Browsing by Author "Mohamad Fuzi, Siti Fatimah Zaharah"

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    Enhanced production of recombinant B-1, 4-endoxylanase by Kluyveromyces lactis GG799
    (Universiti Teknologi Malaysia, 2013) Mohamad Fuzi, Siti Fatimah Zaharah
    Investigation of recombinant ß-1,4-endoxylanase (Xyn2) expression and production strategies in Kluyveromyces lactis GG799 were carried out in this study. Nutrient composition and culture conditions were observed to affect the secretion, production level and stability of the recombinant host and Xyn2. The experiments were performed in shake-flasks followed by bioreactor propagation in batch and fed batch fermentation. Medium developed in this study consists of nitrogen sources such as ammonium sulfate and free amino acid (casamino acid) as well as compounds like MgSO4·7H2O, Na2SO4, ZnSO4·6H2O, MnSO4·4H2O and KH2PO4 and exhibited good support for the growth and recombinant Xyn2 production by K. lactis GG799. Optimization of batch fermentation was carried out using 2 l bioreactor with 1 l working volume. Optimum dissolved oxygen (DO) level and initial glucose concentration were obtained using Response Surface Methodology (RSM) in order to improve both cell growth and recombinant Xyn2 production in K. lactis GG799. Cell concentration and maximum recombinant Xyn2 activity of 7.54 g/l and 75.53 U/ml, respectively were obtained under optimum condition. The productivity and specific activity of the enzyme were also higher in this system. The optimized conditions produced 12 U/ml/h of recombinant Xyn2 compared to fermentation conditions without oxygen supplied (6 U/ml/h). The study of fed batch fermentation was carried out to enhance the biomass and recombinant Xyn2 production in K. lactis GG799. The addition of 0.5 % (w/v) yeast extract in the feeding medium proves to increase the recombinant Xyn2 and biomass production. Exponential feeding with the specific growth rate controlled at 0.05 /h showed highest increment in recombinant Xyn2 and biomass production by 54 % and 25 %, respectively compared with the batch fermentation. Moreover, high cell density cultures of 74.85 g/l was achieved in this study, and productivity of recombinant Xyn2 at 8.1 U/ml/h was also obtained. In conclusion, the present studies show the importance and influence of medium design, DO level and feeding strategy in increasing the performance of K. lactis GG799 as expression host