Utilization of Emulsiflex Prepared Escherichia coli Strain EC4 Oxyrase for Improved Cultivation of Anaerobic Bacteria by using Hungate Technique and Determination of its Kinetic Parameters
Utilization of Emulsiflex Prepared Escherichia coli Strain EC4 Oxyrase for Improved Cultivation of Anaerobic Bacteria by using Hungate Technique and Determination of its Kinetic Parameters
Muhammad Usman Ahmadand Ikram-ul-Haq*
ABSTRACT
Present study was conducted for the utilization of Emulsiflex prepared oxyrase extracted from cytoplasmic of membrane fragments of E. coli. For this purpose, 88 E.coli isolates from 30 intact poultry intestines were cultured and identified preliminary on the basis of microscopy and biochemical testing. E. coli strain EC4 was screened as best oxyrase producer with activity of 0.41±0.008U/mL/min with 41% reduction in dissolved oxygen at pH 7.5, temperature 37°C, 25mM lactate as H+ donor after 20 min. This strain was later on confirmed by 16S ribotyping procedure. Oxyrase was used for improved cultivation of anaerobic bacteria by employing Hungate Technique where its antioxidant potential was compared with common reducing agent Cysteine-HCl (Cys-HCl). Four anaerobic bacterial strains (Anaerobaculum hydrogeniformans OS1, Akkermansia muciniphila, Bilophila wadsworthia, and Roseburia intestinalis) were selected as experimental models for this purpose. Significant improvement in cell density of Anaerobaculum hydrogeniformans culture with maximum OD600nm of 0.80±0.0017 reached after 4 days of incubation when oxyrase used as reducing agent, and maximum OD600nm 0.65±0.0016 in presence of Cys-HCl. Akkermansia muciniphila culture reached maximum OD600nm value of 2.1±0.07 in the presence of oxyrase after 18 h of incubation. While with Cys-HCl, Akkermansia muciniphila culture reached maximum cell density OD600nm of 2.0±0.05 after 27 h of incubation. Oxygen reducing potential of oxyrase also suited the growth of Bilophila wadsworthia because it reached maximum cell density OD600nm of 2.0±0.05 after 73 h of incubation. Whereas, in the presence of Cys-HCl Bilophila wadsworthia cells only achieved maximum OD600nm of 1.54±0.07 after 28 h of incubation. Cultivation studies of Roseburia intestinalis in the presence of oxyrase also exhibited noteworthy improvement in the yield of cell density with OD600nm of 2.1±0.06 after 25 h of incubation. With Cys-HCl, maximum cell density of Roseburia intestinalis with 2.0±0.06 OD600nm was observed after 73 h. Kinetic parameters derived from double reciprocal Lineweaver-Burk plot showed Km values 8.49×10-3 M-1 and 4.75×10-3 M-1 of sonication and Emulsiflex prepared oxyrase membrane fragments, respectively for lactate as substrate.
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June 2019
Vol. 51, Iss. 3, Pages 801-1202
