Study On The 11β-Hydroxylation Of Steroids By Curvularia Lunata

The hydroxylation of steroids by microorganisms belongs to the field of biotransformation. The 11 β -hydroxylation is the most important reaction in biotransformation of steroids, which inserted the oxygen in the 11β -site of the steroid molecule to products having therapeutic activity of anti-inflammatory and mithridatism. As a key biotechnics in the steroid industry, 11 β -hydroxylation by microorganisms has a history with considerable achievements over 40 years. Hydrocortisone, which is the main products of 11 β -hydroxylation reaction, is an adrennocorticosteroids drugs, and is broad utilized to cured the adrenocortical functional deficiency and immunological disease. Hydrocortisone also is an important intermediate to synthesize the other corticosteroid. As producting-hydrocortision strain, Curvularia lunata possesses strong 11 β -hydroxylation. This thesis mainly studied on the breeding of C. lunata with 11 β-hydroxylation from nature, the fermentation process of transformation compound RSA into hydrocortisone, the kinetics of 11β -hydroxylation reaction, the transformation conditions by protoplasts and the effect on conversion rate of polyoxin as inhibitors of cell wall biosynthesis of fungi.One strain of C. lunata C4 with 11β -hydroxylation was obtained form soil. Besides hydrocortisone, several by-products, such as 14 α -OH and 11 α -OH, existed in the fermentation system by TLC and HPLC. As an intermediate, Compound RS was synthesized firstly in the transformation course of compound RSA. The forming speed of 11 β -OH was decided by that of compound RS.The protoplasts from the strain C4 was treated by ultraviolet ray and DES for improving the efficiency of 11 P -hydroxylation. Mutant strain C114 was screened out, which is mutative in the morph. The colony of the strain C114 is flat and with black-green color in the surface and black in the rear on the solid medium, it has short aerial hypha and abundant spores. The effect factors on transformation of compound RSA were investigated. The optimum fermentation medium is composed of 2% glucose, 1% soybeanpowder, 0.5% peptone and 0.5% yeast extract, pH6.5. The suitable pH for growth and hydroxylation were 6.0~6.5 and 6.5, respectively. The aerostation was 160r/min from Oh to 12h, and 200r/min after 12h. The mycelium with 25 hour-old ages has the better vitality. 42.66% of compound RSA was hydroxylated to 11 3 -OH in 48h at the optimum fermentation conditions with strain C114, it's 24.5% higher than that of the initial strain C4.Wherefore, we studied the reaction kinetics of 11β -Hydroxylation of compound RSA by C. lunata, and established the kinetics equation. Parameter Vm' and Km' was 0.793 x 10~2g/h L and 0.160g/L, respectively.The relationship between the speed of hydroxylation and the procent of products was observed deeply by protoplasts of C. lunata C114. At the first, we established the conditions of formatting protoplasts. 0.6mol/L KG1 as the osmotic stabilizers, the protoplasts yield was 16.11 x 106/ml from the mycelia with 16~18 hours-old age which were digested in 1% lywallzyme and 1% celluase at 30C for 4-5 hours. We founded that protoplasts can hydroxylate compound RSA to hydrocortisone, and the speed of conversion is rapid than that of mycelia. The formatting speed of 11β -OH is 1.88-folds than that of the mycelia, and the conversion rate is 41.5%, more over 29% than mycelia. The content of 14 a -OH, a by-product, increase 3.03 times, but the content of 11 a -OH decrease 40%, correspond. It indicated that the cell wall is the barrier of steroid hydroxylation, it affect not only the speed of hydroxylation, but also the percent of products.Basis on the research conclusion of 11β -hydroxylation by protoplasts, we where after studied the effect of polyoxin as inhibitors of cell wall biosynthesis for slacking the cell wall structure on hydroxylation speed and products. The results showed that the hydroxylation speed was 1.3 times and conversion rate increased 10% than that of the contrast experiment, and the percent of...