Optimisation Of A Process For The Preparation Of Alpha-Ketoglutaric Acid By Whole-Cell Transformation

Alpha-ketoglutaric acid(AKG)is a short-chain carboxylic acid molecule that is widely used in various fields as a precursor substance for many important amino acids such as glutamate and glutamine.AKG is not only directly involved in energy supply,but also in a variety of intracellular chemical reactions,and has various physiological effects.Currently,the main production methods of AKG are chemical synthesis,fermentation and whole cell transformation.The chemical synthesis method has safety risks and is likely to cause environmental pollution.The fermentation method has harsh production conditions,and it is difficult to separate the AKG from the fermentation broth because of its complex composition.Whole cell transformation for AKG production has the advantages of low energy consumption,high specificity,relatively few impurities,and environmental friendliness,and this method is a hot topic that attracts a lot of attentions for the production of AKG.We developed and optimized the fermentation enzyme production process,whole cell transformation system and the separation and purification method for the preparation of AKG,and the experimental results were listed as follows:(1)Plackett-Burman(PB)experiments were performed on the fermentation medium system,and the most significant factor affecting the enzyme activity of L-glutamate oxidase(LGOX)was screened to be the glucose concentration of the medium.The optimal glucose concentration was 80 g/L.The fermentation system was scaled up to 5 L for validation,and the enzyme activity of LGOX was 100 U under the condition of 5%inoculum and intermittent replenishment,which was more than double than the enzyme activity before optimization.(2)The conditions of catalase,Triton X-100 and LGOX enzyme concentration in the whole-cell transformation system were optimized by single-factor optimization at the shake flask level.Triton X-100 and catalase were reduced from 2.5% and 1% addition of the original system to 0.06%,and the final concentration of LGOX was reduced from 25 OD to 10 OD.The conversion rate reached 96.47%,and the whole-cell transformation system was scaled up to 5 L for validation.Finally,the total AKG yield was up to 170.6 g/L and the molar conversion rate was increased to 98.3% when the system was scaled up to 5 L at a substrate concentration of 200 g/L.(3)Since the AKG biotransformation solution contains impurities such as residual substrate,heteroproteins,organic heteroacids and pigments,and it is not easily removed by a single separation method approach.This study explored several extraction methods for the separation of AKG,such as organic solvent extraction method,ion exchange method and calcium salt precipitation method for the separation of AKG.Two combinational methods,designated as calcium salt precipitation method combined with ethyl acetate extraction of AKG(Combined method 1)and calcium salt precipitation method combined with ion exchange method(Combined method 2),were developed for more detailed separation of AKG in the transformation solution.Combined method 1: Concentrated hydrochloric acid was used to acidify the conversion solution to pH=2.2 using calcium hydroxide as a precipitant,and the neutralization reaction was carried out at a temperature of 70 ℃ and pH 8.0 to produce calcium α-ketoglutarate(CaAKG)precipitation.The reaction was carried out using concentrated sulfuric acid with CaAKG and acid digestion at pH 1 to produce calcium sulfate precipitate,and AKG was dissociated into the acid solution with a yield of 85.87%.The acid digest was extracted with ethyl acetate at room temperature,and the extraction was left overnight and repeated three times to achieve an extraction yield of 94.57% and an overall yield of 81.21%.The granulation was carried out by evaporation using a rotary evaporator at 50 ℃ with negative 0.095 MPa pressure,and a fixed mass of AKG product was resuspended,and the solid mass ratio of AKG in the solid pellet was calculated as 97.32% according to the weight method.Combination method 2: The acid solution of calcium salt precipitation method was used as ion exchange mother liquor for sample loading.The dynamic loading parameters were as follows: high diameter ratio 2.5:1,loading concentration 30 g/L,loading flow rate2.4 Bv/h,loading to resin penetration of AKG adsorption amount of 30 mg/g.The dynamic elution parameters were listed as below: eluent 0.8 M ammonia,elution flow rate 2 BV/h.The chromatographic purity of the final feed solution was improved from the initial 78.79% to 96.56%,with the yield of 91.57%.The solid mass ratio of AKG in solid particles was 82.5%.Through the exploration and optimization of the two methods,it revealed that the AKG pure product produced by the combined method 1 was closer to the index of industrial production of AKG.Through the optimization of enzyme production,optimization of whole-cell transformation,and development of methods to separate and purify AKG from transformation solution,this study constructs a complete downstream development process for the preparation of AKG product by whole-cell transformation method,which provides a guiding idea for future industrial production of AKG.