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

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 Ni2+. Km and kcat values were 2.91 mM and 1.53 × 102 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.

[1]  N. Fatima,et al.  L-asparaginase produced from soil isolates of Pseudomonas aeruginosa shows potent anti-cancer activity on HeLa cells , 2019, Saudi journal of biological sciences.

[2]  Büşra Başar Gökcen,et al.  Health benefits of fermented foods , 2019, Critical reviews in food science and nutrition.

[3]  S. Khare,et al.  Recent Development in the Uses of Asparaginase as Food Enzyme , 2018, Energy, Environment, and Sustainability.

[4]  K. N. Rajnish,et al.  In silico analysis, molecular cloning, expression and characterization of l-asparaginase gene from Lactobacillus reuteri DSM 20016 , 2017, 3 Biotech.

[5]  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.

[6]  F. F. Dias,et al.  Acrylamide mitigation in French fries using native l-asparaginase from Aspergillus oryzae CCT 3940 , 2017 .

[7]  J. Elmore,et al.  The use of asparaginase to reduce acrylamide levels in cooked food. , 2016, Food chemistry.

[8]  A. Badoei-dalfard,et al.  L-asparaginase production in the pseudomonas pseudoalcaligenes strain JHS-71 isolated from Jooshan Hot-spring , 2016, Molecular biology research communications.

[9]  M. Wakayama,et al.  Effective treatment for suppression of acrylamide formation in fried potato chips using L-asparaginase from Bacillus subtilis , 2015, 3 Biotech.

[10]  Changhee Lee,et al.  Cloning, expression, and characterization of thermophilic L‐asparaginase from Thermococcus kodakarensis KOD1 , 2014, Journal of basic microbiology.

[11]  Jaejoon Jung,et al.  Biochemical characterization of L-asparaginase in NaCl-tolerant Staphylococcus sp. OJ82 isolated from fermented seafood. , 2014, Journal of microbiology and biotechnology.

[12]  Q. Yan,et al.  Biochemical Characterization of a Novel l-Asparaginase with Low Glutaminase Activity from Rhizomucor miehei and Its Application in Food Safety and Leukemia Treatment , 2013, Applied and Environmental Microbiology.

[13]  Z. Rao,et al.  Cloning, expression, and characterization of L-asparaginase from a newly isolated Bacillus subtilis B11-06. , 2013, Journal of agricultural and food chemistry.

[14]  Geert Huys,et al.  Food fermentations: microorganisms with technological beneficial use. , 2012, International journal of food microbiology.

[15]  K. Yoshimune,et al.  Expression in Escherichia coli of a gene encoding type II l-asparaginase from Bacillus subtilis, and characterization of its unique properties , 2011, Annals of Microbiology.

[16]  A. Sumantha,et al.  Production, purification and characterisation of extracellular L-asparaginase from a soil isolate of Bacillus sp. , 2010 .

[17]  P. Mishra,et al.  Structural stability and functional analysis of L-asparaginase from Pyrococcus furiosus , 2010, Biochemistry (Moscow).

[18]  M. Wakayama,et al.  Overexpression of type I L-asparaginase ofBacillus subtilis inEscherichia coli, rapid purification and characterisation of recombinant type I L-asparaginase , 2008, Annals of Microbiology.

[19]  R. Carle,et al.  Acrylamide in cereal products: A review , 2008 .

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

[21]  Bhairab N. Singh,et al.  Extracellular expression and single step purification of recombinant Escherichia coli L-asparaginase II. , 2004, Protein expression and purification.

[22]  F. Escher,et al.  Acrylamide in gingerbread: critical factors for formation and possible ways for reduction. , 2004, Journal of agricultural and food chemistry.

[23]  H. Sather,et al.  Asparaginase Antibody and Asparaginase Activity in Children With Higher-Risk Acute Lymphoblastic Leukemia: Children's Cancer Group Study CCG-1961 , 2004, Journal of pediatric hematology/oncology.

[24]  A. W. Phillips,et al.  Purification and Properties of l-Asparaginase from Serratia marcescens , 1971, Journal of bacteriology.

[25]  J. H. Schwartz,et al.  Localization of the two-L-asparaginases in anaerobically grown Escherichia coli. , 1967, The Journal of biological chemistry.

[26]  Oliver H. Lowry,et al.  Protein measurement with the Folin phenol reagent. , 1951, The Journal of biological chemistry.

[27]  Birgit,et al.  Foodborne antimicrobial resistance as a biological hazard , 2008 .

[28]  D. Kyriakidis,et al.  L-asparaginase of Thermus thermophilus: Purification, properties and identificaation of essential amino acids for its catalytic activity , 2004, Molecular and Cellular Biochemistry.

[29]  C. Derst,et al.  Engineering the substrate specificity of Escherichia coli asparaginase II. Selective reduction of glutaminase activity by amino acid replacements at position 248 , 2000, Protein science : a publication of the Protein Society.

[30]  K. Miura,et al.  PREPARATION OF TRANSFORMING DEOXYRIBONUCLEIC ACID BY PHENOL TREATMENT. , 1963, Biochimica et biophysica acta.