Screening Of Microbial Strain For Biosynthsis Of3-hydroxypropionic Acid And Studies On Its Catalytic Properties

3-hydroxypropionic acid (3-HP) is one of important chemical intermediates in industry,which could be used to synthesize a variety of chemical products with high economic valueand biodegradable materials with excellent functions.3-HP was ranked at the top third amongtwelve platform chemicals derived from biomass released by the US Department of Energybased on its superior features. At now, producing3-HP by constructing genetic engineeringstrains is always the hotspot on the researches. However, there are various factors withobvious drawbacks affecting the process feasibility, such as the poor stability of strains, theenzymes invoved with low activity, the need for expensive coenzyme, purification difficultieswith many by-products. Whole-cell catalysis is one feasible method for synthesis of3-HPwith many advantages, such as high substrate selectivity, simple and efficient catalyticprocess, friendly to the environment, etc.35strains with different colony morphologies were isolated through the transparentcircle method, and their whole-cell catalysis performance of biocatalysis1,3-propanediol(1,3-PDO) to3-HP was measured separately. Finally,3strains with high-capability werescreened out by HPLC and ESI-MS analysis, and were identified as Gluconobacter oxydans,Rhodotorula mucilaginosa and Acetobacter sp. based on morphological analysis, biochemicalproperties and molecular biological identification accordingly. Besides, it is first reported thatR. mucilaginosa and Acetobacter sp. had the capability of biocatalysis1,3-PDO to3-HP.Using5g·L-1cell as biocatalyst, Acetobacter sp. could got7.18g·L-13-HP, while G. oxydansand R. mucilaginosa got6.58g·L-1,3.75g·L-13-HP accordingly under the same conditionsafter5h bioreaction。Acetobacter sp. catalytic specificity showed that the strain had best catalytic effect on1,3-PDO,11.62g·L-13-HP was obtained with the highest molar conversion rate (98.2%) everreported after7h biocatalysis using10g·L-11,3-PDO as substrate. Acetobacter sp. alsoexhibited different degrees of catalytic activity to most compounds with hydroxyl groups,such as glycerol, ethylene glycol, amyl alcohol, benzyl alcohol, phenylethyl alcohol. Whilethe strain showed no catalytic oxidation on lactic acid and1,2-propanediol. The membranecomponents and cytoplasm components catalytic activities on1,3-PDO were detectedaccordingly, and the results showed that the enzymes responsible for the catalytic oxidation of1,3-PDO to3-HP were located on the membrane of the strain. GC-MS results showed that thecatalytic reaction liquid in the m/z74,57,44have characteristic absorption peaks ofintermediate product of3-hydroxypropyl aldehyde (3-HPA). It is concluded that the catalyticreaction pathway of Acetobacter sp. is that:1,3-PDO was first bio-oxidated to3-HPA, andthen3-HPA was bio-oxidated to3-HP finally. The effects of carbon and nitrogen sources, inorganic salts on the biomass and whole-cellbiocatalytic properties of Acetobacter sp. were examined in shake flask level, and the optimalfermentation medium include glucose4g·L-1, sucrose16g·L-1, peptone10g·L-1, MgSO41g·L-1. The culture conditions for Acetobacter sp. were optimized as follows: temperature30℃,the initial pH6.0, liquid volume50mL/500mL, seed age16h, inoculation volume5%.Under the optimal conditions, Acetobacter sp. reached its maximum biomass (4.85g·L-1) andmaximum catalytic properties within24h cultivation.The bioreaction conditions for biosynthesis3-HP from1,3-PDO were also developed.The biocatalysis was conducted at30℃in0.1mol·L-1potassium phosphate buffer (pH6.0)with1%volume oleic acid added. Using5g·L-1whole-cell of Acetobacter sp. as thebiocatalyst,23.25g·L-13-HP was obtained from20g·L-11,3-PDO with molar conversion rateof98.2%after11h biocatalysis in the5mL/50mL bioreaction system. It was also found thatCa2+, Fe3+, Mg2+could promote the catalytic reaction to some extent, while the product3-HPcould inhibit the cell catalytic activity. Acetobacter sp. showed excellent reutilizationcapability when the cells were reused seven times with10g·L-11,3-PDO as substrate, and themolar conversion rate was98.2%-81.3%. The whole-cell of Acetobacter sp. had good storagestability at4℃condition, the strain could still retain93.8%catalytic activity after70hstorage.