农杆菌ATCC 4452降低辅酶Q10发酵液粘度及提高产量的工艺及菌株研究

Pradipta Tokdar, A. Wani, Pratyush Kumar, Prafull V. Ranadive, S. George
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引用次数: 13

摘要

发酵液的黏性对发酵罐的工艺条件和参数有显著的影响。虽然发酵液流变学在工艺研究中有着重要的影响,但对发酵液流体动力学的修正仍然是一个挑战。在农杆菌ATCC 4452生产辅酶q10 (CoQ10)过程中,由于过量合成外多糖,培养液变得高度粘稠。这阻碍了辅酶q10的产量,并使下游过程复杂化。本研究描述了如何通过媒介修饰和突变来解决这个问题。以紫外诱变剂和EMS诱变剂为诱变剂,根据抗菌素耐药性进行合理选择,产生诱变突变体。在以蔗糖为基础的PM-2培养基中筛选这些突变体,紫外诱导的万古霉素抗性突变体M-6在肉汤中的粘度发展显著降低(6.29倍)。突变体M-6(S)是突变体M-6的天然变体,对高底物浓度具有抗性,进一步选择用于辅酶q10的生产。甘蔗糖蜜作为碳源最适合用突变体M-6(S)发酵辅酶q10。用更便宜的甘蔗糖蜜代替蔗糖显著降低了肉汤粘度,提高了特定的辅酶q10含量,从而产生了具有成本效益的发酵工艺。在25°C、500 rpm搅拌、0.2 vvm曝气条件下,采用新配制的甘蔗糖蜜培养基连续补料分批发酵,新开发的突变株CoQ10产量为48.89 mg/L,比CoQ10含量为1.87 mg/g DCW。
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Process and Strain Development for Reduction of Broth Viscosity with Improved Yield in Coenzyme Q10 Fermentation by Agrobacterium tumefaciens ATCC 4452
Viscous nature of the fermentation broth has phenomenal influence on process conditions and parameters in a fermentor. Though broth rheology has attracted significant influence in process research, still there is a challenge to modify fluid dynamics of fermentation broth. During the production of coenzymeQ10 (CoQ10) by Agrobacterium tumefaciens ATCC 4452, the culture broth becomes highly viscous due to excessive synthesis of exopolysaccharides. This hinders the CoQ10 yield and complicates the downstream process. The present study describes how this problem was tackled by media modification and mutation. Induced mutants were generated using UV and EMS as mutagenic agents followed by rational selection based on antibiotic resistance. On screening of these mutants in sucrose based PM-2 medium, UV induced, vancomycin resistant mutant M-6, showed significant reduction (6.29 fold) in viscosity development in the broth. Mutant M-6(S), a natural variant of mutant M-6, resistant to high substrate concentration was further selected for the CoQ10 production. Cane molasses as carbon source was found to be best suitable for CoQ10 fermentation using mutant M-6(S). Replacing sucrose with cheaper cane molasses significantly reduced the broth viscosity with improved specific CoQ10 content, thereby generating cost effective fermentation process. The newly developed mutant strain produced 48.89 mg/L of CoQ10 with specific CoQ10 content of 1.87 mg/g of DCW at 25°C, 500 rpm agitation and 0.2 vvm aeration using continuous fed batch fermentation and newly formulated cane molasses medium.
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