TITLE:
Batch, Fed Batch Production and Characterization of Glutaminase Free L-Asparaginase II of Pectobacterium carotovorum MTCC 1428 in Escherichia coli
AUTHORS:
Rachna Goswami, Krishnamoorthy Hegde, Venkata Dasu Veeranki
KEYWORDS:
Batch Fermentation, Fed-Batch Fermentation, Recombinant L-Asparaginase II, Taguchi’s Method
JOURNAL NAME:
Advances in Microbiology,
Vol.4 No.10,
August
27,
2014
ABSTRACT: The present study describes the production optimization of recombinant
L-asparaginase II of Pectobacterium carotovorum MTCC 1428 in Escherichia coli BL21
(DE3) at batch and fed batch bioreactor level. Production of recombinant
L-asparaginase II in batch and fed batch mode was found to be 1.34 and 5.38
folds higher, respectively as compared to shake flask culture. SDS-PAGE and
native PAGE of the purified enzyme revealed that molecular mass of the subunits
and native enzyme are ~37.5 kDa and ~150 kDa, respectively. Optimum range of pH
and temperature for hydrolysis of L-asparagine were found to be 7.5 - 8.5 and
47°C - 52°C, respectively. The recombinant enzyme is very specific for its
natural substrate, L-asparagine. The activity of recombinant L-asparaginase II
is improved by mono cations and diverse effectors including Na+, K+,
L-cystine, L-histidine, glutathione and 2-mercaptoethanol whereas, it is
moderately inhibited by different divalent cations and thiol group blocking
reagent. The kinetic parameters Km, Vmax, kcat and Km/Kcat of purified recombinant L-asparaginase II
were determined. The purified L-asparaginase II possesses no partial
glutaminase activity, which is prerequisite to reduce the possibility of side
effects during the course of anti-cancer therapy.