Archives

  • 2018-07
  • 2019-04
  • 2019-05
  • 2019-06
  • 2019-07
  • 2019-08
  • 2019-09
  • 2019-10
  • 2019-11
  • 2019-12
  • 2020-01
  • 2020-02
  • 2020-03
  • 2020-04
  • 2020-05
  • 2020-06
  • 2020-07
  • 2020-08
  • 2020-09
  • 2020-10
  • 2020-11
  • 2020-12
  • 2021-01
  • 2021-02
  • 2021-03
  • 2021-04
  • 2021-05
  • 2021-06
  • 2021-07
  • 2021-08
  • 2021-09
  • 2021-10
  • 2021-11
  • 2021-12
  • 2022-01
  • 2022-02
  • 2022-03
  • 2022-04
  • 2022-05
  • 2022-06
  • 2022-07
  • 2022-08
  • 2022-09
  • 2022-10
  • 2022-11
  • 2022-12
  • 2023-01
  • 2023-02
  • 2023-03
  • 2023-04
  • 2023-05
  • 2023-06
  • 2023-08
  • 2023-09
  • 2023-10
  • 2023-11
  • 2023-12
  • 2024-01
  • 2024-02
  • 2024-03
  • 2024-04
  • 2024-05
  • 2024-06
  • 2024-07
  • br Material and methods br

    2024-06-22


    Material and methods
    Results
    Discussion Asparagine, arginine, leucine, methionine, and glutamine are the essential bombesin receptor for cancer cells and intensively investigated for the development of selective targeted therapeutics. Among them, asparagine depleting bacterial enzyme asparaginase is rationally used as an anticancer agent and arginine depleting enzymes arginase and arginine deiminase are under extensive clinical investigations [4], [7], [24], [33]. In this study, out of 116 bacterial isolates, 31 isolates exhibited arginase activity. Among them, one of the most potent isolate IH2 was characterised by morphological, biochemical and 16S rRNA gene sequencing analysis as P. aeruginosa. The arginase from P. aeruginosa IH2 was purified to 95.67 fold which corresponds to specific activity 102.37 IU mg−1. Recently, several researches have been published on purification of arginase from different sources but the purification fold and specific activity of purified arginases are very low [17], [34]. However, proteins or peptides used for therapeutic purposes should be purified upto 95 fold [27]. Hence, the purification fold of our arginase are equal to the purification fold of proteins or peptides used for therapeutic purpose. SDS and native PAGE analysis showed a single band of approximately 37 kDa and 75 kDa respectively, confirming that native enzyme exist as homo-dimer. These findings are in agreement with the earlier reports which stated that arginase from microbial origins showed great molecular polymorphism and exists in dimeric (Aspergillus nidulans) [35], trimeric (Plasmodium falciparum) [36], tetrameric (Bacillus brevis) [23], and hexameric (Rummeliibacillus pycnus) [37] forms. The pI of arginase was found to be 6.2 which was higher than arginase purified from Xanthoria parietina (4.54) [34] and Bacillus brevis (4.8) [23]. Purified arginase had maximum activity at pH 8 and retains more than 80–85% activity between the pH ranges 7–9. The enzyme was optimally active at temperature range of 30–45 °C and showed maximum activity at 35 °C. These biochemical properties suggesting that purified enzyme is a slight alkalic arginase and optimally act near to the pH of human circulating system (pH 7.45) as well as body temperature (37 °C). Among the various metal ions tested, divalent metal ions (Mn2+, Mg2+, Co2+ and Zn2+) highly influence arginase activity and in the presence of Mn2+ arginase activity was increased more than 2 fold, depicting that our purified arginase is manganese dependent metalloenzyme. These findings are similar to the previous observations [5], [16], [17], [18], [23] which stated enzymes that are belongs to the group amidinohydrolases including arginase requires divalent metal ions for their maximal activity. In the presence of various blood sugars arginase activity did not inhibits which indicates that purified arginase is compatible with blood sugars. Purified enzyme exhibited highest activity towards its natural substrate l-arginine and did not hydrolyse other tested substrates more than 5% (except l-alanine and dl-alanine), suggesting that purified arginase showed selective and high substrate specificity. Human body contains several physiological barriers; serum and blood proteolytic enzymes are major of them which neutralize or degrade clinically administrated proteins or peptides [38]. Therefore, anticancer effectiveness of enzymic drugs significantly depends on enzymic properties such as pH, temperature, serum T1/2, and proteolytic tolerance [39]. The in-vitro serum T1/2 of purified arginase was 36 h, which is higher than the T1/2 of therapeutic enzyme (20 h), currently used as an anticancer agent [40]. The proteolytic T1/2 of arginase against trypsin and proteinase-K was 25 and 29 min, respectively, which is almost two fold higher than arginase purified from P. chrysogenum[17]. These observations are collectively suggested that on clinical administration, physiological barriers would not easily hydrolyse our arginase and its bioavailability remains constant for prolong time. Hence, purified arginase could be considered as a drug with longer plasmatic half-life.