Microbes Producing L-Asparaginase free of Glutaminase and Urease isolated from Extreme Locations of Antarctic Soil and Moss

L-Asparaginase (L-asparagine aminohydrolase, E.C. 3.5.1.1) has been proven to be competent in treating Acute Lymphoblastic Leukaemia (ALL), which is widely observed in paediatric and adult groups. Currently, clinical L-Asparaginase formulations are derived from bacterial sources such as Escherichia coli and Erwinia chrysanthemi. These formulations when administered to ALL patients lead to several immunological and hypersensitive reactions. Hence, additional purification steps are required to remove toxicity induced by the amalgamation of other enzymes like glutaminase and urease. Production of L-Asparaginase that is free of glutaminase and urease is a major area of research. In this paper, we report the screening and isolation of fungal species collected from the soil and mosses in the Schirmacher Hills, Dronning Maud Land, Antarctica, that produce L-Asparaginase free of glutaminase and urease. A total of 55 isolates were obtained from 33 environmental samples that were tested by conventional plate techniques using Phenol red and Bromothymol blue as indicators. Among the isolated fungi, 30 isolates showed L-Asparaginase free of glutaminase and urease. The L-Asparaginase producing strain Trichosporon asahii IBBLA1, which showed the highest zone index, was then optimized with a Taguchi design. Optimum enzyme activity of 20.57 U mL−1 was obtained at a temperature of 30 °C and pH of 7.0 after 60 hours. Our work suggests that isolation of fungi from extreme environments such as Antarctica may lead to an important advancement in therapeutic applications with fewer side effects.

[1]  T. Sathish,et al.  l-Asparaginase from Streptomyces griseus NIOT-VKMA29: optimization of process variables using factorial designs and molecular characterization of l-asparaginase gene , 2015, Scientific Reports.

[2]  R. Blanchette,et al.  Distribution and abundance of soil fungi in Antarctica at sites on the Peninsula, Ross Sea Region and McMurdo Dry Valleys , 2011 .

[3]  S. Shivaji,et al.  Sphingobacterium antarcticus sp. nov., a Psychrotrophic Bacterium from the Soils of Schirmacher Oasis, Antarctica , 1992 .

[4]  J. Reeve,et al.  Molecular identification of Bacteria and Eukarya inhabiting an Antarctic cryoconite hole , 2003, Extremophiles.

[5]  N. Russell,et al.  Microorganisms in the atmosphere over Antarctica. , 2009, FEMS microbiology ecology.

[6]  S. Faderl,et al.  Safety, efficacy, and clinical utility of asparaginase in the treatment of adult patients with acute lymphoblastic leukemia , 2017, OncoTargets and therapy.

[7]  V. V. Dasu,et al.  Development of medium for enhanced production of glutaminase-free l-asparaginase from Pectobacterium carotovorum MTCC 1428 , 2009, Applied Microbiology and Biotechnology.

[8]  S. Gummadi,et al.  Screening and Isolation of Novel Glutaminase Free L-asparaginase from Fungal Endophytes , 2014 .

[9]  Sudhir Kumar Jain,et al.  Recent developments in L-asparaginase discovery and its potential as anticancer agent. , 2016, Critical reviews in oncology/hematology.

[10]  A. Pandey,et al.  Cloning and expression of l-asparaginase from E. coli in eukaryotic expression system , 2015 .

[11]  G. L. Costa,et al.  Production of L-asparaginase by filamentous fungi. , 2004, Memorias do Instituto Oswaldo Cruz.

[12]  S. Igarasi,et al.  Asparaginase and glutaminase activities of micro-organisms. , 1973, Journal of general microbiology.

[13]  W. Azmi,et al.  ONE STEP PURIFICATION OF GLUTAMINASE FREE L- ASPARAGINASE FROM ERWINIA CAROTOVORA WITH ANTICANCEROUS ACTIVTY , 2012 .

[14]  A. Demain,et al.  Production of recombinant proteins by microbes and higher organisms. , 2009, Biotechnology advances.

[15]  D. Kumar,et al.  Solid-State Fermentation vs Submerged Fermentation for the Production of l-Asparaginase. , 2016, Advances in food and nutrition research.

[16]  S. Nandi,et al.  Process optimization of lipase catalyzed synthesis of diesters in a packed bed reactor , 2015 .

[17]  S. Fahad,et al.  L-asparaginase as a critical component to combat Acute Lymphoblastic Leukaemia (ALL): A novel approach to target ALL. , 2016, European journal of pharmacology.

[18]  Nikolaos E Labrou,et al.  L-Asparaginase from Erwinia Chrysanthemi 3937: cloning, expression and characterization. , 2007, Journal of biotechnology.

[19]  A. L. Demain.  3.29 – Production of Recombinant Proteins by Microbes and Higher Organisms , 2011 .

[20]  L. Hankin,et al.  Solid media containing carboxymethylcellulose to detect CX cellulose activity of micro-organisms. , 1977, Journal of general microbiology.

[21]  G. Feller,et al.  Kinetic and structural optimization to catalysis at low temperatures in a psychrophilic cellulase from the Antarctic bacterium Pseudoalteromonas haloplanktis. , 2004, The Biochemical journal.

[22]  M. Zucchetti,et al.  L-asparagine depletion and L-asparaginase activity in children with acute lymphoblastic leukemia receiving i.m. or i.v. Erwinia C. or E. coli L-asparaginase as first exposure. , 2000, Annals of oncology : official journal of the European Society for Medical Oncology.

[23]  L. Selbmann,et al.  Fungi in Antarctica , 2007 .

[24]  Evelyne Sernagor,et al.  Dampening Spontaneous Activity Improves the Light Sensitivity and Spatial Acuity of Optogenetic Retinal Prosthetic Responses , 2016, Scientific Reports.

[25]  Susheel Kumar,et al.  Localization and production of novel l-asparaginase from Pectobacterium carotovorum MTCC 1428 , 2010 .

[26]  R. K. Saxena,et al.  Efficient production of L-asparaginase from Bacillus licheniformis with low-glutaminase activity: optimization, scale up and acrylamide degradation studies. , 2012, Bioresource technology.

[27]  C. K. Venil,et al.  Production of L-asparaginase by Serratia marcescens SB08: Optimization by response surface methodology , 2009 .

[28]  C. Rosa,et al.  Endophytic fungi associated with the Antarctic grass Deschampsia antarctica Desv. (Poaceae) , 2009, Polar Biology.

[29]  Dinesh Goyal,et al.  Characterization of microwave-alkali-acid pre-treated rice straw for optimization of ethanol production via simultaneous saccharification and fermentation (SSF) , 2017 .

[30]  S. Deraz,et al.  Purification, characterization, cytotoxicity and anticancer activities of L-asparaginase, anti-colon cancer protein, from the newly isolated alkaliphilic Streptomyces fradiae NEAE-82 , 2016, Scientific Reports.

[31]  R. Solé,et al.  Corrigendum: Variability in mutational fitness effects prevents full lethal transitions in large quasispecies populations , 2015, Scientific Reports.

[32]  T. Bruns,et al.  ITS primers with enhanced specificity for basidiomycetes ‐ application to the identification of mycorrhizae and rusts , 1993, Molecular ecology.

[33]  M. Dhale,et al.  A comparative rapid and sensitive method to screen l-asparaginase producing fungi. , 2014, Journal of microbiological methods.

[34]  V. Sivaramakrishnan,et al.  Preparation and properties of L-asparaginase from green chillies (Capsicum annum L.) , 1980, Journal of Biosciences.

[35]  B. Löwenberg,et al.  The hematologic mission and scope of the Journal in 2013: Blood, a podium for clinical studies and a podium for experimental research in hematology. , 2013, Blood.

[36]  K. Doriya,et al.  Isolation and screening of l-asparaginase free of glutaminase and urease from fungal sp. , 2016, 3 Biotech.

[37]  V. V. Dasu,et al.  Purification and characterization of glutaminase-free L-asparaginase from Pectobacterium carotovorum MTCC 1428. , 2011, Bioresource technology.

[38]  A. Ting,et al.  Endophytic l-asparaginase-producing fungi from plants associated with anticancer properties , 2014, Journal of advanced research.

[39]  Mervat Morsy Abbas Ahmed,et al.  Production, Purification and Characterization of L-Asparaginase from Marine Endophytic Aspergillus sp. ALAA-2000 under Submerged and Solid State Fermentation , 2015 .

[40]  F. F. Dias,et al.  Purification, characterization and antiproliferative activity of l-asparaginase from Aspergillus oryzae CCT 3940 with no glutaminase activity , 2016 .

[41]  W. Vincent.  Evolutionary origins of Antarctic microbiota: invasion, selection and endemism , 2000, Antarctic Science.

[42]  D. M. Greenberg,et al.  EFFECT OF ADMINISTRATION OF THE ENZYME GLUTAMINASE ON THE GROWTH OF CANCER CELLS. , 1964, Cancer research.

[43]  J. Weinstein,et al.  The glutaminase activity of L-asparaginase is not required for anticancer activity against ASNS-negative cells. , 2013, Blood.

[44]  R. K. Saxena,et al.  A rapid plate assay for screening l‐asparaginase producing micro‐organisms , 1997, Letters in applied microbiology.

[45]  C. Robinson.  Cold adaptation in Arctic and Antarctic fungi , 2001 .

[46]  R. Aduri,et al.  Bacterial diversity of a soil sample from Schirmacher Oasis, Antarctica. , 2004, Cellular and molecular biology.