Study On Immobilized Technology Producing Androst-4-ene-3, 17-dione By Mycobacterium Sp. BD696-6

Androst-4-ene-3,17-dione(AD) is the key intermediates of many synthetical steroid drugs.With the depletion of dioscorea resources,the bioconversion of AD is becoming the hotspot of this domain.Mycobacterium sp.BD696—6 could transform sterol to androst-4-ene-3,17-dione,but in the process of microbial transformation there are problems that the biomass concentration is too low,that the period of fermentation is too long and that the conversion rate of AD is low. Studies showed that immobilization could maintain the integrity of the cell membrane,and promote the accumulation of target products.In this paper,the immobilization of Mycobacterium sp.BD696—6 with sodium alginate and celite was studied and its fermentation condition was also optimized,then it was concluded as following.Basing on the single factor test,the immobilization condition for Mycobacterium sp BD696-6 was optimized by orthogonal tests,and the results were as following:sodium alginate 3g/100mL,calcium chloride 1g/100mL,suspended microbial cells 15mL/50mL gel,the immobilization time 1h. Compared with free cells,the fermentation period of immobilization cells was shorted,which changed to 144 hours from 168 hours,and the conversion rate of AD increased by 17.93%.The study on fermentation conditions of Mycobacterium sp.BD696—6 showed that the optimal conditions were as following:cell concentration 20mL sodium alginate/50mL medium,temperature 32℃,pH8.5,rotation speed 230r/min,the sterol concentration 5g/L,and the highest conversion rate of AD was 80.83%.The store stability and repeated fermentation were also presented.Scanning electron microscope showed that nutrientsubstrance could shuttle freely in the immobilized material with calcium alginate as embedded carrier.With celite as the adsorptive carrier,the optimum conditions for strain BD696-6 by orthogonal tests were indicted as following:the celite concentration 7g/L,the inoculation quantity 18%(V/V),the sterol concentration 3g/L.The conversion rate of AD reached to 81.47%after 168 hours,which is 19.82%higher than the free cells system.The optimization of fermentation conditions was presented:rotation speed 230r/rain,temperature 32℃,pH8.7,the quantity of glycine 4g / L.The addition of tween-80 did not accelerate the conversion rate obviously.Addition of metal ions such as Fe²⁺,Zn²⁺,Mg²⁺ clearly promoted the fermentation,the optimal conversion rate of AD reached to 85.64%,with Fe²⁺ 0.1 g/L.The result of 5L bioreactor fermentation showed that:the conversion rate of AD reached to 95.21%,with celite as carrier and 5g/L sterol as substrate.