A novel type IIb L-asparaginase from Latilactobacillus sakei LK-145: characterization and application.

We succeeded in homogeneously expressing and purifying L-asparaginase from Latilactobacillus sakei LK-145 (Ls-Asn1) and its mutated enzymes C196S, C264S, C290S, C196S/C264S, C196S/C290S, C264S/C290S, and C196S/C264S/C290S-Ls-Asn1. Enzymological studies using purified enzymes revealed that all cysteine residues of Ls-Asn1 were found to affect the catalytic activity of Ls-Asn1 to varying degrees. The mutation of Cys196 did not affect the specific activity, but the mutation of Cys264, even a single mutation, significantly decreased the specific activity. Furthermore, C264S/C290S- and C196S/C264S/C290S-Ls-Asn1 almost completely lost their activity, suggesting that C290 cooperates with C264 to influence the catalytic activity of Ls-Asn1. The detailed enzymatic properties of three single-mutated enzymes (C196S, C264S, and C290S-Ls-Asn1) were investigated for comparison with Ls-Asn1. We found that only C196S-Ls-Asn1 has almost the same enzymatic properties as that of Ls-Asn1 except for its increased stability for thermal, pH, and the metals NaCl, KCl, CaCl2, and FeCl2. We measured the growth inhibitory effect of Ls-Asn1 and C196S-Ls-Asn1 on Jurkat cells, a human T-cell acute lymphoblastic leukemia cell line, using L-asparaginase from Escherichia coli K-12 as a reference. Only C196S-Ls-Asn1 effectively and selectively inhibited the growth of Jurkat T-cell leukemia, which suggested that it exhibited antileukemic activity. Furthermore, based on alignment, phylogenetic tree analysis, and structural modeling, we also proposed that Ls-Asn1 is a so-called "Type IIb" novel type of asparaginase that is distinct from previously reported type I or type II asparaginases. Based on the above results, Ls-Asn1 is expected to be useful as a new leukemia therapeutic agent.

Molecular drilling to combat salmonella typhi biofilm using L-Asparaginase via multiple targeting process.

OBJECTIVES: Salmonella Typhibiofilm condition is showing as a major public health problem due to the development of antibiotic resistance and less available druggable target proteins. Therefore, we aimed to identify some more druggable targets of S. Typhibiofilm using computational drilling at the genome/proteome level so that the target shortage problem could be overcome and more antibiofilm agents could be designed in the future against the disease. METHODS: We performed protein-protein docking and interaction analysis between the homological identified target proteins of S.Typhi biofilm and a therapeutic protein L-Asparaginase. RESULTS: We have identified some druggable targets CsgD, BcsA, OmpR, CsgG, CsgE, and CsgF in S.Typhi. These targets showed high-binding affinity BcsA (-219.8 Kcal/mol) >csgF (-146.52 Kcal/mol) >ompR (-135.68 Kcal/mol) >CsgE (-134.66 Kcal/mol) >CsgG (-113.81 Kcal/mol) >CsgD(-95.39 Kcal/mol) with therapeutic enzyme L-Asparaginase through various hydrogen-bonds and salt-bridge. We found six proteins of S. Typhi biofilm from the Csg family as druggable multiple targets. CONCLUSION: This study provides insight into the idea of identification of new druggable targets and their multiple targeting with L-Asparaginase to overcome target shortage in S. Typhibiofilm-mediated infections. Results further indicated that L-Asparaginase could potentially be utilized as an antibiofilm biotherapeutic agent against S.Typhi.

L-asparaginase from Aspergillus oryzae spp.: effects of production process and biochemical parameters.

L-asparaginases prevent the formation of acrylamide, a substance commonly found in foods subjected to heat and that also contains reducing sugars and L-asparagine. This work aimed to select a strain of Aspergillus spp. able to produce L-asparaginase and to optimize the fermentation parameters, the partial purification and biochemical characterization were also performed. The Aspergillus oryzae IOC 3999 was selected due to its greater enzymatic activity: 1443.57 U/mL of L-asparaginase after 48 h of fermentation. The optimized conditions allowed for an increase of 223% on the L-asparaginase production: 2.9% lactose, 2.9% L-asparagine and 0.7% hydrolyzed casein, 0.152% KH2PO4, 0.052% KCl and MgSO4, 0.001% of CuNO3.3H2O, ZnSO4.7H2O and FeSO4.7H2O adjusted to pH 7.0; added a concentration of 5.05x106 spores/mL at 30 °C for 100 rpm. A purification factor of 2.11 was found and the molecular mass was estimated at 20.8 kDa. The enzyme showed optimum activity at 60 °C and pH 5 and stability at 50 °C for 1 h. The enzyme presented desirable biochemical characteristics, mainly the acid pH stability, indicating that the enzyme would work well in food matrices due to the closeness of pH, meaning that it could be a potential option for use in the food industry.

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.

Two-Step Purification of L-Asparaginase from Acrylaway® L

Abstract L-Asparaginase (L-ASNase) is a biopharmaceutical used for acute lymphoblastic leukaemia (ALL) treatment, dramatically increasing the patients' chance of cure. However, its production and distribution in developing countries were disrupted because of its low profitability, which caused great concern among patients. This study evaluates the feasibility of combining fractional precipitation and aqueous two-phase systems (ATPS) to purify L-ASNase from a low-grade product, commercially known as Acrylaway® L. The ATPS purification results were not particularly expressive compared to the two-step purification process composed of ethanol precipitation and gel filtration, which was able to recover the target molecule with a purification factor over 5 fold. Thus, we studied a purification process capable of manufacturing pharmaceutical grade L-ASNase from a commercially available low-grade raw material; however, improvements regarding its throughput must be achieved, and high purity is the first step to apply it as a new biopharmaceutical product. The proposed process could pose as a short-time solution to mitigate its shortage while a cost-effective production plant is being developed.

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.

Optimization of L-asparaginase production from endophytic Fusarium proliferatum using OFAT and RSM and its cytotoxic evaluation.

L-asparaginase from endophytic Fusarium proliferatum (isolate CCH, GenBank accession no. MK685139) isolated from the medicinal plant Cymbopogon citratus (Lemon grass), was optimized for its L-asparaginase production and its subsequent cytotoxicity towards Jurkat E6 cell line. The following factors were optimized; carbon source and concentration, nitrogen source and concentration, incubation period, temperature, pH and agitation rate. Optimization of L-asparaginase production was performed using One-Factor-At-A-Time (OFAT) and Response surface methodology (RSM) model. The cytotoxicity of the crude enzyme from isolate CCH was tested on leukemic Jurkat E6 cell line. The optimization exercise revealed that glucose concentration, nitrogen source, L-asparagine concentration and temperature influenced the L-asparaginase production of CCH. The optimum condition suggested using OFAT and RSM results were consistent. As such, the recommended conditions were 0.20% of glucose, 0.99% of L-asparagine and 5.34 days incubation at 30.50 °C. The L-asparaginase production of CCH increased from 16.75 ± 0.76 IU/mL to 22.42 ± 0.20 IU/mL after optimization. The cytotoxicity of the crude enzyme on leukemic Jurkat cell line recorded IC50 value at 33.89 ± 2.63% v/v. To conclude, the enzyme extract produced from Fusarium proliferatum under optimized conditions is a potential alternative resource for L-asparaginase.

Revealing Escherichia coli type II L-asparaginase active site flexible loop in its open, ligand-free conformation.

Since 1993, when the structure of Escherichia coli type II L-asparaginase (EcAII) in complex with L-aspartate was firstly reported, many structures of the wild type and mutated enzyme have been deposited in the Protein Data Bank. None of them report the full structure of the monomer in its ligand-free, open conformation, mainly because of the high dynamic and flexibility of the active site flexible loop. Here we report for the first time the structure of EcAII wild type in its open conformation comprising, for at least one protomer, clear electron density for the active site flexible loop (PDB ID: 6YZI). The structural element is highly mobile and it is transposed onto the rigid part of the active site upon substrate binding to allow completion of the enzyme catalytic center, thanks to key residues that serve as hinges and anchoring points. In the substrate binding pocket, several highly conserved water molecules are coordinated by residues involved in substrate binding, comprising two water molecules very likely involved in the enzyme catalytic process. We also describe, by molecular dynamics simulations, how the transposition of the loop, besides providing the proximity of residues needed for catalysis, causes a general stabilization of the protein.

A Novel L-Asparaginase from Hyperthermophilic Archaeon Thermococcus sibiricus: Heterologous Expression and Characterization for Biotechnology Application.

L-asparaginase (L-ASNase) is a vital enzyme with a broad range of applications in medicine and food industry. Drawbacks of current commercial L-ASNases stimulate the search for better-producing sources of the enzyme, and extremophiles are especially attractive in this view. In this study, a novel L-asparaginase originating from the hyperthermophilic archaeon Thermococcus sibiricus (TsA) was expressed in Escherichia coli, purified and characterized. The enzyme is optimally active at 90 °C and pH 9.0 with a specific activity of 2164 U/mg towards L-asparagine. Kinetic parameters KM and Vmax for the enzyme are 2.8 mM and 1200 µM/min, respectively. TsA is stable in urea solutions 0-6 M and displays no significant changes of the activity in the presence of metal ions Ni2+, Cu2+, Mg2+, Zn2+ and Ca2+ and EDTA added in concentrations 1 and 10 mmol/L except for Fe3+. The enzyme retains 86% of its initial activity after 20 min incubation at 90 °C, which should be enough to reduce acrylamide formation in foods processed at elevated temperatures. TsA displays strong cytotoxic activity toward cancer cell lines K562, A549 and Sk-Br-3, while normal human fibroblasts WI-38 are almost unsensitive to it. The enzyme seems to be a promising candidate for further investigation and biotechnology application.

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. 16.96 g/L sucrose as carbon source, 1.92 g/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 44.4 kDa. 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%.

Bioprospecting of the agaricomycete Ganoderma australe GPC191 as novel source for L-asparaginase production.

L-Asparaginase is a therapeutically and industrially-competent enzyme, acting predominantly as an anti-neoplastic and anti-cancerous agent. The existing formulations of prokaryotic L-asparaginase are often toxic and contain L-glutaminase and urease residues, thereby increasing the purification steps. Production of L-glutaminase and urease free L-asparaginase is thus desired. In this research, bioprospecting of isolates from the less explored class Agaricomycetes was undertaken for L-asparaginase production. Plate assay (using phenol red and bromothymol blue dyes) was performed followed by estimation of L-asparaginase, L-glutaminase and urease activities by Nesslerization reaction for all the isolates. The isolate displaying the desired enzyme production was subjected to morphological, molecular identification, and phylogenetic analysis with statistical validation using Jukes-Cantor by Neighbour-joining tree of Maximum Likelihood statistical method. Among the isolates, Ganoderma australe GPC191 with significantly high zone index value (5.581 ± 0.045 at 120 h) and enzyme activity (1.57 ± 0.006 U/mL), devoid of L-glutaminase and urease activity was selected. The present study for the first-time reported G. australe as the potential source of L-glutaminase and urease-free L-asparaginase and also is one of the few studies contributing to the literature of G. australe in India. Hence, it can be postulated that it may find its future application in pharmaceutical and food industries.

Pseudomonas aeruginosa recombinant L-asparaginase: Large scale production, purification, and cytotoxicity on THP-1, MDA-MB-231, A549, Caco2 and HCT-116 cell lines.

In previous studies Pseudomonas aeruginosal-ASNase complete coding sequence gene, 984 bp (GenBank accession number KU161101.2) was isolated by PCR, cloned into pET28a(+) vector, expressed in E. coli DE3(BL21) pLysS, purified to apparent homogeneity and biochemically characterized. In the present work we highlight large scale production, affinity purification of the recombinant enzyme, effect of osmolytes on the stability of the l-ASNase and cytotoxicity on different cancer cell lines. Successful overexpression was achieved in E. coli as a 6-His-Tag fusion protein after 18 h of induction with lactose at a concentration of 2 g/L in fermentation medium and at 37 °C. The recombinant enzyme was purified to homogeneity using Ni2+ chelated Fast Flow Sepharose resin with 19758.8 specific activity and 10.28 purification fold. With respect to the effect of osmolytes on the stability of the purified enzyme, the majority of the tested osmolytes namely 5% maltose, 5% mannitol, 30% glycerol and 5% BSA were found to increase the stability of the recombinant l-ASNase as compared to the free enzyme. Triple negative breast cancer cell line, MDA-MB-231 treated with recombinant l-ASNase showed significant morphological changes and the IC50 of the purified enzyme was found to be 3.1 IU. Human leukemia cell line, THP-1 treated with l-ASNase showed apoptotic bodies and morphological changes with IC50 of the purified enzyme 1.75 IU. Moreover, the purified recombinant l-ASNase was found to induced cytotoxic effects on colorectal adenocarcinoma cell line, Caco-2 with IC50 of 68.28 IU. Results of apoptosis assay on THP-1 cells revealed that the purified l-ASNase induced early and late apoptosis at 14.16% and 7.56 respectively as compared to the control untreated cells.

Optimization, Purification and Antitumor Activity of Kodamaea ohmeri ANOMY L-Asparaginase Isolated from Banana Peel.

OBJECTIVE: L-Asparaginase is an important enzyme that converts L-asparagine to L-aspartate and ammonia. Microbial L-asparaginase has important applications as anticancer and food processing agents. METHODS: This study reported the isolation, screening of a local yeast isolate from banana peel for L-asparaginase production using submerged fermentation, optimization of the production, purification, and anticancer assay of L-asparaginase. The yeast isolate was identified as Kodamaea ohmeri ANOMY based on the analysis of nuclear large subunit (26S) rDNA partial sequences. It was a promising L-asparaginase producer with a specific activity of 3059±193 U/mg in a non-optimized medium. The classical one-variable-at-a-time method was used to optimize the production medium components, and it was found that the elimination of K2HPO4 from the medium increased L-asparaginase specific activity (3100.90±180 U/mg). RESULTS: Statistical optimization of L-asparaginase production was done using Plackett-Burman and Box-Behnken designs. The production medium for the maximum L-asparaginase specific activity (8500±578U/mg) was as follows (g/L): L-asparagine (7.50), NaNO3 (0.50), MgSO4.7H2O (0.80), KCl (0.80) associated with an incubation period of 5 days, inoculum size of 5.60 %, and pH (7.0). The optimization process increased L-asparaginase production by 2.78-fold compared to the non-optimized medium. L-Asparaginase was purified using ammonium sulphate precipitation followed by gel filtration on a Sephadex G-100 column. Its molecular weight was 66 KDa by SDS-PAGE analysis. CONCLUSION: The cell morphology technique was used to evaluate the anticancer activity of L-asparaginase against three different cell lines. L-Asparaginase inhibited the growth of HepG-2, MCF-7, and HCT-116 cells at a concentration of 20, 50, and 60 µL, respectively.

Recent Strategies and Applications for l-Asparaginase Confinement.

l-asparaginase (ASNase, EC 3.5.1.1) is an aminohydrolase enzyme with important uses in the therapeutic/pharmaceutical and food industries. Its main applications are as an anticancer drug, mostly for acute lymphoblastic leukaemia (ALL) treatment, and in acrylamide reduction when starch-rich foods are cooked at temperatures above 100 °C. Its use as a biosensor for asparagine in both industries has also been reported. However, there are certain challenges associated with ASNase applications. Depending on the ASNase source, the major challenges of its pharmaceutical application are the hypersensitivity reactions that it causes in ALL patients and its short half-life and fast plasma clearance in the blood system by native proteases. In addition, ASNase is generally unstable and it is a thermolabile enzyme, which also hinders its application in the food sector. These drawbacks have been overcome by the ASNase confinement in different (nano)materials through distinct techniques, such as physical adsorption, covalent attachment and entrapment. Overall, this review describes the most recent strategies reported for ASNase confinement in numerous (nano)materials, highlighting its improved properties, especially specificity, half-life enhancement and thermal and operational stability improvement, allowing its reuse, increased proteolysis resistance and immunogenicity elimination. The most recent applications of confined ASNase in nanomaterials are reviewed for the first time, simultaneously providing prospects in the described fields of application.

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

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 24 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 ß-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 L-asparaginase 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%.

Bioprocess development for L-asparaginase production by Streptomyces rochei, purification and in-vitro efficacy against various human carcinoma cell lines.

In the near future, the demand for L-asparaginase is expected to rise several times due to an increase in its clinical and industrial applications in various industrial sectors, such as food processing. Streptomyces sp. strain NEAE-K is potent L-asparaginase producer, isolated and identified as new subsp. Streptomyces rochei subsp. chromatogenes NEAE-K and the sequence data has been deposited under accession number KJ200343 at the GenBank database. Sixteen different independent factors were examined for their effects on L-asparaginase production by Streptomyces rochei subsp. chromatogenes NEAE-K under solid state fermentation conditions using Plackett-Burman design. pH, dextrose and yeast extract were the most significant factors affecting L-asparaginase production. Thus, using central composite design, the optimum levels of these variables were determined. L-asparaginase purification was carried out by ammonium sulfate followed by DEAE-Sepharose CL-6B ion exchange column with a final purification fold of 16.18. The monomeric molecular weight of the purified L-asparaginase was 64 kD as determined by SDS-PAGE method. The in vitro effects of L-asparaginase were evaluated on five human tumor cell lines and found to have a strong anti-proliferative effects. The results showed that the strongest cytotoxic effect of L-asparaginase was exerted on the HeLa and HepG-2 cell lines (IC50 = 2.16 ± 0.2 and 2.54 ± 0.3 U/mL; respectively). In addition, the selectivity index of L-asparaginase against HeLa and HepG-2 cell lines was 3.94 and 3.35; respectively.

Highly efficient Pyrococcus furiosus recombinant L-asparaginase with no glutaminase activity: Expression, purification, functional characterization, and cytotoxicity on THP-1, A549 and Caco-2 cell lines.

L-Asparaginase (L-ASNase EC 3.5.1.1) is considered as an important biopharmaceutical drug enzyme in the treatment of childhood acute lymphoblastic leukemia (ALL). In the present study, Pyrococcus furiosus L-ASNase gene was cloned into pET26b (+), expressed in E. coli BL21(DE3) pLysS, and purified to homogeneity using Ni2+ chelated Fast Flow Sepharose resin with 5.7 purification fold and 23.9% recovery. The purified enzyme exhibited a molecular weight of ~33,660 Da on SDS-PAGE and showed maximal activity at 50 °C and pH 8.0. It retained 98.3% and 60.7% initial activity after 60 min at 37 °C and 50 °C, respectively. The recombinant enzyme showed highest substrate specificity towards L-ASNase substrate, while no detectable specificity was observed for l-glutamine, urea, and acrylamide at 10 mM concentration. The Km and Vmax of the purified recombinant enzyme as calculated using Lineweaver-Burk plot were determined to be 1.623 mM and 105 µmol min-1 mg-1, respectively. Human leukemia cell line THP-1 treated with recombinant L-ASNase showed significant morphological changes, and the IC50 of the purified enzyme was found to be 0.8 IU. Moreover, the purified recombinant L-ASNase induced cytotoxic effects on lung adenocarcinoma A549 and colorectal adenocarcinoma Caco-2 cell lines with IC50 of 1.78 IU and 30 IU, respectively.

l-asparaginase production and enhancement by Sarocladium strictum: In vitro evaluation of anti-cancerous properties.

AIMS: Utilization of l-asparaginase has been one of the effective strategies for the treatment of lymphoblastic leukaemia. Since the currently used bacterial l-asparaginase causes side effects, searching for new enzyme sources has been an active field of research. This study focuses on the characterization of an l-asparaginase-producing fungal strain. METHODS AND RESULTS: Sarocladium strictum was identified as a potent enzyme-producing strain. For the enhancement of enzyme production, we used two-level factorial design and response surface methodology. The optimization of significant factors showed a 1·84-fold increase in enzyme production. The Km and Vmax values of the enzyme were 9·74 mmol l-1 and 8·19 µmol min-1 . The toxicity of the produced l-asparaginase was measured on K562 and HL60 cancer cell lines and L6 as normal cells. The IC50 values were calculated as 0·4 and 0·5 IU ml-1 for K562 and HL60 respectively and no significant effect was observed in L6. BrdU proliferation and caspase-3 activity assay in l-asparaginase treated HL60 and K562 cells indicated that cell proliferation rates and apoptotic cell death were reduced. CONCLUSIONS: The cytotoxic properties of the produced fungal enzyme indicated significant growth inhibition in cancer cells while having a little toxic effect on normal cells. The possibility of mass production alongside having suitable cytotoxic and kinetic properties suggest the probable use of the produced l-asparaginase for further researches as a potential chemotherapeutic agent. SIGNIFICANCE AND IMPACT OF THE STUDY: The lack of significant l-glutaminase activity and promising toxicity properties in S. strictum and the closer evolutionary relativeness of fungi enzymes to human enzymes compared to bacterial enzymes suggest a new source with lower toxicity and anti-cancerous properties, causing less side effect problems.

Microbial L-asparaginase: purification, characterization and applications.

L-asparaginase (E.C.3.5.1.1) is an important enzyme that has been purified and characterized for over decades to study and evaluate its anti-carcinogenic activity against different lymphoproliferative disorders such as acute lymphoblastic leukemia (ALL) and Hodgkin's lymphoma. The ability of the enzyme to convert L-asparagine into aspartic acid and ammonia is the reason behind its anti-cancerous activity. Apart from its medicinal uses, it is widely used in food industry to tackle acrylamide, a probable human carcinogen and, production in carbohydrate-rich foods cooked at high temperatures. There are variety of organisms including microorganisms such as bacteria, fungi, algae, and plants that produce L-asparaginase. The enzyme obtained from different microbial and plant sources have different physiochemical properties and kinetic parameters. L-asparaginases have an optimum pH range between 6 and 10 and an optimum temperature between 37 and 85 °C. This article has reviewed the lowest molecular mass for L-asparaginase in Yersinia pseudotuberculosis Q66CJ2 which is 36.27 kDa, while the highest for Pseudomonas otitidis which has a molecular mass of 205 ± 3 kDa. This review is an attempt to summarize most of the available sources, their phylogenetic relationships, purification methods, data regarding different physiochemical and kinetic properties of L-asparaginase.

A comprehensive review on microbial l-asparaginase: Bioprocessing, characterization, and industrial applications.

l-Asparaginase (E.C.3.5.1.1.) is a vital enzyme that hydrolyzes l-asparagine to l-aspartic acid and ammonia. This property of l-asparaginase inhibits the protein synthesis in cancer cells, making l-asparaginase a mainstay of pediatric chemotherapy practices to treat acute lymphoblastic leukemia (ALL) patients. l-Asparaginase is also recognized as one of the important food processing agent. The removal of asparagine by l-asparaginase leads to the reduction of acrylamide formation in fried food items. l-Asparaginase is produced by various organisms including animals, plants, and microorganisms, however, only microorganisms that produce a substantial amount of this enzyme are of commercial significance. The commercial l-asparaginase for healthcare applications is chiefly derived from Escherichia coli and Erwinia chrysanthemi. A high rate of hypersensitivity and adverse reactions limits the long-term clinical use of l-asparaginase. Present review provides thorough information on microbial l-asparaginase bioprocess optimization including submerged fermentation and solid-state fermentation for l-asparaginase production, downstream purification, its characterization, and issues related to the clinical application including toxicity and hypersensitivity. Here, we have highlighted the bioprocess techniques that can produce improved and economically viable yields of l-asparaginase from promising microbial sources in the current scenario where there is an urgent need for alternate l-asparaginase with less adverse effects.