Purification and immobilization of l-arginase from thermotolerant Penicillium chrysogenum KJ185377.1; with unique kinetic properties as thermostable anticancer enzyme

Abstract

L-Arginase, hydrolyzing L-arginine to L-ornithine and urea, is a powerful anticancer, L-arginine-depleting agent, against argininosuccinate synthase expressing tumors. Otherwise, the higher antigenicity and lower thermal stability of this enzyme was the main biochemical hurdles. Since, the intrinsic thermal stability of enzymes follow the physiological temperature of their producer, thus, characterization of L-arginase from thermotolerant Penicillium chrysogenum was the objective of this study. L-Arginase (Arg) was purified to its homogeneity from P. chrysogenum by 10.1-fold, with 37.0 kDa under denaturing PAGE, optimum reaction at 50 °C, pH stability (6.8-7.9), with highest molar ratio of constitutional arginine, glutamic acid, lysine and aspartic acid. The purified enzyme was PEGylated and immobilized on chitosan, with 41.9 and 22.1 % yield of immobilization. At 40 °C, the T value of free-Arg, PEG-Arg and Chit-Arg was 10.4, 15.6, 20.5 h, respectively. The free-Arg and Chit-Arg have a higher affinity to L-arginine (K 4.8 mM), while, PEG-Arg affinity was decreased by about 3 fold (K 15.2 mM). The inhibitory constants to the free and PEG-Arg were relatively similar towards HA and PPG. The IC for the free enzyme against HEPG-2 and A549 tumor cells was 0.136 and 0.165 U/ml, comparing to 0.232 and 0.496 U/ml for PEG-Arg, respectively. The in vivo T to the free Arg and PEG-Arg was 16.4 and 20.4 h, respectively as holo-enzyme. The residual L-arginine level upon using free Arg was 156.9 and 144.5 µM, after 6 and 8 h, respectively, regarding to initials at 253.6 µM, while for Peg-Arg the level of L-arginine was nil till 7 h of initial dosing. The titer of IgG was induced by 10-15 % in response to free-Arg after 28 days comparing to IgG titer for PEG-Arg.

Purification, immobilization, and biochemical characterization ofl‐arginine deiminase from thermophilicAspergillus fumigatusKJ434941: Anticancer activityin vitro

“…The cultures were filtered and the fungal growing pellets (∼100 g from 5 L fungal cultures) were collected, washed by sterile potassium phosphate. The enzyme was extracted by grinding the tissues in liquid nitrogen, then dispensing on 50 mM potassium phosphate (pH 7.5) containing 1 mM EDTA, 1 mM 2‐mercaptoethanol, and 1mM PMSF . The enzyme activity and its concentration were determined as follows.…”

“…The biochemical and catalytic properties of the ADIs as pH stability, thermal stability, substrate specificity, inhibitors, and activators were assessed as described by our studies . The effect of suicide inhibitors, active sites analogues, as propargylglycine (PPG), 6‐diazo‐5‐oxo‐norleucine (DON), iodoacetate (IA), and hydroxylamine (HA) on the catalytic efficiency of ADI were determined, in the presence of substrate . The resistance of ADIs for in vitro proteolysis by proteinase K and acid protease was determined …”

Purification and Characterization of Anabaena flos-aquae Phenylalanine Ammonia-Lyase as a Novel Approach for Myristicin Biotransformation

“…The crude enzyme from the selected algal isolates was purified using gel-filtration and ion-exchange chromatographic approaches [59][60][61][62][63][64]. The crude PAL was precipitated with two-fold chilled acetone, and incubated at -20°C for 30 min.…”

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“…Regulates the level of L-ornithine for the production of L-proline and polyamines to form collagen and facilitate cell growth and repair, respectively 4,5 . L-arginase occurs in two forms in mammalian tissues, i.e., L-arginase I and II, which are coded by the genes located on chromosome 6 and 14 respectively 6 L-arginase has been reported to play a crucial role in the treatment of neurological disorders 7 , allergic asthma 8 , rheumatoid arthritis 9 , it is also found to have tumor inhibitory properties and has been accredited substantial consideration due to its extensive range of activity against cancer cells 10 . Certain cancers cannot biosynthesize arginine due to lack of expression of argininosuccinate synthetase-1 (ASS1).…”

“…Keywords: 13 has been characterized well, relatively a very few reports on prokaryotic arginase have been characterized 14 such as Bacillus brevis 15 , B. anthraci 16 , and Helicobacter pylori 17 . Studies of the enzyme from fungi, Neurospora crassa 1 , Aspergillus nidulans 18 and Penicillium chrysogenum 10 have been reported. Microorganisms can be easily cultivated and manipulated due to which they are considered a good source for the production of enzymes 19 .…”