5328
Bigad El-Sayed Mahmoud Ahmed Khalil
GENETIC IMPROVEMENT OF SOME BACTERIAL STRAINS PRODUCING VITAMIN B12
Pseudomonas, vitamin B12, mutation induction, EMS, UV, ISSR
The present investigation aims to improving some bacterial strains for producing vitamin B12 through genetic improvement methods i.e induction of mutation and protoplast fusion. In this study 18 bacterial cultures were isolated from three different areas (Menofia - Domiatt- Cairo) and tested for production of vitamin B12. Three isolates which highly producing vitamin B12 are identified as a Pseudomonas strains by some biochemical tests and 16srDNA technique. The B12 production was determined by spectrophotometric and HPLC methods. Multiple mutagenesis technique by EthylMethanSulphonate (EMS) as a chemical mutagen and Ultraviolet (UV) as a physical mutagen used to increase vitamin B12production of strains. The strains treated with 200 mM EMSfor different exposure time (15, 30, 45, 60 and 90 min). Mutant M 6 from (P. aeruginosa) produced 6 mg/L B12 higher than their parent that produced 4 mg/L that increase the production by 50% in the first step mutation, in the second step mutation the mutant M 6.9 from (P.aeruginosa) produced 12.3 mg/L that increase the production by 207%, and in the third step mutation the mutant M 6.9.9 from (P. aeruginosa) produced 15.5 mg/L that increase the production by 287%. The strains were treated with UV with different exposure time (30, 60, 90, 120, 150 and 180 sec) at fixed distance of 15 cm from light source, mutant coded P1 (M6.9.9.uv7), giving 20.3 mg/L that increase the production by 408 % in first step mutation.In in the second step mutation, the mutant coded P1 (M6.9.9.uv7.5) was the most B12 producer mutant, giving 21 mg/L showed 425% improvement over the wild-type strain (P1). Intra and inter specific protoplast fusion between auxotrophic mutants isolated after treatment with mutagen was carried out, twenty fusants were successfully obtained, fusant F10 produced 7 mg/L, from intra specific protoplast fusion, fusant F4 produced 7.9 mg/L, from inter specific protoplast. Finally ISSR-PCR marker technique were used to differentiation among parents and its mutants.
2017
Ph.d
Ain Shams
Agriculture