Effect Of Hydroxypropyl-β-cyclodextrin On The Degradation Of Phytosterol By Mycobacterium

Androst-4-ene-3,17-dione(AD) and Androst-1,4-diene-3,17-dione(ADD) are the key steroid intermediates,which can be obtained from phytosterols(PS).Phytosterols utilization and the speed rate of conversion reduced because of its low aqueous solubility.And high steroid substrates and products concentration induced the substrates and products inhibition or toxicity.By complexation with hydroxypropyl-β-cyclodextrin(HP-β-CD),steroid solubility improved and the steroid substrates and products inhibition or toxicity reduced. In this paper,the bioconversion reaction of degrading the side chain of phytosterols into AD(D) by Mycobacterium sp.M2 was studieded.A complete transformation process of degradation of phytosterols was established in the present of HP-β-CD.The mechanism of phytosterols transformation in the present of HP-β-CD was preliminarily discussed.A Mycobacterium sp.strain M2-14 with higher bioconversion of PS was screened out by bioconversion tests in shake bottle after separation and purification from 3 strains of M1, M2 and M3.After 168 h conversion,the conversion reached to 63.2%when the phytosterols concentration was 3 g/L.The effect of organic solvent,surfactant and cyclodextrin conversion medium on the degradation of phytosterols into AD(D) by Mycobacterium sp.M2 was studied.A new supramolecular conversion system by adding hydroxypropyl-β-cyclodextrin(HP-β-CD) to the fermentation liquid was developed,where the reaction rate and its conversion were found to be significantly increased.After 168 h conversion,the conversion reached to 89.8%when the phytosterols concentration was 5 g/L,which was about 2.9 times compared with the control experiment.The influence of HP-β-CD on cell growth was established more in detail by the determination of cell growth rates.As a conclusion,HP-β-CD can not be used by Mycobacterium sp.M2 as the carbon source;further more,neither influenced the cell growth rate and biomass.It was found that there was no influence on the product structure when degrading the side chain of phytosterols in the present of HP-β-CD by HPLC,FTIR and NMR.The process of the bioconversion reaction of degrading the side chain of phytosterols in the present of HP-β-CD was established.It was found that the conversion of phytosterols were 85.1%-90.8%after 72 h conversion under the following optimized conditions:the optimum molar ratio of HP-β-CD to PS was 2:1,the adding time was 0 h,the phytosterols concentration was 5 g/L-10 g/L.The fermentation parameters of 7L fermentor were established.When the phytosterols concentration was 5 g/L,the conversion of phytosterols was 99%,and the yield rate of ADD and AD were 80.5%and 3.4%,respectively. The mechanism of HP-β-CD acting on phytosterols biotransformation was preliminarily discussed.The solubility of phytosterols was 27.42 mg/L when the molar ratio of HP-β-CD to PS was 2:1,which was about 41 times compared with the control experiment.HP-β-CD can reduce the product inhibition by including the product.