Comparative Evaluation of the Growth Parameters for Enhanced Extracellular L-Asparaginase Production by Locally Isolated Rossellomorea marisflavi Strain
Iqra Javed, Beenish Maqsood* and Muhammad Khurshid*
School of Biochemistry and Biotechnology, University of the Punjab, Lahore, 54590, Pakistan.
Iqra Javed and Beenish Maqsood first authorship.
Fig. 1.
Selected L-asparaginase producers on M9 agar plates. a, Strain_a showing 2.7 cm pink zone. b, Strain_b showing 3.3 cm pink zone.
Fig. 2.
Phylogenetic tree analysis. A. For Strain_a a phylogenetic tree was constructed through neighborjoining
method using following closely related strains: MF948300.1 (Bacillus sp.), MF062978.1 (Bacillus marisflavi), MF351833.1 (Bacillus marisflavi), MF537093.1 (Bacillus sp.), MG309379.1 (Bacillus sp.), MG309382.1 (Bacillus sp.), MK757929.1 (Bacillus sp.), MK129310.1 (Bacillus marisflavi), MG025780.1 (Bacillus marisflavi), MG049773.1 (Bacillus marisflavi), KR085889.1 (Bacillus marisflavi), LM655317.1 (Bacillus sp.), MH445024.1 (Uncultured bacterium clone_JSP_105), MH445024.1 (Bacillus sp.), MF537085.1 (Bacillus sp.), KY785320.1 (Bacillus aquimaris), KF956699.1 (Bacillus sp.), MK391969.1 (Bacillus marisflavi), MF321816.1 (Bacillus marisflavi), KF956688.1 (Bacillus sp.) and KF956589.1 (Bacillus sp.) Strain_a showed closest homology to MG025780.1 strain and was identified as Bacillus marisflavi, preferably written as Rossellomorea marisflavi. B. For strain_b a phylogenetic tree was constructed through neighborjoining method using following closely related strains: MH683113.1 (Bacillus sp.), KY609479.1 (Uncultured bacterium clone), MG386505.1 (Bacillus cereus), KF641827.1 (Bacillus cereus), MG027636.1 (Bacillus cereus), JN942136.1 (Bacillus sp.), MK064179.1 (Bacillus cereus), HM752769.1 (Bacillus cereus), JF901710.1 (Bacillus sp.), MF967274.1 (Bacillus sp.), JX544748.1 (Bacillus cereus), MK696376.1 (Bacillus sp.), KT259192.1 (Bacillus cereus), MK064179.1 (Bacillus cereus), KM289183.1 (Bacillus Sp.), KY773595.1 (Bacillus cereus) and JF833090.2 (Bacillus cereus). Strain_b showed closest homology to MK064179.1strain and was identified as Bacillus cereus.
Fig. 3.
L-asparaginase production. A, At different pH conditions. L-asparaginase production by Bacillus cereus and Rossellomorea marisflavi was observed to be optimal at neutral pH and pH 8 respectively under suitable temperature conditions. B, At different temperatures (°C). R. marisflavi optimally produced L-asparaginase at 40°C while B. cereus produced it maximally at 37°C under optimal pH conditions. C, Under different incubation times (h). L-asparaginase activity was highest in both strains on the completion of 36 h, under optimal temperature and pH conditions. D, Using different inoculum sizes (%). L-asparaginase production was greatest with 1.5% inoculum size on the completion of 36 h, under optimal by both strains.
Fig. 4.
Change in L-asparaginase production in the presence of different liquid growth media (A), solid substrate (B) and metal ions (C). A, R. marisflavi maximally produced L-asparaginase when grown in modified Czapek-Dox’s medium 2, while TSB proved to be the most suitable media for L-asparginase production by B. cereus. Experiment was performed under optimal conditions. B, strains gave highest L-asparaginase activity when grown on corn cob under optimal conditions. C, the effect of metal ions (0.1 mM final concentration) and EDTA (5 mM final concentration) on L-asparaginase production by both strains showed variable results. Mg2+ was the best L-asparaginase inducer that enhanced the activity to 195.8% (882.35 U/mg) in R. marisflavi and to 148.02% (414.47 U/mg) in B. cereus in contrast to No-salt control (100% L-asparaginase activity).
