Journal of Microbial & Biochemical Technology

Mutagenesis and protoplast fusion of Arthrobacter sp. for improved glucose isomerase production

3^rd World Congress and Expo on Applied Microbiology

November 07-09, 2016 Dubai, UAE

Heba Sayed Mostafa

Cairo University, Egypt

Posters & Accepted Abstracts: J Microb Biochem Technol

Abstract:

In attempts to construct superior glucose isomerase-producing strains, four bacterial strains (i.e., Arthrobacter sp. B-3728, Actinoplanes missouriensis B-3342, Streptomyces phaeochromogenes B-1131 and B-1517) were screened for their glucose isomerase (GI) synthesis. Both Arthrobacter sp. and A. missouriensis were proved as the highest producers (16.8 and 15.6 U.ml-1, respectively). Ultra Violet (UV) and Ethyl Methane Sulfonate (EMS) were used for mutagenesis. Induced mutants having antimicrobial resistance markers generated from Arthrobacter sp. and A. missouriensis (wild types) were screened for their GI production and compared with wild types. About 8 mutants from each treatment and each strain were examined. The mutant EMS 60-28 D generated from Actinoplanes missouriensis exhibited the highest activity (33.6 U.ml-1) amongst the isolated mutants from this strain with 1.99-folds. While, the mutant EMS 60-25 D generated from Arthrobacter sp. exhibited the highest GI production in this study (49.7 U.ml-1) with 3.2 folds improvement than its wild type. Protoplast fusion technology was successfully applied using hyper-producing GI mutants generated from Arthrobacter sp. according to their antimicrobial responses, 4 mutants were selected to perform 6 crosses. Eight fusants were obtained from each cross and their GI activities were determined. The fusant (C 3-2) exhibited the highest GI synthesis (2.75 folds the wild type). For optimal GI synthesis by the mutant EMS 60-25 D and fusant C 3-2, batch fermentation system was optimized. Optimization of production fermentation resulted in an additional 10% improvement in enzyme synthesis by mutant EMS 60-25 D. On the other side, GI of fusant C 3-2 was increased after optimization from 42.4 to 60.1 U.ml-1 with 3.85 times the activity of the wild type. Enhanced glucose conversion ratio (48 and 48.8%), respectively was also noted by the studied strains compared to 35.3% for the wild type. Glucose isomerase of fusant C 3-2 was extracted, then purified by ammonium sulfate fractionation followed by gel filtration on Sepharose 4-B. The total yield was 17.8%. SDS-PAGE of the purified GI showed one band with a molecular weight of 47 kDa. Optimum temperature; pH; substrate and Mg+2 concentration of the purified enzyme were 75 °C, 8500 mM and 0.05 M, respectively. Km value as calculated from Lineweaver-Burk plot was 285 mM. The enzyme was stable for 1 hour at 80-90 °C and pH 5. The highest GI producing fusant C 3-2 was successfully immobilized within K-carrageenan gel, hardened with glutaraldehyde, for the continuous production of HFCS. The immobilized preparation exhibited a maximal glucose conversion of 36% after 12 hours of isomerization at 60 °C and the half life of such beads was 408 hours of continuous operation.

Biography :

Email: Hebabiotech@gmail.com