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Cloning, expression, and characterization of thermophilic L ‐asparaginase from Thermococcus kodakarensis KOD1
Author(s) -
Hong SungJun,
Lee YunHa,
Khan Abdur Rahim,
Ullah Ihsan,
Lee Changhee,
Park Choi Kyu,
Shin JaeHo
Publication year - 2014
Publication title -
journal of basic microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.58
H-Index - 54
eISSN - 1521-4028
pISSN - 0233-111X
DOI - 10.1002/jobm.201300741
Subject(s) - chemistry , enzyme kinetics , molecular mass , enzyme , chromatography , affinity chromatography , biochemistry , thermococcus , sodium dodecyl sulfate , recombinant dna , enzyme assay , polyacrylamide gel electrophoresis , active site , archaea , gene
The present study demonstrates cloning, expression, and characterization of hyperthermostable L ‐asparaginase from Thermococcus kodakarensis KOD1 in Escherichia coli BLR(DE3). The recombinant 6× His‐tagged protein L ‐asparaginase from T. kodakarensis (TkAsn), was purified to homogeneity by heat treatment followed by affinity chromatography using a nickel‐nitrilotriacetic acid (Ni‐NTA) column. The molecular mass of the purified enzyme was found to be approximately 37 kDa by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). The enzymatic properties, such as optimum temperature and pH, were 90 °C and 8.0, respectively. Its appearent K m , V max , and K cat values were 2.6 mM, 1121 µmol min −1 mg −1 , and 694 S −1 , respectively. The enzyme displayed high thermal stability at optimum temperature with an insignificant loss in enzymatic activity, retaining almost 90% of its activity over a time period of 32 h. The relative activity of the enzyme was significantly inhibited by the supplementation of Cu 2+ and Ni 2+ ions, while moderately inhibited by other ions. In contrast, Mg 2+ ions enhanced the relative activity compared to the control. The acrylamide contents in baked dough were reduced to sixty percent after treatment with recombinant TkAsn as compared to the untreated control. Results of the present study revealed that the enzyme was highly active at broader range of temperatures and pH, which reflect the potential of recombinant TkAsn in the food processing industry. In addition, the high thermal stability of the enzyme may facilitates its handling, storage, and transportation.