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Recombinant l-Asparaginase II from Lactobacillus casei subsp. casei ATCC 393 and Its Anticancer Activity.

ABSTRACT: l-asparaginases from bacterial origin are employed extensively in leukemic treatment and food industry. The present study focuses on the characterization of the recombinant l-asparaginase II from Lactobacillus casei subsp. casei ATCC 393 cloned into Escherichia coli expression system and purified using Ni-NTA chromatography. The recombinant l-asparaginase as a monomer had a molecular weight of 35 kDa. The enzyme was active from 10 to 80 °C with the optimum at 40 °C. The enzyme retained its activity at 28 °C and 37 °C up to 24 h. The enzyme had optimum pH of 6 and retained 50% activity till 18 h. The Km of the recombinant enzyme was 0.01235 mM and Vmax 1.576 mM/min. The half life of recombinant l-asparaginase II in human serum was 44 h and trypsin was for 15 min. The LC-MS/MS analysis revealed the molecular weight of 35,050 and pI of 5.64. The secondary structure prediction using CD spectroscopy for the recombinant enzyme showed 33.5% ?-helix, 66.5% turn and 0% ? sheets. The cytotoxicity of the recombinant enzyme was analysed against MOLT 3, Jurkat E6.1 and K-562 with the IC 50 value of 30, 62.5 and 50 µg/ml.

SUBMITTER: Aishwarya SS

PROVIDER: S-EPMC6646611 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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Recombinant l-Asparaginase II from Lactobacillus casei subsp. casei ATCC 393 and Its Anticancer Activity.

Aishwarya S Susan SS Selvarajan E E Iyappan S S Rajnish K N KN

Indian journal of microbiology 20190423 3

l-asparaginases from bacterial origin are employed extensively in leukemic treatment and food industry. The present study focuses on the characterization of the recombinant l-asparaginase II from Lactobacillus casei subsp. casei ATCC 393 cloned into Escherichia coli expression system and purified using Ni-NTA chromatography. The recombinant l-asparaginase as a monomer had a molecular weight of 35 kDa. The enzyme was active from 10 to 80 °C with the optimum at 40 °C. The enzy ...[more]

PMID: 31388208

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Project description:Lactic acid bacteria (LAB) are commonly applied to fish as a means of growth promotion and disease prevention. However, evidence regarding whether LAB colonize the gastrointestinal (GI) tract of fish remains sparse and controversial. Here, we investigated whether Lacticaseibacillus casei ATCC 393 (Lc) can colonize the GI tract of crucian carp. Sterile feed irradiated with 60Co was used to eliminate the influence of microbes, and 100% rearing water was renewed at 5-day intervals to reduce the fecal-oral circulation of microbes. The experiment lasted 47 days and was divided into three stages: the baseline period (21 days), the administration period (7 days: day -6 to 0) and the post-administration period (day 1 to 19). Control groups were fed a sterile basal diet during the whole experimental period, whereas treatment groups were fed with a mixed diet containing Lc (1 × 107 cfu/g) and spore of Geobacillus stearothermophilus (Gs, 1 × 107 cfu/g) during the administration period and a sterile basal diet during the baseline and post-administration periods. An improved and highly sensitive selective culture method (SCM) was employed in combination with a transit marker (a Gs spore) to monitor the elimination of Lc in the GI tract. The results showed that Lc (<2 cfu/gastrointestine) could not be detected in any of the fish sampled from the treatment group 7 days after the cessation of the mixed diet, whereas Gs could still be detected in seven out of nine fish at day 11 and could not be detected at all at day 15. Therefore, the elimination speed of Lc was faster than that of the transit marker. Furthermore, high-throughput sequencing analysis combined with SCM was used to reconfirm the elimination kinetics of Lc in the GI tract. The results show that the Lc in the crucian carp GI tract, despite being retained at low relative abundance from day 7 (0.11% ± 0.03%) to 21, was not viable. The experiments indicate that Lc ATCC 393 cannot colonize the GI tract of crucian carp, and the improved selective culture in combination with a transit marker represents a good method for studying LAB colonization of fish.

Dataset Information

Recombinant l-Asparaginase II from Lactobacillus casei subsp. casei ATCC 393 and Its Anticancer Activity.

Publications

Recombinant l-Asparaginase II from <i>Lactobacillus casei</i> subsp. <i>casei</i> ATCC 393 and Its Anticancer Activity.

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