II (MrAII) and plant-type (MrAIII). The current study is focused on a novel L-ASNase MrAII that was expressed in Escherichia coli, purified and characterized. The enzyme is optimally active at 70 °C and pH 9.3, with a high L-asparaginase activity of 1530 U/mg and L-glutaminase activity ~19% of the activity compared with L-asparagine. The kinetic parameters KM and Vmax for the enzyme were 1.4 mM and 5573 µM/min, respectively. The change in MrAII activity was not significant in the presence of 10 mM Ni2+, Mg2+ or EDTA, but increased with the addition of Cu2+ and Ca2+ by 56% and 77%, respectively, and was completely inhibited by Zn2+, Fe3+ or urea solutions 2–8 M. MrAII displays differential cytotoxic activity: cancer cell lines K562, Jurkat, LnCap, and SCOV-3 were more sensitive to MrAII treatment, compared with normal cells. MrAII represents the first described enzyme of a large group of uncharacterized counterparts from the Chlorobi-Ignavibacteriae-Bacteroidetes clade.

Heterogeneity of bacterial enzymes as nutrient enrichment signature in tropical wetlands: A sustainable environmental management tool

High bacterial activities of urease, L-asparaginase, nitrite and nitrate reductase in some severely ecologically stressed ponds (P2, P4, P6 and P10) with DO (2.65–4.5 mg/L), COD (226.4–322.5 mg/L) and nitrogen enrichment (NH4N:0.109–0.817 mg/L; NO2N: 0.005–0.079 mg/L; NO3N: 0.306–1.511 mg/L) condition indicated nutrient induced enhanced bacterial metabolic pathways. Similarly, high acid phosphatase enzyme activity (0.2865–0.749 U/ml) reflected phosphorus enrichment (0.281–0.949 mg/L) stress in all ponds maximum for agricultural waste received ponds when compared with standards (0.084 mg/L). Various bacterial isolates of ammonifying bacteria (AB), heterotrophic bacteria (HB), phosphate solubilizing bacteria (PSB), nitrifying bacteria (NB), denitrifying bacteria (DNB) groups showed higher activities of urease (AB1P10, HB1-P4, P6), L-asparaginase (HB1P2, NB1P2), acid phosphatase (PSB1-P2, P4, P10), nitrate reductase (NB2P4, NB1P6 and AB1P2) and nitrite reductase (DNB1P2, DNB1P4, AB2P2 and NB1P7). The changed degraded microbial pathways expressed through heterogeneous response of bacterial enzymes has reflected the nutrient enrichment induced altered nutrient cycle in different selected ponds. Thus, this heterogeneity can be used as stress signature assessment of the restoring capacity of the disturbed ecosystem back to ecological resilience.The extent of deviation of water quality parameters (DO 54–126%; COD 1030–1135%; PO4-P 390–1029%) from the standards in relation with the heterogeneity of bacterial enzyme activities were used to categorize the different stress status i.e., severely stress, moderately stress and poorly stress of the studied wetlands as a tool to achieve sustainability.

Purification and anticancer activity of glutaminase and urease-free l-asparaginase from novel endophyte Chaetomium sp.

l-Asparaginase catalyzes the hydrolysis of asparagine into aspartic acid and ammonia. The present work elaborates the isolation and identification of a novel endophytic fungal isolate producing l-glutaminase and urease-free l-asparaginase. Cell growth and enzyme production were investigated for large production. The isolated endophytic fungi were identified at molecular levels and a phylogenetic tree was constructed. The enzyme synthesis was evaluated by cultivating the isolated microorganisms in potato dextrose agar medium. Out of 27 isolated endophytes, nine were producing "l-glutaminase and urease-free l-asparaginase." l-Asparaginase from Chaetomium sp. exhibited superior enzyme activity than from the other isolates. Observed optimal conditions for l-asparaginase activity were 25min of incubation time, 0.5mg of enzyme source, 40°C of temperature, and pH 7.0. l-Asparaginase from Chaetomium sp. exhibited anticancer activity on human blood cancer (MOLT-4) cells. The current study has demonstrated the production of contaminant-free l-asparaginase enzyme from endophytic fungal species. The results showed that: (a) maximum enzyme activity was observed for l-asparaginase from Chaetomium sp., (b) concentration of glucose in the medium as a carbon source suppressed the enzyme production. Chaetomium sp. is a novel source for "l-glutaminase and urease-free l-asparaginase," which may play a major role in pharmacotherapy.

Purification and anticancer activity of glutaminase and urease free intracellular l-asparaginase from Chaetomium sp.

l-asparaginase is a chemotherapeutic drug used in the treatment of acute lymphoblastic leukemia, a malignant disorder in children. l-asparaginase helps in removing acrylamide found in fried and baked foods which is carcinogenic in nature. The search for new therapeutic enzymes is of great interest in both medical and food applications. The present work aims to isolate the intracellular l-asparaginase from endophytic fungi Chaetomium sp. The intracellular enzyme was partially purified by chromatographic techniques. Molecular weight of enzyme was found to be ~66 kDa by SDS PAGE analysis. The enzyme is highly specific for l-asparagine and did not show glutaminase and urease activity. Maximum enzyme activity was found to be 58 ± 5 U/mL at 40 °C, pH 7.0 with 2 μg of protein. Intracellular l-asparaginase from Chaetomium sp. exhibited anticancer activity on human blood cancer (MOLT-4) cells.

Molecular modification and highly efficient expression of L-asparaginase from Rhizomucor miehei

L-asparaginase hydrolyzes L-asparagine to produce L-aspartic acid and ammonia. It is widely distributed in microorganisms, plants and serum of some rodents, and has important applications in the pharmaceutical and food industries. However, the poor thermal stability, low catalytic efficiency and low yield hampered the further application of L-asparaginase. In this paper, rational design and 5' untranslated region (5'UTR) design strategies were used to increase the specific enzyme activity and protein expression of L-asparaginase derived from Rhizomucor miehei (RmAsnase). The results showed that among the six mutants constructed through homology modeling combined with sequence alignment, the specific enzyme activity of the mutant A344E was 1.5 times higher than the wild type. Subsequently, a food-safe strain Bacillus subtilis 168/pMA5-A344E was constructed, and the UTR strategy was used for the construction of recombinant strain B. subtilis 168/pMA5 UTR-A344E. The enzyme activity of B. subtilis 168/pMA5 UTR-A344E was 7.2 times higher than that of B. subtilis 168/pMA5-A344E. The recombinant strain B. subtilis 168/pMA5 UTR-A344E was scaled up in 5 L fermenter, and the final yield of L-asparaginase was 489.1 U/mL, showing great potential for industrial application.

Isolation and Identification of the Potential Novel L-asparaginase producing Bacillus Strains from Soil

L-Asparaginase is a well know enzyme for its antineoplastic potential and is widely used to treat acute lymphoblastic leukemia and lymphosarcoma. The present work describes the isolation and characterization of novel L-asparaginase producing Bacillus strains from soil. Soil samples were collected from three different locations such as fruit garden, dairy farm and agricultural land in Peshawar Khyber Pakhtunkhwa, Pakistan. The isolates were screened to produce L-asparaginase in growth medium supplemented with 1% L-asparagine using a phenol red indicator. Among 30 bacterial isolates, only two strains initially coded as A5 and FG7 showed L-asparaginase activity. Based on biochemical and 16S rRNA sequencing analysis, the isolate A5 and FG7 were identified as Bacillus amyloliquefaciens and Bacillus proteolyticus respectively. Different factors like pH and time were optimized for maximum L-asparaginase activity. Bacillus amyloliquefaciens showed maximum asparaginase activity at pH 7 after 24 hours incubation at 30oC, while Bacillus proteolyticus showed optimum activity at pH 7 after 48 hours of incubation at 30oC. The present study first time reported the production of L-asparginase enzyme from Bacillus amyloliquefaciens and Bacillus proteolyticus. Keywords: L-asparaginase, Bacillusamyloliquefaciens, Bacillus proteolyticus, 16sRNA.

Filamentous Fungi Producing l-Asparaginase with Low Glutaminase Activity Isolated from Brazilian Savanna Soil.

l-asparaginase is an enzyme used as treatment for acute lymphoblastic leukemia (ALL) due to its ability to hydrolyze l-asparagine, an essential amino acid synthesized by normal cells unlike neoplastic cells. The adverse effects of l-asparaginase formulations are associated with its glutaminase activity and bacterial origin; therefore, it is important to find new sources of l-asparaginase-producing eukaryotic microorganisms with low glutaminase activity. This work evaluated the biotechnological potential of filamentous fungi isolated from Brazilian Savanna soil and plants for l-asparaginase production. Thirty-nine isolates were screened for enzyme production using the plate assay, followed by measuring enzymatic activity in cells after submerged fermentation. The variables influencing l-asparaginase production were evaluated using Plackett–Burman design. Cell disruption methods were evaluated for l-asparaginase release. Penicillium sizovae 2DSST1 and Fusarium proliferatum DCFS10 showed the highest l-asparaginase activity levels and the lowest glutaminase activity levels. Penicillium sizovae l-asparaginase was repressed by carbon sources, whereas higher carbon concentrations enhanced l-asparaginase by F. proliferatum. Maximum enzyme productivity, specific enzyme yield and the biomass conversion factor in the enzyme increased after Plackett–Burman design. Freeze-grinding released 5-fold more l-asparaginase from cells than sonication. This study shows two species, which have not yet been reported, as sources of l-asparaginase with possible reduced immunogenicity for ALL therapy.

Biosynthesis and characterization of a novel penicillium janthinellum Biourge L-asparaginase as a diverse biological activities agent

Background and objectives L-asparaginase (L-ASP) is a therapeutic enzyme used in the treatment of certain human cancers, especially acute lymphoblastic leukemia, as a chemotherapeutic agent. Other than as an anticancer agent, it has many applications, including in the treatment of autoimmune disorders, infectious diseases, and antibacterial activity. Microorganisms such as bacteria, fungi, and actinomycetes are very effective producers and a better source of L-ASP because they can be easily cultivated, and it is also easy to extract and purify L-ASP. The aim of this study is to formulate the production medium and to pinpoint the proper growth conditions for the chosen microorganism producing highly active L-ASP enzyme. The general properties of the crude L-ASP enzyme preparation were also determined to define the proper conditions for enzyme action. Under the specified conditions, the opportunity of the crude L-ASP enzyme for antimicrobial and antioxidant activities was determined. Materials and methods Eight recommended microbial isolates were screened for biologically active L-ASP enzyme productivity. Optimization of the cultural conditions for extracellular L-ASP production and also the important properties of the crude L-ASP were duly pinpointed. Finally, biological activities of the crude enzyme were explored. Results and conclusion Among all the screened organisms, the fungal strain Penicillium janthinellum Biourge was the most potent producer of an influential L-ASP enzyme. The maximum L-ASP activity of 17.85±0.579 U/reaction was obtained from medium containing glucose 0.2% (w/v) and L-asparagine 1% (w/v) at 30°C and pH 6.2. The important properties of the crude P. janthinellum Biourge L-ASP were duly pinpointed as follows: optimum enzyme and substrate concentrations were 1 mg/ml and 1% (w/v), respectively, and optimum reaction pH and temperature were 10.7 and 45°C, respectively. Under the specified conditions, at varying concentrations, the enzyme preparation exhibited considerable 2, 2-diphenyl-1-picrylhydrazyl radical scavenging activity accompanied with nonantimicrobial activity, and this pointed out the necessity of partial purification of the crude fungal enzyme for further studies.

Bacteria producing L-asparaginase isolated from Peruvian saline environments

L-asparaginase (EC 3.5.1.1) hydrolyzes L-asparagine in L-aspartic acid and ammonia. Its efficiency is subject to its kinetics and specificity on the substrate, characteristics that vary from one source to another. Thus, microorganisms from saline environments constitute a phylogenetic and metabolically heterogeneous group for the search of new enzymes. Therefore, the objective of this study was the phenotypic and genotypic characterization of bacteria with L-asparaginase activity isolated from Maras, Pilluana and Chilca salterns. The 24 evaluated bacteria were classified as 38% Gram-negative bacilli and 54% positive; and 8% Gram-positive cocci. The majority grew in 5% salt water, pH 7.0 and 37 °C, all assimilated glucose. Of the 24 bacteria that produced L-asparaginase in solid medium, enzymatic activity was determined in submerged cultures by the Nessler method in 14 of them. The CH11, M62, M64, M68, and P19 strains identified as Bacillus sp., by partial sequencing of the 16S ribosomal gene, presented higher L-asparaginase activity and instability due to the presence of proteases. Saline environments bacteria are potential sources for the prospective production of L-asparaginase to use them as a therapeutic agent and in the food industry.

Imatinib Induces Apoptosis of NK/T Cell Neoplasms by Inhibiting Hematopoietic Cell Kinase and NQO2

▪[Background] Natural killer and T (NK/T)-cell malignancies are uncommon in Western countries but relatively frequent in Asia. Although regimens that include L-asparaginase have improved outcomes, these disorders, particularly aggressive NK-cell leukemias/lymphomas, have poor prognoses. No molecular-targeting drugs have been developed so far. Current reports have provided evidence that a subset of NK cell malignancies is of myeloid origin. Some tyrosine kinase inhibitors (TKIs) that target multiple kinases, including LcK involved in the growth of myeloid malignancies, may therefore show inhibitory effects against NK/T cell neoplasms where LcK members are highly expressed. We investigated the potential anti-tumor activities of multitarget TKIs against malignant NK/T cell neoplasms. [Methods and Results] Three TKIs (imatinib, nilotinib, and dasatinib) were tested for their apoptosis-inducing activities against various NK cell lines including KHYG1, NK92, and NKL after 24-h incubation. Therapeutic doses of imatinib (7.5-27 µM) induced variable annexinV(+) cells in 5 out of 7 NK lines, ranging from 27.1%±6.9% to 87.8%±3.3% (Figure 1), while the apoptosis-inducing effects of therapeutic doses of nilobinib and dasatinib were marginal. Membrane array screenings, which can quantitate 43 phosphorylated proteins, showed that hematopoietic cell kinase (HcK) was constitutively active in KHYG1 cells, and treatment with imatinib inhibited pHcK by 80%. Other known imatinib-targets, including PDGFR, LcK, c-kit, and BCR/ABL, were undetectable in the NK cell lines tested. Further immunoblot analyses using anti-pHcK antibodies confirmed pHcK inhibition in imatinib-treated KHYG1 and NKL (Figure 2). Immunohistochemistry studies with anti-pHcK antibodies revealed two of eight extranodal NK/T-cell lymphomas, nasal type, to be strongly positive for pHcK (Figure 3). In addition, immunohistochemistry studies revealed that NAD(P)H dehydrogenase, quinone 2 (NQO2), a lesser-known imatinib target, was highly expressed in tumor tissues of all seven NK/T-cell lymphoma patients examined, including two pHcK(+) patients, and in vitro studies showed 50% NQO2 protein downregulation and 80% inhibition of its enzymatic activity in imatinib-treated cells. Notably, imatinib enhanced the pro-apoptotic and anti-proliferative activities of L-asparaginase against malignant NK and T cells (CI 0.77 and 0.86 respectively). [Conclusions] Imatinib induces the apoptosis of a subset of NK/T cell malignancies by inhibiting pHcK and NQO2. Imatinib and HcK inhibitors may have therapeutic potential against NK/T cell malignancies either alone or in combination with L-asparaginase. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Isolation and Characterization of Probiotic Bacillus subtilis MKHJ 1-1 Possessing L-Asparaginase Activity

The purpose of this study was to isolate functional Bacillus strains from Korean fermented soybeans and to evaluate their potential as probiotics. The L-asparaginase activity of MKHJ 1-1 was the highest among 162 Bacillus strains. This strain showed nonhemolysis and did not produce β-glucuronidase. Among the nine target bacteria, MKHJ 1-1 inhibited the growth of Escherichia coli, Pseudomonas aeruginosa, Shigella sonnei, Shigella flexneri, Klebsiella pneumoniae, Staphylococcus aureus, and Bacillus cereus. 16S rRNA gene sequence analysis resulted in MKHJ 1-1 identified as Bacillus subtilis subsp. stercoris D7XPN1. As a result of measuring the survival rate in 0.1% pepsin solution (pH 2.5) and 0.3% bile salt solution for 3 h, MKHJ 1-1 exhibited high acid resistance and was able to grow in the presence of bile salt. MKHJ 1-1 showed outstanding autoaggregation ability after 24 h. In addition, its coaggregation with pathogens was strong. Therefore, MKHJ 1-1 is a potential probiotic with L-asparaginase activity and without L-glutaminase activity, suggesting that it could be a new resource for use in the food and pharmaceutical industry.

Characterization of Penicillium crustosum L-asparaginase and its acrylamide alleviation efficiency in roasted coffee beans at non-cytotoxic levels.

This work aims at isolating a fungal source for L-asparaginase production to be applied in reducing acrylamide levels in coffee beans at non-cytotoxic levels. An L-asparaginase-producing fungus was isolated from an agricultural soil sample and identified as Penicillium crustosum NMKA 511. A maximum L-asparaginase activity of 19.10 U/mL was obtained by the above-mentioned fungus when grown under optimum conditions (i.e. 16.96g/L sucrose as carbon source, 1.92g/L peptone as nitrogen source, pH 7.7 and 33.5°C). Further, the produced L-asparaginase was purified and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that P. crustosum L-asparaginase was a heterodimer enzyme with molecular weights of approximately 41.3 and 44.4kDa. Also, the purified P. crustosum L-asparaginase was specific towards L-asparagine and showed negligible and no effects towards L-glutamine and D-asparagine, respectively. Additionally, the purified L-asparaginase reduced the acrylamide levels by 80.7% and 75.8% in light and dark roasted coffee beans, respectively. The amount of L-asparaginase used to reduce acrylamide was considered safe when cell viability reached 94.6%.

Bioprocess Optimization of Nutritional Parameters for Enhanced Anti-leukemic L-Asparaginase Production by Aspergillus candidus UCCM 00117: A Sequential Statistical Approach.

Sequential optimization of bioprocess nutritional conditions for production of glutaminase-near-free L-asparaginase by Aspergillus candidus UCCM 00117 was conducted under shake flask laboratory conditions. Catalytic and anti-cancer activities of the poly-peptide were evaluated using standard in vitro biochemical methods. Medium nutrients were selected by one-factor-at-a-time (OFAT) approach while Plackett–Burman design (PBD) screened potential factors for optimization. Path of steepest ascent (PSA) and response surface methodology (RSM) of a Min-Run-Res V fractional factorial of a central composite rotatable design (CCRD) were employed to optimize factor levels towards improved enzyme activity. A multi-objective approach using desirability function generated through predictor importance and weighted coefficient methodology was adopted for optimization. The approach set optimum bioprocess conditions as 49.55 g/L molasses, 64.98% corn steep liquor, 44.23 g/L asparagine, 1.73 g/L potassium, 0.055 g/L manganese and 0.043 g/L chromium (III) ions, at a composite desirability of 0.943 and an L-asparaginase activity of 5216.95U. The Sephadex-200 partially-purified polypeptide had a specific activity of 476.84 U/mg; 0.087U glutaminase activity, 36.46% yield and 20-fold protein purification. Anti-cancer activity potentials of the catalytic poly-peptide were dose-dependent with IC50 (µg/mL): 4.063 (HL-60), 13.75 (HCT-116), 15.83 (HeLa), 11.68 (MCF-7), 7.61 (HepG-2). The therapeutic enzyme exhibited 15-fold more cytotoxicity to myeloid leukemia cell line than to normal (HEK 238 T) cell. Optimum temperature and pH for activity were within physiological range. However, significant interactions between exposure time and levels of each of temperature and pH made interpretations of residual enzyme activities difficult. The manganese-dependent L-asparaginase from Aspergillu s candidus UCCM 00117 is recommended for further anticancer drug investigations.

Production, purification, characterization, antioxidant and antiproliferative activities of extracellular L-asparaginase produced by Fusarium equiseti AHMF4.

L-Asparaginase is an antileukemic agent that depletes L-asparagine “an important nutrient for cancer cells” through the hydrolysis of L-asparagine into L-aspartic acid and ammonia leading to leukemia cell starvation and apoptosis in susceptible leukemic cell populations. Moreover currently, bacterial L-asparaginase has been limited by problems of lower productivity, stability, selectivity and a number of toxicities along with the resistance towards bacterial L-asparaginase. Then the current work aimed to provide pure L-asparaginase with in-vitro efficacy against various human carcinomas without adverse effects related to current L-asparaginase formulations. Submerged fermentation (SMF) bioprocess was applied and improved to maximize L-asparaginase production from Fusarium equiseti AHMF4 as alternative sources of bacteria. The enzyme production in SMF was maximized to reach 40.78 U mL−1 at the 7th day of fermentation with initial pH 7.0, incubation temperature 30 °C, 1.0% glucose as carbon source, 0.2% asparagine as nitrogen source, 0.1% alanine as amino acid supplement and 0.1% KH2PO4. The purification of AHMF4 L-asparaginase yielded 2.67-fold purification and 48% recovery with final specific activity of 488.1 U mg−1 of protein. Purified L-asparaginase was characterized as serine protease enzyme with molecular weight of 45.7 kDa beside stability at neutral pH and between 20 and 40 °C. Interestingly, purified L-asparaginase showed promising DPPH radical scavenging activity (IC50 69.12 μg mL−1) and anti-proliferative activity against cervical epitheloid carcinoma (Hela), epidermoid larynx carcinoma (Hep-2), hepatocellular carcinoma (HepG-2), Colorectal carcinoma (HCT-116), and breast adenocarcinoma (MCF-7) with IC50 equal to 2.0, 5.0, 12.40, 8.26 and 22.8 μg mL−1, respectively. The enzyme showed higher activity, selectivity and anti-proliferative activity against cancerous cells along with tiny cytotoxicity toward normal cells (WI-38) which indicates that it has selective toxicity and it could be applied as a less toxic alternative to the current formulations.

Isolation and identification of marine Bacillus altitudinis KB1 from coastal Kerala: asparaginase producer.

L-asparaginase is a target for many researchers as its properties against cancer, especially leukaemia, and protective agents reduce acrylamide in fried food. In this study, the water samples from Thumba Arattuvazhi Beach in Kerala were screened for l-asparaginase producing microorganisms. This was followed by colourimetric screening using modified M9 media with 0.009% Phenol red dye and using l-asparagine as a sole nitrogen source. Then, the Nessler assay was performed to quantify the enzyme. Molecular identification was made by 16SrRNA sequencing and aligned the sequence with GeneBank for phylogenetic tree construction using BLAST. Seawater was serially diluted for 10-1 to 10-6 using nutrient agar plates. A total of 19 bacterial colonies were isolated. The colonies were evaluated to produce l-asparaginase according to the pink zone around the colonies on the modified M9 medium using a red phenol indicator. The KB1 sample was selected for further studies according to plate colour assay. Nessler assay of L-asparaginase quantified as 2.537 IU/ml. Molecular characterisation showed the sequence association with Bacillus altitudinis the sequence submitted in Genebank as B. altitudinis KB1 strain. The l-asparaginase II gene (AnsB) was amplified based on the entire length of the hypothetical protein of annotated genome with accession number CP022319.2. The l-asparaginase activity in this study was 57% higher than the reference organism B. altitudinis BITHSP010. The l-asparaginase producing bacterium B. altitudinis KB1 from a marine source in Kerala can produce asparaginase, which can be utilised for biotechnology applications.