Studies On Selection Of Heat-resistant Strain Producing Hydroxylase Of Progesterone And Its Transformation Conditions

The hydroxylation of steroids by microorganisms is an important role in biotransformation of steroids. The 11α-hydroxylation is one of the key technologies in the field of steroids medical industry because of the mild conditions, high selectivity and productivity. Rhizopus nigricans is the common microorganism which products 11α-hydroxy-16α,17α-epoxy progesterone at 28℃. A great deal of cooling water was needed to maintain reaction temperature for industrial process during summer, that result in increase the consumption of energy and the cost of production. In this paper, enhancing the heat-resistant of Rhizopus nigricans were investigated.Firstly, effect of various temperatures on 11α-hydroxylation of 16α,17α- epoxy progesterone with Rhizopus nigricans SW3.41 was tested. The results showed that when the temperature over 34℃, the strain grow badly and its 11α-hydroxylated hardly. To get a strain with good conversion capacity at higher temperature, for example 34℃.heat-resistant domestication for Rhizopus nigricans SW3.41 were carried out. Meanwhile, different dosages of ⁶⁰Co irradiation were adopted upon the spore suspension of Rhizopus nigricans SW3.41. The strain SW3.41-11-51 was isolated from the mutagenized spore, which has ability of growth at 34℃and conversion 16α,17α- epoxy progesterone to 11α-hydroxy-16α,17α- epoxy progesterone at same temperature.The fermentation conditions of Rhizopus nigricans SW3.41-11-51 were investigated. Through the single factors and orthogonal test, the optimum fermentation conditions were determined: glucose 30 g/L, corn steep liquor 30 g/L, KH2PO42 g/L, fermentation temperature 34℃, initial pH 4.0, 30 mL medium in 250 mL shaking flask, rotation speed 150 r/min, To increase the conversion yield with substrate concentration of 12 g/L, the transformation conditions, such as solved oxygen, surfactant, ultrasonic irradiation, external electron acceptor and organic solvent, were also researched. The results showed that the conversion yield was increased to some extent. By comparison with original condition, the increment of conversion yield reached 39%, 62.3 %, 38% and 21.1%, respectively when adding dodecane concentration of 5%, surfactant Tween80 concentration of 5%, ultrasonic irradiate for 10 min, external electron acceptor vitamin3 concentration of 5 mg/L. Organic solvent/water two-liquid-phase system was applied to the conversion of 16α,17α-epoxy progesterone preliminary, while dodecane was used as organic phase, the conversion yield was better than others. Surfactant Tween-80 was added to conversion system, the conversion yield reached 43%. That shows it has a good industrial prospect.Wherefore, we studied the reaction kinetics of 11α-Hydroxylation of substrate...