UV Mutagenesis of Leuconostoc mesenteroides NRRL B-640 for Generation of a Mutant (B-640M) with Hyper-Producing Dextransucrase Activity

Order of Publishing in Issue: 
8
Volume :5
Issue :4
October, 2011
Page No: 
1445-1453
Authors: 
Mayur Agrawal, Rishikesh Shukla and Arun Goyal
Address: 
Department of Biotechnology Indian Institute of Technology Guwahati Guwahati 781 039, Assam, India

Abstract
This study reports the strain improvement of Leuconostoc mesenteroides NRRL B-640 by classical mutation technique using ultraviolet radiation to achieve the mutant strain with higher dextransucrase activity. A large number of mutants were generated, cultured and tested for dextransucrase activity. The selected mutant gave considerably higher (46%) dextransucrase activity (0.8 U/mg) as compared to the wild-type strain (0.55 U/mg). Interestingly, the selected mutant yielded 21% higher dextran concentration (8.2 mg/ml) as compared to wild type strain (6.8 mg/ml) in the broth. The selected mutant was characterized for antibiotic sensitivity, carbohydrate fermentation and dextran forming activity and compared with the wild-type strain. The antibiotic sensitivity test showed that, the mutant, unlike the wild-type strain was resistant to kanamycin, claxacin and amikacin. The mutant also showed resistance towards amoxyclav and tobramycin, unlike the wild-type strain which was moderately sensitive to these antibiotics. The carbohydrate fermentation profile showed that the mutant, unlike the wild-type, utilized lactose but did not show any activity towards cellobiose and rhamnose. Dextransucrase from the mutant was purified by PEG fractionation and its analysis by SDS-PAGE revealed the same molecular size of 180 kDa as that of wild-type strain. The analysis of dextransucrase from the mutant by activity staining using Periodic Acid Schiff’s protocol also showed a single band and no change in dextran forming activity when compared to wild-type. 1H NMR and 13C NMR spectroscopic and Scanning Electron Microscopic (SEM) analysis of dextran produced by mutant strain B640M showed no difference from the wild type Leuconostoc mesenteroides NRRL B-640.

Keywords: 
Leuconostoc mesenteroides NRRL B-640, UV mutagenesis, dextransucrase, dextran
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